Cardiovascular Engineering: A Protective Approach 9781260457650, 1260457656, 9781260457643, 1260457648

Table of contents :
Cardiovascular Engineering A Protective Approach
Half Title
Dedication
Title Page
Copyright
Contents
Preface
Chapter 1 Introduction
PART I Foundations of Cardiovascular Protective Engineering
Chapter 2 Development of the Heart, Blood Vessels, and Blood cells
Chapter 3 Stem Cells and Regeneration
Chapter 4 Structure and Function of the Heart
Chapter 5 Structure and Function of Blood Vessels
Chapter 6 Cytokines and Growth Factors in Cardiovascular Disease
Chapter 7 Mechanisms of Disease
Chapter 8 Systems Protective Mechanisms
Chapter 9 Protective Engineering Strategies
PART II Applications of Cardiovascular Protective Engineering
Chapter 10 Systemic Hypertension
Chapter 11 Atherosclerosis
Chapter 12 Arterial Aneurysms
Chapter 13 Coronary Heart Disease
Chapter 14 Cardiomyopathies
Chapter 15 Congenital Heart Defects
Index

Citation preview

Cardiovascular Engineering

Dedicated to my wife Yu Hua and children Diana, David, Charley, Juni, Annie, and Axel.

Cardiovascular Engineering AProtective Approach ShuQ. Liu Biomedical Engineering Department Northwestern University Evanston, IL, USA

New York Chkago San Francisco Athens London Madrid Mexico City Milan New Delhi Singapore Sydney Toronto

Copyright© 2020 McGraw Hill LLC. All rights reserved. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without the prior written permission of the publisher. ISBN: 978-1-26-045765-0 MHID: 1-26-045765-6 The material in this eBook also appears in the print version of this title: ISBN: 978-1-26-045764-3, MHID: 1-26-045764-8. eBook conversion by codeMantra Version 1.0 All trademarks are trademarks of their respective owners. Rather than put a trademark symbol after every occurrence of a trademarked name, we use names in an editorial fashion only, and to the benefit of the trademark owner, with no intention of infringement of the trademark. Where such designations appear in this book, they have been printed with initial caps. McGraw-Hill Education eBooks are available at special quantity discounts to use as premiums and sales promotions or for use in corporate training programs. To contact a representative, please visit the Contact Us page at www.mhprofessional.com. Information contained in this work has been obtained by McGraw-Hill from sources believed to be reliable. However, neither McGraw-Hill nor its authors guarantee the accuracy or completeness of any information published herein, and neither McGrawHill nor its authors shall be responsible for any errors, omissions, or damages arising out of use of this information. This work is published with the understanding that McGraw-Hill and its authors are supplying information but are not attempting to render engineering or other professional services. If such services are required, the assistance of an appropriate professional should be sought. TERMSOFUSE This is a copyrighted work and McGraw-Hill Education and its licensors reserve all rights in and to the work. Use of this work is subject to these terms. Except as permitted under the Copyright Act of 1976 and the right to store and retrieve one copy of the work, you may not decompile, disassemble, reverse engineer, reproduce, modify, create derivative works based upon, transmit, distribute, disseminate, sell, publish or sublicense the work or any part ofit without McGraw-Hill Education's prior consent. You may use the work for your own noncommercial and personal use; any other use of the work is strictly prohibited. Your right to use the work may be terminated if you fail to comply with these terms. THE WORK IS PROVIDED "AS IS." McGRAW-HILL EDUCATION AND ITS LICENSORS MAKE NO GUARANTEES OR WARRANTIES AS TO THE ACCURACY, ADEQUACY OR COMPLETENESS OF OR RESULTS TO BE OBTAINED FROM USING THE WORK, INCLUDING ANY INFORMATION THAT CAN BE ACCESSED THROUGH THE WORK VIA HYPERLINK. OR OTHERWISE, AND EXPRESSLY DISCLAIM ANY WARRANTY, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. McGraw-Hill Education and its licensors do not warrant or guarantee that the functions contained in the work will meet your requirements or that its operation will be uninterrupted or error free. Neither McGraw-Hill Education nor its licensors shall be liable to you or anyone else for any inaccuracy, error or omission, regardless of cause, in the work or for any damages resulting therefrom. McGraw-Hill Education has no responsibility for the content of any information accessed through the work. Under no circumstances shall McGraw-Hill Education and/or its licensors be liable for any indirect, incidental, special, punitive, consequential or similar damages that result from the use of or inability to use the work, even if any of them has been advised of the possibility of such damages. This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise.

Contents 1

Part I

Preface.......................................................

xv

Introduction . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview..................................................... Foundations of Protective Engineering........................ ... . Pathogenic causes and processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naturally occurring protective mechanisms . . . . . . . . . . . . . . . . . . . . . Regional protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distant protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular protective engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enhancing protective impacts by protein administration and gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppressing adverse gene expression. . . . . . . . . . . . . . . . . . . . . . . . . Gene editing-mediated control of gene expression. . . . . . . . . . . . . . Cell-based protective engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue-level protective engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 2 3 3 4 5 5 6 7 8 9 9 10 10 11 12

Foundations of Cardlovascular Protective Engineering 2 Development of the Heart, Blood Vessels, and Blood cells . . . . . . • • • llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early Embryonic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview of embryonic processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fertilization-union of the sperm and ovum. . . . . . . . . . . . . . . . . . . . . Cleavage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formation of blastocyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gastrulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of the Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin and fate of cardiogenic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of heart development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formation of the heart tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Processes of heart tube formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of heart tube formation . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac chamber formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Processes of cardiac chamber formation. . . . . . . . . . . . . . . . . . . . . . .

17 17 18 18 18 19 21 21 22 24 24 25 26 26 26 28 28 y

Yi

Contents

Regulation of cardiac chamber formation . . . . . . . . . . . . . . . . . . . . . Development of the cardiac conduction system . . . . . . . . . . . . . . . . . . Processes of cardiac conduction system development . . . . . . . . . . Regulation of conduction system development . . . . . . . . . . . . . . . . Development of the Vascular System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Origin of vascular cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vasculogenesis and angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of vascular formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Formation of smooth muscle cells and fibroblasts . . . . . . . . . . . . . . . . Development of Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29 30 30 31 31 31 32 33 35 37 40

3

Stem Cells and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Em.bryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Somatic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone marrow stem cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hematopoietic stem cells.................................... Mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resident cardiac stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MDRl-positive resident cardiac stem cells..................... Sca-1-positive resident cardiac stem cells.............. ...... .. Isll-positive cardiac stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

47 47 48 48 51 54 54 55 55 56 57 58 59 59

4

Structure and Function of the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac chambers and great vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Function of the Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Excitation-contraction coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac contractile activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of cardiac performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

67 67 68 68 70 70 73 73 74 77 79

5

Structure and Function of Blood Vessels . . . . . . . . . . . . . . . . . . . . . . . . . 81 Highlights ....................................................... 81 Structure of Blood Vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Functions of Vascular Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Functions of endothelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 84 Barrier function...................................... .... .. Ion transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 95 Protein transport.........................................

Ca nt ent s

Amino acid transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Routes of substance transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of endothelial transport function. . . . . . . . . . . . . . . . . Regulation of pro- and anti-blood coagulation processes . . . . . . . . Regulation of vascular contractility. . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of cell proliferative activities . . . . . . . . . . . . . . . . . . . . . . Regulation of inflammatory processes . . . . . . . . . . . . . . . . . . . . . . . . Generation of extracellular matrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functions of smooth muscle cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contraction and relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of vascular cell proliferative activities. . . . . . . . . . . . . . . Generation of extracellular matrix...................... ..... . Functions of fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of Systemic Blood Pressure. . . . . . . . . . . .. . . . . . . . . . . . . . . . Baroreceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chem.oreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renin-angiotensin-aldosterone system. . . . . . . . . . . . . . . . . . . . . . . . . . Vasopressin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitric oxide................................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

99 100 102 103 103 105 109 111 113 113 113 114 117 117 119 119 121 121 121 122 122 123 123

6 Cytokines and Growth Factors in Cardiovascular Disease. . . . . . . • • .

127

Highlights ...................................................... 127 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Cytoldnes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Interleukins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Interferons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Chemoldnes........................................... ..... . 137 Tumor necrosis factor superfamily factors . . . . . . . . . . . . . . . . . . . . . . . 140 Transforming growth factor ~ family proteins. . . . . . . . . . . . . . . . . . . . 142 Hematopoietins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Growth Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Epidermal growth factor and heparin-binding EGF-like growth factor.......................................... ..... . 146 Fibroblast growth factors...................................... 148 Insulin-like growth factor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Platelet-derived growth factors , ................ , , . . . . . . . . . . . . . 150 Vascular endothelial growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

7 Mechanisms of Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . , , ................ , , ................ , , . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Etiology and Pathogenic Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 161 162 162

vii

viii

Contents

External environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenic viruses....................................... Pathogenic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenic fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenic parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physical factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanical forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Radiation............................................... Body teinperature........................................ Ovemutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein overconsumption................................. Lipid and carbohydrate overconsumption . . . . . . . . . . . . . . . . . . Chemical substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cigarette smoke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alcohol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antigens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prions.................................................. Mental stress.............................................. Genetic defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomal defects ...................................... Addition of a single chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomal deletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Addition of a complete haploid set of chromosomes . . . . . . . . . Simple gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autosomaldominantdisorders .................... .... .... Autosomal recessive disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . X-linked disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Y-linked disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiple gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Senescence.................................................. Pathology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

166 166 167 175 179 181 184 185 196 197 199 199 200 201 201 202 203 204 205 205 205 207 208 208 208 209 209 209 210 212 213 214 214 214 215 216

8 Systems Protective Mechanisms. . • • . . . . . . . • • . . . . . . .. .. . . . . . .. • . . llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regional Cardioprotective Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . Early regional protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . Late regional protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . Protective inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distant Protective Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distant protective factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

225 225 226 229 229 231 232 233 233

Contents

Discovery and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Induction of distant protective factors . . . . . . . . . . . . . . . . . . . . . . . . Actions of distant protective factors . . . . . . . . . . . . . . . . . . . . . . . . . . Significance of distant protective factors . . . . . . . . . . . . . . . . . . . . . . Distant cell mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone marrow cell mobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Splenic cell mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hepatic cell mobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of hepatic cell mobilization . . . . . . . . . . . . . . . . . . . . Protective actions of mobilized hepatic cells . . . . . . . . . . . . . . . . . The Concept of Core Distant Protective Mechanisms. . . . . . . . . . . . . . . . The Concept of Protectome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evolution of Protective Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Systems Protective Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

233 235 236 237 238 238 239 240 240 242 243 245 245 248 249

9 Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Understanding Pathogenic and Protective Mechanisms . . . . . . . . . . . . . Identifying mutant or malfunctioned genes by gene profiling. . . . . . Constructing recombinant genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Testing the function of the gene of interest. . . . . . . . . . . . . . . . . . . . . . . Molecular Protective Engineering Technologies . . . . . . . . . . . . . . . . . . . . Gene transfer technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V:rrus-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Retrovirus-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . Adenovirus-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . Adeno-associated viruses as gene carriers . . . . . . . . . . . . . . . . . . . Receptor-mediated gene transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liposome-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . Calcium phosphate-mediated gene transfer . . . . . . . . . . . . . . . . . . . Electroporation-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . Assessing the expression level of the transferred gene. . . . . . . . . . . Assessing the function of the transferred gene . . . . . . . . . . . . . . . . . Gene editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene editing by zinc finger nucleases. . . . . . . . . . . . . . . . . . . . . . . . . Gene editing by transcription-activator like effector nucleases . . . Gene editing by the CRISPR/ Cas system. . . . . . . . . . . . . . . . . . . . . . Epigenetic modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNAmethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histone modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Histone acetylation................................. ...... Histone methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Post-transcriptional modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

257 257 257 258 258 260 261 261 262 262 262 262 263 264 264 264 265 265 266 266 267 268 269 270 271 272 273 274 275 276

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Small interfering RNA-based mRNA modifications. . . . . . . . . . . . . MicroRNA-based mRNA modifications. . . . . . . . . . . . . . . . . . . . . . . Differences between siRNAs and miRNAs . . . . . . . . . . . . . . . . . . . . Anti-sense oligonucleotide repression of mRNA . . . . . . . . . . . . . . . Cell-Based Protective Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tissue-Level Protective Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Part II

276 277 279 279 280 281 282

Appllcatlons of Cardlovascular Protective Engineering 10

11

Systemic Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Primary hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renovascular hypertension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endocrine hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional Treatment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Protective Engineering Strategies for Primary Hypertension . Boosting vasodilator gene expression. . . . . . . . . . . . . . . . . . . . . . . . . . . Nitric oxide synthase gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natriuretic peptide precursor A gene . . . . . . . . . . . . . . . . . . . . . . . . . Kallikrein and kininogen genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppressing vasoconstrictor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renin-angiotensin-aldosterone system genes . . . . . . . . . . . . . . . . . . Endothelin gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adrenergic receptor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

289 289 290 290 290 295 295 296 297 298 299 299 300 300 300 300 301 301 302

Atherosclerosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

305 305 305 306 306 306 307 307 308 308 308 309 310 310 312 313

llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene mutations responsible for atherogenesis . . . . . . . . . . . . . . . . . . . Lipid metabolism regulatory genes . . . . . . . . . . . . . . . . . . . . . . . . . . Low-density lipoprotein receptor (LDLR) gene . . . . . . . . . . . . . . Low-density lipoprotein genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . LDLR regulatory genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blood pressure regulatory genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Renin-angiotensin system genes . . . . . . . . . . . . . . . . . . . . . . . . . . Sodium regulatory genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atrial natriuretic factor gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adrenergic system genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endothelin genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nitric oxide synthase genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Contents

Inflammation regulatory factor genes . . . . . . . . . . . . . . . . . . . . . . . . Genes regulating vascular cell proliferation and migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atherogenic environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid-rich diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Exposure to cigarette smoke and toxins....................... Regional mechanical factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidant deficiency................................ .... .. Superoxide free radical o;-................................ Reactive nitrogen species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transition metal ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Redox regulatory enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antioxidants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sedentary lifestyle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atherogenic disorders........................................ Obesity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypercholesterolemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypertension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lipid retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell proliferation and migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathological changes during the initial stage. . . . . . . . . . . . . . . . . . . . . Pathological changes during the developing stage. . . . . . . . . . . . . . . . Pathological changes during the advanced stage . . . . . . . . . . . . . . . . . Conventional Treatment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-hyperlipidemia agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-smooth muscle cell proliferative agents. . . . . . . . . . . . . . . . . . . . . Alleviating angina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relieving cardiac workload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angioplasty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stent placement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Arterial reconstruction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular Protective Engineering Strategies....................... siRNA-mediated silencing of inflammatory and mitogenic genes................................................. ..... . Anti-inflammatory and anti-proliferative gene therapy . . . . . . . . . . . Gene editing-based anti-atherogenic strategies . . . . . . . . . . . . . . . . . . Cell- and Tissue-level Protective Engineering Strategies. . . . . . . . . . . . . Cell-integrated extracellular matrix constructs................... Cell-integrated polymeric vascular constructs . . . . . . . . . . . . . . . . . . . Development of arterial constructs in vivo . . . . . . . . . . . . . . . . . . . . . . Mechanical modifications of arterial constructs . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

313 313 315 315 316 316 318 319 319 319 320 320 321 321 321 322 322 322 323 323 325 326 327 327 327 327 328 328 328 329 329 329 330 330 331 331 332 333 333 334 335 336 336 338

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12

Arterial Aneurysms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . ' ' ................ ' ' ................ ' ' . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomechanics of Arterial Aneurysms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular protective engineering strategies ..... , . . . . . . . . . . . . . . . Cell-based protective engineering strategies . . . . . . . . . . . . . . . . . . . . . Tissue-level protective engineering strategies . . . . . . . . . . . . . . . . . . . . References .......................................... ......... .

345 345 346 346 349 349 351 351 353 354 355

13

Coronary- Heart Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . ' ' ................ ' ' ................ ' ' . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coronary artery thrombosis . , ................. , . . . . . . . . . . . . . . . Coronary artery atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Coronary artery embolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology and Pathophysiology , , ................ , , . . . . . . . . . . . . . Acute myocardial infarction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Impairment of myocardial function . . . . . . . . . . . . . . . . . . . . . . . . . . . . Forms of cardiomyocyte death ................. ,........ ....... Necrotic cell death......................................... Apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy .............. , , ................ , , . . . . . . . . . . . . . Acute inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fibrosis and angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic and Adverse Impacts of Blood Reperfusion. . . . . . . . . . . . . Naturally Occurring Mechanisms of Myocardial Protection. . . . . . . . . . Conventional Treatment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardioprotective Engineering Strategies ........... , , . . . . . . . . . . . . . Molecular cardioprotective engineering strategies . . . . . . . . . . . . . . . Protection against cardiomyocyte death....................... Regional cardioprotective factors ........... , , . . . . . . . . . . . . . Distant cardioprotective factors . . . . . . . . . . . . . . . . . . . . . . . . . . . Protection against oxidative stress injury. . . . . . . . . . . . . . . . . . . . . . lschemic preconditioning .. , , ................ , , . . . . . . . . . . . . . Ischemic post-conditioning.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enhancing angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-based protective engineering strategies ..... , . . . . . . . . . . . . . . . Tissue-level cardioprotective engineering strategies . . . . . . . . . . . . . . References....................................................

359 359 360 361 361 361 362 362 362 364 364 365 366 367 368 368 368 369 369 370 370 372 372 373 373 375 376 376 376 377 377

14

Cardiomyopath.ies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Highlights . ' ' ................ ' ' ................ ' ' . . . . . . . . . . . . . 381 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381

Ca nt ent s

15

Dilated Cardiomyopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology and clinical features. . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional treatment strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . Hypertrophic Cardiomyopathies................................. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology and clinical features. . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional treatment strategies.............................. RestrictiveCardiomyopathies ............................. ..... . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology and clinical features. . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional treatment strategies.............................. Arrhythmogenic Right Ventricular Cardiomyopathy. . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathophysiology and clinical features. . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional treatment strategies....................... .... ... Heart Failure.................................................. Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathology and pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional treatment strategies.............................. Molecular Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . Modification of mutant genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Management of ventricular arrhythmia . . . . . . . . . . . . . . . . . . . . . . . . . Prevention of heart failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-Based Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . Cell-mediated delivery of cardiomyocyte supporting factors. . . . . . . Stem cell-based cardiomyocyte regeneration . . . . . . . . . . . . . . . . . . . . Tissue-Level Protective Engineering Strategies. . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

382 382 383 384 385 385 385 387 388 389 389 389 390 391 391 392 392 392 392 393 394 394 394 395 396 396 397 398 399 399 400 400 401 402

Congenital Heart Defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chromosomaldefects ........................................ Single gene mutations........................................ Environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disorders causing congenital heart defects . . . . . . . . . . . . . . . . . . . . . . Pathology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Left to right heart shunts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Atrial septum defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

407 407 407 408 408 410 411 412 413 413 413

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Ventricular septum defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patent ductus arteriosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heart valve defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitral valve defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tricuspid valve defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aortic and pulmonary valve defects . . . . . . . . . . . . . . . . . . . . . . . . . Complex defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional Treatment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Left-to-right heart shunts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heart valve defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complex defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Engineering-Based Interventions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomaterial-based heart valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological tissue-based heart valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell engineering-based heart valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials used for cell engineering-based heart valves . . . . . . . . . . Cell types used for heart valve engineering. . . . . . . . . . . . . . . . . . . . Improving the performance of engineered heart valves . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

414 414 416 416 416 417 417 418 418 419 419 420 420 420 421 421 422 423 424

Ischemic Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis.................................................. Pathology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Naturally Occurring N europrotective Mechanisms . . . . . . . . . . . . . . . . . Regional neuroprotective mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . Distant neuroprotective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . Naturally occurring neuronal regeneration................ ...... Conventional Treatment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroprotective Engineering Strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular neuroprotective engineering strategies . . . . . . . . . . . . . . . . Modulating cell survival signaling mechanisms . . . . . . . . . . . . . . . Modulating cell death signaling mechanisms . . . . . . . . . . . . . . . . . . Preventing secondary injury........................... ...... Inducing neural resident stem cell differentiation . . . . . . . . . . . . . . Preventing fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Engineering distant neuroprotective mechanisms . . . . . . . . . . . . . . Ischemic preconditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell-based engineering strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

429 429

430 431 432 434 435 440 441 441 442 442 442 444 444 445 446 446 447 448 448

In.dex . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . .

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Preface he theme of this book is Protective Engineering, an emerging bioengineering discipline aiming to develop engineering strategies and technologies for inducing and optimizing bio-protective processes and thereby facilitating recovery from injury and disorders. The concept of protective engineering stems largely from the naturally occurring protective mechanisms established against genetic defects and environmental insults through evolution. Although the natural protective mechanisms are critical to the life of organisms, not all these mechanisms are optimized in promptness and effectiveness, supporting the necessity of engineering-based modulations for enhancing protection. Various protective engineering strategies, such as gene transfer, gene editing, gene silencing, cell transplantation, and tissue reconstruction, have been designed and used to induce and modify protective processes and correct natural deficiencies for therapeutic purposes in experimental and clinical research. To date, there is a large amount of information about the naturally occurring protective mechanisms as well as protective engineering strategies in literature with an increasing clinical impact, prompting the establishment of Protective Engineering as a discipline. This book is designed to introduce to students and scientists the principles, foundations, and strategies of protective engineering by using cardiovascular disorders as models. This book includes two parts-Foundations and Applications of Cardiovascular Protective Engineering. The first part covers development of the cardiovascular system, stem cells and regeneration, structure and function of the cardiovascular system, signaling processes of cytokines and growth factors in cardiovascular disease, mechanisms of disease, naturally occurring systems protective mechanisms, and general protective engineering strategies. These aspects are the bases of cardiovascular protective engineering. The second part highlights application of protective engineering to several prevalent cardiovascular disorders, including hypertension, atherosclerosis, arterial aneurysms, coronary heart disease, cardiomyopathies, congenital heart disease, and ischemic stroke. The author hopes that this book helps readers understand the concept of cardiovascular protective engineering. This book cannot be established without the support of the investigators who have made contributions to the field of protective engineering. The author would like to

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Preface express sincere appreciation and gratitude to these investigators. The author would also like to thank Dr. Y. C. Fung, who brought the author to the field of Bioengineering and taught him how to become a teacher and a scientist. ShuQ. Liu July 31, 2019 Evanston, Illinois, USA

CHAPTER

1

Introduction Hlghllghts • Cardiovascular engineering is a broad subject addressing the modulation of the structure and function of the heart and blood vessels at the molecular, cellular, tissue, and organ levels to prevent and treat cardiovascular disease. This book focuses on Gardiovascu/ar Protective Engineering, an emerging discipline of cardiovascular engineering, aiming to understand the naturally occurring protective mechanisms against injury and disorders and developing engineering strategies for inducing and optimizing protective processes, thereby facilitating recovery from disease. • The naturally occurring protective mechanisms are the foundation of protective engineering. There are two types of protective mechanism-regional and distant mechanisms, both activated in response to environmental insults and/or genetic defects. The regional protective mechanisms are those occurring within the disordered organ; whereas the distant protective mechanisms are those activated in remote organs to protect the disordered organ from structural and functional failure. Regional protective mechanisms include disorder-activated expression and/or release of paracrine protective factors (e.g., adenosine, growth factors, and cytokines), inflammatory responses, and resident stem cell differentiation into functional cells. Distant protective mechanisms include upregulation and secretion of endocrine protective proteins and mobilization of distant cells to the disordered organ to discharge protective factors. Both regional and distant mechanisms are collectively defined as systems protective mechanisms. • Protective engineering strategies can be developed and used to optimize and induce protective processes at three levels with currently available technologiesmolecular, cellular, and tissue levels. Molecular protective engineering is to induce and promote protective gene expression, suppress adverse gene expression, and control signaling processes to facilitate recovery from injury and disorders. Cell-based protective engineering is to provide needed cell types for targeted delivery of protective factors and regeneration of functional cells. Tissue-level protective engineering is to provide structural and functional supports to an injured or disordered organ to facilitate recovery and prevent organ failure.

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Chapter One

Overview Cardiovascular engineering is a broad subject addressing the modulation of the structure and function of the heart and blood vessels at the molecular, cellular, tissue, and organ levels by using engineering strategies to prevent and treat cardiovascular disease. This book focuses on Cardiovascular Protective Engineering, an emerging discipline of cardiovascular engineering, aiming to understand the naturally occurring protective mechanisms against cardiovascular disorders and developing engineering strategies for optimizing and inducing protective processes, thereby facilitating recovery from disorders. All organisms possess protective mechanisms against genetic defects and environmental insults. These mechanisms develop during evolution at all structural levels-molecular, cellular, organ, and system levels (Liu et al., 2015; Liu, 2019). Examples of molecular protective mechanisms include homologous recombination for repairing double-strand DNA breaks induced by irradiation and chemical agents ijasin and Rothstein, 2013; Cannan and Pederson, 2016) and protective gene expression in response to injury (Liu et al., 2015; Llu, 2019). Cellular protective mechanisms include cell proliferation and differentiation to prevent organ failure in injury and disorders (Llu, 2007; Llu et al., 2015). At the organ and systems levels, inflammatory responses are a representative example for preventing microorganism infections, stimulating cell regeneration and extracellular matrix generation, and facilitating repairing processes (Rock and Kono, 2008; Chen et al., 2018). However, naturally occurring protective mechanisms are not all optimized in promptness and effectiveness. In selected cases, injured cells and organs cannot be completely regenerated and repaired, resulting in organ failure. For instance, lethal gene mutations occur, causing genetic disorders, in spite of the presence of gene repair mechanisms; the expression of protective genes often lags behind injury, missing the early period of optimal protection (Llu et al., 2015); vital cells, such as the neuron and cardiomyocyte, possess a limited capacity of protection and are largely replaced with fibrotic tissue in the event of injury and death; and inflammatory responses are generally over-activated to cause excessive extracellular matrix production and fibrosis, imposing adverse effects on cell and organ functions (Rock and Kono, 2008; Liu et al., 2015). Protective engineering is developed to correct these natural deficiencies by inducing and optimizing protective processes, thereby maximizing the capacity of protection. Protective engineering is closely related to another bioengineering discipline-regenerative engineering (Liu, 2007; Gardiner, 2018; Laurencin and Khan, 2018). Protective engineering is to prevent cells from death in injury and disorders, whereas regenerative engineering is to reproduce cells after cell death. In nature, protection and regeneration are two continuous, collaborative mechanisms that prevent detrimental consequences in harsh environments, ensuring the survival of disordered cells, organs, and ultimately the entire organism. In a broader sense, regenerative engineering is protective-to protect organs and the organism from death by reproducing cells and tissues. Thus, regenerative engineering can be considered an integral part of protective engineering. The ultimate goals of protective engineering are to alleviate cell injury, support cell survival, promote and control cell regeneration, and restore the structure and function of disordered organs to their natural forms. Protective engineering strategies and technologies can be designed and used to achieve such goals. In this book, the cardiovascular system is used to demonstrate the principles and applications of protective engineering.

Chapter One Cardiovascular disease is the leading cause of human morbidity and mortality. A variety of protective and regenerative engineering strategies and technologies have been established for cardiovascular disorders, such as atherosclerosis, arterial aneurysms, coronary heart disease, and cardiomyopathies. Protective engineering-based interventions, such as protein and gene therapies, cell transplantation, and arterial stenting and reconstruction, have been proven effective in experimental and clinical investigations. This book addresses the foundations, strategies, technologies, and application of protective engineering to cardiovascular disorders. The foundations, strategies, and technologies of protective engineering will be discussed in Chapters 1 through 9, and protective engineering applications will be covered in Chapters 10 through 16. Here, the foundations and general strategies of cardiovascular protective engineering are outlined by using myocardial ischemia-reperfusion injury as a model.

Foundations of Protective Engineering The foundations of protective engineering are the etiology, pathology, and pathophysiology of disease, including the naturally occurring protective mechanisms against disorders. It is essential to understand the pathogenic processes, pathological changes, and protective actions in disorders for the development of protective engineering strategies. In response to a pathogenic cause or process, natural protective mechanisms are activated to protect cells from injury and death, thereby reducing the impact of the disorder. Protective engineering strategies can be designed based on the natural protective mechanisms and used to induce and optimize protective actions, thereby suppressing pathogenic processes and facilitating recovery from the disorder.

Pathogenic causes and processes Adisorderoccursinresponsetopathogeniccause(s),includinggenemutation(s)and/or environmental insult(s). Identifying the pathogenic cause(s) and understanding the pathogenic mechanisms are the first tasks for designing protective engineering strategies. For instance, ischemic myocardial injury is caused largely by coronary artery atherosclerosis and/ or embolism that block coronary blood flow. Once occurring, a coronary intervention, such as coronary angioplasty, stent placement, or reconstruction, can be used to remove the blockade and reperfuse the ischemic myocardium, an effective approach to rescue the ischemic myocardium from infarction. When myocardial injury occurs, preventing cardiomyocyte death becomes the priority of treatment. Various tumor necrosis factor superfamily genes are upregulated in inflammatory responses to ischemia, contributing to cardiomyocyte death. Thus, suppressing the expression of these genes by RNA interference can reduce cardiomyocyte death. Concurrently, growth factors, such as epidermal growth factors (EGFs), fibroblast growth factors (FGFs), and hepatocyte growth factor (HGF), can be used to prevent cardiomyocyte death by activating cell survival signaling networks. When myocardial infarction occurs, the priority of treatment is to induce and promote cardiomyocyte regeneration to replenish the lost myocardium by applying regenerative factors to stimulate the differentiation of cardiac resident stem cells and transplanting exogenous stem cells to boost cardiomyocyte formation. These examples emphasize an important point-understanding the pathogenic causes and processes is the basis for developing protective engineering strategies.

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Chapter One

Naturally accunlng protective mechanisms There exist a variety of natural protective m.echanisms in mammalian systems, as demonstrated through the book, serving as foundations for developing protective engineering strategies. Natural protective m.echanisms evolve in response to genetic defects and environmental insults. Here, myocardial ischemia-reperfusion injury is used as an example to demonstrate how the natural protective m.echanisms act agalnst cardiomyocyte death. Myocardial ischemia causes myocardial infarction, but can also activate innate protective m.echanisms to prevent ca.rdi.omyocyte death. Two types of such mechanism have been identified-regional and distant protective mechanisms (Liu et al., 2015; Liu, 2019), which are collectively defined as systems protective mechanisms (Fig. 1.1). The regional mechanisms include upregulation and release of paracrine protective factors, leukocyte activation and infiltration, resident stem cell activation

Distant

protective I"'-+

mechanisms

+-Kidney

Lung

~-~ '

Intestines

Spleen

'

Bone marrow

F11uR! 1.1 Naturally occurring systems protective mechanisms, including regional protective mechanisms In the lschemlc heart and distant protective mechanisms from non·lnjured organs. The outer oval shows the coverage of cytoklnes and endocrine factors released from the lschemlc cardiac cells and activated leukocytes; the center vertical oval indicates the coverage of distant protective mechanisms, involving endocrine factors and cells mobilized from distant organs: and the top small oval indicates the coverage of regional protective mechanisms from the ischemic heart. The thick arrows represent distant protective mechanisms from organs confirmed In experimental tests, and the thin arrows indicate potential distant protective mechanisms from organs that have not been experimentally confinned. (From Liu, 2019, by permission.)

Chapter One and differentiation, and fibroblast proliferation and extracellular matrix production (Liu et al., 2015; Liu, 2019). The distant mechanisms include upregulation and release of endocrine protective factors and mobilization of protective cells from the bone marrow, spleen, liver, and potentially other organs (Swirski et al., 2009; Liu et al., 2015; Liu, 2019). Both regional and distant cardioprotective mechanisms act in coordination and synergy to protect cardiomyocytes from death and facilitate recovery from ischemic myocardial injury.

Regional protective mechanisms Various protective mechanisms are present within each organ and can be activated to protect cells from injury and death. In the heart, for instance, one mechanism is the expression and release of paracrine protective factors from injured cells, including adenosine, opioids, bradykinin, hepatocyte growth factor (HGF), insulin-like growth factor (ILGF), vascular endothelial growth factor (VEGF), interleukin 6 (IL6), and stromal cellderived factor 1 (SDF1), in response to ischemic myocardial injury (Banai et al., 1994; Liu et al., 2011a; Yellon and Downey, 2003; Granfeldt et al., 2009; Vmten-Johansen and Shi, 2011). Based on the time of action, these factors are divided into two classes: early and late cardioprotective factors. Early factors include adenosine, opioids, and bradykinin that are released from injured cardiac cells during the first several hours following ischemic myocardial injury without the involvement of de novo gene expression, and the others are considered late factors that are produced and released within several days, requiring de novo gene expression (Yellon and Downey, 2003; Vinten-Johansen and Shi, 2011; Liu et al., 2015). The time-dependent actions of these multiple factors provide a wide timeframe of protection. However, the ischemic myocardium may not be well protected during the early period prior to the release and action of the protective factors, and not all protective factors can reach optimal levels for effective protection (Yellon and Downey, 2003; Liu et al., 2015; Liu, 2019). Correction of these deficiencies is a major task of cardioprotective engineering. Distant protective mechanisms Distant protective mechanisms include expression and release of endocrine factors and mobilization of protective cells from distant organs in response to a regional injury or disorder. In ischemic myocardial injury, for instance, endocrine protective factors can be expressed and released from the liver and adipose tissue (Liu et al., 2012, 2013). The liver-derived protective factors include al-acid glycoprotein type 2 (AGP2), bone morphogenetic protein-binding endothelial regulator (BMPER), fibroblast growth factor 21 (FGF21), neuregulin 4 (NRG4), and trefoil factor 3 (TFF3) (Liu et al., 2012), whereas the adipose tissue expresses and releases FGF21 (Liu et al., 2013). These factors are able to reach the ischemic cardiomyocytes via the circulatory system when the endothelial permeability is increased in ischemia-induced inflammation, supporting the cardiomyocyte survival (Liu et al., 2012, 2015; Liu, 2019). Administering each of these factors reduces the rate of myocardial infarction, and administration of all these factors in combination further boosts the impact of these factors, supporting the cardioprotective role of the liver- and adipose tissue-derived endocrine factors (Liu et al., 2012). Cell mobilization from non-injured organs is another distant protective mechanism activated in response to injury. Ischemic myocardial injury has been used as a model to study this type of mechanism. Several organs can mobilize their cells into the circulatory system, including the bone marrow (Ripa et al., 2006; Fazel et al., 2008),

5

6

Chapter One

(

.

""\ IL6

~~ \

HepaUc cells

Leukocytes

F11uRE 1.2 Regulatory mechanisms of hepatic cell moblllzatJon In response to lschemlc myocardial injury.

spleen (Swirski et al., 2009), and liver (Liu et al, 2011b, 2015), in experimental ischemic myocardial injury. The mobilized cells are can e:ngraft to the .isc:hemic myocardium, exerting cardioprotective actions. From the bone marrow, hematopoietic: stem cells and endothelial progenito.rs can be mobilized in isc:hemic myocardial injury an!L once reaching the ischemic: myocardium, can release c:ytokines and growth factors, reducing myocardial infarction (Ripa et al., 2006; Fazel et al., 2008). Bone marrow-derived endothelial proge:nito.r cells can dllfere:ntiate into endothelial cells, facilitating angiogenesis (Shintani et al., 2001). Ischemic myocardial injury can also cause mobilization of splenic mo.nocytes to the circu.latory system and ischemic myocardium to regulate inflammatory .responses and promote recovery from i.schemic: myocardial injury (Sw.irski et al, 2009). Jn addition, the liver can mobilize cells to the c:U:culatory system in response to ischemic myocardial injury (Liu et al, 2011b, 2015). Major cell types mobilized include hepatocytes and biliary epithelial cells (Liu et al., 2011b, 2015). The mobilized hepatic cells can enter the ischemic: myocardium, contributing to myocardial protection and repair by expressing and releasing cardioprotective proteins, as discussed previously (Fig. 1.2). With the understanding of the distant protective mechanisms, protective engineering strategies can be developed and used to modify non-injured organs to maximize the protective impact, an approach. to avoid intervention-induced injury of the ischemic heart.

Protective Engineering Strategies Protective engineering strategies can be developed and used to induce protecti.vepnxesses and optimize protective impacts at three levels with currently available technologiesmolecular, cellular, and tissue levels (Fig. 1.3). Molecular protective engineering is to modify the types, levels, activation timing, and coordination of protective facto.rs by controlled protein delivery, gene transfer, gene editinSt RNA interference, and/ or other molecular modulation strategies, thereby boosting cell protection. Cellular protective engineering is to provide needed cell types for targeted delivery of protective factors

Chapter One Cellular

Molecular Protein delivery

Fibroblasts

~ I

Gene transfer

0

~~

Esc~;esc,

Gene editing

Tissue Guide RNA RNA interference

F11uRE 1.3 Molecular, cellular, and tissue-level protective engineering strategies. The protein structure presented in the Molecular column represents vascular endothelial growth factor (RCSB PDB # 2VPF) (Muller et al., 1997). PAM: Protospacer adjacent motif. RISC: RNMnduced sllenclng complex. slRNA: Small Interfering RNA.ESCs: Embryonic stem cells. IPSCs: Induced pluripotsnt stem cells.

and regeneration of functional cells. Tissue-level protective engineering is to provide structural and functional support to an injured or disordered organ to facilitate recovery and prevent organ failure. Overall, these molecular, cellular, and tissue-level engineering strategies can be designed and used for the precise control of the types, levels, timing, and coordination of systems protective actions, thereby optimizing the protective meclumisms, minimizing cell death, and facilitating recovery from injury and disease.

Molecular protective engineering There are several molecular strategies for protective engineering-protein administration, gene transfer, gene editing, and RNA interference. Proteins can be directly delivered to target cells to initiate rapid protective actions (within seconds) and are comm.only used immediately following the onset of an injury or disorder. Gene transfer is an approach to introduce a gene into target cells to boost and sustain gene expression. Gene editing is a method to modify gene structures and introduce an exogenous

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Chapter One gene into the genome to replace a malfunctioned target gene, resulting in a permanent replacement of the target gene. RNA interference is to temporarily suppress mRNA translation to proteins. These strategies can be used to boost or suppress gene expression and cell activities, depending on the functions of the target genes and the nature of the disorder. Fundamental engineering procedures include mRNA isolation from a cell source, target gene identification by mRNA profiling, mRNA conversion into cDNAs, establishment of recombinant genes, recombinant gene amplification, gene function tests in vitro, gene modifications by gene transfer, gene editing, or RNA interference, and gene function tests in vivo.

Enhancing protectire impacts by protein administtation and gene ttansfer Protein administration and gene transfer represent treatment strategies for initiating prompt protective actions and sustaining protective gene expression, thereby reducing cell death, enhancing cell regeneration, and facilitating recovery from an injury or disorder (Dunbar et al., 2018; Liu, 2019). In ischemic myocardial injury, for instance, a number of protective genes are upregulated and released, including the VEGF, HGF, EGF, and FGF genes (Nakamura et al., 2000; House et al., 2005; Messadi et al., 2014; Folino et al., 2018). Whereas these genes contribute to cardioprotection, their expression (within 1-2 days) lags behind the onset of cardiomyocyte injury and death (within minutes to hours, depending on the degree of injury) and does not reach optimal levels for effective protection. These natural deficiencies can be overcome by targeted delivery of protective proteins immediately following an insult, an approach initiating protective actions prior to cell injury and death, and by transferring genes encoding the protective proteins into injured or disordered cells, an approach sustaining the expression and release of protective proteins. The biological foundation of gene transfer is that mammalian cells are capable of endocytosing genes and the endocytosed genes can be expressed once reaching the nucleus (Liu, 2007; Dunbar et al., 2018). However, the rate of gene endocytosis is low. Thus, plain gene transfer is not effective to produce sufficient proteins for protection. A number of methods have been developed and used to facilitate gene transfer into target cells in vivo, including virus-, liposome-, and receptor-mediated gene transfers (Dunbar et al., 2018; Liu, 2019). Vrruses are the most effective mediators for gene transfer because they are naturally equipped with machineries for host cell infection and viral gene integration into the host genome. Protective genes can be integrated into the viral genome by recombinant biotechnology and introduced to target cells via virus infection. Liposomes are phospholipid vesicles that can bind DNA fragments and fuse into target cells to facilitate gene transfer. Selected protein ligands, such as transferrins, can form complexes with gene fragments via linkers and bind to cognate receptors to cause ligand-gene-receptor internalization and facilitate gene transfer. Protective genecontaining carriers can be delivered into a target tissue and organ by direct injection or mediation of various carriers, such as nanoparticles, capsules, gel patches, biological matrix, or synthetic polymer scaffolds. The carrier-mediated approach can be used to provide controlled and sustained release of proteins and genes. The effectiveness of molecular protective engineering strategies depends on the types, levels, timing, and coordination of protective factors and genes selected for delivery. In principle, genes that are activated in response to an injury or disorder and directly involved in natural protective processes are the candidates of choice. These genes can be identified by a systematic differential gene expression profiling approach

Chapter One such as RNA sequencing (RNA-seq). A challenge for this approach, however, is the cumbersome analysis of a large amount of information with a large number of injuryaltered genes from a gene profiling test. An important task is to identify the most effective genes that can be used for protective therapies. One practical approach is to classify the upregulated or downregulated genes into functional categories, such as secreted protective protein genes (for instance, growth factor and cytokine genes), receptor genes, protein kinase genes, transcription factor genes, and others. The next step is to screen the genes of a selected category by using functional assays in vitro or in vivo. For in vitro assays, each selected gene can be introduced to cultured cells subjected to an insult; and the protective impact is evaluated based on the rate of cell survival or death under a given insult. The most effective protective genes can be selected by comparison analyses between different genes. For in vivo assays, a disorder model such as ischemic myocardial injury can be induced in an animal model, and a similar protocol can be used to identify the most effective protective genes based on various measures, such as the fraction of myocardial infarction, the rate of cardiac cell death, and the relative activities of caspases 3, 8, and 9. Proteins encoded by the selected genes can also be used for these tests with or without concurrent gene transfer.

Supptessint adverse dflne expression A disorder causes expression and release of not only protective genes, but also adverse genes. For instance, ischemic myocardial injury causes expression of not only growth factor genes for cell protection, but also tumor necrosis factor superfamily genes responsible for cell death induction, a mechanism to facilitate the death of severely injured cells that cannot be rescued. An important task of protective engineering is to control the expression of the adverse genes to inhibit their cell death-inducing activities. The RNA interference method by using small interfering RNAs (siRNAs) can be used to silence adverse genes (Xia et al., 2002; Watts and Corey, 2012). An siRNA is a doublestranded RNA sequence about 21-22 base pairs in length, originally discovered in the plant petunias (Napoli et al., 1990; van der Krol et al., 1990) and the nematode worm C. elegens (Gu et al., 2012). siRNAs are capable of recognizing and interacting with specific target mRNAs based on the complementary principle, cleaving or blocking the target mRNAs, and thereby knocking down the synthesis of corresponding proteins (Novina and Sharp, 2004). These features have made RNA interference a useful approach for post-transcriptional silencing of selected target genes. An siRNA can be constructed based on the sequence of a selected gene and delivered to target cells for gene silencing. Once inside the cell, a double-stranded siRNA can form a complex with a protein structure known as RNA-induced silencing complex (RISC). The RISC can separate the two siRNA strands, reject the sense strand, and keep the anti-sense strand. The latter can bind to a target mRNA, and the associated RISC can cleave the target mRNA into small fragments, thereby preventing protein translation.

Gene editint-mediated control of dene expression Gene editing is another potential engineering approach that can be used to enhance the expression of protective genes and suppress the expression of adverse genes. A representative gene editing process is CRISPR/Cas-based gene editing, originally found in bacteria and archaea for protection against viral and plasmid infection (Horvath and Barrangou, 2010). This mechanism can be used to edit the genome of mammalian cells. The principle of action is to cause double strand DNA breaks by a CRISPR/Cas system,

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Chapt er One a process activating homologous recombination and causing insertion of an exogenous gene into the host genome. The CRISPR/Cas9 system has been developed and used for such a purpose (Deltcheva et al., 2011; Jinek et al., 2012). This system consists of three basic elements----CRISPR RNA (crRNA), trans-activating crRNA (trRNA), and Cas9 Oinek et al., 2012). crRNA is able to recognize a target gene to be edited; trRNA is a sequence partially complementary to the crRNA required for Cas9 action Oinek et al., 2012); and Cas9 is an enzyme causing double strand DNA breaks Ginek et al., 2012). Gene editing can be initiated by simply introducing crRNA, trRNA, and Cas9 into the target cells to induce double-strand DNA breaks on a target gene at sites recognized by the gene-specific crRNA (Jinek et al., 2012). In the presence of an exogenous gene (introduced into cells together with CRIPR/Cas9), double strand DNA breaks activate the homologous recombination mechanisms, causing substitution of the introduced exogenous gene for the target gene (Gong et al., 2005; Overballe-Petersen et al., 2013). In the absence of an exogenous gene, double strand DNA breaks cause non-homologous DNA end joining to repair the damage. By using these mechanisms, a protective gene with enhanced expression capacity (induced by using a stronger gene promoter) can be introduced to a specified target gene locus by CRISPR/Cas editing to boost cardioprotective gene expression. A gene imposing an adverse impact on protection can be replaced by a modified gene with reduced expression capacity. Thus, CRISPR/Cas editing is a potentially effective method for molecular protective engineering.

Cell-based protective engineering Cell-based protective engineering strategies can be designed and used to induce and promote cell regeneration and deliver protective factors to target cells. Fundamental engineering procedures include cell identification, isolation from a source structure, expansion in vitro, engineering modifications for expression of selected genes, structural and functional tests in vitro, transplantation into a target organ, and structural and functional tests in vivo. In ischemic myocardial injury, for instance, a fundamental method of cell-based protective engineering is cell transplantation into the ischemic myocardium. Several cell types can be used for cardiac cell protection and regenerationembryonic stem cells, induced pluripotent stem cells, and somatic stem cells. For myocardial regeneration, embryonic and induced pluripotent stem cells are the candidates of choice. Somatic cardiomyocytes are not usually used because these cells have a limited capacity of regeneration and are not able to survive the transplantation procedures and host environment. For myocardial protection, pluripotent stem cells and somatic cells, such as bone marrow progenitor cells, skeletal muscle progenitor cells, and hepatic cells, can be engineered to over-express selected protective gene(s) and transplanted to the ischemic myocardium for regional delivery of protective factors. The effectiveness of cell-based engineering therapies has been demonstrated in numbers of previous investigations (Abdel-Latif et al., 2007; Martin-Rendon et al., 2008; Carvalho et al., 2015).

Tissue-level protective engineering Tissue-level protective engineering strategies can be developed and used to support organ structures and assist in organ performance, preventing organ failure. Fundamental engineering procedures include construction of a biological matrix or synthetic polymer scaffold with the composition, structure, porosity, shape, dimensions, and mechanical

Chapter One properties conforming to the tissue or organ to be replaced; preparation, expansion, and seeding of selected stem or somatic cells into the scaffold; development of the cellseeded scaffold into a functional construct; evaluation of the construct function in vitro; implantation of the construct into a target organ; and evaluation of the construct function in vivo. For instance, in ischemic myocardial injury, the most important tasks of tissue-level protective engineering are to strengthen the ischemic myocardium for preventing myocardial rupture and support the myocardial contractile performance for preventing heart failure. A sheet-like extracellular matrix or synthetic polymer scaffold can be constructed and applied to the external surface of the ischemic myocardium to enhance its strength (Kaiser and Coulombe, 2015; Domenech et al., 2016; Mannhardt et al., 2016; Wang et al., 2018). To assist in myocardial performance, it is necessary to establish a myocardium-mimicking construct integrated with functional cardiomyocytes possessing synchronized contractile activities. The construct can be implanted onto the epicardium to enhance the contractile function of the ischemic myocardium (Kaiser and Coulombe, 2015; Domenech et al., 2016; Mannhardt et al., 2016; Wang et al., 2018). A scaffold can also be used to deliver protective and regenerative factors. To do so, an extracellular matrix or synthetic polymer scaffold can be integrated with cells engineered to express selected genes and implanted onto the exterior surface of the ischemic myocardium. This approach provides controlled and sustained release of protective and regenerative factors from the cells of the implant.

Perspectives Nature has established various mechanisms for cell protection and regeneration in injury and disease; however, not all mechanisms are optimized in promptness and effectiveness. Protective engineering is developed and used to induce and optimize protective processes, thereby correcting natural deficiencies and maximizing the capacity of protection. Various protective engineering strategies have been developed at the molecular, cellular, and tissue levels and used in experimental and clinical investigations for protection against injury and disease; however, not many strategies have exerted a significant clinical impact. The most effective, but not perfect, strategies are those at the tissue level, including ventricular assist device placement, angioplasty, arterial stenting, and arterial reconstruction for the cardiovascular system. Most molecular and cell-level engineering strategies, although effective in experimental tests, have not been successfully used in clinical investigations. One potential obstacle for clinical applications is the lack of complete understanding of the naturally occurring systems protective mechanisms (Llu et al., 2015; Liu, 2019). Most clinical treatment strategies are not designed based on the natural mechanisms of protection (Hausenloy et al., 2017; Reusch, 2017; Davidson et al., 2019). Whereas timedependent multiple protective molecules and cell types are required for the natural form of protection (Llu et al., 2012; Llu, 2019), a single "protective agent'' targeting a selected molecule or pathogenic process is commonly used in clinical tests, a potential problem for the failure of most protective clinical trials (Davidson et al., 2019; Hausenloy et al., 2017; Reusch, 2017). This point is supported by the observation that the activation of multiple protective factors by a preconditioning injury represents the most effective and reproducible treatment strategy for protection against a subsequent injury (Llu et al., 2015; Hausenloy et al., 2017; Reusch, 2017; Davidson et al., 2019). However, a list of

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Chapter One naturally occurring protective factors remains incomplete and their mechanisms of action have not been fully understood. To develop protective engineering strategies that maximize the capacity of cell protection, it is necessary to identify naturally occurring systems protective factors and understand the underlying mechanisms of action. Such information can be used to identify and correct the deficiencies of the naturally occurring protective mechanisms and maximize the capacity of protection by engineering optimization of the types, levels, timing, and coordination of protective actions in injury and disease.

References Abdel-Latif, A., Holli, R., Tleyjeh, l.M., Montori, V.M., Perin, E.C., Hornung, C.A., ZubaSurma, E.K. "Adult Bone Marrow-derived Cells for Cardiac Repair. A Systematic Review and Meta-analysis." Arch Intern Med 167: 989-997, 2007. Banai, S., Shweiki, D., Pinson, A., Chandra, M., Lazarovici, G., Keshet, E. "Upregulation of Vascular Endothelial Growth Factor Expression Induced by Myocardial Ischaemia: Implications for Coronary Angiogenesis." Cardiovasc Res 28: 1176-1179, 1994. Cannan, W.J., and Pederson, D.S., "Mechanisms and Consequences of Double-strand DNA Break Formation in Chromatin." JCell Physiol 231: 3-14, 2016. Carvalho, E., Verma, P., Hourigan, K., Banerjee, R., "Myocardial Infarction: Stem Cell Transplantation for Cardiac Regeneration." Regen Med 10: 1025-1043, 2015. Chen, L., Deng, H., Cui, H., Fang, J., Zuo, Z., Deng, J., Li, Y., Wang, X., Zhao, L. "Inflammatory Responses and Inflammation-Associated Diseases in Organs." Oncotarget. 9: 7204-7218, 2018. Davidson, S.M., Ferdinandy, P., Andreadou, I., Betker, H.E., Heusch, G., Ibanez, B., Ovize, M., Schulz, R., Yellon, D.M., Hausenloy, D.J., and Garcia-Dorado, D. on behalf of the European Union CARDIOPROTECTION COST Action (CA16225). "Multitarget strategies to reduce myocardial ischemia/reperfusion injury." J Am Coll Cardiol 73: 89-99, 2019. Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada, Z. A., Eckert, M. R., Vogel, J., and Charpentier, E. "CRISPR RNA Maturation by Trans-Encoded Small RNA and Host Factor RNase III." Nature 471: 602-607, 2011. Domenech, M., Polo-Corrales, L., Ramirez-Vick, J.E., Freytes, D.O. "Tissue Engineering Strategies for Myocardial Regeneration: Acellular Versus Cellular Scaffolds?" Tissue Eng Part B Rev 22: 438--458, 2016. Dunbar, C.E., High, K.A., Joung, J.K., Kohn, D.B., Ozawa, K., Sadelain, M. "Gene Therapy Comes of Age." Science 359: 175, 2018. Fazel, S.S., Chen, L., Angoulvant, D., Li, S.H., Weisel, R.D., Keating, A., Li, R.K. 2008. "Activation of c-kit is Necessary for Mobilization of Reparative Bone Marrow Progenitor Cells in Response to Cardiac Injury." FASEB J22: 930-940, 2008. Folino, A., Accomasso, L., Giachino, C., Montarolo, P.G., Losano, G., Pagliaro, P., and Rastaldo, R. "Apelin-induced Cardioprotection against Ischaemia/Reperfusion Injury: Roles of Epidermal Growth Factor and Src." Acta Physiol (Oxf) 222: 12924, 2018. Gardiner, D.M. Regenerative Engineering and Developmental Biology: Principles and Applications. In CRC Press Series Regenerative Engineering, lst Edition, Boca Raton, FL: CRC Press, 2018.

Chapter One Gong, C., Bongiorno, P., Martins, A., Stephanou, N. C., Zhu, H., Shuman, S., and Glickman, M.S. "Mechanism of Nonhomologous End-Joining in Mycobacteria: a Low-Fidelity Repair System Driven by Ku, Ligase D and Ligase C." Nat Struct Mol Biol 12: 304-312, 2005. Granfeldt, A., Lefer, D.J., Vinten-Johansen, J. "Protective Ischaemia in Patients: Preconditioning and Postconditioning." Cardiovasc Res 83: 234-246, 2009. Gu, s.,G., Pak, J., Guang, s., Maniar, J., M., Kennedy, s., Fire, A. IIAmplification of siRNA in Caenorhabditis elegans generates a transgenerational sequence-targeted histone H3 lysine 9 methylation footprint." Nature Genetics 44:157-164, 2012. Hausenloy, D.J., Botker, H.E., Engstrom, T., Erlinge, D., Heusch, G., Ibanez, B., Kloner, R.A., Ovize, M., Yellon, D.M., Garcia-Dorado, D. "Targeting Reperfusion Injury in Patients with ST Segment Elevation Myocardial Infarction: Trials and Tribulations." Eur Heart] 38: 935-941, 2017. Heusch, G. "Critical Issues for the Translation of Cardioprotection." Circ Res 120: 14771486, 2017. Horvath, P., and Barrangou, R. "CRISPR/Cas, the Immune System of Bacteria and Archaea." Science 327: 167-170, 2010. House, S.L., Branch, K., Newman, G., Doetschman, T., and Schultz, J.J. "Cardioprotection Induced by Cardiac-Specific Overexpression of Fibroblast Growth Factor-2 is Mediated by the MAPK Cascade." Am JPhysiol Heart Circ Physi.ol 289: H2167-H2175, 2005. Jasin, M., Rothstein, R. "Repair of Strand Breaks by Homologous Recombination." Cold Spring Harb Perspect Biol 5: a012740, 2013. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., Charpentier, E. "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity." Science 337: 816--821, 2012. Kaiser, N.J., and Coulombe, K.L.K. "Physiologically Inspired Cardiac Scaffolds for Tailored In Vivo Function and Heart Regeneration." Bi.omed Mater 10: 034003, 2015. Laurencin, C.T., and Khan, Y. Regenerative Engineering. lst Edition, Boca Raton, FL: CRC Press, 2018. Liu, S.Q., Bioregenerative Engineering: Principles and Applicati.ons. Hoboken, New Jersey: Wiley-Interscience, 2007. Liu, S.Q., Tefft, B.J., Zhang, D., Roberts, D., Schuster, D.J., Wu, A. "Cardioprotective Mechanisms Activated in Response to Myocardial Ischemia." MCB 8: 319-338, 201la. Liu, S.Q., Tefft, B.J., Liu, C., Zhang, B., Wu, Y.H. "Regulation of Hepatic Cell Mobilization in Experimental Myocardial Ischemia." Cell Mol Bioeng 4: 693-707, 2011b. Liu, S.Q., Ren, Y.P., Zhang, L.-Q., Li, Y.C., Phillips, H., Wu, Y.H. "Cardioprotective Proteins Upregulated in the Liver in Response to Experimental Myocardial Ischemia." Am J Physiol Hearl Circ Physiol 303: H1446-1458, 2012. Liu, S.Q., Roberts, D., Kharitonenkov, A., Li, Y.C., Zhang, L.-Q., Wu, Y.H . "Cardioprotective Action of Fibroblast Growth Factor 21 Upregulated and Released from the Liver and Adipose Tissue in Experimental Myocardial Ischemia." Sci Rep 3: 2767, 2013. Liu, S.Q., Ma, X.L., Qin, G.J., Liu, Q.P., Li, Y.C., Wu, Y. H. "Trans-system Mechanisms Against Ischemic Myocardial Injury." Comp Physiol 5: 167-192, 2015. Liu, S.Q. "Cardiac Protective Engineering." JBiomech Eng 141: 090801-1, 2019. Mannhardt, I., Breckwoldt, K., Letuffe-Breniere, D., Schaaf, S., Schulz, H., Neuber, C., Benzin, A., et al. "Human Engineered Heart Tissue: Analysis of Contractile Force." Stem Cell Reports 7: 29--42, 2016.

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Chapt er One Martin-Rendon, E., Brunskill, S.J., Hyde, C.J., Stanworth, S.J., Mathur, A., Watt, S.M. "Autologous Bone Marrow Stem Cells to Treat Acute Myocardial Infarction: A Systematic Review." Eur Heart J29: 1807-1818, 2008. Messadi, E., Aloui, Z., Belaidi, E., Vincent, M.P., Couture-Lepetit, E., Waeckel, L., Decorps, J., et al. "Cardioprotective Effect ofVEGF and Venom VEGF-Like Protein in Acute Myocardial Ischemia in Mice: Effect on Mitochondrial Function."] Cardiovasc Phannacol63:274-28l,2014. Muller, Y.A., Christinger, H.W., Keyt, B.A., de Vos, A.M. "Vascular endothelial growth factor refined to 1.93 angstroms resolution." Structure 5: 1325-1338, 1997. Nakamura, T., Mizuno, S., Matsumoto, K., Sawa, Y., Matsuda, H., Nakamura, T. "Myocardial Protection from Ischemia/Reperfusion Injury by Endogenous and Exogenous HGF." J Clin Invest 106: 1511-1519, 2000. Napoli, C., Lemieux, C., Jorgensen, R. "Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes In Trans." Plant Cell 2: 279-289, 1990. Novina, C.D., and Sharp, P.A. "The RNAi Revolution." Nature 430: 161-164, 2004. Overballe-Petersen, S., Harms, K., Orlando, L.A., Mayar, J.V., Rasmussen, S., Dahl, T. W., Rosing, M.T., et al. "Bacterial Natural Transformation By Highly Fragmented And Damaged DNA." PNAS 110: 19860--19865, 2013. Ripa, R.S., Jergensen, E., Wang, Y., Thune, J.J., Nilsson, J.C., S.0Ildergaard, L., Johnsen, H.E. et al. "Stem Cell Mobilization Induced by Subcutaneous Granulocyte-Colony Stimulating Factor to Improve Cardiac Regeneration after Acute ST-Elevation Myocardial Infarction: Result of the Double-Blind, Randomized, Placebo-Controlled in Myocardial Infarction (STEMMI) Trial." Circulation 113: 1983--1992, 2006. Rock, K.L., and Kono, H. "The Inflammatory Response to Cell Death." Annu Rev Pathol 3: 99-126, 2008. Shintani, S., Murohara, T., Ikeda, H., Ueno, T., Honma, T., Katoh, A., Sasaki, K., Shimada, T., Oike, Y., and Imaizumi, T. "Mobilization of Endothelial Progenitor Cells in Patients with Acute Myocardial Infarction." Circulation 103: 2776--2779, 2001. Swirski, F.K., Nahrendorf, M., Etzrodt, M., Wildgruber, M., Cortez-Retamozo, V., Panizzi, P., Figueiredo, J.L., et al. "Identification of Splenic Reservoir Monocytes and their Deployment to Inflammatory Sites." Science 325: 612-616, 2009. van der Krol, A.R., Mur, L.A., Held, M., Moi, J.N.M., Stuitje A.R. "Flavonoid Genes in Petunia: Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene Expression.'' Plant Cell 2: 291-299, 1990. Vinten-Johansen, J., and Shi, W. "Perconditioning and Postconditioning: Current Knowledge, Knowledge Gaps, Barriers to Adoption, and Future Directions." JCardiovasc Pharmacol Ther 16: 260-266, 2011 Wang, Z., Lee, S. J., Cheng, H-J., Yoo, J. J., Atala A. "3D Bioprinted Functional And Contractile Cardiac Tissue Constructs." Acta Biamater 70: 48-56, 2018. Watts, J.K., and Corey, D.R. "Gene Silencing by Simas and Anti.sense Oligonucleotides in the Laboratory and the Clinic." JPathol 226: 365-379, 2012. Xia, H., Mao, Q., Paulson, H.L., Davidson, B.L. "siRNA-mediated Gene Silencing In Vitro And In Vivo." Nat Biotech 20: 1006--1010, 2002. Yellon, D.M., and Downey, J.M. "Preconditioning the Myocardium: from Cellular Physiology to Clinical Cardiology." Physial Rev 83: 1113--1151, 2003.

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Foundations of Cardiovascular Protective Engineering

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Development of the Heart, Blood Vessels, and Blood cells Hlghllghts • Protective engineering strategies can be established based on the naturally occurring protective mechanisms that evolve during embryogenesis and somatic growth involving developmental processes. Cell regeneration, an integral part of protective mechanisms, shares common regulatory processes with embryonic cell generation. Thus, it is essential to understand developmental biology. • A human individual develops through several processes-ovum fertilization, embryonic cell cleavage, blastocyst formation, gastrulation, and organ formation and maturation, and parturition. The blastocyst inner cell mass contains embryonic stem cells that can be isolated, expanded in vitro, and transplanted into injured or disordered organs to regenerate functional cells with the cell type controlled by environmental factors. Gastrulation is a process generating three germ layers-ectoderm, mesoderm, and endoderm; structures giving rise to distinct cell types and organ systems. • The heart, blood vessels, and blood cells develop from the mesoderm. The mesoderm can also give rise to the skeletal muscle, bone, cartilage, and connective tissue. The ectoderm is the origin of the central and peripheral nerve system, skin, eyes, ears, nose, mouth, hair, and nails. The endoderm gives rise to the lung, liver, stomach, intestines, kidney, and urinary bladder. • Cardiac cells develop from two types of cardiogenic cell-the primary and secondary heart field cardiogenic cells. The primary heart field cells give rise to cardiac cell lineages that form the left and right ventricles, left and right atria, as well as the atrioventricular canals. The secondary heart field cells contribute to the formation of the inflow region (veins}, outflow tract (arteries}, as well as all other cardiac structures except for the left ventricle. • The heart forms via several major stages-creation of the cardiac crescent at about two weeks from fertilization, formation of the heart tube at about three weeks, heart looping at about four weeks, and establishment of the four cardiac

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Part One chambers at about eight weeks. Cyclic cardiac contractile activities occur in the heart-tube stage. • Vascular and blood cells develop from hemangioblasts simultaneously with cardiac development. Hemangioblasts give rise to angioblasts and hematopoietic stem cells. The former develops into vascular cells, including endothelial cells, smooth muscle cells, and fibroblasts; whereas the latter form blood cells, including erythrocytes, leukocytes, and platelets. • Vascular development begins with capillary formation in the blood island from endothelial cells during the early embryonic stage. While the majority of capillaries are connected into networks, selected capillaries are expanded into small arteries and veins by recruiting progenitor cells that give rise to smooth muscle cells and fibroblasts. Selected small arteries and veins develop into larger arteries and veins, respectively. • Blood cell generation is a process continuing through the entire lifespan, beginning in the blood island during the early embryonic stage. The bloodgenerating site moves from the blood island to the aorta-gonad-mesonephroi when blood vessels form in blood islands, moves to the liver when the aorta-gonad-mesonephroi degenerate, and eventually settles in the bone marrow during the late embryonic stage. The bone marrow is the permanent site of hematopoiesis in adults.

Overview Protective engineering strategies can be designed largely based on the naturally occurring protective mechanisms that evolve in response to genetic defects and environmental insults. As these natural mechanisms are established during embryogenesis and somatic growth involving developmental processes, it is essential to understand developmental biology. Furthermore, regenerative engineering, an integral part of protective engineering, is based on the natural mechanisms of cell regeneration in injury and disease. Cell regeneration shares common regulatory mechanisms with embryonic cell generation. We can learn cell regenerative control mechanisms from the generation processes of embryonic cells and structures. For these reasons, the book starts with the concept of cardiovascular development in humans.

Early Embryonic Development Overview of embryonic processes The life of humans begins with fertilization, a process involving fusion of a sperm cell into an ovum to form a zygote. Fertilization initiates several embryonic processes-embryonic cell cleavage, blastocyst formation, gastrulation, organ formation and maturation, and parturition (Liu, 2007). Embryonic development can be divided into several stages-the germinal stage from fertilization to gastrulation (about 2 weeks), organ forming stage from gastrulation to organ formation (from 2 to 8 weeks), and the fetal stage from organ formation to parturition (from 8 to 40 weeks) (Fig. 2.1). During the germinal stage, a fertilized egg is divided continuously to increase the cell population with a decrease in cell volume. When reaching the 16-cell stage, the embryonic cells are prompted to form a ball-shaped structure known as blastocyst at about seven days.

Chapter Two

Inner

Mesoderm

cell

""~·\ T

Fertilization FIGURE

Blastocyst formation 1week

Gastrulation 2 weeks

' Fetus formation 8 weeks

2.1 Processes of early embryonic development.

The blastocyst further develops into a three-layered structure defined as gastrula, composed of the ectoderm, mesoderm, and endoderm at about two weeks. The process of gastrula formation is referred to as gastrulation. During the organ formation stage, the ectoderm develops into the brain, spinal cord, ganglia, peripheral nerves, and the epidermal layer of the skin, ears, nose, and teeth. The mesoderm gives rise to the heart, blood vessels, blood cells, soft connective tissues, skeletal muscles, bones, and cartilages. The endoderm develops into the lung, gastrointestinal tract, liver, pancreas, and urinary bladder. During the organ formation and fetal stages, tissues and organs develop rapidly and gain size and functionality. By the time of parturition, most tissues and organs are ready to function.

Fertilization-union of the sperm and ovum Fertilization is a process by which a sperm fuses into an ovum to develop into an embryo (Liu, 2007). A sperm cell is composed of a haploid nucleus, a propulsion apparatus, and protein enzymes, responsible for interactions with the ovum. Two sperm structures, the acrosome and flagellum, are critical to sperm-ovum interactions. The acrosome contains enzymes necessary for degrading the external layer zona pellucida of the ovum, allowing union of the sperm with the ovum. The flagellum is responsible for sperm movement, driven by the motile apparatus axoneme, a structure composed of microtubules and the motor protein dynein. Dynein is attached to the microtubules and contains an enzyme that hydrolyzes ATP, providing energy necessary for dynein motility, flagellar propulsion, and sperm movement. An ovum consists of a haploid nucleus and a large cytoplasm for storage of nutrients to support the embryo growth. The ovum cell membrane is surrounded by an extracellular matrix layer known as zona pellucida. Outside the zona pellucida, there is a thick cellular structure defined as cumulus, composed of a large number of ovarian

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Part One follicular cells, which provide soluble factors and mechanical protection to the ovum. When a female individual reaches maturity, ova develop from oocytes (developing ova) through meiosis, a process starting at -13 years of age and ending at the age of -50. About 400 eggs can be produced through the lifespan (Liu, 2007). Fertilization involves several processes, including chemotactic attraction of sperm cells to an ovum, penetration of a sperm cell through the exterior layers of the ovum, sperm fusion into the ovum, and integration of two gamete nuclei. Sperm cells can be attracted to an ovum in the oviduct by chemical gradient-directed cell movement, a process known as chemotaxis. Ova are capable of expressing and releasing the spermattracting protein resact that interacts with cognate receptors in sperm cells, causing directed movement of sperm cells toward an ovum. This process is species-dependentsperm cells can only move to the ovum of the same species because of the specificity of chemotactic molecules (Liu, 2007). Upon the interaction of sperm cells with the exterior layer of an ovum, the acrosomal vesicle of the sperm is activated. A sperm cell can go through the follicular cell layer of the ovum and release proteolytic enzymes when reaching the zona pellucida. The enzymes are responsible for degrading the zona pellucida, allowing the sperm approaching the ovum membrane. A zona pellucida protein known as zona protein 3 (ZP3) controls acrosomal vesicle exocytosis by activating the sperm membrane G protein-coupled receptor galactosyltransferase-1. This process stimulates a signaling process, causing calcium release into and elevation in the cytosol. Calcium in turn activates processes that cause acrosomal vesicle exocytosis, regulating sperm union with an ovum. As all the regulatory ligands and receptors are animal species-specific, the sperm-ovum union occurs only in the same species (Llu, 2007). A sperm cell, when passing through the zona pellucida, can attach to the ovum membrane, inducing membrane fusion of both cells, introducing the sperm contents into the ovum. This process is controlled by several proteins, including CD9 and Izumo protein. CD9 contributes to the regulation of sperm-ovum membrane fusion. Genetic modifications of the CD9 gene result in impairment of the sperm-ovum interaction, potentially causing infertility. Introduction of wild-type CD9 mRNA into ova with CD9 gene deficiency reverses the infertility. Izumo protein is responsible for regulating the sperm-ovum membrane fusion. When the lzumo gene is deficient in the mouse, sperm cells, while capable of growing to maturity and going across the zona pellucida, are not able to fuse into an ovum (Liu, 2007). Once sperm-ovum union occurs, the ovum becomes resistant to interactions with other sperm cells. This activity is controlled by short- and long-term mechanisms. The short-term mechanisms involve cell membrane potentials. A sperm cell can fuse into an ovum at the normal resting membrane potential. The resting membrane potential of ova is about -90 m V, a level allowing sperm-ovum interactions and union. Spermovum membrane fusion triggers rapid ovum cell membrane depolarization, reversing the membrane potential to about 20 m V by opening the sodium channels and inducing sodium flux into the ovum. The positive membrane potential prevents other sperm cells from interaction with the ovum. Depolarization is a short process lasting about a minute. The long-term preventive process is controlled by sperm-ovum interaction proteins. The ovum is equipped with proteolytic enzyme-containing granules. Spermovum fusion causes calcium release and cytosolic calcium elevation, stimulating enzyme release from the ovum granules into the zona pellucida. The released enzymes cleave

Chapter Two the sperm-ovum interaction proteins, including zona pellucida glycoprotein 3 (ZP3), thereby preventing other sperm cells from entering the sperm-fused ovum (Liu, 2007). Sperm-ovum union triggers several processes-DNA synthesis, mitosis, protein synthesis, and plasma membrane formation. Calcium is an important ion involved in the regulation of these processes with the activity depending on its cytosolic level. In WlStimulated cells, the concentration of cytosolic calcium is about 0.0001 mM. Sperm interaction with an ovum causes rapid elevation in cytosolic calcium, a process regulated by a G protein-coupled receptor signaling system. The presence of sperm cells activates zona protein 3 (ZP3) in the ovum. ZP3 can bind to cognate G protein-coupled receptors in the sperm, causing activation of G protein and subsequently phospholipase C. Phospholipase C is an enzyme capable of cleaving phosphatidylinositol biphosphate (PIP2) to form inositol triphosphate (IP3 ) and diacylglycerol. IP3 causes calcium release from the endoplasmic reticulum to the cytosol by opening the calcium ion channels. Calcium in turn triggers signaling processes leading to mitosis, DNA synthesis, protein synthesis, and membrane generation. Diacylglycerol is able to activate protein kinase C, a key signaling molecule participating in the regulation of DNA and protein syntheses-essential processes for the development of a fertilized ovum. These regulatory processes eventually result in the degradation of the sperm mitochondria and flagellum, migration of the sperm nucleus to the ovum nucleus, and fusion of the sperm and ovum nuclei (Liu, 2007).

Cleavage Cleavage is zygote division accomplished by karyokinesis (mitotic nucleus division) and cytokinesis (cytoplasmic division). Karyokinesis occurs immediately after spermovum union followed by cytokinesis, generating two nucleated cells defined as blastomeres. These blastomeres divide further in a symmetrical manner to form morula-a solid ball-like structure containing blastomeres. Occasionally, asymmetric cleavage may occur, but it is not well understood how the cleavage pattern is controlled. Early embryonic cleavage is regulated by a protein known as mitosis-promoting factor (MPF), which is activated in response to sperm-ovum union. The level of MPF changes periodically during embryonic mitosis-increasing during the M phase and decreasing during the S phase. Blastomere divisions are controlled by cyclic changes in the level of MPF (Liu, 2007).

Formation of blastocyst The blastocyst is an early embryonic structure developed from the morula within about a week following fertilization. This structure consists of the inner cell mass and trophoblast. The inner cell mass is composed of about 30 cells defined as embryonic stem cells, which give rise to all specified functional cells. The trophoblast is a single layer of cells that encloses the inner cell mass and develops into the chorion and the placenta that support and protect the embryo. The formation of the blastocyst is considered a milestone of embryogenesis as the embryonic stem cells of the inner cell mass are the origin of all cells, tissues, and organs of an individual. During the short life of the blastocyst, about one week, the embryonic stem cells of the inner cell mass undergo dynamic asymmetrical differentiation, generating specified stem cells for the formation of distinct organ systems (Liu, 2007). It is interesting to note that the inner cell mass

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Part One embryonic stem cells all appear identical in morphology during the blastocyst stage and are capable of differentiating to structurally and functionally distinct cells within several days. It remains poorly understood how such a process occurs and what signaling mechanisms are involved.

Gastrulatlon Gastrulation is a process by which the inner cell mass of the blastocyst grows into three distinct layers-the ectoderm, mesoderm, and endoderm within about two weeks from fertilization (Fig. 2.2). These layers eventually give rise to specified organ systems. Several intermediate structures develop during gastrulation. The inner cell mass first gives rise to epiblast and hypoblast. The former develops into the embryonic epiblast and amniotic ectoderm, and the latter develops into extraembryonic endoderm. The embryonic epiblast is the primitive form of the three germ layers. It gives rise to the embryonic ectoderm and primitive streak, a short-lived structure that is rapidly transformed into embryonic endoderm and mesoderm. The amniotic ectoderm from the epiblast forms the amnion, a membrane enclosing the embryo. The extraembryonic endoderm from the hypoblast develops into the yolk sac, which contains nutrients for embryo growth. The trophoblast is the origin of the extraembryonic chorion and placenta that are located outside the amnion (Liu, 2007). The epiblast-derived ectoderm, mesoderm, and endoderm give rise to specified tissues and organs during the remaining embryonic period. The ectoderm develops into an intermediate structure consisting of the surface ectoderm, neural crest, and neural tube. The surface ectoderm is the origin of the epidermis, nails, hair, sebaceous glands, mucous membrane of the mouth and anus, tooth enamel, lens and cornea, and anterior pituitary. The neural crest develops into the peripheral ganglia, Schwann cells, and sympathetic and parasympathetic nerves, adrenal medulla, melanocytes, and tooth dentine. The neural tube gives rise to the brain, spinal cord, retina, and inner ear (Liu, 2007). The mesoderm is the origin of the heart, blood vessels, blood cells, skeletal muscle, bone, cartilage, connective tissue, lymphatic system, kidney, and gonads. From the beginning of gastrulation, the mesoderm develops into a structure with four distinct regions--the notochord, paraxial mesoderm, intermediate mesoderm, and lateral plate mesoderm. The notochord is a transient structure mediating the formation of the neural tube, determining the anterior-posterior axis of the embryo, and contributing

A

F111uRE

Trophoblast

B Mesoderm Ectoderm Endoderm

2.2 Embryonic blastocyst {A) and germ layers--ectoderm, mesodenm, and endoderm (B).

Chapter Two to the formation of the endodermal primitive gut. The paraxial mesoderm is organized into two parallel columns along the lateral sides of the neural tube and is the origin of the somites (segmented blocks). These somites give rise to dermis, skeletal muscle, ribs, and vertebrae. The intermediate mesoderm participates in the formation of the kidney, ureter, bladder, urethra, and reproductive system. The lateral plate mesoderm consists of the somatic mesoderm and splanchnic mesoderm. The somatic mesoderm is adjacent to the ectoderm and the splanchnic mesoderm is next to the endoderm. These mesodermal structures develop into the heart, blood vessels, and blood cells. There is a gap between the somatic and splanchnic mesoderm layers, which is defined as the coelom-the origin of the pleural, pericardia!, and peritoneal cavities (Liu, 2007). The endoderm is the layer that gives rise to the epithelial cell-based structures of the lung, airways, gastrointestinal tract, liver, and pancreas. The respiratory, digestive, and hepatic systems develop from an early endodermal structure known as primitive gut. In the human, the primitive gut forms at about 16 days following the conception and is composed of three parts during the early stage: the foregut, midgut, and hindgut. At about 22 days, the liver bud forms from the foregut and is the presumptive structure for the formation of the liver. At about 28 days, the anterior end of the foregut forms the oral opening, which is the presumptive structure of the mouth. The foregut also gives rise to the pharynx, esophagus, thyroid bud, lung bud, and stomach at about the same time. The midgut and hindgut are the presumptive structures for the formation of the small and large intestines. The endodermal cells also regulate the formation of mesoderm-derived tissues and organs by secreting soluble mediating factors (Liu, 2007). The liver develops from the liver bud that sprouts from the foregut of the endodermal primitive gut. The liver bud grows into the surrounding mesodermal tissue, which produces and releases regulatory factors to stimulate liver bud cell differentiation into hepatocytes and ductular epithelial cells. Mesoderm-derived vascular endothelial cells proliferate and migrate into the primitive liver, forming the hepatic vascular system, an essential process for liver development. The removal of vascular endothelial cells results in the failure of liver formation. Gallbladder, an affiliated structure of the liver, develops from the hepatic drainage duct (Liu, 2007). The pulmonary system develops from the lung rudiment sprouted from the foregut. The lung rudiment first grows into the trachea, which bifurcates into the left and right bronchi, subsequently establishing the left and right lungs. During the lung development, endodenn-derived epithelial cells work together with mesodermal cells to form an integrated respiratory and circulatory system, establishing the structural basis for gas exchange. It is interesting to note that, when embryonic tracheal epithelial cells are cultured in the absence of mesodermal cells, the epithelial cells will not develop into airway structures. In contrast, airway-like structures develop when lung epithelial cells are cultured in the presence of mesodermal cells. Thus, mesodennal cells play a role in regulating the differentiation of endoderm-derived pulmonary epithelial cells. Regional differences in the structure and function of the mesodermal cells may determine the specification of pulmonary cells and control lung formation. During the embryonic stage, although the pulmonary system is established, it is not functional for gas exchange. The embryo and fetus obtain oxygen and remove carbon dioxide via the placenta. The lung initiates the gas exchange function immediately after birth (Liu, 2007).

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Part One

Development of the Heart Origin and fate of canliogenic cells The heart is the first organ to develop following gastrulation because of the necessity of blood circulation for the growing embryo (Liu, 2007). Cardiac development undergoes four major stages-creation of the cardiac crescent at about two weeks, formation of the heart tube at about three weeks, heart looping at about four weeks, and establishment of the four cardiac chambers at about eight weeks (Fig. 2.3). The heart is composed of various cell types, including cardiomyocytes, endocardial cells, epicardial cells, fibroblasts, valvular cells, vascular endothelial cells, and smooth muscle cells. These cardiac cells originate from a set of embryonic progenitor cells known as cardiogenic cells or presumptive cardiac cells in the mesoderm. There are two types of cardiogenic cells defined on the basis of their origins and lineage specifications: primary and secondary heart field cardiogenic cells (Buckingham et al., 2005; Chien et al., 2008). The primary heart field cardiogenic cells arise from the primitive streak and migrate to the anterior lateral mesoderm to form two cardiogenic mesodermal clusters symmetrically with reference to the primitive streak. The two cardiogenic cell clusters move toward each other and form two endocardial primordia, the initial form of the heart. Cells from the endocardial primordia extend across the midline of the embryo and form a crescent-shaped structure, known as the cardiac crescent, at about embryonic day 14. The primary heart field cells in the cardiac crescent give rise to cardiac cell lineages that form the left and right ventricles, left and right atria, as well as the atrioventricular canals. The secondary heart field cardiogenic cells arise from the pharyngeal and splanchnic mesoderm, migrate and merge into the primary heart field structures, and contribute to the formation of the inflow region (veins), outflow tract (arteries), as well as all other cardiac structures except for the left ventricle (Buckingham et al., 2005; Laugwitz et al., 2005; Martin-Puig et al., 2008). Overall, the left and right atria, right ventricle, aortic root, and pulmonary trunk develop from both the primary and secondary heart fields, whereas the left ventricle arises from the primary heart field.

Cardiac crescent 2 weeks

Cardiac tube 3weeks

Cardiac loop 4 weeks

IV Cardiac chambers Sweeks

F1auRE 2.3 The four stages of heart development. AS: Aortic sac; RA: Right atrium; LA: Left atrium; RV: Right ventricle; LV: Left ventricle; SV: Superior vena cava; IV: Inferior vena cava; AO: Aorta; PA: Pulmonary artery.

Chapter Two

Regulatlon of heart development The development of the cardiac cell lineages is regulated by signaling molecules expressed in a time- and location-dependent manner. The early cardiogenic cells express several transcription factors, including mesoderm posterior protein 1 (MESPl) and mesoderm posterior protein 2 (MESP2) (Kitajima et al., 2003). Both MESPl and MESP2 have been shown to regulate the specification of early cardiogenic cells. MESPl is a class C basic helix-loop-helix protein (268 amino acids, -29 kDa) and can induce expression of a secretary protein known as dickkopf-related protein-1 (Dkk-1, 266 amino acids, -29 kDa). Dkk-1 inhibits the activity of the Wnt (wingless-type M:MTV integration site family member) signaling pathway, which negatively regulates cardiogenesis. Thus, MESPl stimulates cardiogenic cell specification by suppressing the activity of the Wnt inhibitory signaling pathway (David et al., 2008). MESP2 is also a class C basic helix-loop-helix protein (397 amino acids, -42 kDa). This molecule works in coordination with MESPl in the regulation of cardiogenesis (Kitajima et al., 2003). The determination of the early cardiogenic cell lineages is regulated by paracrine factors that change in the level of expression temporarily and spatially. While a complete list of signaling factors is not available, several extracellular soluble protein ligands have been implicated in regulating the fate of cardiogenic cells. Major soluble protein ligands include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), and Wnt proteins (Mummery et al., 2012). BMPs and FGFs serve as cues for the specification of the primary heart field cardiogenic cells and induce activation of the transcription factor Nkx-2.5 (NK2 transcription factor- related, 324 amino acids, -35 kDa) (Brand, 2003; Buckingham et al., 2005). Nkx-2.5 controls the formation of the cardiac crescent from the primary heart field cardiogenic cells. Nkx-2.5 mutations induce early impairment of cardiogenesis (Lyons et al., 1995, Tanaka et al., 1999), resulting in congenital heart abnormalities, such as atrial septal defects (Reamon-Buettner and Barlak, 2004; Hirayama-Yamada et al., 2005), ventricular septal defects (Benson et al., 1999; Zhu et al., 2000), atrioventricular conduction abnormalities (Schott et al., 1998), and tetralogy of Fallot (I'OF) (Benson et al., 1999; Goldmuntz et al., 2001), a disorder characterized by right ventricular hypertrophy, ventricular septal defect, dextroposition of the aorta, and pulmonary stenosis. In contrast to the BMP and FGF signaling pathways, the Wnt signaling pathway exerts an inhibitory effect on the activity of Nkx2.5 (Brand, 2003; Buckingham et al., 2005). Coordinated interaction of the BMP- and FGF-dependent stimulatory pathways with the Wnt-mediated inhibitory pathway possibly determines the morphogenesis of the cardiac crescent. The signaling pathways involving BMPs, FGFs, Nkx-2.5, and Wnt are also responsible for the regulation of the morphogenic activities of the secondary heart field cells (Kelly et al., 2001; Cai, 2003). Another important factor that regulates the specification of cardiogenic cells is GATA4 (GATA-binding protein 4, 442 amino acids, -45 kDa), a member of the GATA family of zinc-finger transcription factors (White et al., 1995). GATA4 recognizes and binds to the sequence 5'-AGATAG-3' present in the promoter region of target genes. This molecule is expressed in the pre-cardiogenic cells in the splanchnic mesoderm and has been known to regulate the differentiation of cardiogenic cells to various cardiac cell lineages in coordination with Nkx-2.5 (Molkentin et al., 1997). The secreted proteins BMPs and FGFs regulate the activity of GATA4 (Buckingham et al., 2005). A missense mutation of GATA4, a type of point mutation with replacement of a single nucleotide, resulting in a change in a single amino acid (G296S, or glycine-to-serine substitution

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Part One at site 296 due to G-to-A transition on nucleotide 886), causes impairment of GATA4 interaction with T-box protein-5 (Tbx5, 518 amino acids, -58 kDa), a transcription factor involved in the regulation of cardiac development (Garg et al., 2003). Such impairment negatively influences GATA4 binding to target genes responsible for cardiac development. These GATA4 mutation-induced changes often result in cardiac septal defects (Garg et al., 2003). Another type of GATA4 gene mutation, E359del or glutamic acid deletion at site 359, results in the loss of GATA4 transcription function, causing cardiac septal defects (Garg et al., 2003). While the primary heart field cardiogenic cells are known to generate functional cells in most cardiac compartments, these cardiogenic cells have been poorly characterized and their downstream cell lineages have not been clearly identified. In contrast, there is considerable information about the developmental lineages of the secondary heart field, containing multipotent cardiogenic cells characterized by the expression of the LIM-homeodomain transcription factor Isletl (Isll), an insulin gene enhancing protein (Cai, 2003; Laugwitz et al., 2005). These cardiogenic cells can develop into various cell lineages, including Isll +/TNT+ (troponin T), Isll +/ smMHC+ (smooth muscle myosin heavy chain), and Isll + /HCN4+ (hyperpolarization activated cyclic nucleotidegated eation channel 4) progenitor cells. The Isll +/TNT+ progenitor cells can give rise to cardiomyocytes, the Isll+I smMHC+ cells develop into smooth muscle cells, whereas the Isll+/HCN4+ progenitor cells form conduction system cells (Cai, 2003; Laugwitz et al., 2005). The expression of the muscular marker TNT is indicative of the specification of cardiogenic cells to cardiomyocyte progenitors. Similarly, the expression of the smooth muscle marker smMHC and the conduction cell marker HCN4 indicates the differentiation of cardiogenic cells to smooth muscle and conductive cell progenitors, respectively.

Formation of the heart tube Proce55es of heart tube formation Following the formation of the cardiac crescent from the primary heart field, cells from the cardiac crescent are specified to form a linear tube structure known as the heart tube through crescent fusion at about embryonic day 21 (Fig. 2.3) (Srivastava and Olson, 2000). The heart tube consists of an inner endothelial cell layer and an outer myocardial layer with extracellular matrix between the two layers (Baldwin, 1996; Brand, 2003). These layers are derived from cardiac crescent progenitor cells. A subset of cardiogenic cells differentiates to endothelial cells, whereas others give rise to cardiomyocytes. The endothelial cells further develop into the endocardium, as well as several other structures, including the endocardial cushions (giving rise to the cardiac valves) and the membrane portions of the atrial and ventricular septum (Abu-Issa and Kirby et al., 2007). The myocardial layer of the heart tube gives rise to the ventricular and atrial walls. The endocardial cells play an important role in regulating the formation of the trabecular myocardium (Wagner and Siddiqui, 2007; Stankunas et al., 2008) and differentiation of cardiomyocytes to Purkinje fiber cells (Srivastava and Olson, 2000). At about three weeks, the cardiac tube displays peristaltic contraction activities in the human embryo.

Regulation of heart tube formation The formation of the heart tube is controlled by complicated molecular signaling processes. Although it remains incompletely understood how heart tube development is

Chapter Two regulated, the transcription factor GATA4-based signaling pathways play a critical role. GATA4 is known to regulate the migration and morphogenesis of the mesodermal precardiogenic cells, processes critical to the formation of the heart tube. In homozygous GATA4 null mice, the primitive myocardial tube can no longer be established during embryogenesis (Molkentin et al., 1997). When the GATA4 gene is knocked out (GATA4-1-) in mice, the embryo dies from 8.5 to 10.5 days post coitum (Kuo et al., 1997). GATA4deficient embryos exhibit a lack of the linear myocardial tube and pericardia! cavity. In these embryos, although cardiogenic cells are able to form cardiomyocytes expressing contractile proteins, cardiomyocytes are not able to form a heart tube (Kuo et al., 1997). These observations suggest that GATA4 is a critical transcription factor for regulating the morphogenesis of the heart tube. An important process for cardiac development is heart looping, by which the linear heart tube undergoes rightward bending and twisting, forming a C-shaped cardiac loop at about four weeks (Manner 2000). This process results in a change in the left-right symmetrical heart tube into a left-right asymmetrical structure suitable for establishing the four cardiac chambers (Fig. 2.3). Prior to heart looping, the primitive cardiac tube is positioned in the anterior-posterior direction with the outflow tract/ventricular region at the anterior pole and the inflow tract/ atrial region at the posterior pole. During heart looping, the posterior atrial region bends rightward and gradually moves to the top of the ventricular region. Following heart looping, the heart tube develops into a structure with several distinct regions, including the primitive atrium, left ventricle, and right ventricle. The primitive atrium is connected at the top to the inflow region (sinus venosus), which eventually gives rise to the vena cava and pulmonary veins. The primitive right ventricle is connected to the outflow tract (aortic sac), which develops eventually to the aortic root and pulmonary trunk. Between these structures, there are four rings, including the sinoatrial ring, the atrioventricular ring or canal, the primary ring or fold, and ventriculoarterial ring. Heart looping is regulated by complex signaling pathways. Whereas the exact signaling and biomechanical mechanisms for heart looping remain to be investigated, it has been hypothesized that location-dependent differential cell proliferation, migration, and death, along with non-uniformly developed biomechanical factors, such as interstitial pressure and myocardial tension, may contribute to the asymmetrical morphogenic process of heart looping (Taber et al., 1995; Manner 2000). Asymmetrical expression and activities of signaling molecules may contribute to the regulation of heart looping. As demonstrated in investigations by using the chick model system, the signaling molecules activin-(3B, activin receptor, and bone morphogenetic protein (BMP) 4 are asymmetrically expressed in the early embryonic structures, Hensen's node, and perinodal area, thus playing a role in regulating asymmetrical heart looping (Levin et al., 1995). Activin-[3B and activin receptor are predominantly expressed in the right side of the Hensen's node. Activin-f3B (15.2 kDa), a TGFf3 protein superfamily member, forms a homodimer (activin BB) with another activin-(3B or a heterodimer (activin AB) with activin-f3A by a single covalent disulfide bond, and acts on its receptors to regulate cardiac development (Schmelzer et al., 1990). Activated activin-[38 inhibits the expression of sonic hedgehog (462 amino acids, -50 kDa), a morphogen expressed symmetrically around the Hensen's node. This action restrains sonic hedgehog expression to the left side of the Hensen's node (Hoyle et al., 1992; Levin et al., 1995). Sonic hedgehog participates in the regulation of cell activities during heart looping, inducing left-side expression of the Nodal gene in the lateral plate mesoderm, from which the heart develops.

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Part One Nodal (347 amino acids, -40 kDa) is a member of the TGF~ family, playing a critical role in regulating asymmehical heart looping (Schlange et al., 2001). The function of Nodal is mediated by several downstream signaling molecules, including paired-like homeodomain transcription factor 2 (Pitx2, 317 amino acids, -35 kDa) and Homeobox protein NK-3 homolog B (Nkx-3.2, 333 amino acids, -35 kDa) (Logan et al., 1998; Ryan et al., 1998). Nodal activates Pitx2 and Nkx-3.2, which in tum regulate cell activities involved in heart looping. Pitx2 is a transcription factor essential to asymmehical morphogenesis of the heart tube. Genetically induced ectopic expression of Pitx2 on the right side of the lateral plate mesoderm results in abnormally randomized directionality of heart looping instead of rightward bending and twisting (Yu et al., 2001). Expression of Pitx2 to the right side of the lateral plate mesoderm, in association with suppression of the left side expression of Pix2, induces reversed directionality of heart looping (Logan et al., 1998). These investigations have provided evidence for the role of Pitx2-related signaling pathways in the regulation of heart looping.

Cardiac chamber formation Processes of cardiac chamber fonnatlon The cardiac tube undergoes dynamic morphogenetic changes following heart looping. Among these changes are primitive cardiac chamber formation and cardiac septation. Cardiac chamber formation takes place immediately after heart looping. The right and left ventricles form through expansion of the looped heart tube in the middle region. The right and left atria develop through expansion of the looped heart tube near the venous pole. These processes occur at about three weeks in the human embryo. At this time, the heart remains a single channel without separation of the atrial and ventricular chambers. Cardiac chamber formation is immediately followed by cardiac septation, a process for the development of separated cardiac chambers with atrial and ventricular septa and valves. Cardiac septation is usually accomplished at about eight weeks in the human embryo (Lamers and Moorman, 2002). During cardiac septation, there form several important structures that separate the single heart tube into four chambers, with inflow and outflow blood vessels. These structures include the atrioventricular endocardial cushion, outflow tract endocardial ridge, atrial septum, and ventricular septum. The atrioventricular endocardial cushion arises from the endocardium at the location of the ahioventricular ring, separates the atrium from the ventricle, and gives rise to the ahiovenhicular valves. The formation of the ahiovenhicular cushion involves endothelial to mesenchymal transformation, i.e., differentiation of endothelial cell-derived endocardial cells to mesenchymal cells, such as fibroblasts that generate extracellular matrix for valve construction. At about the same time, the atrial septum develops from the atrial portion of the heart tube, while the ventricular septum grows from the ventricular portion. Both septa grow toward the atrioventricular cushion and separate the myocardial tube into a four-chambered structure. The outflow tract endocardial ridge arises from the endocardium along the outflow tract and separates the outflow tract into the aortic root and pulmonary trunk. These large blood vessels are derived primarily from a set of cardiogenic cells of the secondary heart field. Cells from the neural crest also contribute to the formation of the large blood vessels. The neural crest cells migrate to the outflow tract, and transform into vascular smooth muscle cells and fibroblasts, conhibuting to the formation of the ascending aorta and pulmonary trunk (Epstein and Parmacek, 2005). The inflow

Chapter Two region develops into the vena cava and pulmonary veins. With further morphogenic development, the heart valves are established, the aortic root and pulmonary trunk are connected to the left and right ventricle, respectively, and the vena cava and pulmonary veins are connected to the right and left atria, respectively. A functional four-chambered heart is established at about eight weeks in the human embryo (Fig. 2.3).

Regulation of cardiac chamber formation The formation of the cardiac chambers and large blood vessels is controlled by complex signaling mechanisms. Although the exact mechanisms remain poorly understood, prior investigations have identified several signaling molecules responsible for regulating the cardiac chamber formation. These molecules include the transcription factors eHAND (heart and neural crest derivatives expressed 1) and dHAND (heart and neural crest derivatives-expressed protein 2). These two molecules have been shown to regulate the formation of the left and right ventricular chambers, respectively. eHAND is a 215 amino acid protein (-24 kDa), also known as HANOI, belongs to the basic helix-loop-helix family of transcription factors, and is expressed primarily in the primitive segment of the left ventricle (Russell et al., 1998; Knofler et al., 1999). In response to extracellular cues, eHAND regulates the morphogenesis of the cardiogenic cells that form the left ventricle. eHAND-deficient cardiogenic cells fail to develop into the left ventricle, causing embryo death (Firulli et al., 1998; Riley et al., 1998). The activity of eHAND is regulated by Nkx-2.5. Mice with Nkx-2.5 deficiency exhibit impaired expression of eHAND in association with lethal left ventricular defects, suggesting that Nkx-2.5 and eHAND act coordinately in the regulation of left ventricular formation (Biben and Harvey, 1997). dHAND is a 217 amino acid protein (-24 kDa), also known as HAND2, and is another member of the basic helix-loop-helix family of transcription factors. This protein is expressed predominantly in the primitive segment of the right ventricle and has been shown to regulate the morphogenesis of the right ventricle (Srivastava and Olson, 2000). dHAND-deficient mice display reduced cardiomyocyte development and impaired right ventricular formation (Srivastava et al., 1997). Another factor that regulates right ventricular development is forkhead box protein Hl (FOXH1), a transcription activator capable of binding to the DNA sequence 5'-TGT[GT] [GT]ATT-3'. FOXH1 can respond to transforming growth factor~ and act in coordination with SMADs, a family of transcription factors that form homomeric and heteromeric complexes and regulate the transcription of target genes. Deletion of the FOXHl gene results in the failure of right ventricular formation (von Both et al., 2004). During the linear cardiac tube stage, the primitive ventricular chamber is connected to the outflow tract, and the atrial chamber is connected to the inflow region, also known as the venous pole. The outflow tract and inflow region are largely derived from the secondary heart field cardiogenic cells. Several signaling molecules have been shown to regulate the formation of the outflow tract and inflow region. One of such molecules is FGF8, a FGF family member with 233 amino acids (-27 kDa). FGFS is required for cell proliferation in the secondary heart field and development of the left and right outflow tract myocardium (Waldo et al., 2001). FGFS mutations result in defects of the outflow tract as well as the aorta and pulmonary artery trunk (Abu-Issa et al., 2002). Conditional deletion of FGF receptor 1 (FGFRl) and FGFR2, which interact with FGFS, in the secondary heart field results in a similar phenotype (Rochais et al., 2009). FGFS expression is possibly regulated by the FOXC1 and FOXC2 (forkhead box protein Cl and C2, respectively) signaling pathways. FOXCl (553 amino acids, -57 kDa) and FOXC2

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Part One (501 amino acids, -54 kDa) belong to the forkhead family of transcription factors, characterized by the presence of the DNA-binding forkhead domain. These transcription factors may serve as upstream activators of T-box 1 transcription factor C (Tbxl), a 398 amino acid protein (-43 kDa) with a T-box DNA-binding domain (Buckingham etal., 2005). Tbxl caninduceexpressionofFGF8, which in turn regulates the proliferation of the secondary heart field cells. Genetically induced Tbxl gene overexpression causes FGF8 upregulation in the secondary heart field and overgrowth of the outflow tract myocardium (Hu et al., 2004). Deficiency of the Tbxl gene results in impaired cell proliferation in the secondary heart field that gives rise to the outflow tract (Xu et al., 2004). A clinical consequence of Tbxl gene mutation is outflow tract defects, known as DiGeorge syndrome. Several forms of Tbxl gene mutation have been identified in DiGeorge syndrome families (Epstein and Parmacek, 2005). Taken together, experimental and clinical investigations have suggested that the FOXC1/FOXC2, Tbxl, and FGF8 signaling pathways act coordinately to regulate the formation of the outflow tract. Any mutation of genes encoding these factors may cause congenital outflow tract defects. The development of the left and right atria is regulated by a set of signaling molecules. One particular signaling pathway involves the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), also known as nuclear receptor subfamily 2 group F member 2 (NR2F2). This protein (414 amino acids, -46 kDa) is predominantly expressed in atrial cardiogenic cells and has been shown to regulate the morphogenesis of the left and right atria (Pereira et al., 1999). Another molecule that regulates atrial formation is LIM/homeodomain Isll transcription factor (Isll, 349 amino acids, -39 kDa), expressed in regions of developing atria derived from the secondary heart field (Cai, 2003). Embryos with Isll mutation display reduced growth of the posterior atrial myocardium (Buckingham et al., 2005). Several other signaling molecules, including Nkx-2.5 and Tbx5, have also been implicated in regulating atrial development. Involvement of these molecules was demonstrated based on genetic analyses of congenital heart defects. In humans, congenital atrial septa! defects are associated with Nkx-2.5 mutations (Schott et al., 1998). Tbx5 is a transcription factor (518 amino acids, 58 kDa) belonging to the T-box family of proteins. This factor regulates atrial septum formation via interaction with Nkx-2.5 (Epstein and Parmacek, 2005). Overall, the development of the left and right atria is regulated through coordinated mechanisms involving multiple signaling molecules. However, the exact signaling pathways remain to be identified.

Development of the cardiac conduction system Processes of cardiac conduction system development The cardiac conduction system, composed of the sinus node, atrioventricular node, and ventricular conduction network (including the atrioventricular bundle, bundle branches, and Purkinje fibers), initiates and propagates action potentials that induce myocardial contraction (Mohan et al., 2018). Cells in the conduction system are specified cardiomyocytes. The sinus and atrioventricular nodes contain a subset of cardiomyocytes characterized by the ability of automatic initiation of cyclic action potentials. The conduction fibers contain cardiomyocytes capable of conducting rapid action potentials. Under physiological conditions, action potentials are generated in the sinus node, propagates through the atrial myocardium, atrioventricular node, and ventricular conduction system. Whereas a ventricular conduction fiber network is present,

Chapter Two there is no such fiber network found in the atria. It is likely that the atrial wall myocardium serves as a conduction system. When the function of the sinus node is impaired due to a pathological disorder, such as ischemia, lower levels of conduction cells are capable of initiating action potentials that control myocardial contractile activities. During embryonic development, the cardiac conduction system arises from the myocardium. A specified set of cardiomyocytes is capable of differentiating into conduction cells. In particular, the sinus node arises from the inflow tract, the atrioventricular node originates from the atrioventricular canal, whereas the ventricular conduction subsystem develops from the trabecular ventricular cardiomyocytes (Moorman et al., 1998). The formation of a pace making region starts in the inflow tract approximately at about 20 days from ovum fertilization, followed by the establishment of other conduction subsystems. The sinus node can be recognized morphologically at about five weeks. Other conduction system components are identifiable at about the same time. The conduction system cells remain certain cardiomyocyte phenotypes, such as expression of actin and myosin contractile filaments and presence of sarcoplasmic reticulum. However, the nodal cells are usually smaller than the contractile cardiomyocytes, the actin and myosin filaments are poorly organized, and the sarcoplasmic reticulum are not well developed (Moorman et al., 1998; Mohan et al., 2018). These contractile myocytic characteristics demonstrate that the conduction system cells develop through transdifferentiation of cardiomyocytes.

Regulation of conduction system development The formation of the cardiac conduction system is regulated by complicated signaling pathways (van Weerd and Christoffels, 2016). One example is the endothelin-1 signaling pathway, playing a role in the regulation of Purkinje fiber formation (Hyer et al., 1999). Endothelin-1 (212 amino acids, -24 kDa) is produced and released from vascular endothelial cells. This protein is known to induce vasoconstriction. During cardiac development, endothelin-1 has been shown to induce transdifferentiation of a subset of specialized cardiomyocytes, localized to the developing coronary arteries, to conduction system cells (Hyer et al., 1999). The conduction system cells express a high level of Nkx-2.5 and homeodomain-only protein (HOP, 73 amino acids, -8 kDa). The latter interacts with serum response factor (SRF, 508 amino acids, -52 kDa), a transcription factor capable of binding to the serum response element (SRE) of genes and modulating SRF-dependent gene expression in the developing myocardium. Nkx-2.5 is known to regulate the expression of HOP, which in turn mediates the formation of the conduction system. Genetically induced mutation of these factors results in defects of the atrioventricular node and the proximal His conduction fibers (Chen et al., 2002; Pashmforoush et al., 2004). These observations support the role of Nkx-2.5 and HOP in regulating the formation of the cardiac conduction system.

Development of the Vascular System Origin of vascular cells The vascular system consists of arteries, capillaries, and veins. The arteries conduct blood flow from the heart to the capillaries where gas and nutrient exchange takes place. The veins drain blood from the capillary network and return blood to the heart. The arteries and veins contain several cell types, including endothelial cells, smooth

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Hemangioblasts · -

/ Capillary

Smooth muscle cells

F111uRE

2.4 Development of blood vessels. SMC: Smooth muscle cell.

muscle cells, and fibroblasts, and extracellular matrix components, including collagen fibers, elastic laminae or fibers, and proteoglycans. The capillaries contain only a single layer of endothelial cells and a layer of extracellular basement membrane. All vascular cell types arise from vascular progenitor cells originated from the splanchnic mesoderm. Formation of blood vessels occurs almost simultaneously with cardiac development. As the first step, specified mesodermal cells differentiate into hemangioblasts, which are stem cells for vascular and blood cells. Hemangioblasts migrate to designated tissues and organs, where they form cell clusters known as blood islands. A blood island is composed of two types of cell-angioblasts (outer cells of the blood island) and hematopoietic stem cells (inner cells). Angioblasts give rise to vascular cells, including endothelial cells, smooth muscle cells, and fibroblasts (Fig. 2.4), whereas hematopoietic stem cells develop into blood cell types, including erythrocytes, leukocytes, and platelets.

Yasculogenesls and anglogenesls There are two distinct forms of vascular development - vasculogenesis and angiogenesis. Vasculogenesis is the formation of primary endothelial cells, capillaries, arteries, and veins, during the embryonic stage, whereas angiogenesis is the formation of capillaries, small arteries, and veins, based on existing capillaries, which occurs in the embryo during development as well as somatic tissues in response to injury (Folkman and D'Amore, 1996). For vasculogenesis, angioblasts differentiate into endothelial cells, which subsequently form tube-shaped capillaries. The capillaries are organized into a capillary network, from which capillaries extend and arteries and veins grow via angiogenesis. Newly established blood vessels can extend into avascular regions of the embryo (Folkman and D'Amore, 1996; Potente et al., 2011). During vasculogenesis and angiogenesis, angioblasts can also differentiate into pericytes around the established capillaries. The pericytes are recruited to newly established capillaries to transform into smooth muscle cells and fibroblasts, leading to the formation of arteries and veins.

Chapter Two In adults, angiogenesis is activated in response to injury, a process establishing micro-

vascular networks in injured and regenerated tissues to facilitate wound healing (Potente et al., 2011).

Regulatlon of vascular formation Vasculogenesis and angiogenesis are controlled by mechanisms involving various signaling networks during the embryonic development. A key process is cell differentiation regulated by a combination of growth and morphogenetic factors released from selected embryonic cell types within and surrounding the blood islands in a temporally and spatially controlled manner. Several regulatory factors have been identified, including vascular endothelial growth factors (VEGFs), angiopoietins, and epherins. Coordinated and synergic actions of these factors are required for the formation of functional blood vessels. VEGFs are a family of endothelial cell-derived proteins, including VEGF-A, B, C, and D and placental growth factor (PLGF or PGF). Among these factors, VEGF-A (232 amino acids, -27 kDa) was the first member discovered and is well known for its role in regulating vasculogenesis and angiogenesis. VEGF-A can bind to several receptors, including VEGF receptor 1 (VEGFRl, 1338 amino acids, -151 kDa), VEGFR2 (1356 amino acids, -152 kDa), and VEGFR3 (1298 amino acids, -146 kDa). VEGFR 1 is also known as Fms-like tyrosine kinase 1 (Fltl); VEGFR2 as kinase insert domain receptor (KOR) or Petal liver kinase 1 (Flkl); and VEGFR3 as Fms-like tyrosine kinase 4 (Flt4). These receptors all contain protein tyrosine kinases in their intracellular domain and are referred to as protein tyrosine kinase receptors. VEGFs and their receptors regulate cell survival, proliferation, and differentiation via the mediation of the Ras-MAPK signaling pathways. VEGFRl and VEGFR2 are expressed in vascular endothelial cells, whereas VEGFR3 is largely expressed in lymphatic endothelial cells, but can also be found in vascular endothelial cells (Kukk et al., 1996; Ferrara et al., 2003). VEGF-A is the most important factor regulating vascular morphogenesis, whereas the other VEGFs play a less important role. VEGF-B (207 amino acids, -22 kDa) interacts with VEGFRl, contributing to the regulation of vascular cell survival and stability (Zhang et al., 2009). VEGF-C (419 amino acids, -47 kDa) binds to VEGFR2 and VEGFR3, plays a role in regulating angiogenesis and vascular wall permeability, and also contributes to the formation and maintenance of lymphatic vessels (Gale and Yancopoulos, 1999). VEGF-D (354 amino acids, -40 kDa) interacts with VEGFR2 and VEGFR3 (Achen et al., 1998; Gale and Yancopoulos, 1999) and is similar to VEGF-C in structure and function. PLGF (221 amino acids, -25 kDa) interacts with VEGFRl and stimulates endothelial cell proliferation and migration (Gale and Yancopoulos, 1999). VEGF-A and its receptors VEGFRl and VEGFR2 have been studied extensively for their role in regulating embryonic vasculogenesis and angiogenesis. The VEGF-AVEGFR2 signaling pathway has been shown to stimulate endothelial cell proliferation, migration, and sprouting, critical processes for embryonic vascular morphogenesis (Cleaver and Melton, 2003). Genetically induced homozygous knockout of VEGF-A or even the loss of a single VEGF-A allele in the mouse results in embryonic lethality due to failure of vascular formation (Carmeliet et al., 1996; Ferrara et al., 1996). Similarly, genetic deletion of VEGFR2 in the mouse induces severe impairment of vascular morphogenesis, resulting in embryo death (Gale and Yancopoulos, 1999).

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Part One These observations suggest a critical role for VEGF-A and VEGFR2 in regulating vasculogenesis and angiogenesis. It is interesting to note that VEGFRl may exert a different effect on vascular morphogenesis compared with VEGFR2. Genetically induced deficiency of VEGFRl in the mouse is associated with increased formation of endothelial cells (Fong et al., 1995), suggesting that VEGFRl may down-regulate the activity of VEGF-A and inhibit endothelial cell formation. Thus, in response to VEGF-A, the stimulatory receptor VEGFR2 and the inhibitory receptor VEGFRl may act in coordination to ensure an appropriate level of endothelial cell formation and vascular morphogenesis (Gale and Yancopoulos, 1999). Another type of secreted factor that regulates endothelial cell formation is the family of angiopoietins, including angiopoietin 1 (Angl, 498 amino acids, -58 kDa), Ang2 (496 amino acids, -57 kDa), Ang3 (491 amino acids, -57 kDa), and Ang4 (503 amino acids, -57 kDa) (Davis et al., 1996; Suri et al., 1996; Valenzuela et al., 1999; Cleaver and Melton, 2003). These angiopoietins are capable of binding to tunica interna endothelial cell kinase receptor (Tie2, 1124 amino acids, -126 kDa), but exert different effects. Angl and Ang4 can activate the Tie2 signaling pathway, whereas Ang2 and Ang3 exert an opposite effect (Davis et al., 1996; Gale and Yancopoulos, 1999). Endothelial cells express another angiopoietin receptor tyrosine kinase with immunoglobulin-like and EGF-like domains (Tiel, 1138 amino acids, -125 kDa). However, it remains unclear whether angiopoietins interact with Tiel. Among the angiopoietin molecules, Angl and Ang2 have been studied extensively for their roles in regulating vascular morphogenesis. Angl is largely expressed in vascular pericytes and smooth muscle cells and acts on Tie2 in endothelial cells via a paracrine mechanism (Davis et al., 1996; Suri et al., 1996). Unlike VEGF-A, Angl may not directly stimulate endothelial cell differentiation and vascular formation, but regulates the interaction of endothelial cells with pericytes or smooth muscle cells, enhancing the stability of the established primitive vasculature. Angl may coordinate with VEGF-A in regulating vascular morphogenesis. While VEGF-A stimulates early endothelial cell differentiation and vascular formation, Angl acts in a later stage to maintain the integrity of the established vasculature (Gale and Yancopoulos, 1999). In mice with Angl or Tie2 deficiency, primitive capillary networks can form during the early embryonic stage, but endothelial cells fail to attach to and interact with pericytes and extracellular matrix, resulting in abnormal vascular morphology (Sato et al., 1995; Suri et al, 1996). These observations support the role of Angl and Tie2 in regulating the maturation and stability of the vascular system. Ang2 is an antagonist to Angl, although both act on the tyrosine kinase receptor Tie2. In mice with Ang2 overexpression, the embryo dies due to impairment of vascular development, a phenomenon similar to that seen in mice with Angl or Tie2 deficiency (Davis et al., 1996; Suri et al., 1996). Taken the roles of Angland Ang2 together, it seems that the stimulatory and inhibitory angiopoietins act in coordination to ensure an appropriate level of vascular development and stability. Whereas Angl strengthens the developing vasculature, Ang2 reverts blood vessels to a relatively unstable state and liberates endothelial cells, possibly a necessary process for blood vessel sprouting. Such a mechanism is supported by the observation that Ang2 is upregulated at sites with a high level of angiogenesis, which is usually associated with upregulation of angiogenic factors such as VEGFs (Davis et al., 1996; Suri et al., 1996). Ang2-induced vascular alterations may render the vasculature ready for VEGF-mediated induction of endothelial cell proliferation and new capillary formation. The coordinated actions of

Chapter Two Angl and Ang2 represent an example of regulatory processes in biosystems involving factors with opposing functions. In addition to VEGFs and angiopoietins, there is a third family of proteins that participate in the regulation of embryonic vascular morphogenesis. These proteins are epherins or Eph family receptor interacting proteins, including two structurally different subfamilies----epherin class A and epherin class B (Gale and Yancopoulos, 1999). Epherins are ligands for a family of tyrosine kinase receptors known as Eph receptors (the term Eph was derived from the cell line erythropoietin-producing hepatocellul.ar carcinoma, from which the first Eph receptor was discovered) (Hirai et al., 1987). The epherin class A subfamily includes at least six members, epherins A1-A6, which attach to the cell member by interacting with glycosylphosphatidylinositol molecules. The epherin class B subfamily includes three members, epherins Bl, B2, and B3, which are all transmembrane glycoproteins. With several exceptions, class A epherins usually bind to class A Eph receptors, including EpMl-AlO, and class B epherins interact with class B Eph receptors, including EphB1-B4 (Pandey et al. 1995; Pasquale, 1997). As epherins are cell membrane-anchored molecules, cell-cell interactions are required for epherin-Eph receptor binding. In response to epherin binding, Eph receptors undergo autophosphorylation on the tyrosine residues of the cytoplasmic domain, resulting in activation of the tyrosine kinase receptor signaling pathways (Bruckner et al., 1997; Gale and Yancopoulos, 1999). Given that the class B epherins are transmembrane proteins, they can serve as ligands as well as receptors. Binding of epherin B ligands to Eph receptors may induce bidirectional signaling, resulting in activation of both epherinand Eph receptor-borne cells. Indeed, the epherin-B ligands may be phosphorylated in their cytoplasmic domains and induce intracellular signaling events (Holland et al., 1996; Bruckner et al., 1997). Among the epherins and Eph receptors, epherin-Al (205 amino acids, -24 kDa), epherin-B2 (333 amino acids, -37 kDa), and the Eph receptor EphB4 (987 amino acids, -108 kDa) have been implicated in the regulation of embryonic vascular formation. Epherin-Al is expressed at sites of embryonic vasculogenesis (Flenniken et al., 1996; McBride and Ruiz, 1998), inducing directed endothelial cell migration and capillary formation (Pandey et al., 1995; Daniel et al., 1996). Epherin-B2 and its receptor EphB4 are expressed in endothelial cells. The interaction of epherin-B2 ligands with cognate receptor EphB4 mediates arterial and venous formation (Wang et al., 1998). In mice with genetically induced deficiency of epherin-B2, endothelial cells fail to interact with pericytes, resulting in impairment of arterial and venous formation (Wang et al., 1998). It is interesting to note that, although primitive arterial and venous systems develop in mice with epherin-B2 deficiency, they display abnormal morphology with a lack of arterial and venous inter-digitation (Wang et al., 1998). These observations suggest that endothelial cell activation through epherin-B2-EphB4 interactions is an important process for regulating the development and stability of arteries and veins.

Formation of smooth muscle cells and flbroblasts Smooth muscle cells are a cell type localized to the tunica media of the arteries and veins, whereas fibroblasts are found in the tunica adventitia. Although both cell types arise from the same embryonic origin (mesoderm), they exhibit different phenotypes. Smooth muscle cells express ~-actin and myosin filaments and are capable of contraction and relaxation, contributing to the control of blood vessel diameter and resistance

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Part Ou to blood flow. Smooth muscle cells also produce extracellular matrix proteins including collagen, tropoelastin, and proteoglycans. In contrast, fibroblasts do not exhibit apparent contractile activities. The primary function of fibroblasts is to produce extracellular matrix. The formation of distinct vascular cell types may be regulated by cell-specific m.orphogenetic factors. One such factor is platelet-derived growth factor (PDGF)-B (PDGF-B, 241 amino acids, -27 kDa). This growth factor is present in the dimeric form (PDGF-BB) and has been shown to regulate specifically smooth muscle cell formation from pericytes recruited to newly formed blood vessels (Hellstrom et al, 1999; Owens et al., 2004). PIX;F-BB can bind to PIX;F receptor-13 and activate the receptor tyrosine kinase signaling pathways, contn'buting to the determination of smooth muscle cells (Lindahl et al., 1997). Extracellular matrix components aze known to mediate cell survival, differentiation, and proliferation (Liu et al., 2005, 2008). Smooth muscle cells reside within an elastic lamina-rich extracellular matrix in the vascular tunica media, whereas fibroblasts reside within a collagen-rich matrix in the tunica adventitia. The distinct matrix components may differentially influence the fate and development of vascular cells. Elastic laminae or fibers have been shown to inhibit smooth muscle cell proliferation and migration, and prevent leukocyte adhesion, whereas the collagen matrix exerts an opposite effect (Llu et al., 2005, 2008). In transgenic mice with elastin deficiency and incomplete development of elastic laminae, excessive SMC proliferation occurs during the embryonic stage, resulting in intimal hyperplasia and arterial stenosis (Li et al, 1998). I days

10days

20days

CD34+ o.-actin+ cells on elastic laminae

CD34+ C)(-8ctln+ cells on collagen matrix

F11uRE 2.5 Differentiation of bone marrow-derived CD34•a-ectrn• cells to smooth muscl«Hlke cells when cultured on arterial elastic laminae, but not on arterial collagen matrix in vitro. Green: Smootll muscle OM1ctin filaments. Blue: Cell nuclei. Scale: 10 11m. (From LJu et al., 2008 by permission.)

Chapter Two Similar pathological changes have been found in human arterial disorders with incomplete development of elastic laminae, such as supravalvular stenosis and William's syndrome (Mari et al., 1995; Morris and Mervis, 2000). Furthermore, exposure to elastic fibers or laminae enhances differentiation of vascular progenitor cells to smooth muscle cells and contributes to the maintenance of the smooth muscle cell contractile phenotype (Liu et al., 2008). Arterial medial elastic laminae can induce transformation of bone marrow-derived CD34+o.-actin+ cells to smooth muscle-like cells, whereas arterial adventitial collagen-rich matrix does not cause such a change (Fig. 2.5) (Liu et al., 2008). These observations support the role of elastic fibers and laminae in the regulation of smooth muscle cell formation and maintenance. In the arterial and venous tunica adventitia, the co-existence of fibroblasts and collagen matrix suggests a possible role for collagen matrix in regulating fibroblast formation. However, this role has not been confirmed in experimental tests. The mechanisms of elastic lamina-mediated smooth muscle-like cell formation has been studied by using in vitro and in viva experimental model systems. When bone marrow-derived CD34+o.-actin+ cells are cultured on arterial elastic laminae, the inhibitory receptor signal regulatory protein o. (SIR.Pc., 503 amino acids, -55 kDa) was phosphorylated. This receptor is a transmembrane glycoprotein that antagonizes cell proliferative activities, such as cell migration and proliferation, induced by receptor tyrosine kinase signaling pathways. These observations suggest that elastin components from the elastic laminae may serve as SIR.Po. ligands, causing phosphorylation and activation of SIR.Pc.. Further studies have demonstrated that phosphorylated SIRPn can recruit and activate SH2 domain-containing protein tyrosine phosphatase 1 (SHPl, 595 amino acids, -68 kDa), which dephosphorylates and represses protein tyrosine kinases, including receptor tyrosine kinases, Src kinases, and JAK kinases (Liu et al., 2005). As activation of these protein tyrosine kinases causes a transition of the contractile phenotype to the proliferative phenotype of smooth muscle cells, repression of these protein tyrosine kinases supports the maintenance of the smooth muscle contractile phenotype. Inhibition of SIR.Po. and SHPl expression by siRNA-mediated gene silencing or deletion of the SHP-1 gene in a mouse model system results in significantly reduced formation of smooth muscle-like cells from bone marrow-derived CD34+o.-actin+ cells on arterial elastic laminae (Liu et al., 2008). These investigations support the role of elastic laminae in regulating the smooth muscle cell formation and maintenance via the mediation of the SIRPo.-SHPl signaling network. However, it remains to be determined how the SHPl signaling pathway controls smooth muscle cell differentiation. Overall, soluble morphogenic factors and extracellular matrix may act coordinately to control the development of vascular cells.

Development of Blood Cells Blood cells include erythrocytes, leukocytes, and platelets. Erythrocytes are responsible for transport of oxygen and carbon dioxide; leukocytes participate in immune and inflammatory responses, and platelets are involved in blood coagulation. Leukocytes and platelets contribute to the development of vascular disorders, including thrombosis and atherosclerosis. Leukocytes consist of two groups-granulocytes and non-granulocytes. The granulocyte group is composed of neutrophils, basophils, and eosinophils, characterized by the presence of enzyme-containing granules or lysosomes in

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Part One the cytoplasm. These cells are responsible for attacking and breaking down bacteria and pathogens and participate in inflanunatory and allergic responses. The nongranulocyte group includes monocytes and lymphocytes (f- and B-cells). Monocytes can migrate into the tunica intima of blood vessels and the interstitial compartment of organs and transform into macrophages, a cell type involved in inflanunatory responses. Macrophages can also carry and present foreign antigens or pathogens together with their cell membrane major histocompatibility complexes (MHCs) to T-lymphocytes for immune reactions. T- and B-cells are major cell types of the immune system. A fraction of lymphocytes is circulating in the blood, while others reside in lymphoid organs, including the lymph nodes and spleen. T-lymphocytes can recognize antigens presented by macrophages through specific receptors, triggering processes of antigen destruction. B-lymphocytes are capable of recognizing specific antigens, producing antibodies that form complexes with and neutralize antigens. During embryonic development, blood cells arise from hematopoietic stem cells derived from the mesodermal hemangioblasts (Liu, 2007). The hemangioblasts can migrate to designated tissues and organs, forming cell clusters known as blood islands. In a blood island, hemangioblasts give rise to two cell types-hematopoietic stem cells and angioblasts. Hematopoietic stem cells develop into blood cell types, a process known as hematopoiesis; whereas angioblasts give rise to vascular cells. There are two forms of hematopoiesis---embryonic and somatic hematopoiesis, which occur during and after the embryonic stage, respectively. During the embryonic stage, hematopoiesis initially occurs in the blood islands. The blood islands are not permanent sites for hematopoiesis. These sites change in location for several times during the embryonic period. The first change takes place when blood vessels form in blood islands. The sites of hematopoiesis are moved from the blood islands to temporary embryonic structures near the aorta known as aorta-gonad-mesonephroi. With the degeneration of the mesonephroi, the sites of hematopoiesis are shifted to the liver. During the late embryonic stage, hematopoiesis takes place in the bone marrow. During the somatic stage, the bone marrow is the permanent site of hematopoiesis. Blood cells die constantly through the lifespan and are replenished with newly generated cells from the bone marrow. In the adult bone marrow, there exist multipotent hematopoietic stem cells, which can self-renew, proliferate, and differentiate into all mature blood cell types through the entire lifespan (Liu, 2007). The population of hematopoietic stem cells in the bone marrow is about 0.01% of the total cells. Hematopoietic stem cells can differentiate into lineage progenitor cells, including B- and T-lymphocyte progenitor cells and myeloid progenitor cells (Fig. 2.6). The B-lymphocyte progenitor cells can differentiate into B-lymphocytes and plasma cells through several intermediate cell lineages, including the pro-B cell and pre-B cell lineages. The T-lymphocyte progenitor cells can differentiate into T-lymphocytes through several similar stages. The myeloid progenitor cells can differentiate into six cell lineages-erythroid precursors, platelet precursors, monocyte precursors, neutrophil precursors, basophil precursors, and eosinophil precursors. The erythroid precursors can differentiate subsequently into proerythroblasts, erythroblasts, reticulocytes(withnucleiremoved),anderythrocytes. Theplateletprecursorscandevelop into megakaryoblasts and megakaryocytes, which produce platelets by cell membrane and cytoplasmic fragmentation. The monocyte precursors can give rise to monoblasts and subsequently monocytes, which transform into macrophages when migrating into the blood vessel wall and interstitial space. The neutrophil, basophil, and eosinophil precursors can differentiate into neutrophils, basophils, and eosinophils, respectively.

Chlphr Twa

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6.3 Signaling mechanisms of interleukin 3 {IL3). IL3R: IL3 receptor. JAK: Janus kinase. STAT: Signal transducer and activator of transcription.

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complex, autophosphorylation of the receptor-associated protein tyrosine kinases JAKl and JAI800 protein-encoding genes, whereas the Y chromosome contains only ..2, 137 IFN>-.3, 137 IFNw, 137 IFN~. 137 IGFl, 149 IGFlR, 149 IK.B, 140, 239, 311 IL. See Interleukins (ILs) ILl,186 ILl subfamily of cytokines, 129f, 130-131 ILlo.,130,313,436 ILl~, 130, 436 ILlRl, 131, 436 IL1R2, 131 ILlRa, 130 ILlRAcP, 131, 437 112, 131, 132/, 313 IL2Ra, 131

Index IL2Rj'.3, 131 IL3, 131, 133/, 313 IL3Ro., 131 IlA, 131, 313 IL5, 131 IL5Ro., 131 IL6,133,186,240,241f,313,437 IL6 subfamily of cytokines, 129/, 133, 134/ IL6Ro., 133, 437 IL7, 131 IL8, 186 IL9, 131 ILlO, 135, 135/, 313 ILlO subfamily of cytokines, 129/, 135, 135/ ILll, 133 ILllRo., 133 IL12, 135, 136/, 313 IL12 subfamily of cytokines, 129/, 135-136, 136/ IL13, 131 IL15, 131 IL17 subfamily of cytokines, 129/, 136-137 IL17A, 136 IL17B, 136 IL17C, 136 IL17D, 136 IL17E, 136 IL17F, 136 IL18, 130 IL19, 135 IL20, 135 IL22, 135 IL23, 135 IL24, 135 IL25, 136 IL26, 135 IL27, 135, 313 IL28A, 135, 137 IL28B, 135, 137 IL29, 135, 137 IL33, 130 IL35, 135, 313 IL36a, 130 IL36f3, 130 IL36'Y, 130 IL36Ra, 130 IL37, 130 IL38, 130 ILGF,232 Illnesses. See Diseases and disorders lliumina "sequencing by synthesis" method, 259, 259f

Immunoglobulin light chain, 390 In situ preconditioning injury, 228/ Induced DNA methylation, 273 Induced mild-level preconditioning injury, 228/ Induced mild-level remote preconditioning injury, 228/ Induced pluripotent stem cells (iPSCs), 51-54, 280, 400, 448 Inducible nitric oxide synthase (iNOS), 106 Induction of cell apoptosis, 244 Induction of distant protective factors, 235--236 Infectious disease, 166 Inferior vena cava, 69/, 83 InflaIIUilation,215-216,232,313 InflaIIlIIlation-repressing cytokines, 313 InflaIIlIIlation-stimulating cytokines, 313 InflaIIUilatory processes, 111-113 InflaIIUilatory regulatory factor gene mutations, 313 InflaIIUilatory response, 232, 325-326, 368 Influenza virus, 170, 171/ Inherited familial dilated cardiomyopathy, 382 Inherited gene mutations, 208 Inner mass cell, 19/, 21 iNOS. See Inducible nitric oxide synthase (iNOS) Inositol triphosphate. See IP3 Insertion mutation, 165/, 208 Insulin-like growth factor 1. See IGFl Interferons (IFNs), 129/, 137, 138f See also individual interferons Interleukins (ILs), 129/, 130-137. See also individual interleukins common f3 subfamily, 129/, 131, 133, 133/ common 'Y subfamily, 129/, 131, 132/ ILl subfamily, 129/, 130-131 IL6 subfamily, 129/, 133, 134f ILlO subfamily, 129/, 135, 135/ IL12 subfamily, 129/, 135-136, 136/ IL17 subfamily, 129/, 136-137 Intermediate mesoderm, 23 Internal elastic lamina, 83 Internal environmental insults, 162-163 Internal mechanical forces, 187-196 Interstitial colloid osmotic pressure, 95 Interstitial hydrostatic pressure, 95 Intima (tunica intima), 83 Intra-aneurysmal wall cell injection of stem cells, 354

467

468

Index Intracellular antioxidant enzymes, 320 Intracranial atherosclerosis, 442 Intrapericardial injection (cardiomyopathies), 397 Intravascular stenting, 442 Intravenous cell administration of stem cells, 354

Intrinsic cardiac control mechanism, 77-78 Inward rectifier K+ channel, 92, 93/ Ion channel blockers, 444 Ion channels, 84 Ion-dependent amino acid transport system, 99-100, 99/ Ion-independent amino acid transport system, 100 Ion pumps and exchangers, 84 Ion transport, 84--95 Ca2+channels, 85-87 c1- channels, 92-95 K+ channels, 92, 93/ Na+ channels, 88-92 Ionizing radiation, 196

IPs Ca2+channels, 85, 87, 107, 108, 114, 310 calcium release, 21, 148, 239, 310 IP3. SeeIP3 iPSCs. See Induced pluripotent stem cells (iPSCs) IRAI