1. Cardiovascular Disease

2. Blood vessels
The cardiovascular system has three types of blood vessels:
Arteries (and arterioles) – carry blood away from the heart
Veins (and venules) – carry blood toward the heart.
Capillaries – where nutrient and gas exchange occur
The walls of arteries and veins have three layers. The inner layer is composed largely of endothelium, with a basement membrane that has elastic fibers; the middle layer is smooth muscle tissue; the outer layer is connective tissue (largely collagen fibers). Arteries (on left) have a thicker wall than veins because they have a larger middle layer than veins. Capillary walls (center) are one-cell-thick endothelium. Veins (on right) are larger in diameter than arteries, so that collectively veins have a larger holding capacity than arteries.

3. The Arteries The Capillaries The Veins
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.
Venules drain blood 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 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.
The inner layer of an artery wall is a simple squamous epithelium called endothelium with a connective tissue basement membrane with elastic fibers. The outer layer is fibrous connective tissue near the middle layer, but it becomes loose connective tissue at its periphery.

4. Anatomy of a capillary bed
A capillary bed forms a maze of capillary vessels that lies between an arteriole and a venule. When sphincter muscles are relaxed, the capillary bed is open, and blood flows through the capillaries. When sphincter muscles are contracted, blood flows through a shunt that carries blood directly from an arteriole to a venule. As blood passes through a capillary in the tissues, it gives up its oxygen (O2). Therefore, blood goes from being O2-rich in the arteriole (red color) to being O2-poor in the vein (blue color).

5. The Heart
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.

6. External heart anatomy
The superior vena cava and the pulmonary trunk are attached to the right side of the heart. The aorta and pulmonary veins are attached to the left side of the heart. The right ventricle forms most of the ventral surface of the heart, and the left ventricle forms most of the dorsal surface.

7. 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.
Oxygen rich and oxygen poor blood never mix.

8. Internal view of the heart
The heart has four valves. The atrioventricular valves allow blood to pass from the atria to the ventricles, and the semilunar valves allow blood to pass out of the heart.

9. Blood Pressure
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 cross-sectional area of arteries and then arterioles increases.

10. Path of blood through the heart
This diagrammatic representation of the heart allows you to trace the path of the blood through the heart.

11. Intrinsic Control of Heartbeat
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.
If the SA node fails to work properly, the heart still beats due to impulses generated by the AV node, but the beat is slower (40 to 60 beats per minute). To correct this condition, it is possible to implant an artificial pacemaker, which automatically gives an electrical stimulus to the heart.

12. Conduction system of the heart
The SA node sends out a stimulus, which cause the atria to contract. When this stimulus reaches the AV node, it signals the ventricles to contract. Impulses pass down the two branches of the atrioventricular bundle to the Purkinje fibers, and thereafter the ventricles contract.

13. Extrinsic Control of Heartbeat The Electrocardiogram
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.

14. Electrocardiogram
A normal ECG (top) indicates that the heart is functioning properly. The P wave occurs just prior to atrial contraction; the QRS complex occurs just prior to ventricular contraction; and the T wave occurs when the ventricles are recovering from contraction.
Ventricular fibrillation (bottom) produces an irregular electrocardiogram due to irregular stimulation of the ventricles. Ventricular fibrillation is of special interest because it can be caused by an injury or drug overdose. It is the most common cause of sudden cardiac death in a seemingly healthy person over age 35. Once the ventricles are fibrillating, they have to be defibrillated by applying a strong electrical current for a short period of time. Then the SA node may be able to reestablish a coordinated beat.

15. The Vascular Pathways The cardiovascular system includes two circuits:
Pulmonary circuit which circulates blood through the lungs
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.
Systemic circuit which circulates blood to the rest of the body
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.

16. Cardiovascular system diagram
The blue-colored vessels carry O2-poor blood, and the red-colored vessels carry O2-rich blood; the arrows indicate the flow of blood. Capillaries are present in all parts of the body, so no cell is located far from a capillary.

17. Major arteries and veins of the systemic circuit
A realistic representation of the major blood vessels of the systemic circuit shows how the systemic arteries and veins are arranged in the body.

18. Blood Flow Blood Flow in Arteries Blood Flow in Capillaries
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
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 arterioles and arteries.
Blood Flow in Capillaries
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
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.
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.
A portal system begins and ends with capillaries. The hepatic portal system begins with capillaries in the small intestine, continues with the hepatic portal vein leading to the liver, and ends with capillaries in the liver.

19. Cross-sectional area as it relates to blood pressure and velocity
Blood pressure and blood velocity drop off in capillaries because capillaries have a greater cross-sectional area than arteries.

20. Capillary Exchange
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.
Osmotic pressure in the blood is created by the presence of salts and the plasma proteins.

21. Capillary exchange
At the arterial end of a capillary, the blood pressure is higher than the osmotic pressure; therefore, water tends to leave the bloodstream. In the midsection, oxygen and carbon dioxide follow their concentration gradients. At the venous end of a capillary, the osmotic pressure is higher than the blood pressure; therefore, water tends to enter the bloodstream.

22. Coronary artery circulation
The coronary arteries and cardiac veins pervade cardiac muscle. The coronary arteries bring oxygen and nutrients to cardiac cells, which derive no benefit from blood coursing through the heart.

23. Blood
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).

24. Composition of blood Coagulation Factors!
When blood is transferred to a test tube and prevented from clotting, it forms tw layers. The transparent, yellow top layer is plasma, the liquid portion of the blood. The formed elements are the bottom layer.

25. The Red Blood Cells
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 iron-containing group that transports oxygen in the blood.
Hemoglobin is a red pigment, thus imparting a red color to red blood cells. A hemoglobin molecule contains four polypeptide chains that make up the protein globin.

26. Red Blood Cell
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.

27. Bone Marrow Stem Cells
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 use of adult stem cells does not require that an embryo be sacrificed.

28. The White Blood Cells
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.

29. Macrophage engulfing bacteria
Monocyte-derived macrophages are the body’s scavengers. They engulf microbes and debris in the body’s fluids and tissues, as illustrated in this colorized scanning electron micrograph. The macrophage is in red, and the bacteria are in green.

30. The Platelets and Blood Clotting
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.
Vitamin K, found in green vegetables and also formed by intestinal bacteria, is necessary for the production of prothrombin. Hemorrhagic disorders might develop if this vitamin is missing.

31. Cardiovascular Disease
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
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

32. Coronary Heart Disease and Heart Failure
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:
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
Women often have different symptoms of heart attack from men. The most common symptoms reported by women are:
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:
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:
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

33. Cerebrovascular Disease (Stroke)
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.

34. Risk Factors For CVD Obesity Tobacco Use
Psychological & Social Factors
Heredity
Aging
Male factor
Ethnicity
Syndrome X
Tobacco Use
Hypertension
High Levels of Cholesterol
Physical inactivity
Diabetes
High Triglyceride Levels

35. Atherosclerosis
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.
Thromboembolism is a clot that has moved and is now stationary in a new blood vessel where it can cause damage.

36. Coronary bypass operation
A coronary bypass operation involves removing a segment of another blood vessel and replacing a clogged coronary artery.
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.
During this operation, the surgeon grafts segments of another vessel, usually a small vein from the leg, between the aorta and the coronary vessels, bypassing areas of blockage. Patients who require surgery often receive two to five bypasses in a single operation.

37. Clearing Clogged Arteries
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

38. Dissolving Blood Clots
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.

39. Heart Transplants and Artificial Hearts
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.

40. Hypertension
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
Pre-hypertension
80-89
Hypertension – Stage 1
90-99
Hypertension – Stage 2
>160
>100

41. Risk Factors of Hypertension
Family history of hypertension
Excess Consumption of Sodium Chloride
Less active
Overweight
Dietary
Alcohol consumption
Smoking
African-Americans have a greater prevalence of hypertension (38% men; 39% of women) than non-Hispanic whites (33% of men; 25% women).
The risk of developing elevated BP is 2-6 times higher in overweight than in normal-weight individuals.
Weight management is a treatment for HBP.
Prevalence of of Hispanic population is similar to the non-Hispanic whites.

42. Prevention of Hypertension
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.
The relationship between obesity and BP appears to be linear and exists throughout the non-obese range. But the strength of the association of obesity with hypertension varies among different racial and ethnic groups. Generally, risk estimates suggest that approximately 75 and 65 percent of the cases of hypertension in men and women, respectively, are directly attributable to an overweight condition and obesity.
Most Americans consume about 4, ,000 mg sodium/day.

43. Regulation of Cardiovascular System

44. Multifactor/Multigene Effector
Genetic
Single gene disease
Multigene disease, e.g., SNP
Nongenetic
Factors affect one gene
Factors affect many genes

45. Epigenetic Regulation of cardiovascular system
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.

46. Diagrammatic representation of chromatin and chromatin-mediated gene regulation.
Diagrammatic representation of chromatin and chromatin-mediated gene regulation. TF indicates transcription factor.
Bruneau B G Circulation Research 2010;107:

47. Molecular Mechanisms of Epigenetic Gene Regulation
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 higher-order 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

48. Figure 1. Epigenetic mechanisms of gene regulation.
Figure 1. Epigenetic mechanisms of gene regulation. Epigenetic mechanisms important in the control of gene expression comprise 3 highly interrelated pathways: DNA methylation, histone posttranslational modifications, and RNA-based pathways. (1) DNA methylation (depicted as red balls) involves the covalent modification of cytosine in the context of CpG dinucleotides to define the “fifth base of DNA,” 5-methyl-cytosine. (2) Posttranslational modifications of the histone amino terminal tails (depicted as light and dark blue balls) are myriad and can importantly affect the physical properties and higher-order compaction of chromatin. (3) RNA-based mechanisms have recently emerged as important regulators of chromatin structure and gene expression (depicted as red strands coating chromatin).
Matouk C C , Marsden P A Circulation Research 2008;102:
Copyright © American Heart Association

49. DNA Methylation
DNA methylation involves the postsynthetic, covalent modification of the 5-position 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.

50. CpG Island
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

51. DNA Methylation and Gene Expression
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

52. Histon Modification
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.

53. Histone Methylation
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-domain-containing 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

54. RNA-Based Mechanisms
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 RNA-based mechanisms will have far-reaching influence in the regulation of mammalian gene expression.

55. Epigenetic Regulation of Vascular Endothelial Gene Expression
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.

56. Endothelial Cell-specific Regulation
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 cell-restricted gene expression is presently available.
Alternative mechanisms may contribute to the cell-specific expression of endothelial genes.

57. Transient Transfection of eNOS Promoter-Reporter Constructs Suggests Epigenetic Mechanisms of Gene Regulation
eNOS exists as a single copy in the haploid genome, contains 26 exons, spans approximately 21 kb of genomic DNA, maps to chromosome 7q35-36, 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 cis-regulatory 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

58. Demonstration of Epigenetic Regulation of eNOS
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

59. Transient transfection of eNOS promoter-reporter constructs into expressing and nonexpressing cell types.
Figure 3. Transient transfection of eNOS promoter-reporter constructs into expressing and nonexpressing cell types. The expression of eNOS is exquisitely cell-restricted to the vascular endothelium. A, In expressing cultured human endothelial cells, the eNOS promoter directs eNOS expression at the endogenous locus (center), as well as reporter expression from episomal eNOS promoter-reporter constructs (left). 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. B, 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. These data suggested that epigenetic, chromatin-based pathways may be relevant in the cell-specific expression of the eNOS gene. Refer to Figure 4 for a more detailed description of the chromatin and its modifications.

60. The Role of DNA Methylation in eNOS
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).

61. In Vivo Differential Regulation of eNOS
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.

62. Vascular Endothelium Proteins
Restricted expression of eNOS steady-state RNA and protein, respectively, to the endothelium, especially large- and medium-sized arteries.
von Willebrand factor (VWF), vascular-endothelial 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).

63. Endothelial NOS (eNOS)
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.

64. Genetic and Epigenetic Cardiovascular Disease
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.

65. DNA Methylation at iNOS Promoter
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.
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.

66. Histone posttranslational modifications contribute to the transcriptional silencing of iNOS in cultured human cells
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.

67. Epigenetic Pathways in Vascular Development and Endothelial Differentiation
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.

68. Hint of Vascular Genes
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.

69. Epigenetic Pathways in Postnatal Angiogenesis
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

70. Epigenetic Pathways in Inflammation and the Endothelial Response to Blood Flow
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.

71. Epigenetic mechanisms in diabetic vascular complications
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.

72. DNA methylation: relation to diabetes and vascular complications
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 (PGC-1α) 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

73. Methylation and Cardiovascular Diseases
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.

74. miRNAs in vascular complications
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.

75. Use of Vascular Endothelial Growth Factor (VEGF) as a Treatment for End Stage Coronary Artery Disease (CAD)

76. VEGF is involved in 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:
Embryogenesis (called vasculogenesis)
Wound healing
Tumor growth and metastasization
Rheumatoid arthritis
Ischemic heart disease
Ischemic peripheral vascular disease

77. 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

78. Why use VEGF to Promote Angiogenesis?
VEGF (vascular endothelial growth factor)
Specific for only endothelial cells
May inhibit smooth muscle growth…reduce restenosis
FGF (fibroblast growth factor)
Associated with tumor angiogenesis
Can stimulate growth in other cells besides endothelial cells
Not as specific as VEGF
TGF- (transforming growth factor ß)
Indirect angiogenesis effect
Possibly induces VEGF expression (Protein Kinase C pathway)
PDGF (platelet derived growth factor)
Not well characterized in angiogenesis

79. Other VEGF Characteristics
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

80. How to Deliver VEGF Protein Therapy Direct injection of protein
Time delay delivery
Local intercoronary bolus
Gene Therapy
Adenovirus vector
Excellent specificity for endothelial cells
Extended expression of VEGF
Direct gene transfer
Involves direct injection of eukaryotic plasmid DNA containing VEGF cDNA
Should VEGF administration prove effective, it is likely that VEGF/VEGF DNA will be delivered on a catheter platform

81. 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
Prior Bypass and/or angioplasty
Class 3-4 Angina
No longer respond to additional treatment

82. Animal Data:
Charles Mack et al. New York Hospital-Cornell Medical Center
Administration VEGF gene through Adenovirus mediated gene therapy

83. Model:
Pig with a constrictor band around circumflex artery to induce myocardial infarction and ischemia
Eventually results in complete occlusion of circumflex artery
Vector:
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

84. 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

85. Why it works?
VEGF stimulates growth of “collateral” vessels?