Index
AAVs (adeno-associated viruses), 87–88 Action potential generation, currents
underlying, 146–147 Action potentials in human fetal
ventricle and atrium, 145–146 Adeno-associated viruses (AAVs), 87–88 Adenoviral vectors, 87
Affinity matrix, 5 Affymetrix, 7
Agarose gel electrophoresis, 6 AGENT 3, 191
AGENT 4, 191 AGENT trial, 183
AGFs (angiogenic growth factors), 173–174 Angiogenesis, 175–176
gene therapy for, 171–196 therapeutic, 95, 175
Angiogenic growth factors (AGFs), 173–174 Angiograms, 179
Angiography, 181 Angioscopy, 220
ANGUS and shear stress, 227–228 Arterial cytoprotection, 95, 97
Arterial graft generation, in vivo, 209–210 Arterogenesis, 175
Atherogenesis, prevention of, 97 Atherosclerosis, 171–175, 205 Atherosclerotic lesions, 35
Baculovirus, 88 Beads-based techniques, 4 Bioinformatics, 11–12, 55, 104
in cardiovascular research, 111–114 in genomic medicine, 103–114 Biological databases, public, 104–108 BMPs (bone morphogenetic proteins), 136–137 Bone marrow, 158
Bone marrow cells, 135–136 Bone marrow-derived stem cells, 162
Bone morphogenetic proteins (BMPs), 136–137
CABG (coronary artery bypass graft), 182 CaGE (Cardiac Gene Expression knowledge
base), 112
Cardiac Gene Expression knowledge base (CaGE), 112
Cardiac hypertrophy, 36
Cardiac progenitor cells (CPCs), 165–166 Cardiomyoblasts, 165–166
Cardiomyocytes, 133
electrophysiology of, 143–148 human fetal atrial, 147–148 Cardiomyoplasty, cellular, 213–215 Cardiovascular angiogenesis
gene transfer for, 186–187 recombinant protein for, 184–185 Cardiovascular disease, 112
animal models for studying, 94–97 expression profiling in, using microarrays,
3–39
Cardiovascular genomics, 30 Cardiovascular medicine
genomic, future of, 114
impact of genomic variation on, 113–114 Cardiovascular proteonomics, 65–72 Cardiovascular research
basic, proteonomics in, 66–67
genomics and bioinformatics in, 111–114 new technologies in gene therapy in, 85–98 cDNA arrays, 7
Cell-based products, 208–209 Cell fusion, 164
Cell recruitment, in vivo, 209 Cellular cardiomyoplasty, 213–215 CETP (cholesteryl ester transfer protein), 113 Cholesteryl ester transfer protein (CETP), 113 Chromosomes, 4
Cluster analysis, 24–26 Collagen gel scaffolds, 207–208 Concatemers, 5
Coomassie stains, 51
Coronary artery bypass graft (CABG), 182 235
236 Index
CPCs (cardiac progenitor cells), 165–166 Cross-sectional experiments, time course
experiments versus, 17 Cytokine cascade, 159
DAPK (death-associated protein kinase), 35 Database standardization and ontologies,
107–108
Data interpretation, 22–24 dbSNP database, 105
Death-associated protein kinase (DAPK), 35 D3 embryonic stem cell line, 158
Dendrimers, 86 dHAND, 142
Diabetic peripheral neuropathy, 173 Dicer processes, 122
Differential display, 4–5
Disease classification, profile analysis and, 16–17
DNA, 4
DNA amplification, 77 DNA microarrays, 4–7, 109–110 DNA polymerase, 76
Double balloon catheter, 93–94 dsRNA, 122
Dual-label hybridization techniques, 10
Early growth response factor-1 (Egr-1), 33–35 EC function, shear stress and, 32–33 Echolucent zones, 220
Egr-1 (early growth response factor-1), 33–35 eHAND, 142
Elastography, intravascular, 221–222 Electrophysiology of cardiomyocytes, 143–148 Embryonic stem (ES) cells, 134
Endogenous stem cells, mobilization of, 159–160
Endothelial dysfunction, 171–175 Endothelium, 30
ES (embryonic stem) cells, 134
Essential hypertension, systematic changes in, 38–39
ESTs (expressed sequence tags), 8, 15
Fluorescent quantification nonspecific, 77–78 specific, 79–80
Fluorescent stains and dyes, 51–52 FUT-175 (Futhan/nafamstat mesilate), 70 Futhan/nafamstat mesilate (FUT-175), 70
GATA family, 139–141
G-CSF (granulocyte-colony stimulating factor), 159–160
Genechip series, 8
Gene delivery, intravascular, 92–94 Gene expression analysis, 3
Gene Ontology (GO) Consortium, 26–27 Genes, number of, 4
Genespring package, 29 Gene therapy, 85
for angiogenesis, 171–196 proteonomics relevance for, 45–61 vectors for, 86–89
Gene transfer (GTx)
for cardiovascular angiogenesis, 186–187 perivascular, 91–92
principles of, 86 safety concerns, 192–194 Gene transfer routes, 90
Genomic cardiovascular medicine, future of, 114
Genomic medicine, 110–111 bioinformatics in, 103–114 Genomics, in cardiovascular research,
111–114
Genomics experiments, experimental design for, 108–110
Genomic variation, impact of, on cardiovascular medicine, 113–114
Glass arrays, 7
GO (Gene Ontology) Consortium, 26–27 Graft restenosis, prevention of, 95, 97 Granulocyte-colony stimulating factor (G-CSF),
159–160 GTx, see Gene transfer
Index 237
Heart valves, 210–212 Synergraft, 211
Heat shock proteins (HSPs), 67–69 Hematopoietic stem cells (HSCs), 163–164 hES2 cell line, 147–148
HHCy (hyperhomocysteinemia), 31–32 Housekeeping genes, 12
HP-2DPAGE, 66 HSC-2DPAGE, 66
HSCs (hematopoietic stem cells), 163–164 HSPs (heat shock proteins), 67–69 Human fetal ventricle and atrium, action
potentials in, 145–146 Human Transcriptome Map, 106 Hybridization probes, 82 Hypercholesterolemia, 97
Hyperhomocysteinemia (HHCy), 31–32
IHD (ischemic heart disease), 171–173 Immobilized pH gradients (IPGs), 50 Injured heart, injection of stem cells into,
160–162 IN sequence, 67
Intravascular coronary ultrasound (IVUS), 220–221
Intravascular elastography, 221–222 Intravascular gene delivery, 92–94 Intramyocardial gene injections, 90–91 IPGs (immobilized pH gradients), 50 Ischemic heart disease (IHD), 171–173 Isoform switching, 67
IVUS (intravascular coronary ultrasound), 220–221
KAT (Kuopio Angiogenesis Trial), 189 Kuopio Angiogenesis Trial (KAT), 189
Left ventricular (LV) dysfunction, 174 Left ventricular electromechanical mapping
(LV EMM), 190 Lentiviruses, 88
Library-based yeast two-hybrid screening method, 59
Linkage rule, 24–25 Lipid depositions, 220 Liposomes, 86
L-type calcium currents, 144, 146 LV (left ventricular) dysfunction, 174 LV EMM (left ventricular electromechanical
mapping), 190
MAGE-ML (Microarray and Gene Expression Markup Language), 107
Magnetic resonance imaging (MRI), 226–227 Mammalian cells, RNA interference in, 124–126 MAPCs (multipotent adult precursor cells), 162 Mass fingerprinting, 53
Massively parallel signature sequencing, 8–9 Mass spectroscopy, 53–55
MEF-2 family, 141–142
Mesenchymal stem cells (MSCs), 162–163 MGED (Microarray Gene Expression Data)
Society, 12
MIAME (Minimum Information About a Microarray Experiment), 12–13, 107–108 Microarray and Gene Expression Markup
Language (MAGE-ML), 107 Microarray experiments, sources of error in,
19–20
Microarray Gene Expression Data (MGED) Society, 12
Microarrays, 4–7 classification of, 7–9
expression profiling in cardiovascular disease using, 3–39
Minimum Information About a Microarray Experiment (MIAME), 12–13, 107–108 Molecular beacon, 80
Mononuclear cell fraction, 160
MRI (magnetic resonance imaging), 226–227 MSCs (mesenchymal stem cells), 162–163 Multipotent adult precursor cells (MAPCs), 162 Myocardial graft, 212–215
Myocardial infarction, 37, 159 Myocardial tissue, somatic stem cells
regenerating, 157–167
Near-infrared (NIR) spectroscopy, 226 Neovascularization, 175–176 NIR (near-infrared) spectroscopy, 226 Nkx 2–5, 141
NO actions, 31 NOGA system, 90–91 Northern blot analysis, 75–76
OCT (optical coherence tomography), 224–225 O’Farrell’s lysis buffer, 49
OMIN (Online Mendelian Inheritance in Man), 105
Online Mendelian Inheritance in Man (OMIN), 105
Open reading frames (ORFs), 58
238 Index
Optical coherence tomography (OCT), 224–225 ORFs (open reading frames), 58
Palpography, 221–222 Pathway analysis, 16
PCR (polymerase chain reaction), 75 Percutaneous CB electromechanical mapping,
181
Perivascular gene transfer, 91–92 Phage display method, 60 Pi gene locus, 80 Plaque(s), 219
vulnerable, visualization of, see Visualization of vulnerable plaque
Plasmid DNA, 86 Polyethyleneimine, 86
Polymerase chain reaction (PCR), 75 Polymeric scaffolds, synthetic, 208
Profile analysis, disease classification and, 16–17 Protein expression, 3
Protein prefractionation, methods for, 49 Protein-protein interactions, 56–57 Protein separation by 2D-PAGE, 48–52 Proteome, 46
Proteonomics, 45, 46
of animal models of heart diseases, 69–72 in basic cardiovascular research, 66–67 cardiovascular, 65–72
importance of, 46–47
relevance for gene therapy, 45–61 Pulse wave velocity (PWV), 35 Pump function, 143
PWV (pulse wave velocity), 35
Quantitative real-time PCR, 75–82
Raman spectroscopy, 225–226 RAT HEART-2DPAGE, 66 Recombinant protein (RP), 173, 183
for cardiovascular angiogenesis, 184–185 Resistance vessels, 30
Restenosis, 172, 206
in mammalian cells, 124–126
mechanisms and therapeutic applications, 121–128
as therapeutic tool, 127
RNA isolation, labeling, and hybridization, 9–10 RP, see Recombinant protein
SAGE (serial analysis of gene expression), 4, 5–7
Satellite cells, 214
SCF (stem cell factor), 159–160
Serial analysis of gene expression (SAGE), 4, 5–7
Shear stress, EC function and, 32–33 Side population (SP) cells, 160–161 Silver stain, 51
siRNA, 89–90 Skeletal myoblasts, 214 Somatic stem cells, 157, 158–159
differentiation capacity of, 162–165 expression of, 161
isolation of, 160
regenerating myocardial tissue, 157–167 SP (side population) cells, 160–161 Spectroscopic techniques, 225–226 Spot finding operation, 10–11 Spotted oligonucleotide microarrays, 8 Staining techniques, comparison of, 51 Statistical algorithms, development of, 108 Stem cell factor (SCF), 159–160 Stem cell research, 158 Stem cells, 134–136
bone marrow-derived, 162
endogenous, mobilization of, 159–160 hematopoietic, 163–164
injection of, into injured heart, 160–162 mesenchymal, 162–163
somatic, see Somatic stem cells tissue repair and, 135–136 Stiletto catheter, 90–91 SYBR green, 78
Synergraft heart valves, 211 Systems biology, 28–29
Index 239
Therapeutic angiogenesis, 175 Thermography, 222–224 Threshold cycle, 81 Thrombosis, 219
Time course experiments, cross-sectional experiments versus, 17
Tissue engineering, 205–215 Tissue repair, stem cells and, 135–136 Tissue samples, 48
TKRs (tyrosine kinase receptors), 177 TRAFFIC trial, 194
Transcription factor binding sites (TFBSs), 27–28
Transcription factors, 139
Transplantation, perspectives for, 148 Tumorigenicity, 127
2D-PAGE, 49–51
protein separation by, 48–52 software for analysis of, 53 Tyrosine kinase receptors (TKRs), 177
Ubiquitin carboxyl-terminal hydrolase (UCH), 69–70
UCH (ubiquitin carboxyl-terminal hydrolase), 69–70
UMLS (Unified Medical Language System), 107 Unified Medical Language System (UMLS), 107 Unusual ratio method, 22
Validation approach, 15 Valvular pathology, 210
Vascular endothelial growth factors (VEGFs), 95 Vascular grafts, 205–206
acellular, 209
Vascular permeability factor (VEGF), 174 Vascular prosthesis, ideal, 206
Vascular smooth muscle cell (VSMC), 30–31 Vasculogenesis, 175–176
Vasculotropin, 174
VEGF (vascular permeability factor), 174 VEGFs (vascular endothelial growth factors), 95 Viral vectors, 87
Visualization of vulnerable plaque, 219–229 by angioscopy, 220
by ANGUS and shear stress, 227–228 by intravascular coronary ultrasound,
220–221
by intravascular elastography/palpography, 221–222
by magnetic resonance imaging, 226–227 by optical coherence tomography, 224–225 by spectroscopic techniques, 225–226 by thermography, 222–224
VIVA trial, 191, 193, 194
VSMC (vascular smooth muscle cell), 30–31 Vulnerable plaque, 219
visualization of, see Visualization of vulnerable plaque
Wnts, 137–138
Yeast two-hybrid system, 57–60
mRNA mRNA
cDNA cDNA
microarray surface DNA of 1 clone
Sample 1 Sample 2
reverse transcriptase
Labeling Hybridization
microarray surface
microarray surface
DNA of 1 clone DNA of 1 clone
Laser PMT Laser PMT
“3” “6”
Scanning Quantification
FIGURE 1 (page 6). Overview of the microarray procedure.
FIGURE 5 (page 21). Image of a microarray and the corresponding table with the log2ratios. No location-related ratios can be detected.
2
FIGURE 1 (page 46). The ways in which gene expression can be regulated or modified from tran- scription to posttranslation.
FIGURE 3 (page 48). Laser capture microdissection. A transparent polymer film is placed in direct contact with the surface of a heterogeneous tissue section. Laser energy is used to activate the polymer directly over the selected cells. The activated region captures the selected cells, which can be lifted away from unwanted tissue.
FIGURE 4 (page 50). Silver-stained 2d-page gels of a mouse heart (left ventricle).
FIGURE 6 (page 56). Annotated protein in swiss-2dpage and swiss-prots databases.
4
FIGURE 7 (page 58). Analysis of protein–protein interactions. The protein of interest is expressed as a fusion protein with a cleavable affinity tag to identify interacting proteins. It is immobilized onto agarose beads using a glutathione S-transferase tag. Nuclear cell extracts are incubated with the beads and the beads washed extensively. Thrombin is used to cleave between the glutathione S-transferase and the
“bait” protein, which results in the elution of all proteins that are specifically bound to “bait” (Pandey and Mann, 2000). The eluted proteins are resolved by 2d-page and analyzed by ms. The success of the above-mentioned strategies relies on sufficient affinity of the protein complex to the bait and on optimized conditions for purifications steps.
FIGURE 8 (page 59). The yeast two-hybrid system. (a) Schematic representation of the yeast two- hybrid system. (1) The two separated domains of a transcription factor are not functional and therefore do not induce transcription of the reporter gene. (2) The DBD and AD are fused to two proteins of interest and co-expressed in a yeast reporter strain. (3) If DBD-X and AD-Y interact, the fusion proteins are assembled at the binding site of the reporter gene, which leads to activation of transcription. (b) Library- based yeast two-hybrid screening method. In this strategy, two different yeast strains containing two different cDNA libraries are prepared. In one case, the open reading frames (ORFs) are expressed as GAL 4–BD fusions and in other case, they are expressed as GAL 4–AD fusions. The two yeast strains are then mated and diploids selected on deficient media. Thus, only the yeast cells expressing interacting proteins survive. The inserts from both the plasmids are then sequenced to obtain a pair of interacting genes. (Modified from Pandey and Mann, 2000).
6
FIGURE 9 (page 60). Affinity-selection of ligands from phage display libraries. Different peptide sequences can be screened for binding to a specific target molecule. After several rounds only the clones with the peptides that bind to the target are isolated.
FIGURE 2 (page 72). Coomassie-stained 2D gels of proteins isolated from normal (left) and infarcted left ventricle (right). Spots displaying significant differences in staining intensity between normal and infarcted left ventricle are framed (interrupted frames indicate the positions of spots of reduced or even undetectable intensities).
FIGURE 3 (page 79). Schematic picture of the TaqManr assay and the molecular beacon approach.
(A) In the TaqManr assay, the probe is labeled at the 5 end with a fluorescent reporter (R) and at the 3 end with a quencher molecule (Q). The fluorescence of the reporter will be absorbed by the quencher when the probe is still intact. During the extension phase the Taq polymerase cleaves the hybridized probe and the reporter molecule is released from the quencher. The fluorescent signal can now be detected and will be increased during amplification when more and more probe will be bound and will be cleaved. (B) The molecular beacon is a hairpin-loop-shaped oligonucleotide with a fluorescent reporter (R) to one arm and a quencher molecule to the complementary other arm. When the probe binds to the template the reporter and quencher are separated and the fluorescent signal can be monitored. During amplification more and more probe will bind and an increased fluorescent signal is observed.
FIGURE 1 (page 91). Intramyocardial gene delivery catheters. (A) Stiletto r catheter for intramy- ocardial injection. A microscopic needle is on the tip of the catheter for the penetration of the endocar- dial border and for the infiltration of genes inside the myocardium. (B) NOGA r electromechanical mapping and injection catheter. The electrical measurements and the myocardial movements can be detected with the same catheter as the gene injection. A small needle is pushed out and pulled in during the gene injection.
8
FIGURE 2 (page 92). Extravascular gene delivery methods. (A) A biologically inert collar installed around the rabbit carotid artery. Genes are injected into the collar so that they are in close contact with the vascular wall. (B) Biodegradable collar releasing therapeutic agents into the vessel wall. (C) Direct injection of genetic material into blood vessel.
FIGURE 3 (page 93). Intravascular gene delivery catheters. (A) Channel balloon r with microscopic
infusion–perfusion
FIGURE 4 (page 96). In vivo examples of angiogenic gene therapy. (A) An animal model of a rabbit hind limb before (1) and 35 days after (2) intramuscular injections of VEGF gene in an adenovirus vector (Laitinen et al., 1997). (B) Human truncus of leg arteries before (1), 3 months after (2), and 9 months after (3) angioplasty combined with local intra-arterial VEGF gene transfer (Laitinen et al., 1998). (C) Myocardial scintigraphy of a human heart before and 6 months after local intracoronary adenoviral VEGF gene transfer performed during coronary angioplasty (Hedman et al., 2003).
10
FIGURE 1 (page 123). Mechanism of RNAi. dsRNA is cleaved into 19–23 nucleotide fragments:
siRNA. The RNA duplex is presented to the inactive RISC. RISC is activated by the reduction of ATP and causes unwinding of the RNA double helix. The antisense strand is incorporated in the RISC and is responsible for the recognition of sequence-specific mRNA. The sense strand is discarded. Upon recognition, the mRNA is cleaved and degraded.
FIGURE 2 (page 125). Construction of siRNA by means of hairpin-expressing vectors. A 19 nu- cleotide DNA sequence is designed in homology to the gene of interest. A construct is created with this sequence followed by a short spacer and the same sequence in the antisense direction (inverted repeat) and a poly-T fragment. This construct is ligated into an expression vector containing a RNA polymerase III promoter. Transfection of the vector into eukaryotic cells initiates transcription of the construct into single-stranded RNA (ssRNA). The palindromic nature of the ssRNA leads to back folding of the ssRNA into shRNA. Dicer processes shRNA into functional siRNA.
12
FIGURE 1 (page 176). Neovascularization encompasses both angiogenesis and vasculogenesis. An- giogenesis represents the classic paradigm for new vessel growth, as mature, differentiated endothelial cells (ECs) break free from their basement membrane and migrate as well as proliferate to form sprouts from parental vessels. Vasculogenesis involves participation of bone marrow-derived endothelial pro- genitor cells (BM-EPCs), which circulate to sites of neovascularization where they differentiate in situ into mature ECs. Growth factors, cytokines, or hormones released endogenously in response to tissue ischemia, or administered exogenously for therapeutic neovascularization, act to promote EPC proliferation, differentiation, and mobilization from BM (via the peripheral circulation) to finally home and incorporate into neovascular foci.
FIGURE 4 (page 189). Modified mapping-injecting catheter (BiosenseTM, Johnson & Johnson).
(A) The electrode in the distal tip of the catheter allows annotation of electromechanical maps to document the sites of gene transfer by intramyocardial injection. (B) The 27-gauge needle has been advanced out of the distal tip of the catheter to simulate myocardial engagement in preparation for injection. (C) Posteroanterior view recorded during cine-fluoroscopy shows the distal tip of the catheter (arrow) against the endocardium of the left ventricular lateral wall in preparation for injection. The 27-gauge needle advanced into the myocardium is not visible.
FIGURE 5 (page 190). NOGATMleft ventricular electromechanical mapping (LV EMM): Unipolar images (A) showing normal voltages suggestive of viable myocardium (purple/pink/blue/green) and linear local shortening (LLS) map (B) with large zone of abnormal wall motion (red, arrow) that represents electromechanical uncoupling suggestive of ischemic or hibernating myocardium involving the inferoseptal region, from a 48-year-old man before phVEGF165GTx. The red lines represent the long axis through apex. Vertical and horizontal axes (x, y, and z) are presented as white lines. Sixty days after GTx, unipolar (C) and LLS (D) images show complete resolution of ischemia (10.08 cm2 before GTx vs 0.00 cm2after GTx) that corresponds to changes observed on SPECT scan. Persantine SPECT-sestamibi myocardial perfusion scanning images: White and yellow colors depict maximal uptake of radionuclide, and red depicts impaired uptake. Selected short-axis stress and resting images were taken before (E, F) and after (G, H) phVEGF165 GTx in the same patient. Pre-GTx scans (top) show reversible inferoseptal defect (arrows). Post-GTx scans (bottom) show complete normalization of resting perfusion and perfusion after pharmacological stress.
14
1(page225).OCTimagesobtainedduringapullbackofanatheroscleroticrabbitaorta.Bloodlessconditionswerecreatedbyballoonocclusionandflushing rate.Indicatedareanormalsegment(panelA),aplaquehavingfeaturesoffibrotictissue(panelB),andacrosssectionofaplaquecontainingalipid-rich C).
FIGURE 3 (page 229). Three-dimensional reconstruction of human coronary blood vessels based upon a combination of angiography and IVUS (ANGUS). Indicated are three vessels from three different patients.
FIGURE 4 (page 230). Indication of the method to obtain a shear stress mapping. Panel A shows the filling of the lumen by finite elements, panel B the resulting velocity field for a cross section, panel C shows the envelope of the vectors, and panel D the resulting shear stress mapping on the endothelium of the 3D reconstructed blood vessel.
