Cardiovascular Disease
Blood vessels
The cardiovascular
system has three
types of blood
vessels:
1. Arteries (and
arterioles) – carry
blood away from the
heart
2. Veins (and venules) –
carry blood toward
the heart.
3. Capillaries – where
nutrient and gas
exchange occur
The Arteries
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Arteries and
arterioles take blood
away from the heart.
The largest artery is
the aorta.
The middle layer of
an artery wall
consists of smooth
muscle that can
constrict to regulate
blood flow and
blood pressure.
Arterioles can
constrict or dilate,
changing blood
pressure.
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The Veins The Capillaries
Venules drain blood
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from capillaries, then
join to form veins that
take blood to the heart.
Veins have much less 
smooth muscle and
connective tissue than
arteries.
Veins often have valves
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that prevent the
backward flow of blood
when closed.
Veins carry about 70%
of the body’s blood and
act as a reservoir during
hemorrhage.
Capillaries have walls only
one cell thick to allow
exchange of gases and
nutrients with tissue fluid.
Capillary beds are present
in all regions of the body
but not all capillary beds
are open at the same
time.
Contraction of a sphincter
muscle closes off a bed
and blood can flow
through an arteriovenous
shunt that bypasses the
capillary bed.
Anatomy of a capillary bed
The Heart
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The heart is a cone-shaped, muscular organ
located between the lungs behind the sternum.
The heart muscle forms the myocardium, with
tightly interconnect cells of cardiac muscle tissue.
The pericardium is the outer membranous sac with
lubricating fluid.
The heart has four chambers: two upper, thin-walled
atria, and two lower, thick-walled ventricles.
The septum is a wall dividing the right and left sides.
Atrioventricular valves occur between the atria and
ventricles – the tricuspid valve on the right and the
bicuspid valve on the left; both valves are reenforced by
chordae tendinae attached to muscular projections within
the ventricles.
External heart anatomy
Passage of Blood Through
the Heart
Blood follows this sequence through the heart:
superior and inferior vena cava → right
atrium → tricuspid valve → right ventricle
→ pulmonary semilunar valve → pulmonary
trunk and arteries to the lungs →
pulmonary veins leaving the lungs → left
atrium → bicuspid valve → left ventricle →
aortic semilunar valve → aorta → to the
body.
Internal view of the heart
Blood Pressure
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The pumping of the heart sends out blood under
pressure to the arteries.
Blood pressure is greatest in the aorta; the wall
of the left ventricle is thicker than that of the
right ventricle and pumps blood to the entire
body.
Blood pressure then decreases as the crosssectional area of arteries and then arterioles
increases.
Path of blood through the heart
Intrinsic Control of Heartbeat
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Each heartbeat is called a cardiac cycle.
When the heart beats, the two atria contract together,
then the two ventricles contract; then the whole heart
relaxes.
Systole is the contraction of heart chambers; diastole is
their relaxation.
The heart sounds, lub-dup, are due to the closing of
the atrioventricular valves, followed by the closing of
the semilunar valves.
The SA (sinoatrial) node, or pacemaker, initiates the
heartbeat and causes the atria to contract on average
every 0.85 seconds.
The AV (atrioventricular) node conveys the stimulus and
initiates contraction of the ventricles.
The signal for the ventricles to contract travels from the
AV node through the atrioventricular bundle to the
smaller Purkinje fibers.
Conduction system of the
heart
Extrinsic Control of Heartbeat
The Electrocardiogram
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A cardiac control center in the medulla oblongata speeds up or
slows down the heart rate by way of the autonomic nervous
system branches: parasympathetic system (slows heart rate) and
the sympathetic system (increases heart rate).
Hormones epinephrine and norepinephrine from the adrenal
medulla also stimulate faster heart rate.
An electrocardiogram (ECG) can record of the electrical
changes that occur in the myocardium during a cardiac
cycle.
Atrial depolarization creates the P wave, ventricle
depolarization creates the QRS wave, and repolarization
of the ventricles produces the T wave.
Electrocardiogram
The Vascular Pathways
The cardiovascular system includes two circuits:
Pulmonary circuit which circulates blood through the
lungs
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Systemic circuit which circulates blood to the rest of
the body
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The pulmonary circuit begins with the pulmonary trunk from the
right ventricle which branches into two pulmonary arteries that
take oxygen-poor blood to the lungs.
In the lungs, oxygen diffuses into the blood, and carbon dioxide
diffuses out of the blood to be expelled by the lungs.
Four pulmonary veins return oxygen-rich blood to the left atrium.
The systemic circuit starts with the aorta carrying O2-rich blood
from the left ventricle.
The aorta branches with an artery going to each specific organ.
Generally, an artery divides into arterioles and capillaries which
then lead to venules.
Both circuits are vital to homeostasis.
Cardiovascular system diagram
Major arteries and veins of
the systemic circuit
Blood Flow
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The beating of the heart is necessary to homeostasis because it creates pressure that propels blood in
arteries and the arterioles.
Blood Flow in Arteries
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Venous blood flow is dependent upon:
skeletal muscle contraction,
presence of valves in veins, and
respiratory movements.
Compression of veins causes blood to move forward past a valve that then prevents it from returning
backward. Changes in thoracic and abdominal pressure that occur with breathing also assist in the return of blood.
The coronary arteries serve the heart muscle itself; they are the first branch off the aorta.
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Blood moves slowly in capillaries because there are more capillaries than arterioles.
This allows time for substances (gas and nutrients) to be exchanged between the blood and tissues.
Blood Flow in Veins
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arterioles and arteries.
Blood Flow in Capillaries
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Blood pressure due to the pumping of the heart accounts for the flow of blood in the arteries.
Systolic pressure is high when the heart expels the blood.
Diastolic pressure occurs when the heart ventricles are relaxing.
Both pressures decrease with distance from the left ventricle because blood enters more and more
Since the coronary arteries are so small, they are easily clogged, leading to heart disease.
The hepatic portal system carries blood rich in nutrients from digestion in the small intestine to the liver,
the organ that monitors the composition of the blood.
Varicose veins develop when the valves of veins become weak.
Hemorrhoids (piles) are due to varicose veins in the rectum.
Phlebitis is inflammation of a vein and can lead to a blood clot and possible death if the clot is dislodged
and is carried to a pulmonary vessel.
Cross-sectional area as it relates to
blood pressure and velocity
Capillary Exchange
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At the arteriole end of a capillary, water moves out of
the blood due to the force of blood pressure.
At the venule end, water moves into the blood due to
osmotic pressure of the blood.
Substances that leave the blood contribute to tissue fluid,
the fluid between the body’s cells.
In the midsection of the capillary, nutrients diffuse out
and wastes diffuse into the blood.
Since plasma proteins are too large to readily pass out of
the capillary, tissue fluid tends to contain all components
of plasma except it has lesser amounts of protein.
Excess tissue fluid is returned to the blood stream as
lymph in lymphatic vessels.
Capillary exchange
Coronary artery circulation
Blood
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Blood separates into two main parts: plasma
(Molecular) and formed elements (Cellular).
Plasma (Liquid) accounts for 55% and formed
elements (Centrifuge precipitates) 45% of blood
volume.
Plasma contains mostly water (90–92%) and
plasma proteins (7–8%), but it also contains
nutrients and wastes.
Albumin is a large plasma protein that transports
bilirubin; globulins are plasma proteins that
transport lipoproteins.
Formed elements are mostly red blood cells,
platelets and white cells (leukocytes).
Composition of blood
Coagulation Factors!
The Red Blood Cells
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Red blood cells
(erythrocytes or RBCs)
are made in the red bone
marrow of the skull, ribs,
vertebrae, and the ends
of long bones.
Normally there are 4 to 6
million RBCs per mm3 of
whole blood.
Red blood cells contain
the pigment hemoglobin
for oxygen transport;
hemogobin contains
heme, a complex ironcontaining group that
transports oxygen in the
blood.
Red Blood Cell
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Red blood cells lack a nucleus and have a 120 day life
span.
When worn out, the red blood cells are dismantled in the
liver and spleen.
Iron is reused by the red bone marrow where stem cells
continually produce more red blood cells; the remainder of
the heme portion undergoes chemical degradation and is
excreted as bile pigments into the bile.
The kidneys produce the hormone erythropoietin to
increase blood cell production when oxygen levels are low.
VEGF regulates erythropoietin secretion from kidney.
Lack of enough hemoglobin results in anemia.
The air pollutant carbon monoxide combines more readily
with hemoglobin than does oxygen, resulting in oxygen
deprivation and possible death.
Bone Marrow Stem Cells
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A stem cell is capable
of dividing into new
cells that differentiate
into particular cell
types.
Bone marrow is
multipotent, able to
continually give rise
to many particular
types of blood cells.
The skin and brain
also have stem cells,
and mesenchymal
stem cells give rise to
connective tissues
including heart
muscle.
Blood cell formation in red bone marrow
The White Blood Cells
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White blood cells (leukocytes) have nuclei, are fewer in
number than RBCs, with 5,000 – 10,000 cells per mm3, and
defend against disease.
Leukocytes are divided into granular and agranular based on
appearance.
Granular leukocytes (neutrophils, eosinophils, and basophils)
contain enzymes and proteins that defend the body against
microbes.
The agranular leukocytes (monocytes and lymphocytes)
have a spherical or kidney-shaped nucleus.
Monocytes can differentiate into macrophages that
phagocytize microbes and stimulate other cells to defend the
body.
Lymphocytes are involved in immunity.
An excessive number of white blood cells may indicate an
infection or leukemia; HIV infection drastically reduces the
number of lymphocytes.
Macrophage engulfing bacteria
The Platelets and Blood Clotting
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Red bone marrow produces large cells called megakaryocytes
that fragment into platelets at a rate of 200 billion per day;
blood contains 150,000–300,000 platelets per mm3.
Dozens of clotting factors in the blood help platelets form
blood clots.
Injured tissues release Tissue factor to blood and activate
FVIIa, which activates FX to FXa.
FXa/FVa are called prothrombin activator, which converts
prothrombin into thrombin.
Thrombin, in turn, acts as an enzyme and converts fibrinogen
into insoluble threads of fibrin.
Platelets are also activated and aggregate to form plug at
injury site.
These conversions require the presence of calcium ions (Ca2+)
and phospholipid.
Trapped red blood cells make a clot appear red.
Cardiovascular Disease
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Cardiovascular Diseases (CVD) Affect the Heart and the Circulatory System.
Coronary artery disease is the build-up of plaque in the arteries supplying blood to the
heart (also ischaemic heart disease or Coronary heart disease).
Peripheral artery disease is the build-up of plaque in the arteries supplying blood to the arms and
legs.
Cardiac Diseases
Heart Failure
Hypertensive heart disease - diseases of the heart secondary to high blood pressure
Cardiomyopathy - diseases of cardiac muscle, A myocardial infarction, or heart attack, occurs when a
portion of heart muscle dies due to lack of oxygen.
Cor pulmonale - a failure of the right side of the heart.
Cardiac dysrhythmias - abnormalities of heart rhythm.
Inflammatory heart disease
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Endocarditis – inflammation of the inner layer of the heart, the endocardium. The structures most commonly
involved are the heart valves.
Inflammatory cardiomegaly
Myocarditis – inflammation of the myocardium, the muscular part of the heart.
Valvular heart disease
Vascular Diseases of brain and kidney
Cerebrovascular Disease (Stroke)
Carotid artery disease is the build-up of plaque in the arteries that supply blood to the brain.
Hypertension
Atherosclerosis
Coronary Heart Disease and
Heart Failure
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Coronary Heart Disease (CHD) is the most common form of heart disease.
It occurs when the arteries supplying blood to the heart narrow or harden from the build-up of plaque.
Plaque is made up of fat, cholesterol and other substances found in the blood.
This plaque build-up is also known as atherosclerosis.
The site of the plaque determines the type of heart disease.
The decrease in blood flow due to plaque build-up can lead to chest pain, also called angina, or progress to a heart attack.
The five most common symptoms of a heart attack are:
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Women often have different symptoms of heart attack from men. The most common symptoms reported by women are:
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Unusual fatigue
sleep disturbance
shortness of breath
Indigestion Heart failure is a condition that occurs slowly over time.
Heart failure occurs after an injury to the heart muscle, usually caused by uncontrolled high blood pressure, a heart attack,
or a heart valve that does not work properly.
The weakened heart muscle has to work overtime to keep up with the body's demands, which can leave a person tired.
Some of the symptoms of heart failure:
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Chest pressure or pain
Shortness of breath
Pain or discomfort in the arms or shoulder
Pain or discomfort in the jaw, neck or back
Feeling weak, lightheaded, or nauseous
Shortness of breath
Difficulty breathing when lying down
Swelling in the legs, ankles, and feet
General fatigue and weakness.
Risk factors that increase your chances of developing heart failure:
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High blood pressure
Heart attack
Damage to a heart valve or a history of a murmur
Enlargement of the heart or a family history of an enlarged heart
Diabetes
Cerebrovascular Disease (Stroke)
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A stroke, or cerebrovascular accident (CVA), is the rapid loss of brain function(s) due
to disturbance in the blood supply to the brain.
This can be due to ischemia (lack of blood flow) caused by blockage (thrombosis, arterial
embolism), or a hemorrhage.
As a result, the affected area of the brain cannot function, which might result in an inability
to move one or more limbs on one side of the body, inability
to understand or formulate speech, or an inability to see one side of the visual field.
A stroke is a medical emergency and can cause permanent neurological damage,
complications, and death.
Risk factors for stroke include old age, high blood pressure, previous stroke or transient
ischemic attack (TIA), diabetes, high cholesterol, tobacco smoking and atrial fibrillation.
High blood pressure is the most important modifiable risk factor of stroke.
It is the second leading cause of death worldwide.
An ischemic stroke is occasionally treated in a hospital with thrombolysis (also known as a
"clot buster"), and some hemorrhagic strokes benefit from neurosurgery.
Treatment to recover any lost function is termed stroke rehabilitation, ideally in a stroke
unit and involving health professions such as speech and language therapy, physical therapy
and occupational therapy.
Prevention of recurrence may involve the administration of antiplatelet drugs such as aspirin
and dipyridamole, control and reduction of high blood pressure, and the use of statins.
Selected patients may benefit from carotid endarterectomy and the use of anticoagulants.
Risk Factors For CVD
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Tobacco Use
Hypertension
High Levels of
Cholesterol
Physical inactivity
Diabetes
High Triglyceride
Levels
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Obesity
Psychological & Social
Factors
Heredity
Aging
Male factor
Ethnicity
Syndrome X
Atherosclerosis
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Atherosclerosis is due to a build-up of fatty material (plaque),
mainly cholesterol, under the inner lining of arteries.
The plaque can cause a thrombus (blood clot) to form.
The thrombus can dislodge as an embolus and lead to
thromboembolism.
Partial blockage of a coronary artery causes angina pectoris,
or chest pain.
An aneurysm is a ballooning of a blood vessel, usually in the
abdominal aorta or arteries leading to the brain.
Death results if the aneurysm is in a large vessel and the
vessel bursts.
Atherosclerosis and hypertension weaken blood vessels over
time, increasing the risk of aneurysm.
Coronary bypass operation
1. A coronary bypass
operation involves
removing a segment of
another blood vessel
and replacing a
clogged coronary
artery.
2. It may be possible to
replace this surgery
with gene therapy that
stimulates new blood
vessels to grow where
the heart needs more
blood flow.
Clearing Clogged Arteries
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Angioplasty uses a long
tube threaded through
an arm or leg vessel to
the point where the
coronary artery is
blocked; inflating the
tube forces the vessel
open.
Small metal stents are
expanded inside the
artery to keep it open.
Stents are coated with
heparin to prevent
blood clotting and with
chemicals to prevent
arterial closing.
Angioplasty
Dissolving Blood Clots
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Medical treatments for dissolving blood
clots include use of t-PA (tissue
plasminogen activator) that converts
plasminogen into plasmin, an enzyme that
dissolves blood clots, but can cause brain
bleeding.
Aspirin reduces the stickiness of platelets
and reduces clot formation and lowers the
risk of heart attack.
Heart Transplants and
Artificial Hearts
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Heart transplants are routinely
performed but immunosuppressive drugs
must be taken thereafter.
There is a shortage of human organ
donors.
Work is currently underway to improve
self-contained artificial hearts, and
muscle cell transplants may someday be
useful.
Hypertension
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Hypertension (high blood
pressure) is present when
systolic pressure is 140 or
greater or diastolic pressure
is 100 or greater; diastolic
pressure is emphasized when
medical treatment is
considered.
A genetic predisposition for
hypertension occurs in those
who have a gene that codes
for angiotensinogen, a
powerful vasoconstrictor.
Category
Systolic
Blood
Pressure
Diastolic
Blood
Pressure
Normal
< 120
<80
Prehypertension
120-139
80-89
Hypertension
– Stage 1
140-159
90-99
Hypertension
– Stage 2
>160
>100
Risk Factors of Hypertension
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Family history of hypertension
Excess Consumption of Sodium Chloride
Less active
Overweight
Dietary
Alcohol consumption
Smoking
Prevention of Hypertension
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Maintain a healthy weight.
Be more physically active.
Balanced nutrition
Drink less alcoholic beverages.
Reduce the intake of salt and sodium in
the diet to approximately 2400 mg/day.
Regulation of Cardiovascular
System
Multifactor/Multigene Effector
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Genetic
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Single gene disease
Multigene disease, e.g., SNP
Nongenetic
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Factors affect one gene
Factors affect many genes
Epigenetic Regulation of
cardiovascular system
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Epigenetics refers to chromatin-based mechanisms important in the
regulation of gene expression that do not involve changes to the
DNA sequence
Epigenetic regulation through histone modifications is an important
aspect of gene regulation at chromatin level.
The unstructured tails of histones (the proteins that assemble into
the nucleosomes around which chromosomal DNA is wound) are
subject to myriad chemical modifications, including acetylation,
methylation, phosphorylation, ubiquitinylation, and sumoylation.
In combination, these modifications are thought to result in a
histone “code” that is read and translated into signals for activation
or repression of associated genes.
For example, certain histone modifications are most often
associated with repressed genes, and others with active genes.
Diagrammatic representation of chromatin and chromatin-mediated gene regulation.
Bruneau B G Circulation Research 2010;107:324-326
Molecular Mechanisms of
Epigenetic Gene Regulation
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DNA is packaged as a DNA–protein complex that is conserved across all
eukaryotic genomes.
The fundamental repeating unit of this structure, termed chromatin, is the
nucleosome comprising an octamer of core histone proteins around which is
wrapped 146 bp of DNA.
Each nucleosome comprises 2 molecules of H2A, H2B, H3, and H4.
Adjacent nucleosome particles are separated by shorter species-specific
lengths of linker DNA associated with a fifth histone protein, histone H1.
This linker histone facilitates further compaction of chromatin into higherorder chromatin structures that enable the packaging of extraordinary
lengths of DNA into the tight confines of the cell nucleus.
Over the last 20 years, 3 highly interconnected epigenetic pathways have
emerged that impact on the structure and accessibility of chromatin.
Each of these pathways is important in the regulation of gene expression:
DNA methylation, histone posttranslational modifications, and RNA-based
mechanisms
Figure 1. Epigenetic mechanisms of gene regulation.
Matouk C C , Marsden P A Circulation Research
2008;102:873-887
Copyright © American Heart Association
DNA Methylation
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DNA methylation involves the postsynthetic, covalent modification of the 5position of cytosine to define the “fifth base of DNA,” 5-methyl-cytosine.
In mammals, DNA methylation is almost exclusively restricted to CpG
dinucleotides.
DNA methylation is catalyzed by 3 different DNA methyltransferases
(DNMTs) encoded by different genes on distinct chromosomes: DNMT1,
DNMT3a, and DNMT3b.
De novo methylation is catalyzed by the latter 2 enzymes and is important
in the establishment of DNA methylation patterns in the early embryo and
during development.
In contrast, DNMT1 serves a maintenance function and is responsible for
the propagation of DNA methylation patterns following DNA replication
during mitotic cell division.
DNA methylation is a remarkably stable epigenetic modification.
Its dynamic regulation has been clearly demonstrated during
embryogenesis, cellular differentiation, and carcinogenesis.
CpG Island
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Approximately 70% to 90% of CpG dinucleotides, representing 3% to 6%
of all cytosines, are methylated in healthy somatic cells.
Surprisingly, CpG dinucleotides are relatively depleted in the mammalian
genome, ie, occur at a frequency less than would be expected based on the
GC content of the genome
Although variably defined, these relatively (G+C)- and CpG-rich regions are
commonly referred to as CpG islands.
They account for approximately 7% of CpG dinucleotides genome-wide and
are associated with the 5′-regulatory regions of ≈40% to 60% of human
genes.
Typically, these CpG dinucleotides are unmethylated.
A significant proportion of CpG dinucleotides also occur in the context of
intergenic, repetitive DNA sequences, such as Alu elements.
In contrast to those comprising CpG islands, these CpG dinucleotides are
usually densely methylated.8
DNA Methylation and Gene Expression
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DNA methylation is a repressive mark associated with transcriptional silencing.
It has been strongly implicated in a growing number of integral cellular functions,
including the silencing of repetitive (parasitic) sequences, X chromosome
inactivation, genomic imprinting, mammalian embryonic development, and lineage
specification.
Its dysregulation is also characteristic of a growing number of human diseases,
most prominently, cancer.
A strikingly similar pattern is also observed on the inactive X chromosome.
DNA methylation itself can impede the binding of transcription factors to CpG
dinucleotide-containing cis-DNA binding elements.
A family of methyl-CpG binding proteins has been described that can specifically
recognize the mammalian methylation mark
These include 4 proteins containing a homologous methyl-CpG-binding domain
(MBD1, MBD2, MBD4, and the founding member, MeCP2) and a recently
characterized, nonhomologous protein, Kaiso, which is capable of binding a
methylated CpG dinucleotide doublet
These methyl-CpG-binding proteins can directly repress transcription, prevent the
binding of activating transfactors, or recruit enzymes that catalyze histone
posttranslational modifications and chromatin-remodeling complexes that alter the
structure of chromatin and actively promote transcriptional repression.
A transcriptional activator that specifically recognizes unmethylated CpG
dinucleotides, human CpG binding protein (hCGBP) is also identified
Histon Modification
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More than 60 distinct modification sites have been described which include lysine
acetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine
ubiquitylation, and lysine sumoylation, among others.
Two classes of histone posttranslational modifications, in particular, have well-established
roles in the control of mammalian gene expression: lysine acetylation and lysine
methylation.
Lysine acetylation involves the transfer of acetyl groups from acetyl-coenzyme A
molecules to the lysine ε-amino groups of histone tails.
In mammalian cells, this reaction is catalyzed by 3 principal families of histone
acetyltransferases (HATs): GNAT, MYST, and CBP/p300.
A number of transcriptional coactivators have intrinsic HAT activity
HATs demonstrate poor specificity for individual histone tail lysine residues and are also
capable of acetylating many nonhistone proteins important in the regulation of
transcription; for example, c-Jun, E2F, MyoD, nuclear factor (NF)-κB, p53, pRb, and YY1,
among others
Removal of histone lysine acetylation is catalyzed by 4 families of mammalian histone
deacetylases (HDACs): class I (HDAC1-3, HDAC8), class II (HDAC4-7, HDAC9-10), class
III sirtuins (SIRT1-7), and class IV (HDAC11)
HDACs evidence poor specificity for individual histone lysine residues and are also active
on many nonhistone proteins.
The acetyl-lysine mark is read by a group of chromatin-associated proteins, including
several HATs and chromatin-remodeling enzymes, that contain bromodomains.
The interplay between HATs, HDACs, and bromodomain-containing readers allows for a
highly dynamic gene transcriptional control pathway.
Histone Methylation
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A lysine residue can either be acetylated or methylated.
Histone lysine acetylation is tightly correlated with transcriptional activation, the impact of
histone lysine methylation on gene expression is context-dependent.
For example, histone H3 lysine 4 (H3K4) methylation is strongly associated with
transcriptional activation.
This epigenetic mark is written by the family of trithorax group (Trx-G) proteins.
A single lysine residue can be mono-, di-, or trimethylated.
These more subtle epigenetic modifications are likely to be functionally relevant.
This contention is supported by a recent genome-wide survey that demonstrated
preferential localization of trimethylated H3K4 to active promoters and monomethylated
H3K4 to enhancers.
The characterization of specific di- and trimethylated H3K4 readers, the mammalian ING
(inhibitor of growth) family proteins (ING1-5), provides further compelling evidence.
Conversely, di- and trimethylated histone H3 lysine 9 (H3K9) are strongly correlated with
transcriptional repression.
These epigenetic marks are catalyzed by an increasingly large family of SET-domaincontaining histone lysine methyltransferases, including SUV39H1, SUV39H2, PRDM2/RIX1,
and G9A/BAT8.
Heterochromatin 1 (HP1) proteins (HP1α, HP1β, and HP1γ) specifically bind di- and
trimethylated H3K9 via interactions with their methyl-binding chromodomain and are crucial
for the formation of heterochromatin and transcriptional silencing.
Jumonji-domain-containing proteins (JMJD) is histone lysine demethylases
RNA-Based Mechanisms
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There is mounting evidence that noncoding RNAs and the RNA interference
machinery are fundamental determinants of chromatin-based gene expression.
In mammalian systems, the best characterized examples include the role of XIST
RNA in X chromosome inactivation, Air RNA at the murine imprinted Igf2r locus,
and as of yet unidentified RNAs in the assembly of centromeric heterochromatin.
These examples importantly involve coordinated epigenetic activities including
DNA methylation and histone posttranslational modifications.
In contrast, micro-RNAs and short interfering (si)RNAs, ≈ 21 to 26 nucleotide
small RNA species, are well-known mediators of cytoplasmic, posttranscriptional
gene silencing as components of the RNA-induced silencing complex (RISC).
Micro-RNAs are derived from nuclear transcripts with characteristic stem–loop
structures and transported to the cytoplasm.
Alternatively, siRNAs are derived from long double-stranded RNA precursors
delivered exogenously to cells or that arise naturally within cells.
Exogenously administered siRNAs directed at promoter regions can effectuate
transcriptional gene silencing in mammalian cells by inducing site-specific DNA
methylation and repressive histone posttranslational modifications.
Given that at least 15% to 20% of mouse and human genes, respectively,
demonstrate cis-encoded natural antisense transcripts, it is anticipated that RNAbased mechanisms will have far-reaching influence in the regulation of
mammalian gene expression.
Epigenetic Regulation of Vascular
Endothelial Gene Expression
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Epigenetics refers to chromatin-based pathways important in the
regulation of gene expression
Includes 3 distinct, but highly interrelated mechanisms: DNA
methylation, histone density and posttranslational modifications,
and RNA-based mechanisms.
They offer a newer perspective on transcriptional control paradigms
in vascular endothelial cells and provide a molecular basis for how
the environment impacts the genome to modify disease
susceptibility.
Using endothelial nitric oxide synthase (NOS3) as an example,
examine the growing body of evidence implicating epigenetic
pathways in the control of vascular endothelial gene expression in
health and disease.
Endothelial Cell-specific Regulation
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Although an as of yet uncharacterized endothelial master regulator
may exist, the present cis/trans paradigm supports a model for the
cooperative activity of several ubiquitously expressed transcription
factors in the constitutive expression of endothelial-restricted genes.
These include Ets family members, GATA-2, Sp1, activator protein-1,
and octamer transcription factors.
Indeed, a majority of endothelial-restricted genes possess cis-DNA
binding elements for these factors in their 5′-regulatory regions.
The eNOS gene is a representative example.
Specificity could be achieved by a unique combination of transcription
factors in endothelial compared with nonendothelial cell types.
Additionally, unique posttranslational modifications or alternatively
spliced mRNA species may be relevant.
However, little direct evidence for these models of endothelial cellrestricted gene expression is presently available.
Alternative mechanisms may contribute to the cell-specific expression
of endothelial genes.
Transient Transfection of eNOS Promoter-Reporter
Constructs Suggests Epigenetic Mechanisms of Gene
Regulation
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eNOS exists as a single copy in the haploid genome, contains 26 exons,
spans approximately 21 kb of genomic DNA, maps to chromosome 7q3536, and directs the expression of a single major transcript measuring
4052 nucleotides.
A single major transcription initiation site was defined by primer
extension, S1 nuclease protection, and 5′-RACE (rapid amplification of 5′
cDNA ends).
The human eNOS promoter lacks a canonical TATA box and does not
contain a proximal CpG island.
Detailed molecular characterization of the human eNOS promoter using
deletion analysis and linker-scanning mutagenesis defined 2 clustered cisregulatory regions: positive regulatory domain I (PRD I) (−104/−95
relative to transcription initiation) and PRD II (−144/−115).
In the vascular endothelium, these regions bind multiprotein activator
complexes including Sp1, Sp3, and Ets1 transcription factors, among
others.
Transgenic eNOS promoter-reporter mice faithfully recapitulate
expression of the native eNOS gene
Demonstration of Epigenetic
Regulation of eNOS
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In expressing cultured human endothelial cells, the eNOS promoter directs eNOS
expression at the endogenous locus, as well as reporter expression from episomal eNOS
promoter-reporter constructs.
ChIP experiments demonstrated enrichment of the transcription factors Sp1, Sp3, and Ets1
at the endogenous eNOS proximal promoter as well as recruitment of RNA polymerase II.
In nonexpressing cultured human vascular smooth muscle cells, eNOS is not expressed at
the endogenous locus.
However, episomal eNOS promoter-reporter constructs demonstrated robust activity,
similar to transient transfection of episomal constructs into human endothelial cells.
ChIP experiments demonstrated no enrichment of Ets1, Sp1, and Sp3 at the proximal
promoter of the endogenous eNOS gene despite similar global levels of these transcription
factors in endothelial and vascular smooth muscle cells by Western blotting.
RNA polymerase II was not recruited to the eNOS proximal promoter.
Episomal constructs demonstrated robust promoter activity in a majority of nonexpressing
cell types.
These results from transient transfection experiments were in stark contrast to the
endothelial cell-restricted expression of stably integrated promoter-reporter constructs in
transgenic eNOS promoter-reporter mice.
Data demonstrated that nonexpressing cell types possess the requisite transcriptional
machinery to direct eNOS expression.
These data suggested that epigenetic, chromatin-based pathways may be relevant in the
cell-specific expression of the eNOS gene
Transient transfection of eNOS promoter-reporter constructs into expressing and
nonexpressing cell types.
The Role of DNA Methylation in eNOS
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Using Southern hybridization with methylation-sensitive isoschizomer
mapping and nucleotide-resolution bisulfite genomic sequencing, a
differentially methylated region (DMR) was demonstrated in the native
eNOS proximal promoter (−361/+3) in expressing (endothelial) and
nonexpressing cell types.
Genomic DNA isolated from endothelial cells was unmethylated or lightly
methylated, whereas genomic DNA isolated from nonendothelial cells
was heavily methylated at the eNOS proximal promoter.
Importantly DNA methylation was determined to be symmetrical
(occurring on both sense and antisense strands) and restricted to CpG
dinucleotides.
Methylation further upstream (−4912/−4587) in a region corresponding
to an enhancer or further downstream in a CpG island located at the 3′end of the gene failed to demonstrate differential methylation in
expressing and nonexpressing cell types.
The eNOS proximal promoter DMR was confirmed in vivo by performing
bisulfite genomic sequencing of the eNOS proximal promoter in
endothelial and vascular smooth muscle cells isolated by laser-capture
microdissection from the murine aorta (a majority of CpG sites is
conserved between mouse and man).
In Vivo Differential Regulation of eNOS
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To explore the functional relevance of the eNOS proximal promoter DMR,
chromatin immunoprecipitation (ChIP) combined with quantitative real-time PCR
was performed to assess the binding of relevant transfactors to the native eNOS
proximal promoter in human endothelial and vascular smooth muscle cells.
These experiments demonstrated preferential recruitment of Sp1, Sp3, and Ets1
transcription factors to the eNOS proximal promoter in endothelial cells despite
the presence of these factors in vascular smooth muscle cells, as determined by
Western blotting.
Consistent with these results, the transcriptional machinery (RNA polymerase II)
was also preferentially bound to the eNOS proximal promoter in endothelial cells.
MeCP2, a methyl-CpG-binding protein associated with transcriptional repression,
was preferentially recruited to the eNOS proximal promoter in nonexpressing
vascular smooth muscle cells.
Treatment of nonexpressing cell types with 5-azacytidine, a DNMT inhibitor,
demethylated the eNOS promoter in various nonendothelial cell types and
increased expression of the eNOS mRNA.
Several groups have previously demonstrated DMRs in the proximal promoters of
cell-restricted genes, for example, human maspin (SERPINB5) and erythropoietin
(EPO).
Human eNOS represents the first example of a constitutively expressed gene in
the vascular endothelium whose cell-restricted pattern of expression is
determined, at least in part, by DNA methylation pathways.
Vascular Endothelium Proteins
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Restricted expression of eNOS steady-state RNA
and protein, respectively, to the endothelium,
especially large- and medium-sized arteries.
von Willebrand factor (VWF), vascularendothelial cadherin (VE-cadherin) (CDH5),
intercellular adhesion molecule-2 (ICAM-2), the
angiopoietin receptors (TIE1 and TIE2), and the
vascular endothelial growth factor (VEGF)
receptors (FLT-1/VEGFR1 and FLK-1/VEGFR2).
Endothelial NOS (eNOS)
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In mammals, the production of nitric oxide is catalyzed by 3 isoforms of nitric oxide
synthase (NOS) encoded by separate genes on 3 different chromosomes: neuronal NOS
(NOS1), inducible NOS (iNOS) (NOS2), and endothelial NOS (eNOS) (NOS3).
These NOS isoforms differ in their regulation and cell-specific distribution.
The latter isoform, eNOS, is constitutively expressed and responsible for the majority of
nitric oxide produced by the vascular endothelium and, therefore, represents the
dominant source of bioactive endothelium-derived relaxing factor.
Here, nitric oxide plays important antithrombotic and antiatherogenic roles characterized
by the inhibition of platelet aggregation, leukocyte–endothelium adhesion, and vascular
smooth muscle cell proliferation.
Its fundamental role in cardiovascular physiology is underscored by the phenotype of
eNOS-null mice.
These eNOS-deficient animals demonstrate systemic and pulmonary hypertension,
abnormal vascular remodeling, defective angiogenesis, pathological healing in response to
vascular injury, and impaired mobilization of stem and progenitor cells.
In human disease, eNOS deficiency has been documented in the lungs of patients with
pulmonary hypertension and in the neointimal covering of advanced atheromatous
plaques.
Given its prominent role as a signaling molecule in the cardiovascular system, much
attention has been focused on deciphering the regulation of eNOS both in vitro and in
vivo.
Important transcriptional, posttranscriptional, and posttranslational mechanisms have
been defined.
Genetic and Epigenetic
Cardiovascular Disease
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Heritability estimates have been calculated for
common cardiovascular diseases and range from
30% to 50%.
The recent characterization of evolutionarily
conserved, epigenetic mechanisms has offered a
fundamentally new paradigm for understanding
mammalian gene regulation.
Although best characterized in cancer and
developmental biology, these mechanisms provide
the molecular substrate for the improved
understanding of complex, non-Mendelian diseases
including common diseases of the human vascular
system.
DNA Methylation at iNOS Promoter
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iNOS is a cytokine-inducible gene whose expression is implicated in a number of
human diseases, in particular, chronic inflammatory conditions, e.g., iNOS mRNA
and protein in the neointima of atherosclerotic human blood vessels.
Consistent with a role for promoter DNA methylation in transcriptional repression,
heavy methylation of CpG dinucleotides in the human iNOS proximal promoter of
nonresponsive human endothelial cells was documented.
In contrast, the highly homologous mouse iNOS promoter in responsive cell types
was only lightly methylated.
DNA methylation was symmetrical and restricted to CpG dinucleotides.
The percentage of methylation of CpG dinucleotides in the core iNOS promoter
was well correlated with gene expression, as determined by quantitative real-time
RT-PCR.
The functional relevance of these findings was demonstrated by treating human
cells in culture with 5-azacytidine, a pharmacological DNMT inhibitor.
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In minimally responsive DLD-1 cells, the iNOS promoter was completely demethylated
by treatment with 5-azacytidine and associated with increased iNOS steady-state mRNA
after cytokine stimulation.
In contrast, the iNOS promoter in human endothelial cells remained hypermethylated
and refractory to cytokine stimulation, suggesting additional layers of epigenetic control.
Taken together, these data demonstrate a prominent role for DNA methylation in
the transcriptional silencing of the human iNOS promoter in nonresponsive human
endothelial cells in culture.
Histone posttranslational modifications contribute to the
transcriptional silencing of iNOS in cultured human cells
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Using ChIP combined with real-time PCR, differential recruitment of the methyl-binding
domain protein MeCP2 to the heavily methylated iNOS promoter in human endothelial
cells but not to the highly cytokine-inducible VCAM-1 promoter were detected.
Because MeCP2 can mediate transcriptional silencing by recruiting corepressor complexes
with associated HDAC and H3K9 methyltransferase activities, the corresponding histone
posttranslational modifications at the iNOS and VCAM-1 core promoters in a variety of cell
types before and after cytokine stimulation were analyzed.
Cytokine stimulation was associated with increased recruitment of RNA polymerase II at
the VCAM-1 promoter, but not at the iNOS promoter, in human endothelial cells.
Importantly, di- and trimethylated H3K9 marks were enriched at the iNOS proximal
promoter before and after cytokine stimulation in human endothelial cells.
These repressive marks were not detected at the cytokine-inducible VCAM-1 promoter.
Similar results have been reported for E-selectin, another cytokine-inducible endothelial
gene.
These data establish a role for epigenetic pathways, in particular, DNA and histone H3K9
methylation, in the transcriptional silencing of the human iNOS gene in cultured human
endothelial cells.
It is tempting to speculate that dysregulation of these epigenetic pathways in disease
leads to aberrant iNOS expression in endothelial cells, for example, as observed in human
atherosclerosis.
Epigenetic Pathways in Vascular
Development and Endothelial
Differentiation
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Epigenetic pathways have received increasing attention in embryonic
development and cellular differentiation.
Global and endothelial cell-specific knockout of a class II HDAC, HDAC7, is
embryonic lethal and required for the development of a normal vasculature.
Specifically, these mice demonstrated defects in endothelial–endothelial and
endothelial–smooth muscle cell contacts, resulting in vascular dilatation and
rupture.
This phenotype is the result of a dysregulated MMP10/TIMP1 axis, resulting
in pathological degradation of the extracellular matrix.
In addition, HDAC activity has been shown by multiple independent
laboratories to be critical for endothelial differentiation of embryonic stem
cells and adult endothelial progenitor cells.
HDAC3 activity appears to be particularly relevant.
HDAC3 and HDAC7 are specifically implicated in endothelial development
and differentiation, colocalize in vivo and are constituents of the same
multiprotein, corepressor complexes, SMRT and N-CoR.
Whether HDAC activity is also required for maintenance of the mature
endothelial phenotype is presently not known.
Hint of Vascular Genes
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The histone methyltransferase WHSC1 interacts
with an important cardiac transcription factor,
Nkx2-5, to regulate the normal development of
the heart.
Jarid2, also known as Jumonji, is an integral
component of the Polycomb repressor complex,
which deposits repressive histone marks. Jarid2
has long been known to function in heart
development, but its mechanism of action was
unknown.
Epigenetic Pathways in
Postnatal Angiogenesis
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In the mature vascular system, HDACs regulate postnatal angiogenesis in
response to various pathological cues.
The utility of pharmacological HDAC inhibitors as cancer chemotherapeutics is
related, in part, to their potent antiangiogenic activity.
Similar findings were reported in VEGF-induced, hypoxia-induced, and other
models of postnatal angiogenesis and endothelial cell migration.
HDAC1 has been implicated in hypoxia-induced angiogenesis.
Specific knockdown of HDAC7 using an siRNA strategy inhibited cell migration and
angiogenesis in mature primary endothelial cells in culture.
Potente et al demonstrated a fundamental role for SIRT1, a class III HDAC, in
angiogenic signaling both in vitro and in vivo.
The mechanistic basis for the requirement of HDAC activity in the angiogenic
response is presently not clear and likely involves the regulation of the acetylation
status of various transcription factors, such as the forkhead transcription factor,
Foxo1, and other proteins, including eNOS itself.
It remains to be defined whether global or locus-specific histone modifications
important in the chromatin-based control of gene expression are also relevant.
Perhaps not surprisingly other epigenetic pathways are beginning to emerge as
important regulators of postnatal angiogenesis and include DNA methylation,
histone methylation, and the RNA interference machinery
Epigenetic Pathways in Inflammation and
the Endothelial Response to Blood Flow
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Hyperacetylation of histones H3 and H4 in the 5′-regulatory regions of genes is strongly associated with
transcriptional activation.
Treatment with HDAC inhibitors induce gene expression at sensitive promoters.
HDAC inhibitors have emerged as an important new class of potent antiinflammatory agents in a number
of cell types, including endothelial cells.
HDAC inhibitors have shown early promise in the treatment of a growing number of chronic inflammatory
diseases such as inflammatory bowel disease, systemic lupus erythematosus, and rheumatoid arthritis.
To date, the mechanism of action remains unclear but may involve modulation of NF-κB transcriptional
activity.
In human cultured endothelial cells, HDAC inhibitors inhibited TNF-α–induced monocyte adhesion in vitro
and in vivo via suppression of the VCAM-1 gene.
Interestingly, other cytokine-inducible genes, ICAM-1 and E-selectin, were not suppressed, suggesting a
direct effect on the VCAM-1 promoter as opposed to a general inhibition of the NF-κB signaling pathway.
Similar results have recently been reported for the repression of cytokine-induced tissue factor in human
endothelial cells by a variety of structurally distinct HDAC inhibitors.
What accounts for this unique promoter specificity is presently not known.
The recent demonstration that the administration of the pharmacological HDAC inhibitor, TSA, to
atherosclerosis-prone, Ldlr−/− mice exacerbates neointimal lesions emphasizes the need for a better
understanding of epigenetic pathways in animal models of atherosclerosis.
It is of great interest that epigenetic pathways in human endothelial cells are responsive to the physical
forces of blood flow, in particular, laminar shear stress.
Pronounced changes in global and locus-specific histone posttranslational modifications in cultured
human endothelial cells in response to laminar shear stress.
It is interesting to speculate that epigenetic pathways, in part, determine the susceptibility of different
regions of the vascular system to atherosclerosis.
Relative reduction of eNOS transcription in atherosclerosis prone regions of the mouse aorta.
Epigenetic mechanisms in
diabetic vascular complications
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Increasing evidence suggests that epigenetic factors play a key role
in the complex interplay between genes and the environment.
Actions of major pathological mediators of diabetes and its
complications such as hyperglycaemia, oxidant stress, and
inflammatory factors can lead to dysregulated epigenetic
mechanisms that affect chromatin structure and gene expression.
Furthermore, persistence of this altered state of the epigenome may
be the underlying mechanism contributing to a ‘metabolic memory’
that results in chronic inflammation and vascular dysfunction in
diabetes even after achieving glycaemic control.
Further examination of epigenetic mechanisms by also taking
advantage of recently developed next-generation sequencing
technologies can provide novel insights into the pathology of
diabetes and its complications in vasculature.
DNA methylation: relation to
diabetes and vascular complications
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Regulation of Agouti gene expression by DNA methylation plays an
important role in the development of obesity and diabetes in mice
The islet dysfunction and development of diabetes in rats is
associated with epigenetic silencing via promoter DNA methylation
of Pdx1, a key transcription factor that regulates β-cell
differentiation and insulin gene expression
Peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α) regulates insulin production in pancreatic β-cells.
Studies with T2D animals showed that DNA hypermethylation at the
promoter of its gene PPARGC1A reduces PGC-1α expression and
inhibits insulin production
Methylation and Cardiovascular Diseases
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The role of DNA methylation in the pathogenesis of cardiovascular diseases
(CVDs) is not completely understood.
Atherosclerosis was associated with global hypomethylation in SMCs of
atherosclerotic lesions from humans, and animal models such as high-fat diet-fed
ApoE null mice and balloon-injured rabbits.
Furthermore, altered DNA methylation of several candidate genes linked with
atherosclerosis was identified in both VSMCs and ECs, and in mouse models.
These include hypoxia-inducible factor-1α, c-fos, p53 and oestrogen receptor,
growth factors, arachidonic acid-metabolizing enzymes (15-lipoxygenase),
vasodilator endothelial nitric oxide synthase, and matrix metalloproteinases.
Alterations in genomic DNA methylation were also demonstrated in leucocytes
derived from ApoE null mice preceding the development of atherosclerosis.
Other CVD risk factors such as hyperhomocysteinaemia, hypercholesterolaemia,
and inflammation have also been implicated in DNA methylation changes
associated with atherosclerosis.
Altered global DNA methylation was noted in peripheral blood monocytes of
patients with increased risk for CVDs.
Risk for CVDs and diabetes increases with age, and ageing is associated with
hypomethylation of genomic DNA.
miRNAs in vascular complications
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The 22-nucleotide small non-coding miRNAs play important roles in diverse
biological processes and disease conditions such as cancer, diabetes, DN,
cardiogenesis, angiogenesis, and vascular development by post-transcriptional
mechanisms.
Interaction of mature miRNAs with specific binding sites in the 3′ untranslated
regions of target mRNAs in the RNA-induced silencing complex leads to either
mRNA degradation or inhibition of translation.
Each miRNA can regulate multiple targets including signal transduction
components, transcription and epigenetic factors, and provide another level of
epigenetic mechanism to fine tune gene regulation in response to environmental
stimuli.
Evidence shows that miRNAs can affect the function of both ECs and VSMCs
relevant to vascular diseases.
miRNAs are implicated in phenotypic switching, proliferation, migration, and
neointimal thickening in VSMCs, as well as capillary formation, migration,
senescence, expression of adhesion molecules, and angiogenic growth and
transcription factors in ECs.
In monocytes and macrophages, miRNAs regulate inflammation, response to
oxidized lipids, oxidative stress, immune function, cholesterol homeostasis, and
differentiation.
Use of Vascular
Endothelial Growth Factor
(VEGF) as a Treatment for
End Stage Coronary Artery
Disease (CAD)
VEGF is involved in Angiogenesis
Angiogenesis
The formation of new blood vessels from existing
microvessels
VEGF contributes to the preservation of ischemic tissue
and myocardial pump function after myocardial
infarction
VEGF is important in:
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Embryogenesis (called vasculogenesis)
Wound healing
Tumor growth and metastasization
Rheumatoid arthritis
Ischemic heart disease
Ischemic peripheral vascular disease
Inducing Angiogenesis
Need a stimulus
Hypoxic tissue, Ischemic tissue, Mechanically damaged tissue
Need expression of angiogenic molecules to initiate
angiogenesis
VEGF, FGF, TGF, PDGF
Need angiogenesis to occur
1. Proliferation and migration of endothelial cells from the
microvasculature
2. Controlled expression of proteolytic enzymes
3. Breakdown and reassembly of extracellular matrix
4. Morphogenic process of endothelial tube formation
Mechanism of Angiogenesis not completely known
Why use VEGF to Promote
Angiogenesis?
VEGF (vascular endothelial growth factor)
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Specific for only endothelial cells
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May inhibit smooth muscle growth…reduce restenosis
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FGF (fibroblast growth factor)
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Associated with tumor angiogenesis
Can stimulate growth in other cells besides endothelial cells
Not as specific as VEGF
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TGF- (transforming growth factor ß)
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Indirect angiogenesis effect
Possibly induces VEGF expression (Protein Kinase C pathway)
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PDGF (platelet derived growth factor)
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Not well characterized in angiogenesis
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Other VEGF Characteristics
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VEGF expressed by Macrophages, fibroblasts, smooth
muscle cells, endothelial cells (all are present in the
heart)
Action is direct because of the exclusive specificity for
receptors (flt-1 and flk-1)
Receptors only found on endothelial cells
Causes activation of many other genes involved in
angiogenic response
How to Deliver VEGF
Protein Therapy
Direct
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injection of protein
Time delay delivery
Local intercoronary bolus
Gene Therapy
Adenovirus
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vector
Excellent specificity for endothelial cells
Extended expression of VEGF
Direct
gene transfer
Involves direct injection of eukaryotic plasmid DNA
containing VEGF cDNA
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Should VEGF administration prove effective, it is likely that
VEGF/VEGF DNA will be delivered on a catheter platform
Case Studies
Injection of naked VEGF cDNA contained in
an Eukaryotic Expression Vector
Jeffery Isner et al. St. Elizabeth’s Medical Center
Phase I clinical trial…designed to assess safety
and bioactivity of treatment methods
Limited sample…only 5 patients involved
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Prior Bypass and/or angioplasty
Class 3-4 Angina
No longer respond to additional treatment
Animal Data:
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Charles Mack et al. New York HospitalCornell Medical Center
Administration VEGF gene through
Adenovirus mediated gene therapy
Model:
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Pig with a constrictor band around circumflex artery to induce
myocardial infarction and ischemia
Eventually results in complete occlusion of circumflex artery
Vector:
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Adenovirus vector in E1a-, partial E1b-, and partial E3- mutations
(makes them replication deficient)
Adenovirus used because of the natural selectivity for endothelial
cells
Minimal inflammation detected in animals 4 weeks post therapy
In vivo conformation of expression confirmed by ELISA 3 days
after injection
Results
 Treatment
Resulted in significantly reduced ischemic
area (area of oxygen starved tissue) and
 Ischemic maximum (severity of ischemia) in treated
animals
 Strength of heartbeat returned in treated animals
more than untreated animals
 More vessels visible angiographically in treated
animals vs. untreated animals
 Treated animals seemed to route around the
occlusion as demonstrated by the filling of branching
arteries
Why it works?
VEGF stimulates growth of “collateral” vessels?