Cardiovascular Engineering A Protective Approach Shu Q Liu Full Chapter

Cardiovascular Engineering: A
Protective Approach Shu Q. Liu

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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

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Contents
Preface....................................................... xv
1 Introduction . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . . . . . . . • • . 1
llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Overview..................................................... 2
Foundations of Protective Engineering........................ ... . 3
Pathogenic causes and processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Naturally occurring protective mechanisms . . . . . . . . . . . . . . . . . . . . . 4
Regional protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Distant protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Molecular protective engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Enhancing protective impacts by protein administration
and gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Suppressing adverse gene expression. . . . . . . . . . . . . . . . . . . . . . . . . 9
Gene editing-mediated control of gene expression. . . . . . . . . . . . . . 9
Cell-based protective engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Tissue-level protective engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Part I Foundations of Cardlovascular Protective Engineering

2 Development of the Heart, Blood Vessels, and Blood cells . . . . . . • • • 17
llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Early Embryonic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Overview of embryonic processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Fertilization-union of the sperm and ovum. . . . . . . . . . . . . . . . . . . . . 19
Cleavage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Formation of blastocyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Gastrulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Development of the Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Origin and fate of cardiogenic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Regulation of heart development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Formation of the heart tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Processes of heart tube formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Regulation of heart tube formation . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Cardiac chamber formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Processes of cardiac chamber formation. . . . . . . . . . . . . . . . . . . . . . . 28
y
Yi Contents
Regulation of cardiac chamber formation . . . . . . . . . . . . . . . . . . . . . 29

Development of the cardiac conduction system . . . . . . . . . . . . . . . . . . 30
Processes of cardiac conduction system development . . . . . . . . . . 30
Regulation of conduction system development . . . . . . . . . . . . . . . . 31
Development of the Vascular System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Origin of vascular cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Vasculogenesis and angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Regulation of vascular formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Formation of smooth muscle cells and fibroblasts . . . . . . . . . . . . . . . . 35
Development of Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3 Stem Cells and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Em.bryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Somatic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Bone marrow stem cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Hematopoietic stem cells.................................... 55
Mesenchymal stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Resident cardiac stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
MDRl-positive resident cardiac stem cells..................... 57
Sca-1-positive resident cardiac stem cells.............. ...... .. 58
Isll-positive cardiac stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4 Structure and Function of the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Structure of the Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Cardiac chambers and great vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Cardiac vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Cardiac cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Function of the Heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Excitation-contraction coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Cardiac contractile activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Regulation of cardiac performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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
Barrier function...................................... .... .. 84
Ion transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Protein transport......................................... 95
Ca nt ent s vii
Amino acid transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Glucose transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Routes of substance transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Evaluation of endothelial transport function. . . . . . . . . . . . . . . . . 103
Regulation of pro- and anti-blood coagulation processes . . . . . . . . 103
Regulation of vascular contractility. . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Regulation of cell proliferative activities . . . . . . . . . . . . . . . . . . . . . . 109
Regulation of inflammatory processes . . . . . . . . . . . . . . . . . . . . . . . . 111
Generation of extracellular matrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Functions of smooth muscle cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Contraction and relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Regulation of vascular cell proliferative activities. . . . . . . . . . . . . . . 117
Generation of extracellular matrix...................... ..... . 117
Functions of fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Regulation of Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Regulation of Systemic Blood Pressure. . . . . . . . . . . .. . . . . . . . . . . . . . . . 121
Baroreceptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Chem.oreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Renin-angiotensin-aldosterone system. . . . . . . . . . . . . . . . . . . . . . . . . . 122
Vasopressin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
Nitric oxide................................................. 123
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Highlights . , , ................ , , ................ , , . . . . . . . . . . . . . 161
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Etiology and Pathogenic Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
viii Contents
External environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Pathogenic viruses....................................... 167
Pathogenic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Pathogenic fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Pathogenic parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Physical factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Mechanical forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Radiation............................................... 196
Body teinperature........................................ 197
Ovemutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Protein overconsumption................................. 199
Lipid and carbohydrate overconsumption . . . . . . . . . . . . . . . . . . 200
Chemical substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Cigarette smoke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Alcohol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Biological factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Antigens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Prions.................................................. 205
Mental stress.............................................. 207
Genetic defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Chromosomal defects ...................................... 208
Addition of a single chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Chromosomal deletion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Addition of a complete haploid set of chromosomes . . . . . . . . . 209
Simple gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Autosomaldominantdisorders .................... .... .... 210
Autosomal recessive disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
X-linked disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Y-linked disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Multiple gene mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Senescence.................................................. 214
Pathology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
8 Systems Protective Mechanisms. . • • . . . . . . . • • . . . . . . .. .. . . . . . .. • . . 225
llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
Regional Cardioprotective Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Early regional protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Late regional protective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Protective inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Distant Protective Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Distant protective factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Contents ix
Discovery and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Induction of distant protective factors . . . . . . . . . . . . . . . . . . . . . . . . 235
Actions of distant protective factors . . . . . . . . . . . . . . . . . . . . . . . . . . 236
Significance of distant protective factors . . . . . . . . . . . . . . . . . . . . . . 237
Distant cell mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Bone marrow cell mobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Splenic cell mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Hepatic cell mobilization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Mechanisms of hepatic cell mobilization . . . . . . . . . . . . . . . . . . . . 240
Protective actions of mobilized hepatic cells . . . . . . . . . . . . . . . . . 242
The Concept of Core Distant Protective Mechanisms. . . . . . . . . . . . . . . . 243
The Concept of Protectome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Evolution of Protective Mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
Systems Protective Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
9 Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Understanding Pathogenic and Protective Mechanisms . . . . . . . . . . . . . 258
Identifying mutant or malfunctioned genes by gene profiling. . . . . . 258
Constructing recombinant genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Gene amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Testing the function of the gene of interest. . . . . . . . . . . . . . . . . . . . . . . 261
Molecular Protective Engineering Technologies . . . . . . . . . . . . . . . . . . . . 262
Gene transfer technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
V:rrus-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Retrovirus-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . 262
Adenovirus-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . 263
Adeno-associated viruses as gene carriers . . . . . . . . . . . . . . . . . . . 264
Receptor-mediated gene transfer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
Liposome-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
Calcium phosphate-mediated gene transfer . . . . . . . . . . . . . . . . . . . 265
Electroporation-mediated gene transfer . . . . . . . . . . . . . . . . . . . . . . 265
Assessing the expression level of the transferred gene. . . . . . . . . . . 266
Assessing the function of the transferred gene . . . . . . . . . . . . . . . . . 266
Gene editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Gene editing by zinc finger nucleases. . . . . . . . . . . . . . . . . . . . . . . . . 268
Gene editing by transcription-activator like effector nucleases . . . 269
Gene editing by the CRISPR/ Cas system. . . . . . . . . . . . . . . . . . . . . . 270
Epigenetic modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
DNAmethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Histone modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Histone acetylation................................. ...... 274
Histone methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Post-transcriptional modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
x Contents
Small interfering RNA-based mRNA modifications. . . . . . . . . . . . . 276

MicroRNA-based mRNA modifications. . . . . . . . . . . . . . . . . . . . . . . 277
Differences between siRNAs and miRNAs . . . . . . . . . . . . . . . . . . . . 279
Anti-sense oligonucleotide repression of mRNA . . . . . . . . . . . . . . . 279
Cell-Based Protective Engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Tissue-Level Protective Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
Part II Appllcatlons of Cardlovascular Protective Engineering

10 Systemic Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Primary hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Renovascular hypertension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Endocrine hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Conventional Treatment Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Molecular Protective Engineering Strategies for Primary Hypertension . 298
Boosting vasodilator gene expression. . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Nitric oxide synthase gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Natriuretic peptide precursor A gene . . . . . . . . . . . . . . . . . . . . . . . . . 300
Kallikrein and kininogen genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Suppressing vasoconstrictor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Renin-angiotensin-aldosterone system genes . . . . . . . . . . . . . . . . . . 300
Endothelin gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Adrenergic receptor genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
11 Atherosclerosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
llighlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
Gene mutations responsible for atherogenesis . . . . . . . . . . . . . . . . . . . 306
Lipid metabolism regulatory genes . . . . . . . . . . . . . . . . . . . . . . . . . . 306
Low-density lipoprotein receptor (LDLR) gene . . . . . . . . . . . . . . 307
Low-density lipoprotein genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
LDLR regulatory genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Blood pressure regulatory genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Renin-angiotensin system genes . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Sodium regulatory genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Atrial natriuretic factor gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
Adrenergic system genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
Endothelin genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Nitric oxide synthase genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Contents xi
Inflammation regulatory factor genes . . . . . . . . . . . . . . . . . . . . . . . . 313

Genes regulating vascular cell proliferation and
migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Atherogenic environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Lipid-rich diets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Exposure to cigarette smoke and toxins....................... 316
Regional mechanical factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
Antioxidant deficiency................................ .... .. 318
Superoxide free radical o;-................................ 319
Reactive nitrogen species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Transition metal ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Redox regulatory enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Antioxidants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Sedentary lifestyle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Atherogenic disorders........................................ 321
Obesity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Hypercholesterolemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Hypertension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Pathogenic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Lipid retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Cell proliferation and migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
Pathological changes during the initial stage. . . . . . . . . . . . . . . . . . . . . 327
Pathological changes during the developing stage. . . . . . . . . . . . . . . . 327
Pathological changes during the advanced stage . . . . . . . . . . . . . . . . . 327
Anti-hyperlipidemia agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
Anti-smooth muscle cell proliferative agents. . . . . . . . . . . . . . . . . . . . . 328
Alleviating angina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Relieving cardiac workload . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Angioplasty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Stent placement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Arterial reconstruction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Molecular Protective Engineering Strategies....................... 331
siRNA-mediated silencing of inflammatory and mitogenic
genes................................................. ..... . 331
Anti-inflammatory and anti-proliferative gene therapy . . . . . . . . . . . 332
Gene editing-based anti-atherogenic strategies . . . . . . . . . . . . . . . . . . 333
Cell- and Tissue-level Protective Engineering Strategies. . . . . . . . . . . . . 333
Cell-integrated extracellular matrix constructs................... 334
Cell-integrated polymeric vascular constructs . . . . . . . . . . . . . . . . . . . 335
Development of arterial constructs in vivo . . . . . . . . . . . . . . . . . . . . . . 336
Mechanical modifications of arterial constructs . . . . . . . . . . . . . . . . . . 336
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
xii Contents
12 Arterial Aneurysms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

Highlights . ' ' ................ ' ' ................ ' ' . . . . . . . . . . . . . 345
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
Biomechanics of Arterial Aneurysms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Molecular protective engineering strategies ..... , . . . . . . . . . . . . . . . 351
Cell-based protective engineering strategies . . . . . . . . . . . . . . . . . . . . . 353
Tissue-level protective engineering strategies . . . . . . . . . . . . . . . . . . . . 354
References .......................................... ......... . 355
13 Coronary- Heart Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Highlights . ' ' ................ ' ' ................ ' ' . . . . . . . . . . . . . 359
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Coronary artery thrombosis . , ................. , . . . . . . . . . . . . . . . 361
Coronary artery atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Coronary artery embolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Pathology and Pathophysiology , , ................ , , . . . . . . . . . . . . . 362
Acute myocardial infarction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Impairment of myocardial function . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Forms of cardiomyocyte death ................. ,........ ....... 364
Necrotic cell death......................................... 365
Apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
Autophagy .............. , , ................ , , . . . . . . . . . . . . . 367
Acute inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
Fibrosis and angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
Therapeutic and Adverse Impacts of Blood Reperfusion. . . . . . . . . . . . . 368
Naturally Occurring Mechanisms of Myocardial Protection. . . . . . . . . . 369
Cardioprotective Engineering Strategies ........... , , . . . . . . . . . . . . . 370
Molecular cardioprotective engineering strategies . . . . . . . . . . . . . . . 370
Protection against cardiomyocyte death....................... 372
Regional cardioprotective factors ........... , , . . . . . . . . . . . . . 372
Distant cardioprotective factors . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
Protection against oxidative stress injury. . . . . . . . . . . . . . . . . . . . . . 373
lschemic preconditioning .. , , ................ , , . . . . . . . . . . . . . 375
Ischemic post-conditioning.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
Enhancing angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
Cell-based protective engineering strategies ..... , . . . . . . . . . . . . . . . 376
Tissue-level cardioprotective engineering strategies . . . . . . . . . . . . . . 377
References.................................................... 377
14 Cardiomyopath.ies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Highlights . ' ' ................ ' ' ................ ' ' . . . . . . . . . . . . . 381
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Ca nt ent s xiii
Dilated Cardiomyopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382

Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Pathophysiology and clinical features. . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Conventional treatment strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Hypertrophic Cardiomyopathies................................. 385
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Conventional treatment strategies.............................. 389
RestrictiveCardiomyopathies ............................. ..... . 389
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Arrhythmogenic Right Ventricular Cardiomyopathy. . . . . . . . . . . . . . . . 392
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
Pathology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
Conventional treatment strategies....................... .... ... 393
Heart Failure.................................................. 394
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Pathology and pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Clinical features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Molecular Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . . 396
Modification of mutant genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
Management of ventricular arrhythmia . . . . . . . . . . . . . . . . . . . . . . . . . 398
Prevention of heart failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Cell-Based Protective Engineering Strategies . . . . . . . . . . . . . . . . . . . . . . 399
Cell-mediated delivery of cardiomyocyte supporting factors. . . . . . . 400
Stem cell-based cardiomyocyte regeneration . . . . . . . . . . . . . . . . . . . . 400
Tissue-Level Protective Engineering Strategies. . . . . . . . . . . . . . . . . . . . . 401
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
15 Congenital Heart Defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
Chromosomaldefects ........................................ 408
Single gene mutations........................................ 410
Environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
Disorders causing congenital heart defects . . . . . . . . . . . . . . . . . . . . . . 412
Left to right heart shunts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Atrial septum defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
xiv Contents
Ventricular septum defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

Patent ductus arteriosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
Heart valve defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Mitral valve defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Tricuspid valve defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416
Aortic and pulmonary valve defects . . . . . . . . . . . . . . . . . . . . . . . . . 417
Complex defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Left-to-right heart shunts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Heart valve defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Complex defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Engineering-Based Interventions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
Biomaterial-based heart valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
Biological tissue-based heart valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
Cell engineering-based heart valves . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
Materials used for cell engineering-based heart valves . . . . . . . . . . 421
Cell types used for heart valve engineering. . . . . . . . . . . . . . . . . . . . 422
Improving the performance of engineered heart valves . . . . . . . . . 423
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
16 Ischemic Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Highlights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
Pathogenesis.................................................. 431
Naturally Occurring N europrotective Mechanisms . . . . . . . . . . . . . . . . . 434
Regional neuroprotective mechanisms. . . . . . . . . . . . . . . . . . . . . . . . . . 435
Distant neuroprotective mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Naturally occurring neuronal regeneration................ ...... 441
Neuroprotective Engineering Strategies. . . . . . . . . . . . . . . . . . . . . . . . . . . 442
Molecular neuroprotective engineering strategies . . . . . . . . . . . . . . . . 442
Modulating cell survival signaling mechanisms . . . . . . . . . . . . . . . 442
Modulating cell death signaling mechanisms . . . . . . . . . . . . . . . . . . 444
Preventing secondary injury........................... ...... 444
Inducing neural resident stem cell differentiation . . . . . . . . . . . . . . 445
Preventing fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
Engineering distant neuroprotective mechanisms . . . . . . . . . . . . . . 446
Ischemic preconditioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Cell-based engineering strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
In.dex . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . . . . . . . • . . 453
Preface
he theme of this book is Protective Engineering, an emerging bioengineering disci-
T pline 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 environmen-
tal 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 enhanc-
ing 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 thera-
peutic purposes in experimental and clinical research. To date, there is a large amount
of information about the naturally occurring protective mechanisms as well as protec-
tive 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 pro-
tective 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 sys-
tem, stem cells and regeneration, structure and function of the cardiovascular system,
signaling processes of cytokines and growth factors in cardiovascular disease, mecha-
nisms of disease, naturally occurring systems protective mechanisms, and general pro-
tective 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
xv
xvi 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 car-
diovascular 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 technologies-
molecular, 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.
1
2 Chapter One
Overview
Cardiovascular engineering is a broad subject addressing the modulation of the struc-
ture 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 dis-
ease. This book focuses on Cardiovascular Protective Engineering, an emerging discipline
of cardiovascular engineering, aiming to understand the naturally occurring protec-
tive mechanisms against cardiovascular disorders and developing engineering strat-
egies 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 struc-
tural 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 protec-
tion 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 strate-
gies 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 protec-
tive engineering.
Chapter One 3
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 interven-
tions, such as protein and gene therapies, cell transplantation, and arterial stenting
and reconstruction, have been proven effective in experimental and clinical investiga-
tions. 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 pathophysi-
ology of disease, including the naturally occurring protective mechanisms against dis-
orders. 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 acti-
vated to protect cells from injury and death, thereby reducing the impact of the disor-
der. 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 strat-
egies. 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 reconstruc-
tion, 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 inflam-
matory 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.
4 Chapter One
Naturally accunlng protective mechanisms

There exist a variety of natural protective m.echanisms in mammalian systems, as dem-
onstrated through the book, serving as foundations for developing protective engineer-
ing 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 car-
diomyocyte 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 mecha-
nisms (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
~-~
' Spleen
Intestines
' 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 5
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 mar-
row, 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 ade-
nosine, opioids, bradykinin, hepatocyte growth factor (HGF), insulin-like growth factor
(ILGF), vascular endothelial growth factor (VEGF), interleukin 6 (IL6), and stromal cell-
derived 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 bradyki-
nin 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 pro-
vide 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 mor-
phogenetic 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 cardio-
myocyte 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 com-
bination 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 mecha-
nism 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),
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 reach-
ing the ischemic: myocardium, can release c:ytokines and growth factors, reducing myo-
cardial 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 addi-
tion, the liver can mobilize cells to the c:U:culatory system in response to ischemic myo-
cardial 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 express-
ing 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 technologies-
molecular, 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 7
Molecular Cellular
Protein delivery
Fibroblasts
~
Gene transfer
I
0
Gene editing
~~
Esc~;esc,
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 recov-
ery and prevent organ failure. Overall, these molecular, cellular, and tissue-level engi-
neering strategies can be designed and used for the precise control of the types, levels,
timing, and coordination of systems protective actions, thereby optimizing the pro-
tective meclumisms, minimizing cell death, and facilitating recovery from injury and
disease.
Molecular protective engineering

There are several molecular strategies for protective engineering-protein admin-
istration, 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 expres-
sion. Gene editing is a method to modify gene structures and introduce an exogenous
8 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 expres-
sion 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 reduc-
ing 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 min-
utes 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 protec-
tive 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 suffi-
cient 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 trans-
fer 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 gene-
containing 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 9
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 injury-
altered genes from a gene profiling test. An important task is to identify the most effec-
tive 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 double-
stranded 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 pro-
teins (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 repre-
sentative 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,
10 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 cardio-
protective gene expression. A gene imposing an adverse impact on protection can be
replaced by a modified gene with reduced expression capacity. Thus, CRISPR/Cas edit-
ing 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, struc-
tural 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 regeneration-
embryonic stem cells, induced pluripotent stem cells, and somatic stem cells. For myo-
cardial regeneration, embryonic and induced pluripotent stem cells are the candidates
of choice. Somatic cardiomyocytes are not usually used because these cells have a lim-
ited 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 trans-
planted 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 11
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 cell-
seeded 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 func-
tion in vivo. For instance, in ischemic myocardial injury, the most important tasks of
tissue-level protective engineering are to strengthen the ischemic myocardium for pre-
venting 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 cardiomyo-
cytes 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 capac-
ity of protection. Various protective engineering strategies have been developed at
the molecular, cellular, and tissue levels and used in experimental and clinical inves-
tigations 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, arte-
rial 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 time-
dependent 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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BUSH-FIRE ON THE MAKONDE PLATEAU
This ascent, though barren of results in other respects, has

produced one small monument of African art, a drawing of our
climbing caravan, which is here offered to the reader’s inspection.
The native artist has quite correctly indicated the steepness of the
mountain by the vertical line representing the road. The confusion of
circles and curves at the lower end stands for the buildings of the
Mission station:—the foundations of a church vast enough, should it
ever be finished, to hold all the converted heathen of Africa and the
adjacent continents; the ci devant cowhouse, in which the two aged
clergymen have found a primitive refuge after the destruction of their
beautiful buildings by the Majimaji, the boys’ school and the girls’
school—two large bamboo huts in the native style; and the dwellings
of the native teachers and boarders. The curly labyrinth at the upper
end of the line is the top of the mountain with its gneiss blocks. The
two uppermost climbers are the kirongozi or guide and one of our
men, the third is Captain Ewerbeck, and the fourth myself. The
District Commissioner is readily recognizable by the epaulettes with
the two stars denoting his military rank, which belong to the uniform
worn on duty by this class of officials. Of all attributes of the white
man this seems to make the greatest impression on the native mind,
since, in every drawing in my possession where
officers are represented, their rank is invariably (and
always correctly) indicated by the number of stars. In
the same way the native draughtsman never makes a
mistake with regard to the stripes on the sleeves of
non-commissioned officers, black or white. The
advantages of a well-developed corporation are here
evident! Ewerbeck and Seyfried are about the same
age as myself, and our chest and other measurements
are pretty nearly identical. This I suppose must be
the reason why the inhabitants of Lindi, and later on
those of the interior, have promoted me to the rank
of captain; at Lindi I went by the name of Hoffmani
mpya, “the new captain” (Hauptmann). The drawing
here reproduced is evidence of my promotion, the
artist having bestowed the epaulettes on me as well
as on Ewerbeck. The figures behind us are of no
importance, they are only the rest of our party. Now,
however, comes the psychologically noteworthy
point; I figure in the picture twice over, first
laboriously climbing the mountain, and then in
majestic pose at the top, in the act of photographing
OUR ASCENT the African landscape. You must know that the tripod
OF MTANDI
MOUNTAIN. shown in the drawing is that of my 13 × 18 cm.
DRAWN BY camera, the zig-zags between its legs are the brass
JUMA struts which keep it rigid; the long snake-like line is
the rubber tube for the release of the instantaneous
shutter—of which, as a matter of fact, I could make
no use on account of the mist,—and the photographer is, as above
stated, myself. The men behind me are my personal attendants to
whom the more fragile parts of the apparatus are usually entrusted.
The graphic reproduction of this ascent is no great achievement on
the part of the native intellect, but nevertheless it is a very important
document for the beginnings of art in general and for the African
point of view in particular. To the ethnographer, of all men, the most
apparently insignificant matters are not without importance, and
this is why the prospect of working undisturbed for many months in
these surroundings is such a delight to me.
Our ascent of Mtandi was concluded, at any rate for the present, by
a ceremonious breakfast, to which the two missionaries had kindly
invited us. Englishmen, as is well known, live extremely well in their
own country; but abroad, too, even in the far interior of a continent,
they know how to make the best of things. I was here impressed with
the fact that Masasi must be a “very nourishing district,” as Wilhelm
Raabe would say. We had no champagne, it is true—Archdeacon
Carnon had set it before us on the previous day, in a huge water-jug,
apologizing for the absence of champagne glasses. We showed him
that we were able to appreciate his hospitality, even in the absence of
such refinements.
The merriest part of our whole Mtandi expedition, however, was
the ride home, with the Mission pupils trotting along beside us. The
little fellows looked warlike enough with their bows and arrows, and
seemed desirous of shouting each other down. I could not at first
make out what they wanted, but on reaching home, that is to say, our
police-post, I soon understood that their object was nothing less than
to offer me the whole of their martial equipment for my ethnographic
collection. But not as a present—giving things away for nothing is not
in the negro’s line, and in this he resembles our German rustics. On
the contrary, these young people demanded fancy prices for the bows
which they had made on purpose to sell them to the mzungu, that
remarkable character who buys all sorts of native rubbish. I
purchased such of their wares as seemed suitable for my objects, and
thought it advisable to prevent disappointment to those whose offers
had been refused by giving each a copper or two out of the famous jar
of which we shall hear again later on. Before doing so, however, I
instituted a pleasing experiment, instructive for myself and highly
enjoyable for the youth of Masasi, in the shape of an archery
competition.
Comparative ethnography has for a long time past busied itself
with the task of classifying and analyzing all the technical and mental
activities of man. Thus some decades ago, the American, Morse,[12]
ascertained that all men who shoot, or ever have shot, with the bow,
have certain definite ways of drawing it. There are about half-a-
dozen distinct methods, which are so distributed over the globe that,
in some places the same release (or “loose” as it is technically called)
is known to be common to the whole of a large area, while elsewhere
the most abrupt contrasts may be observed between contiguous
nations or tribes. It might be supposed that there could be no
possible differences in so simple an action as that of drawing a bow;
but experiment shows otherwise, and this experiment I have made
over and over again in the course of my lectures.
It is a thousand to one that any German (leaving out of
consideration the English and the Belgians, who still practise archery
according to the rules of the game, and can distinguish a good “loose”
from a bad one), when he has taken the bow in his left hand and
grasped the arrow and the string in his right, will hold the notch as it
rests on the string between his thumb and forefinger, and thus only
indirectly draw the string by means of the arrow. This, which is the
“loose” we used on the little toy bows of our boyhood, is the very
worst conceivable, as anyone who understands the other methods
can convince himself by every shot he tries. It is obvious that the
arrow must slip from the fingers if a moderately strong pull is given.
The best proof of the inferiority of this particular “loose” is the fact
that it is very seldom found among those sections of mankind who
still use the bow as a serious and effective weapon, whether in war or
hunting. These handle it after a very different fashion. Only where
the bow is a mere survival, and only used as a toy by children (the
most conservative class in the community), as for instance among
ourselves, this method, quite useless for an effective shot, is
practised simply because no better is known.
If I felt compelled to take the boys at Masasi Mission as a standard
for estimating the culture of the race, I should have to say that here
too the bow is a survival, for nine-tenths of the whole multitude shot
in the same way as our boys at home, but with one difference; we
hold the bow horizontally, the African boys held it vertically, the
arrow lying on the left side of the string between the index and
middle finger. Only one-tenth of the whole number used a different
“loose,” and these, significantly enough, were older boys, who
therefore had evidently taken over with them into their Christianity a
considerable dose of old African conservatism.
My competition was arranged with a view, not so much of
registering the number of hits and misses, as of observing the
method of drawing; but, notwithstanding, I must say that the little
archers acquitted themselves by no means contemptibly. It is true
that the distances were short, and my mark was scarcely a small one,
being a copy of the Tägliche Rundschau; but the greater number sent
their arrows inside the rings I had hastily drawn on this improvised
target. They were proud of their success, too; and when I praised a
good shot it was good to see the triumphant looks that the little black
hero cast round on his admiring companions.
As to the other methods, if I were asked the question in my Leipzig
lecture-room, I should have to answer it at once. As it is, I am
enabled to claim the privilege of the investigator and excuse myself
from giving further information till I have collected sufficient
material by a series of fresh observations. I hope to gratify my
readers’ thirst for knowledge when I have traversed the whole plain
north of the Rovuma, and, encamped on the cool heights of the
Makonde plateau, find leisure to look back and take stock of my
studies. Till then—Au revoir, Messieurs!
MNYASA HUNTER WITH DOG. DRAWN BY SALIM MATCLA
CHAPTER VI
NATIVE LIFE SEEN FROM THE INSIDE
Masasi, end of July, 1906.
Every normal human being is a walking demonstration of the theory

of adaptation to environment. I have been in Africa barely two
months, and only as yet a fraction of a month in the interior, and yet
I feel quite at home already. After all, I could scarcely do otherwise.
On the 21st, when we had only lived together a few days, Mr.
Ewerbeck marched away before daybreak, by the light of a lantern
borne before him through the darkness of the tropic night, to attend
to higher duties at Lindi, viz., the reception of the eight delegates
from the Reichstag, now fairly embarked on that desperate
adventure which for many months past has kept our daily press busy
celebrating their heroism.
Nils Knudsen remains behind as the last relic of civilization. His
name alone is sufficient to indicate his Scandinavian origin, and he
is, in fact, a fair-haired descendant of the Vikings. He joined the
expedition so unobtrusively that at first I scarcely noticed the
presence of a third European. While Ewerbeck and I marched
proudly at the head of our long line of followers, Knudsen usually
brought up the rear, and in camp he remained modestly in the
background. Now that we have fixed our headquarters at Masasi, he
has become prominent by virtue of his office; he is supposed to keep
things straight here and exercise some supervision over the native
local authorities. Whether this is necessary, I am at present unable to
judge, having as yet no insight into the difficulties of internal
administration in a large district like Lindi. However, a man who
knows the country as well as Ewerbeck does, would hardly have
taken such a measure without good reason. In the meantime I have
persuaded Knudsen to quit his tent—which, to judge by its venerable
appearance, must have been left behind as too far gone to take away,
by Vasco Da Gama when he landed in this part of Africa—and come
to live with me in the rest-house. Now he is installed, with his scanty
possessions—two old tin trunks, which do not even appear to be full
—on one side of the spacious apartment, while I with my princely
outfit reside on the other. He is, however, abundantly compensated
for the niggardliness with which fortune has treated him by goodness
of heart and fineness of feeling. Knudsen’s life has been adventurous
enough, and recalls to some extent the fate of that English sailor who
was wrecked among the aborigines of South-East Australia, and had
to live as a savage among savages. My fair-haired neighbour did not
fare quite so badly as that; but he has had plenty of time to “go Fanti”
had he been so disposed. So far as I have yet ascertained anything
about his personal affairs, he started life as cabin-boy on board a
merchant vessel, from which he ran away about ten years ago, when
it was anchored in a harbour of Madagascar. He wandered about this
island for some years, and at last found his way across to the
mainland and into the hinterland of Lindi. He says that he never
learnt a trade, but professes to know something of a great many, and
can act on occasion as mason, builder, carpenter, and locksmith.
Indeed he erected all the buildings at the Luisenfelde mines, far
south near the Rovuma, which I may yet be able to visit, and was
general factotum there as long as they continued working. Since then
the municipality of Lindi has appointed him head instructor at the
industrial school, from which post he is at present on leave of
absence.
THROUGH THE BUSH ON A COLLECTING EXCURSION
Our manner of life here is, of course, essentially different from that
followed on the march. Life on the march is always full of charm,
more especially in a country quite new to one; and mine has so far
been entirely without drawbacks. In African travel-books we find
that almost every expedition begins with a thousand difficulties. The
start is fixed for a certain hour, but no carriers appear, and when at
last the leader of the expedition has, with infinite pains, got his men
together, they have still endless affairs to settle, wives and
sweethearts to take leave of, and what not, and have usually vanished
from the traveller’s ken on the very first evening. In my case
everything went like clockwork from the start. I can blame no one
but myself for the quarter of an hour’s delay in starting from Lindi,
which was caused by my being late for breakfast. On the second
morning the askari could not quite get on with the folding of the
tent, and Moritz with the best will in the world failed to get my
travelling-lamp into its case, which was certainly a very tight fit. But
with these exceptions we have all behaved as if we had been on the
road for months. Anyone who wants a substantial breakfast first
thing in the morning, after the English fashion, should not go
travelling in Africa. I have given directions to wake me at five.
Punctually to the minute, the sentinel calls softly into the tent,
“Amka, bwana” (“Wake up, sir”). I throw both feet over the high
edge of the trough-like camp bed, and jump into my khaki suit. The
water which Kibwana, in the performance of his duties as
housemaid, has thoughtfully placed at the tent door overnight, has
acquired a refreshing coolness in the low temperature of a tropic
night in the dry season. The shadow of the European at his toilet is
sharply outlined on the canvas by the burning lamp, which, however,
does not confine its illumination to its owner, but radiates a circle of
light on the shining brown faces of the carriers and the askari. The
former are busy tying up their loads for the march, while the soldiers
are ready to rush on the tent like a tiger on his prey, so soon as the
white man shall have finished dressing and come out. In the
twinkling of an eye the tent is folded, without a word spoken, or a
superfluous movement; it is division of labour in the best sense of
the word, faultlessly carried out. Meanwhile the traveller goes to his
camp-table, takes a hurried sip of tea, cocoa, or whatever his
favourite beverage may be, eating at the same time a piece of bread
baked by himself, and now stands ready for the march. “Tayari?”
(“Ready?”) his voice rings out over the camp. “Bado” (“Not yet”) is
the invariable answer. It is always the same lazy or awkward
members of the party who utter this word beloved of the African
servant. The beginner lets himself be misled by it at first, but in a few
days he takes no more notice of the “Bado,” but fires off his “Safari!”
(literally “Journey!”) or (as speedily introduced by me), “Los!”[13] at
the band in general, flourishes his walking-stick boldly in the air,
thereby indicating to the two leading askari the direction of the
march, and the day’s work has begun.
I do not know how other tribes are accustomed to behave at the
moment of starting, but my Wanyamwezi are certainly neither to
hold nor to bind on these occasions. With evident difficulty each one
has got his load lifted to head or shoulder, and stands in his place
bending under the weight. At the word of command arises an uproar
which baffles description. All the pent-up energy of their throats
rings out into the silent forest; stout sticks rattle in a wild, irregular
rhythm on the wooden cases, and, alas! also on the tin boxes, which
furnish only too good a resonator. The noise is infernal, but it is a
manifestation of joy and pleasure. We are off! and, once on the
march, the Wanyamwezi are in their element. Before long the chaos
of noise is reduced to some order; these men have an infinitely
delicate sense of rhythm, and so the din gradually resolves itself into
a kind of march sung to a drum accompaniment, whose charm even
the legs of the askari—otherwise too dignified for such childish
goings-on—cannot resist.
READY FOR MARCHING (MASASI)
Oh! the beauty of these early mornings in the tropics! It is now

getting on for six o’clock; the darkness of night has quickly yielded to
the short twilight of dawn; the first bright rays gild the light clouds
floating in the sky, and suddenly the disc of the sun rises in its
wonderful majesty above the horizon. With swift, vigorous strides,
and still in close order, the procession hastens through the dew-
drenched bush, two soldiers in the van, as if in a military expedition;
then, after an interval we Europeans, immediately followed by our
personal servants with guns, travelling-flask and camp-stool. Then
comes the main body of the soldiers followed by the long line of
carriers and the soldiers’ boys, and, lastly, to keep the laggards up to
the mark, and also to help any who have to fall out from exhaustion
or illness, two soldiers bringing up the rear. An admirable figure is
the mnyampara or headman. His position is in a sense purely
honorary, for he receives not a farthing more wages than the lowest
of his subordinates. Perhaps even this expression should not be used;
he is rather primus inter pares. The mnyampara is everywhere. He
is in front when the master sends for him, and he is back at the very
end of the line (which becomes longer with every hour of the march)
if there is a sick man needing his help. In such a case he carries the
man’s load himself, as a matter of course, and brings him safely to
camp. It seems to me that I have made an unusually happy choice in
Pesa mbili. He is young, like the great majority of my men, probably
between 23 and 25, of a deep black complexion, with markedly
negroid features, and a kind of feline glitter in his eyes; he is only of
medium height, but uncommonly strong and muscular; he speaks
shocking Swahili—far worse than my own—and withal he is a
treasure. It is not merely that he is an incomparable singer, whose
pleasant baritone voice never rests whether on the march or in camp,
but he thoroughly understands the organization of camp life, the
distribution of tasks and the direction of his men. The demands
made on such a man by the end of the day’s march are arduous
enough.
The delicious coolness of the morning has long since given place to
a perceptibly high temperature; the white man has exchanged his
light felt hat or still lighter travelling-cap for the heavy tropical
helmet, and the naked bodies of the carriers are coated with a
shining polish. These, who have been longing for the day to get warm
ever since they awoke shivering round the camp fire at four, have
now reached the goal of their desires; they are warm—very warm—
and the white man will do well to march at the head of the caravan,
otherwise he will find opportunities more numerous than agreeable
for studying the subject of “racial odours.” After two hours, or two
hours and a half, comes the first halt. The European shouts for his
camp-stool and sits watching the long string of loads coming up and
being lowered to the ground. A frugal breakfast of a couple of eggs, a
piece of cold meat, or a few bananas, here awaits the traveller, but
the carriers, who started without a meal, steadily fast on. It seems
incomprehensible that these men should be able to march for many
hours with a load of sixty or seventy pounds, while practising such
abstinence, but they are quite content to have it so. In the later hours
of the day, it is true, they begin to flag, their steps become slower and
shorter, and they lag more and more behind the personal “boys” who
have no heavy loads to carry. Yet when they reach camp at last, they
are as merry and cheerful as they were in the early morning. The
same noise—though now with quite different words from the throats
of the singers—overwhelms the European, who has long been seated
at the halting-place. My company seem to be obsessed by the
“Central-Magazin” at Dar es Salam, where they entered my service;
they are celebrating this spacious building in the closing song of their
day’s march.
CAMP AT MASASI
The duties of my followers—whether boys, askari, or porters—are

by no means over when they have reached camp. By the time they
come up, the leader of the expedition has looked round for a place to
pitch his tent, a matter which seems to me to require special gifts.
The fundamental principles to bear in mind are: that it should be
within reach of good drinking water and free from noxious insects,
such as ticks, mosquitoes, and jiggers. The second point, but one by
no means to be overlooked, is the position of the tent-pole with
regard to the course of the sun, and the next the shade of leafy trees,
if that is attainable. I find it simplest to draw the outline of the tent
on the sandy ground, after the spot has been carefully swept,
indicating the place where I want the door to be by a break in the
line. That is quite enough for my corporal in command. Scarcely have
the two unfortunates, whose shoulders are weighed down by my
heavy tent, come up panting and gasping for breath, when the loads
are unrolled, and in a twinkling every warrior has taken up his
position. “One, two, three!” and the two poles are in their places, and
the next moment I hear the blows of the mallet on the tent-pegs.
While this is going on, the two boys, Moritz and Kibwana, are
amusing themselves with my bed. This occupation seems to
represent for them the height of enjoyment, for it seems as if they
would never be done. Neither scolding nor threats can avail to hasten
their movements. It seems as if their usually slow brains had become
absolutely torpid. Mechanically they set up the bedstead;
mechanically they spread the cork mattress and the blankets over it;
in the same dull, apathetic way they finally set up the framework of
the mosquito-net. The soldiers have taken their departure long
before my two gentlemen condescend to carry the bed into the tent.
My carriers meanwhile have found all sorts of work to do. Water
has to be fetched for the whole caravan, and fires to be made, and the
sanitary requirements of the camp provided for; and noon is long
past by the time their turn comes and they can live their own life for
an hour or two. Even now they cannot be said to revel in luxury. This
southern part of the German territory is very poor in game, and in
any case I have no time for shooting, so that meat is almost an
unknown item in my people’s menu. Ugali, always ugali—stiff
porridge of millet, maize or manioc, boiled till it has almost a
vitreous consistency, and then shaped with the spoon used for
stirring into a kind of pudding—forms the staple of their meals day
after day.
INTERIOR OF A NATIVE HUT IN THE ROVUMA VALLEY
Here at Masasi the tables are turned; my men have a good time,
while I can scarcely get a minute to myself. My escort are quite
magnificently housed, they have moved into the baraza or council-
house to the left of my palatial quarters and fitted it up in the native
way. The negro has no love for a common apartment; he likes to
make a little nest apart for himself. This is quickly done: two or three
horizontal poles are placed as a scaffolding all round the projected
cabin, then a thick layer of long African grass is tied to them, and a
cosy place, cool by day and warm by night, is ready for each one. The
carriers, on the other hand, have built themselves huts in the open
space facing my abode, quite simple and neat, but, to my
astonishment, quite in the Masai style—neither circular hut nor
tembe. The circular hut I shall discuss in full later on, but in case
anyone should not know what a tembe is like, I will here say that the
best notion of it can be got by placing three or four railway carriages
at right angles to one another, so that they form a square or
parallelogram, with the doors inward. This tembe is found
throughout most of the northern and central part of German East
Africa, from Unyamwezi in the west to the coast on the east, and
from the Eyasi and Manyara basin in the north to Uhehe in the
south. The Masai hut, finally, can best be compared with a round-
topped trunk. Though the Masai, as everyone knows, usually stand
well over six feet, their huts, which (quite conformably with the
owners’ mode of life as cattle-breeders par excellence) are neatly and
fragrantly plastered with cowdung, are so low that even a person of
normal stature cannot stand upright in them. My Wanyamwezi,
however, never attempt to stand up in their huts; on the contrary,
they lie about lazily all day long on their heaps of straw.
My activities are all the more strenuous. The tropical day is short,
being only twelve hours from year’s end to year’s end, so that one has
to make the fullest possible use of it. At sunrise, which of course is at
six, everyone is on foot, breakfast is quickly despatched, and then the
day’s work begins. This beginning is curious enough. Everyone who
has commanded an African expedition must have experienced the
persistence of the natives in crediting him with medical skill and
knowledge, and every morning I find a long row of patients waiting
for me. Some of them are my own men, others inhabitants of Masasi
and its neighbourhood. One of my carriers has had a bad time. The
carrier’s load is, in East Africa, usually packed in the American
petroleum case. This is a light but strong wooden box measuring
about twenty-four inches in length by twelve in width and sixteen in
height, and originally intended to hold two tins of “kerosene.” The
tins have usually been divorced from the case, in order to continue a
useful and respected existence as utensils of all work in every Swahili
household; while the case without the tins is used as above stated.
One only of my cases remained true to its original destination, and
travelled with its full complement of oil on the shoulders of the
Mnyamwezi Kazi Ulaya.[14] The honest fellow strides ahead sturdily.
“It is hot,” he thinks. “I am beginning to perspire. Well, that is no
harm; the others are doing the same.... It is really very hot!” he
ejaculates after a while; “even my mafuta ya Ulaya, my European
oil, is beginning to smell.” The smell becomes stronger and the
carrier wetter as the day draws on, and when, at the end of the
march, he sets down his fragrant load, it is with a double feeling of
relief, for the load itself has become inexplicably lighter during the
last six hours. At last the truth dawns on him and his friends, and it
is a matter for thankfulness that none of them possess any matches,
for had one been struck close to Kazi Ulaya, the whole man would
have burst into a blaze, so soaked was he with Mr. Rockefeller’s
stock-in-trade.
Whether it is to be accounted for by a strong sense of discipline or
by an almost incredible apathy, the fact remains that this man did
not report himself on the first day when he discovered that the tins
were leaking, but calmly took up his burden next morning and
carried it without a murmur to the next stopping place. Though once
more actually swimming in kerosene, Kazi Ulaya’s peace of mind
would not even now have been disturbed but for the fact that
symptoms of eczema had appeared, which made him somewhat
uneasy. He therefore presented himself with the words a native
always uses when something is wrong with him and he asks the help
of the all-powerful white man—“Dawa, bwana” (“Medicine, sir”),
and pointed significantly, but with no sign of indignation, to his
condition. A thorough treatment with soap and water seemed
indicated in the first instance, to remove the incrustation of dirt
accumulated in seven days’ marching. It must be said, in justice to
the patient, that this state of things was exceptional and due to
scarcity of water, for Kazi Ulaya’s personal cleanliness was above the
average. I then dressed with lanoline, of which, fortunately, I had
brought a large tin with me. The patient is now gradually getting over
his trouble.
Another case gives a slight idea of the havoc wrought by the jigger.
One of the soldiers’ boys, an immensely tall Maaraba from the
country behind Sudi, comes up every morning to get dawa for a
badly, damaged great toe. Strangely enough, I have at present
neither corrosive sublimate nor iodoform in my medicine chest, the
only substitute being boric acid tabloids. I have to do the best I can
with these, but my patients have, whether they like it or not, got
accustomed to have my weak disinfectant applied at a somewhat
high temperature. In the case of such careless fellows as this
Maaraba, who has to thank his own lazy apathy for the loss of his
toe-nail (which has quite disappeared and is replaced by a large
ulcerated wound), the hot water is after all a well-deserved penalty.
He yells every time like a stuck pig, and swears by all his gods that
from henceforth he will look out for the funsa with the most
unceasing vigilance—for the strengthening of which laudable
resolutions his lord and master, thoroughly annoyed by the childish
behaviour of this giant, bestows on him a couple of vigorous but
kindly meant cuffs.
As to the health of the Masasi natives, I prefer to offer no opinion
for the present. The insight so far gained through my morning
consultations into the negligence or helplessness of the natives as
regards hygiene, only makes me more determined to study other
districts before pronouncing a judgment. I shall content myself with
saying here that the negro’s power of resisting the deleterious
influences of his treacherous continent is by no means as great as we,
amid the over-refined surroundings of our civilized life, usually
imagine. Infant mortality, in particular, seems to reach a height of
which we can form no idea.
Having seen my patients, the real day’s work begins, and I march
through the country in the character of Diogenes. On the first few
days, I crawled into the native huts armed merely with a box of
matches, which was very romantic, but did not answer my purpose. I
had never before been able to picture to myself what is meant by
Egyptian darkness, but now I know that the epithet is merely used on
the principle of pars pro toto, and that the thing belongs to the whole
continent, and is to be had of the very best quality here in the plain
west of the Makonde plateau. The native huts are entirely devoid of
windows, a feature which may seem to us unprogressive, but which is
in reality the outcome of long experience. The native wants to keep
his house cool, and can only do so by excluding the outside
temperature. For this reason he dislikes opening the front and back
doors of his home at the same time, and makes the thatch project
outward and downward far beyond the walls. My stable-lantern,
carried about the country in broad daylight by Moritz, is a great
amusement to the aborigines, and in truth our proceeding might well
seem eccentric to anyone ignorant of our object. In the darkness of a
hut-interior, however, they find their complete justification. First
comes a polite request from me, or from Mr. Knudsen, to the owner,
for permission to inspect his domain, which is granted with equal
politeness. This is followed by an eager search through the rooms
and compartments of which, to my surprise, the dwellings here are
composed. These are not elegant, such a notion being as yet wholly
foreign to the native consciousness; but they give unimpeachable
testimony to the inmates’ mode of life. In the centre, midway
between the two doors is the kitchen with the hearth and the most
indispensable household implements and stores. The hearth is
simplicity itself: three stones the size of a man’s head, or perhaps
only lumps of earth from an ant-heap, are placed at an angle of 120°
to each other. On these, surrounded by other pots, the great earthen
pot, with the inevitable ugali, rests over the smouldering fire. Lying
about among them are ladles, or spoons, and “spurtles” for stirring
the porridge. Over the fireplace, and well within reach of the smoke,
is a stage constructed out of five or six forked poles. On the cross-
sticks are laid heads of millet in close, uniform rows, and under
them, like the sausages in the smoke-room of a German farmhouse,
hang a great number of the largest and finest cobs of maize, by this
time covered with a shining layer of soot. If this does not protect
them from insects, nothing else will; for such is the final end and aim
of the whole process. In the temperate regions of Europe, science
may be concerned with preserving the seed-corn in a state capable of
germination till sowing-time; but here, in tropical Africa, with its all-
penetrating damp, its all-devouring insect and other destroyers, and,
finally, its want of suitable and permanent building material, this
saving of the seed is an art of practical utility. It will be one, and not
the least welcome, of my tasks, to study this art thoroughly in all its
details.
As to the economy of these natives, their struggle with the
recalcitrant nature of the country, and their care for the morrow, I
am waiting to express an opinion till I shall have gained fuller
experience. In the literature dealing with ethnology and national
economy, we have a long series of works devoted to the classification
of mankind according to the forms and stages of their economic life.
It is a matter of course that we occupy the highest stage; all authors
are agreed on one point, that we have taken out a lease of civilization
in all its departments. As to the arrangement of the other races and
nations, no two authors are agreed. The text-books swarm with
barbarous and half-barbarous peoples, with settled and nomadic,
hunter, shepherd, and fisher tribes, migratory and collecting tribes.
One group carries on its economic arts on a basis of tradition,
another on that of innate instinct, finally, we have even an animal
stage of economics. If all these classifications are thrown into a
common receptacle, the result is a dish with many ingredients, but
insipid as a whole. Its main constituent is a profound contempt for
those whom we may call the “nature-peoples.”[15] These books
produce the impression that the negro, for instance, lives direct from
hand to mouth, and in his divine carelessness takes no thought even
for to-day, much less for to-morrow morning.
The reality is quite otherwise, here and elsewhere, but here in an
especial degree. In Northern Germany, the modern intensive style of
farming is characterized by the barns irregularly distributed over the
fields, and in quite recent times by the corn-stacks, both of which,
since the introduction of the movable threshing-machine, have made
the old barn at the homestead well-nigh useless. Here the farming
differs only in degree, not in principle; here, too, miniature barns are
irregularly scattered over the shambas, or gardens; while other food-
stores which surprise us by their number and size are found close to
and in the homestead. If we examine the interior of the house with a
light, we find in all its compartments large earthen jars, hermetically
sealed with clay, containing ground-nuts, peas, beans, and the like,
and neatly-made bark cylinders, about a yard long, also covered with
clay and well caulked, for holding maize, millet and other kinds of
grain. All these receptacles, both outdoor and indoor, are placed to
protect them from insects, rodents and damp, on racks or platforms
of wood and bamboo, from fifteen inches to two feet high, plastered
with clay, and resting on stout, forked poles. The outdoor food-stores
are often of considerable dimensions. They resemble gigantic
mushrooms, with their thatched roofs projecting far beyond the
bamboo or straw structure, which is always plastered with mud
inside and out. Some have a door in their circumference after the
fashion of our cylindrical iron stoves; others have no opening
whatever, and if the owner wishes to take out the contents, he has to
tilt the roof on one side. For this purpose he has to ascend a ladder of
the most primitive construction—a couple of logs, no matter how
crooked, with slips of bamboo lashed across them a yard apart. I
cannot sketch these appliances without a smile, yet, in spite of their
primitive character, they show a certain gift of technical invention.
The keeping of pigeons is to us Europeans a very pleasing feature
in the village economy of these parts. Almost every homestead we
visit has one or more dovecotes, very different from ours, and yet
well suited to their purpose. The simplest form is a single bark
cylinder, made by stripping the bark whole from the section of a
moderately thick tree. The ends are fastened up with sticks or flat
stones, a hole is cut in the middle for letting the birds in and out, and

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Cardiovascular Engineering: A

Protective Approach Shu Q. Liu

New York Chkago San Francisco

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Part I Foundations of Cardlovascular Protective Engineering

Regulation of cardiac chamber formation . . . . . . . . . . . . . . . . . . . . . 29

Amino acid transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

External environmental factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Discovery and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Small interfering RNA-based mRNA modifications. . . . . . . . . . . . . 276

Part II Appllcatlons of Cardlovascular Protective Engineering

Inflammation regulatory factor genes . . . . . . . . . . . . . . . . . . . . . . . . 313

12 Arterial Aneurysms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

Dilated Cardiomyopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382

15 Congenital Heart Defects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407

Ventricular septum defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 414

Cardiovascular disease is the leading cause of human morbidity and mortality. A

Foundations of Protective Engineering

Pathogenic causes and processes

Naturally accunlng protective mechanisms

and differentiation, and fibroblast proliferation and extracellular matrix production

Regional protective mechanisms

Distant protective mechanisms

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

Protective Engineering Strategies

and regeneration of functional cells. Tissue-level protective engineering is to provide

Molecular protective engineering

Enhancing protectire impacts by protein administtation and gene ttansfer

Supptessint adverse dflne expression

Gene editint-mediated control of dene expression

a process activating homologous recombination and causing insertion of an exogenous

Cell-based protective engineering

Tissue-level protective engineering

properties conforming to the tissue or organ to be replaced; preparation, expansion,

This ascent, though barren of results in other respects, has

Masasi, end of July, 1906.

Every normal human being is a walking demonstration of the theory

READY FOR MARCHING (MASASI)

Oh! the beauty of these early mornings in the tropics! It is now

The duties of my followers—whether boys, askari, or porters—are

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