Cardiovascular Cardiovascular DiseaseDisease

Blood vesselsBlood vessels

The The cardiovascular cardiovascular systemsystem has three has three types of blood types of blood vessels:vessels:

1.1. ArteriesArteries (and (and arteriolesarterioles) – carry ) – carry blood away from the blood away from the heartheart

2.2. VeinsVeins (and (and venulesvenules) – ) – carry blood toward carry blood toward the heart.the heart.

3.3. CapillariesCapillaries – where – where nutrient and gas nutrient and gas exchange occurexchange occur

The ArteriesThe Arteries

ArteriesArteries and and arterioles take arterioles take blood away from blood away from the heart. the heart.

The largest artery The largest artery is the is the aortaaorta. .

The middle layer The middle layer of an artery wall of an artery wall consists of consists of smooth musclesmooth muscle that can constrict that can constrict to regulate blood to regulate blood flow and blood flow and blood pressure. pressure.

ArteriolesArterioles can can constrict or constrict or dilate, changing dilate, changing blood pressure.blood pressure.

VenulesVenules drain blood drain blood from capillaries, then from capillaries, then join to form join to form veinsveins that take blood to that take blood to the heart. the heart.

Veins have much Veins have much less smooth muscle less smooth muscle and connective and connective tissue than arteries. tissue than arteries.

Veins often have Veins often have valvesvalves that prevent that prevent the backward flow of the backward flow of blood when closed.blood when closed.

Veins carry about Veins carry about 70% of the body’s 70% of the body’s blood and act as a blood and act as a reservoirreservoir during during hemorrhage.hemorrhage.

The The VeinsVeins

The The CapillariesCapillaries

CapillariesCapillaries have walls have walls only one cell thick to only one cell thick to allow exchange of allow exchange of gases and nutrients gases and nutrients with tissue fluid. with tissue fluid.

Capillary beds are Capillary beds are present in all regions present in all regions of the body but not all of the body but not all capillary beds are capillary beds are open at the same open at the same time. time.

Contraction of a Contraction of a sphinctersphincter musclemuscle closes off a bed and closes off a bed and blood can flow through blood can flow through an an arteriovenous shuntarteriovenous shunt that bypasses the that bypasses the capillary bed.capillary bed.

Anatomy of a capillary Anatomy of a capillary bedbed

The The heartheart is a cone-shaped, muscular organ is a cone-shaped, muscular organ located between the lungs behind the sternum.located between the lungs behind the sternum.

The heart muscle forms the The heart muscle forms the myocardiummyocardium, with , with tightly interconnect cells of tightly interconnect cells of cardiac musclecardiac muscle tissue. tissue.

The The pericardiumpericardium is the outer membranous sac is the outer membranous sac with lubricating fluid.with lubricating fluid.

The heart has four chambers: two upper, thin-walled The heart has four chambers: two upper, thin-walled atriaatria, and two lower, thick-walled , and two lower, thick-walled ventriclesventricles..

The The septumseptum is a wall dividing the right and left sides. is a wall dividing the right and left sides. Atrioventricular valvesAtrioventricular valves occur between the atria and occur between the atria and

ventricles – the ventricles – the tricuspid valvetricuspid valve on the right and the on the right and the bicuspid valvebicuspid valve on the left; both valves are reenforced on the left; both valves are reenforced by by chordae tendinaechordae tendinae attached to muscular projections attached to muscular projections within the ventricles.within the ventricles.

The HeartThe Heart

External heart External heart anatomyanatomy

Passage of Blood Passage of Blood Through the HeartThrough the Heart

Blood follows this sequence through the Blood follows this sequence through the heart: heart:

superior and inferior vena cava → right superior and inferior vena cava → right atrium → tricuspid valve → right atrium → tricuspid valve → right ventricle → pulmonary semilunar valve ventricle → pulmonary semilunar valve → pulmonary trunk and arteries to the → pulmonary trunk and arteries to the lungslungs → → pulmonary veins leaving the pulmonary veins leaving the lungs → left atrium → bicuspid valve → lungs → left atrium → bicuspid valve → left ventricle → aortic semilunar valve left ventricle → aortic semilunar valve → aorta → to the body.→ aorta → to the body.

Internal view of the Internal view of the heartheart

Blood PressureBlood Pressure

The pumping of the heart sends out blood The pumping of the heart sends out blood under pressure to the arteries.under pressure to the arteries.

Blood pressureBlood pressure is greatest in the aorta; the is greatest in the aorta; the wall of the left ventricle is thicker than wall of the left ventricle is thicker than that of the right ventricle and pumps blood that of the right ventricle and pumps blood to the entire body.to the entire body.

Blood pressure then decreases as the Blood pressure then decreases as the cross-sectional area of arteries and then cross-sectional area of arteries and then arterioles increases.arterioles increases.

Path of blood through the Path of blood through the heartheart

Intrinsic Control of Intrinsic Control of HeartbeatHeartbeat

Each heartbeat is called a Each heartbeat is called a cardiac cyclecardiac cycle.. When the heart beats, the two atria contract When the heart beats, the two atria contract

together, then the two ventricles contract; then together, then the two ventricles contract; then the whole heart relaxes.the whole heart relaxes.

SystoleSystole is the contraction of heart chambers; is the contraction of heart chambers; diastolediastole is their relaxation. is their relaxation.

The The heart soundsheart sounds, lub-dup, are due to the , lub-dup, are due to the closing of the atrioventricular valves, followed closing of the atrioventricular valves, followed by the closing of the semilunar valves. by the closing of the semilunar valves.

The The SASA ((sinoatrialsinoatrial) ) nodenode, or , or pacemakerpacemaker, initiates , initiates the heartbeat and causes the atria to contract on the heartbeat and causes the atria to contract on average every 0.85 seconds. average every 0.85 seconds.

The The AVAV ( (atrioventricularatrioventricular) ) nodenode conveys the conveys the stimulus and initiates contraction of the ventricles. stimulus and initiates contraction of the ventricles.

The signal for the ventricles to contract travels The signal for the ventricles to contract travels from the AV node through the from the AV node through the atrioventricular atrioventricular bundlebundle to the smaller to the smaller Purkinje fibersPurkinje fibers..

Conduction system of Conduction system of the heartthe heart

Extrinsic Control of HeartbeatExtrinsic Control of Heartbeat The ElectrocardiogramThe Electrocardiogram

A A cardiac control centercardiac control center in the medulla oblongata speeds in the medulla oblongata speeds up or slows down the heart rate by way of the autonomic up or slows down the heart rate by way of the autonomic nervous system branches: nervous system branches: parasympathetic systemparasympathetic system (slows heart rate) and the (slows heart rate) and the sympathetic systemsympathetic system (increases heart rate). (increases heart rate).

Hormones Hormones epinephrineepinephrine and and norepinephrinenorepinephrine from the from the adrenal medulla also stimulate faster heart rate.adrenal medulla also stimulate faster heart rate.

An An electrocardiogramelectrocardiogram ( (ECGECG) can record of the ) can record of the electrical changes that occur in the myocardium electrical changes that occur in the myocardium during a cardiac cycle. during a cardiac cycle.

Atrial depolarizationAtrial depolarization creates the creates the P waveP wave, , ventricle ventricle depolarizationdepolarization creates the creates the QRS waveQRS wave, and , and repolarizationrepolarization of the ventricles produces the of the ventricles produces the T T wavewave..

ElectrocardiogramElectrocardiogram

The Vascular PathwaysThe Vascular Pathways The cardiovascular system includes two circuits:The cardiovascular system includes two circuits:• Pulmonary circuitPulmonary circuit which circulates blood through which circulates blood through

the lungsthe lungs The The pulmonary circuitpulmonary circuit begins with the begins with the pulmonary trunkpulmonary trunk from from

the right ventricle which branches into two the right ventricle which branches into two pulmonary pulmonary arteriesarteries that take oxygen-poor blood to the lungs. that take oxygen-poor blood to the lungs.

In the lungs, oxygen diffuses into the blood, and carbon In the lungs, oxygen diffuses into the blood, and carbon dioxide diffuses out of the blood to be expelled by the lungs.dioxide diffuses out of the blood to be expelled by the lungs.

Four Four pulmonary veinspulmonary veins return oxygen-rich blood to the left return oxygen-rich blood to the left atrium.atrium.

• Systemic circuitSystemic circuit which circulates blood to the rest which circulates blood to the rest of the bodyof the body

The The systemic circuitsystemic circuit starts with the aorta carrying O starts with the aorta carrying O22-rich -rich blood from the left ventricle. blood from the left ventricle.

The aorta branches with an artery going to each specific The aorta branches with an artery going to each specific organ. organ.

Generally, an artery divides into arterioles and capillaries Generally, an artery divides into arterioles and capillaries which then lead to venules.which then lead to venules.

• Both circuits are vital to homeostasis.Both circuits are vital to homeostasis.

Cardiovascular system Cardiovascular system diagramdiagram

Major arteries and veins Major arteries and veins of the systemic circuitof the systemic circuit

The beating of the heart is necessary to homeostasis because it creates pressure that The beating of the heart is necessary to homeostasis because it creates pressure that propels blood in arteries and the arterioles.propels blood in arteries and the arterioles.

Blood Flow in ArteriesBlood Flow in Arteries Blood pressureBlood pressure due to the pumping of the heart accounts for the flow of blood in the arteries. due to the pumping of the heart accounts for the flow of blood in the arteries. Systolic pressureSystolic pressure is high when the heart expels the blood. is high when the heart expels the blood. Diastolic pressureDiastolic pressure occurs when the heart ventricles are relaxing. occurs when the heart ventricles are relaxing. Both pressures decrease with distance from the left ventricle because blood enters more and Both pressures decrease with distance from the left ventricle because blood enters more and

more arterioles and arteries.more arterioles and arteries. Blood Flow in CapillariesBlood Flow in Capillaries

Blood moves slowly in capillaries because there are more capillaries than arterioles. 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 This allows time for substances (gas and nutrients) to be exchanged between the

blood and tissues.blood and tissues. Blood Flow in VeinsBlood Flow in Veins

Venous blood flow is dependent upon:Venous blood flow is dependent upon:1)1) skeletal muscle contraction, skeletal muscle contraction, 2)2) presence of valves in veins, and presence of valves in veins, and 3)3) respiratory movements. respiratory movements. Compression of veins causes blood to move forward past a valve that then prevents it from Compression of veins causes blood to move forward past a valve that then prevents it from

returning backward. returning backward. Changes in thoracic and abdominal pressure that occur with breathing also Changes in thoracic and abdominal pressure that occur with breathing also assist in the return of blood. assist in the return of blood.

The The coronary arteriescoronary arteries serve the heart muscle itself; they are the first branch off the aorta. 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.Since the coronary arteries are so small, they are easily clogged, leading to heart disease.

The The hepatic portal systemhepatic portal system carries blood rich in nutrients from digestion in the small carries blood rich in nutrients from digestion in the small intestine to the liver, the organ that monitors the composition of the bloodintestine to the liver, the organ that monitors the composition of the blood ..

Varicose veinsVaricose veins develop when the valves of veins become weak. develop when the valves of veins become weak. HemorrhoidsHemorrhoids (piles) are due to varicose veins in the rectum. (piles) are due to varicose veins in the rectum. PhlebitisPhlebitis is inflammation of a vein and can lead to a blood clot and possible death if the clot 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.is dislodged and is carried to a pulmonary vessel.

Blood FlowBlood Flow

Cross-sectional area as it Cross-sectional area as it relates to blood pressure and relates to blood pressure and

velocityvelocity

Capillary ExchangeCapillary Exchange At the arteriole end of a capillary, water moves out At the arteriole end of a capillary, water moves out

of the blood due to the force of of the blood due to the force of blood pressureblood pressure. . At the venule end, water moves into the blood due At the venule end, water moves into the blood due

to to osmotic pressureosmotic pressure of the blood. of the blood. Substances that leave the blood contribute to Substances that leave the blood contribute to

tissue fluidtissue fluid, the fluid between the body’s cells., the fluid between the body’s cells. In the midsection of the capillary, nutrients diffuse In the midsection of the capillary, nutrients diffuse

out and wastes diffuse into the blood. out and wastes diffuse into the blood. Since plasma proteins are too large to readily pass Since plasma proteins are too large to readily pass

out of the capillary, tissue fluid tends to contain all out of the capillary, tissue fluid tends to contain all components of plasma except it has lesser components of plasma except it has lesser amounts of protein.amounts of protein.

Excess tissue fluid is returned to the blood stream Excess tissue fluid is returned to the blood stream as as lymphlymph in in lymphatic vesselslymphatic vessels..

Capillary exchangeCapillary exchange

Coronary artery Coronary artery circulationcirculation

BloodBlood Blood separates into two main parts: Blood separates into two main parts: plasma plasma

(Molecular) and (Molecular) and formed elements formed elements (Cellular). (Cellular). Plasma (Liquid) accounts for 55% and Plasma (Liquid) accounts for 55% and

formed elements (Centrifuge precipitates) formed elements (Centrifuge precipitates) 45% of blood volume.45% of blood volume.

Plasma contains mostly water (90–92%) and Plasma contains mostly water (90–92%) and plasma proteins (7–8%), but it also contains plasma proteins (7–8%), but it also contains nutrients and wastes. nutrients and wastes.

AlbuminAlbumin is a large plasma protein that is a large plasma protein that transports bilirubin; transports bilirubin; globulinsglobulins are plasma are plasma proteins that proteins that transporttransport lipoproteins. lipoproteins.

Formed elements are mostly red blood cells, Formed elements are mostly red blood cells, platelets and white cells (leukocytes). platelets and white cells (leukocytes).

Composition of bloodComposition of blood

Coagulation Factors!

The Red Blood CellsThe Red Blood Cells Red blood cellsRed blood cells

((erythrocytes erythrocytes oror RBCs RBCs) ) are made in the are made in the red red bone marrowbone marrow of the of the skull, ribs, vertebrae, skull, ribs, vertebrae, and the ends of long and the ends of long bones.bones.

Normally there are 4 to Normally there are 4 to 6 million RBCs per 6 million RBCs per mmmm33 of whole blood. of whole blood.

Red blood cells contain Red blood cells contain the pigment the pigment hemoglobinhemoglobin for oxygen for oxygen transport; hemogobin transport; hemogobin contains contains hemeheme, a , a complex iron-complex iron-containing group that containing group that transports oxygen in transports oxygen in the blood. the blood.

Red Blood CellRed Blood Cell Red blood cells lack a nucleus and have a 120 day Red blood cells lack a nucleus and have a 120 day

life span. life span. When worn out, the red blood cells are dismantled in When worn out, the red blood cells are dismantled in

the liver and spleen.the liver and spleen. Iron is reused by the red bone marrow where stem Iron is reused by the red bone marrow where stem

cells continually produce more red blood cells; the cells continually produce more red blood cells; the remainder of the heme portion undergoes chemical remainder of the heme portion undergoes chemical degradation and is excreted as bile pigments into the degradation and is excreted as bile pigments into the bile. bile.

The kidneys produce the hormone erythropoietin to The kidneys produce the hormone erythropoietin to increase blood cell production when oxygen levels increase blood cell production when oxygen levels are low.are low.

VEGF regulates erythropoietin secretion from kidney. VEGF regulates erythropoietin secretion from kidney. Lack of enough hemoglobin results in anemia.Lack of enough hemoglobin results in anemia. The air pollutant carbon monoxide combines more The air pollutant carbon monoxide combines more

readily with hemoglobin than does oxygen, resulting readily with hemoglobin than does oxygen, resulting in oxygen deprivation and possible death. in oxygen deprivation and possible death.

Bone Marrow Stem Bone Marrow Stem CellsCells

A A stem cellstem cell is is capable of dividing capable of dividing into new cells that into new cells that differentiate into differentiate into particular cell particular cell types.types.

Bone marrow is Bone marrow is multipotentmultipotent, able to , able to continually give rise continually give rise to many particular to many particular types of blood cells. types of blood cells.

The skin and brain The skin and brain also have stem also have stem cells, and cells, and mesenchymal stem mesenchymal stem cellscells give rise to give rise to connective tissues connective tissues including heart including heart muscle. muscle.

Blood cell formation in red bone marrowBlood cell formation in red bone marrow

The White Blood CellsThe White Blood Cells White blood cellsWhite blood cells ( (leukocytesleukocytes) have nuclei, are fewer ) have nuclei, are fewer

in number than RBCs, with 5,000 – 10,000 cells per in number than RBCs, with 5,000 – 10,000 cells per mmmm33, and defend against disease. , and defend against disease.

Leukocytes are divided into Leukocytes are divided into granulargranular and and agranularagranular based on appearance.based on appearance.

Granular leukocytes (Granular leukocytes (neutrophilsneutrophils, , eosinophilseosinophils, and , and basophilsbasophils) contain enzymes and proteins that defend ) contain enzymes and proteins that defend the body against microbes.the body against microbes.

The agranular leukocytes (The agranular leukocytes (monocytesmonocytes and and lymphocyteslymphocytes) have a spherical or kidney-shaped ) have a spherical or kidney-shaped nucleus.nucleus.

Monocytes can differentiate into Monocytes can differentiate into macrophagesmacrophages that that phagocytizephagocytize microbes and stimulate other cells to microbes and stimulate other cells to defend the body.defend the body.

Lymphocytes are involved in immunity.Lymphocytes are involved in immunity. An excessive number of white blood cells may An excessive number of white blood cells may

indicate an infection or indicate an infection or leukemialeukemia; HIV infection ; HIV infection drastically reduces the number of lymphocytes.drastically reduces the number of lymphocytes.

Macrophage engulfing Macrophage engulfing bacteriabacteria

The Platelets and Blood ClottingThe Platelets and Blood Clotting Red bone marrow produces large cells called Red bone marrow produces large cells called

megakaryocytesmegakaryocytes that fragment into that fragment into plateletsplatelets at a rate at a rate of 200 billion per day; blood contains 150,000–of 200 billion per day; blood contains 150,000–300,000 platelets per mm300,000 platelets per mm33. .

Dozens of Dozens of clotting factorsclotting factors in the blood help platelets in the blood help platelets form blood clots.form blood clots.

Injured tissues release Tissue factor to blood and Injured tissues release Tissue factor to blood and activate FVIIa, which activates FX to FXa. activate FVIIa, which activates FX to FXa.

FXa/FVa are called FXa/FVa are called prothrombin activatorprothrombin activator, which , which converts prothrombin into thrombin.converts prothrombin into thrombin.

Thrombin, in turn, acts as an enzyme and converts Thrombin, in turn, acts as an enzyme and converts fibrinogen into insoluble threads of fibrinogen into insoluble threads of fibrinfibrin..

Platelets are also activated and aggregate to form Platelets are also activated and aggregate to form plug at injury site.plug at injury site.

These conversions require the presence of calcium These conversions require the presence of calcium ions (Caions (Ca2+2+) and phospholipid.) and phospholipid.

Trapped red blood cells make a clot appear red.Trapped red blood cells make a clot appear red.

Cardiovascular DiseaseCardiovascular Disease Cardiovascular Diseases (CVD) Affect the Heart and the Circulatory System. Cardiovascular Diseases (CVD) Affect the Heart and the Circulatory System. Coronary artery diseaseCoronary artery disease is the build-up of plaque in the arteries supplying blood to the  is the build-up of plaque in the arteries supplying blood to the

heart  (also heart  (also ischaemic heart disease or   or  Coronary heart disease).). Peripheral artery diseasePeripheral artery disease is the build-up of plaque in the arteries supplying blood to the  is the build-up of plaque in the arteries supplying blood to the

arms and legs.arms and legs. Cardiac DiseasesCardiac Diseases Heart Failure Hypertensive heart disease - diseases of the heart secondary to high blood pressureHypertensive heart disease - diseases of the heart secondary to high blood pressure Cardiomyopathy - diseases of cardiac muscle, A Cardiomyopathy - diseases of cardiac muscle, A myocardial infarctionmyocardial infarction, or , or heart attackheart attack, ,

occurs when a portion of heart muscle dies due to lack of oxygen.occurs when a portion of heart muscle dies due to lack of oxygen. Cor pulmonale - a failure of the right side of the heart.Cor pulmonale - a failure of the right side of the heart. Cardiac dysrhythmias - abnormalities of heart rhythm.Cardiac dysrhythmias - abnormalities of heart rhythm. Inflammatory heart diseaseInflammatory heart disease

Endocarditis – Endocarditis – inflammation of the inner layer of the  of the inner layer of the heart, the , the endocardium. The structures most . The structures most commonly involved are the commonly involved are the heart valves..

Inflammatory Inflammatory cardiomegaly Myocarditis –  – inflammation of the  of the myocardium, the muscular part of the heart., the muscular part of the heart.

Valvular heart diseaseValvular heart disease Vascular Diseases of brain and kidneyVascular 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.Carotid artery disease is the build-up of plaque in the arteries that supply blood to the brain. HypertensionHypertension AtherosclerosisAtherosclerosis

Coronary Heart Disease and Coronary Heart Disease and

Heart FailureHeart Failure Coronary Heart Disease (CHD) is the most common form of heart disease. 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. 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. Plaque is made up of fat, cholesterol and other substances found in the blood. This plaque build-up is also known as atherosclerosis. This plaque build-up is also known as atherosclerosis. The site of the plaque determines the type of heart disease.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 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:a heart attack. The five most common symptoms of a heart attack are: Chest pressure or painChest pressure or pain Shortness of breathShortness of breath Pain or discomfort in the arms or shoulderPain or discomfort in the arms or shoulder Pain or discomfort in the jaw, neck or backPain or discomfort in the jaw, neck or back Feeling weak, lightheaded, or nauseousFeeling weak, lightheaded, or nauseous

Women often have different symptoms of heart attack from men. The most common symptoms reported by Women often have different symptoms of heart attack from men. The most common symptoms reported by women are:women are:

Unusual fatigueUnusual fatigue sleep disturbancesleep disturbance shortness of breathshortness of breath Indigestion Heart failure is a condition that occurs slowly over time. 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, 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. 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 The weakened heart muscle has to work overtime to keep up with the body's demands, which can leave a person tired. person tired.

Some of the symptoms of heart failure:Some of the symptoms of heart failure: Shortness of breathShortness of breath Difficulty breathing when lying downDifficulty breathing when lying down Swelling in the legs, ankles, and feetSwelling in the legs, ankles, and feet General fatigue and weakness.General fatigue and weakness.

Risk factors that increase your chances of developing heart failure:Risk factors that increase your chances of developing heart failure: High blood pressureHigh blood pressure Heart attackHeart attack Damage to a heart valve or a history of a murmurDamage to a heart valve or a history of a murmur Enlargement of the heart or a family history of an enlarged heartEnlargement of the heart or a family history of an enlarged heart DiabetesDiabetes

Cerebrovascular Disease (Stroke) A A strokestroke, or , or cerebrovascular accidentcerebrovascular accident ( (CVACVA), is the rapid loss of ), is the rapid loss of brain function(s) due to disturbance in the  function(s) due to disturbance in the blood supply to the brain.  to the brain.

This can be due to This can be due to ischemia (lack of blood flow) caused by blockage ( (lack of blood flow) caused by blockage (thrombosis, , arterial embolism), or a ), or a hemorrhage..

As a result, the affected area of the brain cannot function, which might result in an 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  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. 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, A stroke is a medical emergency and can cause permanent neurological damage, complications, and death. complications, and death. 

Risk factors for stroke include old age, high blood pressure, previous stroke Risk factors for stroke include old age, high blood pressure, previous stroke or transient ischemic attack (TIA), diabetes, high cholesterol, tobacco or transient ischemic attack (TIA), diabetes, high cholesterol, tobacco smoking and atrial fibrillation.smoking and atrial fibrillation.

High blood pressure is the most important modifiable risk factor of stroke.High blood pressure is the most important modifiable risk factor of stroke. It is the second leading cause of death worldwide.It is the second leading cause of death worldwide. An ischemic stroke is occasionally treated in a hospital with thrombolysis (also An ischemic stroke is occasionally treated in a hospital with thrombolysis (also

known as a "clot buster"), and some hemorrhagic strokes benefit from known as a "clot buster"), and some hemorrhagic strokes benefit from neurosurgery. neurosurgery.

Treatment to recover any lost function is termed stroke rehabilitation, ideally in Treatment to recover any lost function is termed stroke rehabilitation, ideally in a stroke unit and involving health professions such as speech and language a stroke unit and involving health professions such as speech and language therapy, physical therapy and occupational therapy. therapy, physical therapy and occupational therapy.

Prevention of recurrence may involve the administration of antiplatelet drugs such Prevention of recurrence may involve the administration of antiplatelet drugs such as aspirin and dipyridamole, control and reduction of high blood pressure, and the as aspirin and dipyridamole, control and reduction of high blood pressure, and the use of statins. use of statins.

Selected patients may benefit from carotid endarterectomy and the use of Selected patients may benefit from carotid endarterectomy and the use of anticoagulants.anticoagulants.

Risk Factors For CVDRisk Factors For CVD

Tobacco UseTobacco Use HypertensionHypertension High Levels of High Levels of

CholesterolCholesterol Physical inactivityPhysical inactivity DiabetesDiabetes High Triglyceride High Triglyceride

LevelsLevels

ObesityObesity Psychological & Psychological &

Social FactorsSocial Factors HeredityHeredity AgingAging Male factorMale factor EthnicityEthnicity Syndrome XSyndrome X

AtherosclerosisAtherosclerosis AtherosclerosisAtherosclerosis is due to a build-up of fatty material is due to a build-up of fatty material

((plaqueplaque), mainly cholesterol, under the inner lining of ), mainly cholesterol, under the inner lining of arteries. arteries.

The plaque can cause a The plaque can cause a thrombusthrombus (blood clot) to form. (blood clot) to form. The thrombus can dislodge as an The thrombus can dislodge as an embolusembolus and lead to and lead to

thromboembolismthromboembolism.. Partial blockage of a coronary artery causes Partial blockage of a coronary artery causes angina angina

pectorispectoris, or chest pain. , or chest pain. An An aneurysmaneurysm is a ballooning of a blood vessel, usually is a ballooning of a blood vessel, usually

in the abdominal aorta or arteries leading to the brain.in the abdominal aorta or arteries leading to the brain. Death results if the aneurysm is in a large vessel and Death results if the aneurysm is in a large vessel and

the vessel bursts.the vessel bursts. Atherosclerosis and hypertension weaken blood vessels Atherosclerosis and hypertension weaken blood vessels

over time, increasing the risk of aneurysm.over time, increasing the risk of aneurysm.

Coronary bypass Coronary bypass operationoperation

1.1. A A coronary bypass coronary bypass operationoperation involves involves removing a segment of removing a segment of another blood vessel another blood vessel and replacing a and replacing a clogged coronary clogged coronary artery. artery.

2.2. It may be possible to It may be possible to replace this surgery replace this surgery with with gene therapygene therapy that that stimulates new blood stimulates new blood vessels to grow where vessels to grow where the heart needs more the heart needs more blood flow.blood flow.

Clearing Clogged Clearing Clogged ArteriesArteries

AngioplastyAngioplasty uses a uses a long tube threaded long tube threaded through an arm or leg through an arm or leg vessel to the point vessel to the point where the coronary where the coronary artery is blocked; artery is blocked; inflating the tube inflating the tube forces the vessel forces the vessel open. open.

Small metal Small metal stentsstents are are expanded inside the expanded inside the artery to keep it open.artery to keep it open.

Stents are coated with Stents are coated with heparinheparin to prevent to prevent blood clotting and blood clotting and with chemicals to with chemicals to prevent arterial prevent arterial closing.closing.

AngioplastyAngioplasty

Dissolving Blood ClotsDissolving Blood Clots

Medical treatments for dissolving Medical treatments for dissolving blood clots include use of blood clots include use of t-PAt-PA ( (tissue tissue plasminogen activatorplasminogen activator) that converts ) that converts plasminogen into plasmin, an plasminogen into plasmin, an enzyme that dissolves blood clots, enzyme that dissolves blood clots, but can cause brain bleeding. but can cause brain bleeding.

AspirinAspirin reduces the stickiness of reduces the stickiness of platelets and reduces clot formation platelets and reduces clot formation and lowers the risk of heart attack.and lowers the risk of heart attack.

Heart Transplants and Heart Transplants and Artificial HeartsArtificial Hearts

Heart transplantsHeart transplants are routinely are routinely performed but immunosuppressive performed but immunosuppressive drugs must be taken thereafter. drugs must be taken thereafter.

There is a shortage of human organ There is a shortage of human organ donors. donors.

Work is currently underway to Work is currently underway to improve self-contained improve self-contained artificial artificial heartshearts, and muscle cell transplants , and muscle cell transplants may someday be usefulmay someday be useful..

HypertensionHypertension

Hypertension (Hypertension (high blood high blood pressurepressure) is present when ) is present when systolic pressure is 140 or systolic pressure is 140 or greater or diastolic greater or diastolic pressure is 100 or pressure is 100 or greater; diastolic pressure greater; diastolic pressure is emphasized when is emphasized when medical treatment is medical treatment is considered.considered.

A genetic predisposition A genetic predisposition for hypertension occurs in for hypertension occurs in those who have a gene those who have a gene that codes for that codes for angiotensinogenangiotensinogen, a , a powerful vasoconstrictor.powerful vasoconstrictor.

CategoryCategory Systolic Systolic Blood Blood PressurePressure

Diastolic Diastolic Blood Blood PressurePressure

NormalNormal < 120< 120 <80<80

Pre-Pre-hypertensiohypertensionn

120-139120-139 80-8980-89

HypertensioHypertension n –– Stage 1 Stage 1

140-159140-159 90-9990-99

HypertensioHypertension n –– Stage 2 Stage 2

>>160160 >>100100

Risk Factors of HypertensionRisk Factors of Hypertension

Family history of hypertensionFamily history of hypertension Excess Consumption of Sodium ChlorideExcess Consumption of Sodium Chloride Less active Less active OverweightOverweight DietaryDietary Alcohol consumptionAlcohol consumption SmokingSmoking

Prevention of HypertensionPrevention of Hypertension

Maintain a healthy weight.Maintain a healthy weight. Be more physically active.Be more physically active. Balanced nutritionBalanced nutrition Drink less alcoholic beverages.Drink less alcoholic beverages. Reduce the intake of salt and Reduce the intake of salt and

sodium in the diet to sodium in the diet to approximately 2400 mg/day.approximately 2400 mg/day.

Regulation of Regulation of Cardiovascular SystemCardiovascular System

Multifactor/Multigene Multifactor/Multigene EffectorEffector

GeneticGenetic Single gene diseaseSingle gene disease Multigene disease, e.g., SNPMultigene disease, e.g., SNP

NongeneticNongenetic Factors affect one geneFactors affect one gene Factors affect many genesFactors affect many genes

Epigenetic Regulation of Epigenetic Regulation of cardiovascular system cardiovascular system

Epigenetics refers to chromatin-based mechanisms Epigenetics refers to chromatin-based mechanisms important in the regulation of gene expression that do not important in the regulation of gene expression that do not involve changes to the DNA sequenceinvolve changes to the DNA sequence

Epigenetic regulation through histone modifications is an Epigenetic regulation through histone modifications is an important aspect of gene regulation at chromatin level.important aspect of gene regulation at chromatin level.

The unstructured tails of histones (the proteins that The unstructured tails of histones (the proteins that assemble into the nucleosomes around which chromosomal assemble into the nucleosomes around which chromosomal DNA is wound) are subject to myriad chemical DNA is wound) are subject to myriad chemical modifications, including acetylation, methylation, modifications, including acetylation, methylation, phosphorylation, ubiquitinylation, and sumoylation. phosphorylation, ubiquitinylation, and sumoylation.

In combination, these modifications are thought to result in In combination, these modifications are thought to result in a histone “code” that is read and translated into signals for a histone “code” that is read and translated into signals for activation or repression of associated genes.activation or repression of associated genes.

For example, certain histone modifications are most often For example, certain histone modifications are most often associated with repressed genes, and others with active associated with repressed genes, and others with active genes. genes.

Diagrammatic representation of chromatin and chromatin-mediated gene regulation.

Bruneau B G Circulation Research 2010;107:324-326

Molecular Mechanisms of Molecular Mechanisms of Epigenetic Gene RegulationEpigenetic Gene Regulation

DNA is packaged as a DNA–protein complex that is conserved across all DNA is packaged as a DNA–protein complex that is conserved across all eukaryotic genomes. eukaryotic genomes.

The fundamental repeating unit of this structure, termed chromatin, is The fundamental repeating unit of this structure, termed chromatin, is the nucleosome comprising an octamer of core histone proteins around the nucleosome comprising an octamer of core histone proteins around which is wrapped 146 bp of DNA. which is wrapped 146 bp of DNA.

Each nucleosome comprises 2 molecules of H2A, H2B, H3, and H4. Each nucleosome comprises 2 molecules of H2A, H2B, H3, and H4. Adjacent nucleosome particles are separated by shorter species-specific Adjacent nucleosome particles are separated by shorter species-specific

lengths of linker DNA associated with a fifth histone protein, histone H1. lengths of linker DNA associated with a fifth histone protein, histone H1. This linker histone facilitates further compaction of chromatin into This linker histone facilitates further compaction of chromatin into

higher-order chromatin structures that enable the packaging of higher-order chromatin structures that enable the packaging of extraordinary lengths of DNA into the tight confines of the cell nucleus.extraordinary lengths of DNA into the tight confines of the cell nucleus.

Over the last 20 years, 3 highly interconnected epigenetic pathways Over the last 20 years, 3 highly interconnected epigenetic pathways have emerged that impact on the structure and accessibility of have emerged that impact on the structure and accessibility of chromatin. chromatin.

Each of these pathways is important in the regulation of gene Each of these pathways is important in the regulation of gene expression: DNA methylation, histone posttranslational modifications, expression: DNA methylation, histone posttranslational modifications, and RNA-based mechanismsand 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 MethylationDNA Methylation

DNA methylation involves the postsynthetic, covalent modification DNA methylation involves the postsynthetic, covalent modification of the 5-position of cytosine to define the “fifth base of DNA,” 5-of the 5-position of cytosine to define the “fifth base of DNA,” 5-methyl-cytosine. methyl-cytosine.

In mammals, DNA methylation is almost exclusively restricted to In mammals, DNA methylation is almost exclusively restricted to CpG dinucleotides. CpG dinucleotides.

DNA methylation is catalyzed by 3 different DNA DNA methylation is catalyzed by 3 different DNA methyltransferases (DNMTs) encoded by different genes on methyltransferases (DNMTs) encoded by different genes on distinct chromosomes: DNMT1, DNMT3a, and DNMT3b. distinct chromosomes: DNMT1, DNMT3a, and DNMT3b.

De novo methylation is catalyzed by the latter 2 enzymes and is De novo methylation is catalyzed by the latter 2 enzymes and is important in the establishment of DNA methylation patterns in the important in the establishment of DNA methylation patterns in the early embryo and during development. early embryo and during development.

In contrast, DNMT1 serves a maintenance function and is In contrast, DNMT1 serves a maintenance function and is responsible for the propagation of DNA methylation patterns responsible for the propagation of DNA methylation patterns following DNA replication during mitotic cell division. following DNA replication during mitotic cell division.

DNA methylation is a remarkably stable epigenetic modification. DNA methylation is a remarkably stable epigenetic modification. Its dynamic regulation has been clearly demonstrated during Its dynamic regulation has been clearly demonstrated during

embryogenesis, cellular differentiation, and carcinogenesis. embryogenesis, cellular differentiation, and carcinogenesis.

CpG IslandCpG Island

Approximately 70% to 90% of CpG dinucleotides, representing 3% Approximately 70% to 90% of CpG dinucleotides, representing 3% to 6% of all cytosines, are methylated in healthy somatic cells.to 6% of all cytosines, are methylated in healthy somatic cells.

Surprisingly, CpG dinucleotides are relatively depleted in the Surprisingly, CpG dinucleotides are relatively depleted in the mammalian genome, ie, occur at a frequency less than would be mammalian genome, ie, occur at a frequency less than would be expected based on the GC content of the genome expected based on the GC content of the genome

Although variably defined, these relatively (G+C)- and CpG-rich Although variably defined, these relatively (G+C)- and CpG-rich regions are commonly referred to as CpG islands.regions are commonly referred to as CpG islands.

They account for approximately 7% of CpG dinucleotides genome-They account for approximately 7% of CpG dinucleotides genome-wide and are associated with the 5′-regulatory regions of ≈40% to wide and are associated with the 5′-regulatory regions of ≈40% to 60% of human genes. 60% of human genes.

Typically, these CpG dinucleotides are unmethylated. Typically, these CpG dinucleotides are unmethylated. A significant proportion of CpG dinucleotides also occur in the A significant proportion of CpG dinucleotides also occur in the

context of intergenic, repetitive DNA sequences, such as Alu context of intergenic, repetitive DNA sequences, such as Alu elements. elements.

In contrast to those comprising CpG islands, these CpG In contrast to those comprising CpG islands, these CpG dinucleotides are usually densely methylated.8 dinucleotides are usually densely methylated.8

DNA Methylation and Gene DNA Methylation and Gene ExpressionExpression

DNA methylation is a repressive mark associated with transcriptional DNA methylation is a repressive mark associated with transcriptional silencing. silencing.

It has been strongly implicated in a growing number of integral cellular It has been strongly implicated in a growing number of integral cellular functions, including the silencing of repetitive (parasitic) sequences, X functions, including the silencing of repetitive (parasitic) sequences, X chromosome inactivation, genomic imprinting, mammalian embryonic chromosome inactivation, genomic imprinting, mammalian embryonic development, and lineage specification.development, and lineage specification.

Its dysregulation is also characteristic of a growing number of human Its dysregulation is also characteristic of a growing number of human diseases, most prominently, cancer. diseases, most prominently, cancer.

A strikingly similar pattern is also observed on the inactive X chromosome.A strikingly similar pattern is also observed on the inactive X chromosome. DNA methylation itself can impede the binding of transcription factors to DNA methylation itself can impede the binding of transcription factors to

CpG dinucleotide-containing CpG dinucleotide-containing ciscis-DNA binding elements. -DNA binding elements. A family of methyl-CpG binding proteins has been described that can A family of methyl-CpG binding proteins has been described that can

specifically recognize the mammalian methylation mark specifically recognize the mammalian methylation mark These include 4 proteins containing a homologous methyl-CpG-binding These include 4 proteins containing a homologous methyl-CpG-binding

domain (MBD1, MBD2, MBD4, and the founding member, MeCP2) and a domain (MBD1, MBD2, MBD4, and the founding member, MeCP2) and a recently characterized, nonhomologous protein, Kaiso, which is capable of recently characterized, nonhomologous protein, Kaiso, which is capable of binding a methylated CpG dinucleotide doublet binding a methylated CpG dinucleotide doublet

These methyl-CpG-binding proteins can directly repress transcription, These methyl-CpG-binding proteins can directly repress transcription, prevent the binding of activating transfactors, or recruit enzymes that prevent the binding of activating transfactors, or recruit enzymes that catalyze histone posttranslational modifications and chromatin-remodeling catalyze histone posttranslational modifications and chromatin-remodeling complexes that alter the structure of chromatin and actively promote complexes that alter the structure of chromatin and actively promote transcriptional repression. transcriptional repression.

A transcriptional activator that specifically recognizes unmethylated CpG A transcriptional activator that specifically recognizes unmethylated CpG dinucleotides, human CpG binding protein (hCGBP) is also identified dinucleotides, human CpG binding protein (hCGBP) is also identified

Histon ModificationHiston Modification More than 60 distinct modification sites have been described which include More than 60 distinct modification sites have been described which include

lysine acetylation, lysine and arginine methylation, serine and threonine lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitylation, and lysine sumoylation, among others. phosphorylation, lysine ubiquitylation, and lysine sumoylation, among others.

Two classes of histone posttranslational modifications, in particular, have well-Two classes of histone posttranslational modifications, in particular, have well-established roles in the control of mammalian gene expression: lysine established roles in the control of mammalian gene expression: lysine acetylation and lysine methylation. acetylation and lysine methylation.

Lysine acetylation involves the transfer of acetyl groups from acetyl-coenzyme Lysine acetylation involves the transfer of acetyl groups from acetyl-coenzyme A molecules to the lysine ε-amino groups of histone tails. A molecules to the lysine ε-amino groups of histone tails.

In mammalian cells, this reaction is catalyzed by 3 principal families of histone In mammalian cells, this reaction is catalyzed by 3 principal families of histone acetyltransferases (HATs): GNAT, MYST, and CBP/p300. acetyltransferases (HATs): GNAT, MYST, and CBP/p300.

A number of transcriptional coactivators have intrinsic HAT activity A number of transcriptional coactivators have intrinsic HAT activity HATs demonstrate poor specificity for individual histone tail lysine residues HATs demonstrate poor specificity for individual histone tail lysine residues

and are also capable of acetylating many nonhistone proteins important in the and are also capable of acetylating many nonhistone proteins important in the regulation of transcription; for example, c-Jun, E2F, MyoD, nuclear factor (NF)-regulation of transcription; for example, c-Jun, E2F, MyoD, nuclear factor (NF)-κB, p53, pRb, and YY1, among others κB, p53, pRb, and YY1, among others

Removal of histone lysine acetylation is catalyzed by 4 families of mammalian Removal of histone lysine acetylation is catalyzed by 4 families of mammalian histone deacetylases (HDACs): class I (HDAC1-3, HDAC8), class II (HDAC4-7, histone deacetylases (HDACs): class I (HDAC1-3, HDAC8), class II (HDAC4-7, HDAC9-10), class III sirtuins (SIRT1-7), and class IV (HDAC11) HDAC9-10), class III sirtuins (SIRT1-7), and class IV (HDAC11)

HDACs evidence poor specificity for individual histone lysine residues and are HDACs evidence poor specificity for individual histone lysine residues and are also active on many nonhistone proteins. also active on many nonhistone proteins.

The acetyl-lysine mark is read by a group of chromatin-associated proteins, The acetyl-lysine mark is read by a group of chromatin-associated proteins, including several HATs and chromatin-remodeling enzymes, that contain including several HATs and chromatin-remodeling enzymes, that contain bromodomains. bromodomains.

The interplay between HATs, HDACs, and bromodomain-containing readers The interplay between HATs, HDACs, and bromodomain-containing readers allows for a highly dynamic gene transcriptional control pathway.allows for a highly dynamic gene transcriptional control pathway.

Histone MethylationHistone Methylation A lysine residue can either be acetylated or methylated. A lysine residue can either be acetylated or methylated. Histone lysine acetylation is tightly correlated with transcriptional activation, the Histone lysine acetylation is tightly correlated with transcriptional activation, the

impact of histone lysine methylation on gene expression is context-dependent.impact of histone lysine methylation on gene expression is context-dependent. For example, histone H3 lysine 4 (H3K4) methylation is strongly associated with For example, histone H3 lysine 4 (H3K4) methylation is strongly associated with

transcriptional activation. transcriptional activation. This epigenetic mark is written by the family of trithorax group (Trx-G) proteins.This epigenetic mark is written by the family of trithorax group (Trx-G) proteins. A single lysine residue can be mono-, di-, or trimethylated. A single lysine residue can be mono-, di-, or trimethylated. These more subtle epigenetic modifications are likely to be functionally relevant. These more subtle epigenetic modifications are likely to be functionally relevant. This contention is supported by a recent genome-wide survey that demonstrated This contention is supported by a recent genome-wide survey that demonstrated

preferential localization of trimethylated H3K4 to active promoters and preferential localization of trimethylated H3K4 to active promoters and monomethylated H3K4 to enhancers.monomethylated H3K4 to enhancers.

The characterization of specific di- and trimethylated H3K4 readers, the The characterization of specific di- and trimethylated H3K4 readers, the mammalian ING (inhibitor of growth) family proteins (ING1-5), provides further mammalian ING (inhibitor of growth) family proteins (ING1-5), provides further compelling evidence.compelling evidence.

Conversely, di- and trimethylated histone H3 lysine 9 (H3K9) are strongly Conversely, di- and trimethylated histone H3 lysine 9 (H3K9) are strongly correlated with transcriptional repression.correlated with transcriptional repression.

These epigenetic marks are catalyzed by an increasingly large family of SET-These epigenetic marks are catalyzed by an increasingly large family of SET-domain-containing histone lysine methyltransferases, including SUV39H1, domain-containing histone lysine methyltransferases, including SUV39H1, SUV39H2, PRDM2/RIX1, and G9A/BAT8. SUV39H2, PRDM2/RIX1, and G9A/BAT8.

Heterochromatin 1 (HP1) proteins (HP1α, HP1β, and HP1γ) specifically bind di- Heterochromatin 1 (HP1) proteins (HP1α, HP1β, and HP1γ) specifically bind di- and trimethylated H3K9 via interactions with their methyl-binding chromodomain and trimethylated H3K9 via interactions with their methyl-binding chromodomain and are crucial for the formation of heterochromatin and transcriptional and are crucial for the formation of heterochromatin and transcriptional silencing.silencing.

Jumonji-domain-containing proteins (JMJD) is histone lysine demethylasesJumonji-domain-containing proteins (JMJD) is histone lysine demethylases

RNA-Based MechanismsRNA-Based Mechanisms There is mounting evidence that noncoding RNAs and the RNA interference There is mounting evidence that noncoding RNAs and the RNA interference

machinery are fundamental determinants of chromatin-based gene machinery are fundamental determinants of chromatin-based gene expression.expression.

In mammalian systems, the best characterized examples include the role of In mammalian systems, the best characterized examples include the role of XISTXIST RNA in X chromosome inactivation, RNA in X chromosome inactivation, AirAir RNA at the murine imprinted RNA at the murine imprinted Igf2rIgf2r locus, and as of yet unidentified RNAs in the assembly of centromeric locus, and as of yet unidentified RNAs in the assembly of centromeric heterochromatin.heterochromatin.

These examples importantly involve coordinated epigenetic activities These examples importantly involve coordinated epigenetic activities including DNA methylation and histone posttranslational modifications. including DNA methylation and histone posttranslational modifications.

In contrast, micro-RNAs and short interfering (si)RNAs, ≈ 21 to 26 nucleotide In contrast, micro-RNAs and short interfering (si)RNAs, ≈ 21 to 26 nucleotide small RNA species, are well-known mediators of cytoplasmic, small RNA species, are well-known mediators of cytoplasmic, posttranscriptional gene silencing as components of the RNA-induced posttranscriptional gene silencing as components of the RNA-induced silencing complex (RISC). silencing complex (RISC).

Micro-RNAs are derived from nuclear transcripts with characteristic stem–loop Micro-RNAs are derived from nuclear transcripts with characteristic stem–loop structures and transported to the cytoplasm. structures and transported to the cytoplasm.

Alternatively, siRNAs are derived from long double-stranded RNA precursors Alternatively, siRNAs are derived from long double-stranded RNA precursors delivered exogenously to cells or that arise naturally within cells.delivered exogenously to cells or that arise naturally within cells.

Exogenously administered siRNAs directed at promoter regions can Exogenously administered siRNAs directed at promoter regions can effectuate transcriptional gene silencing in mammalian cells by inducing site-effectuate transcriptional gene silencing in mammalian cells by inducing site-specific DNA methylation and repressive histone posttranslational specific DNA methylation and repressive histone posttranslational modifications.modifications.

Given that at least 15% to 20% of mouse and human genes, respectively, Given that at least 15% to 20% of mouse and human genes, respectively, demonstrate demonstrate ciscis-encoded natural antisense transcripts, it is anticipated that -encoded natural antisense transcripts, it is anticipated that RNA-based mechanisms will have far-reaching influence in the regulation of RNA-based mechanisms will have far-reaching influence in the regulation of mammalian gene expression. mammalian gene expression.

Epigenetic Regulation of Epigenetic Regulation of Vascular Endothelial Gene Vascular Endothelial Gene

ExpressionExpression Epigenetics refers to chromatin-based pathways important Epigenetics refers to chromatin-based pathways important

in the regulation of gene expressionin the regulation of gene expression Includes 3 distinct, but highly interrelated mechanisms: Includes 3 distinct, but highly interrelated mechanisms:

DNA methylation, histone density and posttranslational DNA methylation, histone density and posttranslational modifications, and RNA-based mechanisms. modifications, and RNA-based mechanisms.

They offer a newer perspective on transcriptional control They offer a newer perspective on transcriptional control paradigms in vascular endothelial cells and provide a paradigms in vascular endothelial cells and provide a molecular basis for how the environment impacts the molecular basis for how the environment impacts the genome to modify disease susceptibility. genome to modify disease susceptibility.

Using endothelial nitric oxide synthase (Using endothelial nitric oxide synthase (NOS3NOS3) as an ) as an example, examine the growing body of evidence example, examine the growing body of evidence implicating epigenetic pathways in the control of vascular implicating epigenetic pathways in the control of vascular endothelial gene expression in health and disease. endothelial gene expression in health and disease.

Endothelial Cell-specific Endothelial Cell-specific RegulationRegulation

Although an as of yet uncharacterized endothelial master Although an as of yet uncharacterized endothelial master regulator may exist, the present regulator may exist, the present ciscis//transtrans paradigm supports a paradigm supports a model for the cooperative activity of several ubiquitously model for the cooperative activity of several ubiquitously expressed transcription factors in the constitutive expression of expressed transcription factors in the constitutive expression of endothelial-restricted genes. endothelial-restricted genes.

These include Ets family members, GATA-2, Sp1, activator These include Ets family members, GATA-2, Sp1, activator protein-1, and octamer transcription factors.protein-1, and octamer transcription factors.

Indeed, a majority of endothelial-restricted genes possess Indeed, a majority of endothelial-restricted genes possess ciscis--DNA binding elements for these factors in their 5′-regulatory DNA binding elements for these factors in their 5′-regulatory regions. regions.

The eNOS gene is a representative example.The eNOS gene is a representative example. Specificity could be achieved by a unique combination of Specificity could be achieved by a unique combination of

transcription factors in endothelial compared with transcription factors in endothelial compared with nonendothelial cell types. nonendothelial cell types.

Additionally, unique posttranslational modifications or Additionally, unique posttranslational modifications or alternatively spliced mRNA species may be relevant. alternatively spliced mRNA species may be relevant.

However, little direct evidence for these models of endothelial However, little direct evidence for these models of endothelial cell-restricted gene expression is presently available.cell-restricted gene expression is presently available.

Alternative mechanisms may contribute to the cell-specific Alternative mechanisms may contribute to the cell-specific expression of endothelial genes. expression of endothelial genes.

Transient Transfection of eNOS Promoter-Reporter Transient Transfection of eNOS Promoter-Reporter Constructs Suggests Epigenetic Mechanisms of Constructs Suggests Epigenetic Mechanisms of

Gene RegulationGene Regulation

eNOS exists as a single copy in the haploid genome, contains 26 eNOS exists as a single copy in the haploid genome, contains 26 exons, spans approximately 21 kb of genomic DNA, maps to exons, spans approximately 21 kb of genomic DNA, maps to chromosome 7q35-36, and directs the expression of a single chromosome 7q35-36, and directs the expression of a single major transcript measuring 4052 nucleotides. major transcript measuring 4052 nucleotides.

A single major transcription initiation site was defined by primer A single major transcription initiation site was defined by primer extension, S1 nuclease protection, and 5′-RACE (rapid extension, S1 nuclease protection, and 5′-RACE (rapid amplification of 5′ cDNA ends).amplification of 5′ cDNA ends).

The human eNOS promoter lacks a canonical TATA box and does The human eNOS promoter lacks a canonical TATA box and does not contain a proximal CpG island. not contain a proximal CpG island.

Detailed molecular characterization of the human eNOS Detailed molecular characterization of the human eNOS promoter using deletion analysis and linker-scanning promoter using deletion analysis and linker-scanning mutagenesis defined 2 clustered mutagenesis defined 2 clustered ciscis-regulatory regions: positive -regulatory regions: positive regulatory domain I (PRD I) (−104/−95 relative to transcription regulatory domain I (PRD I) (−104/−95 relative to transcription initiation) and PRD II (−144/−115).initiation) and PRD II (−144/−115).

In the vascular endothelium, these regions bind multiprotein In the vascular endothelium, these regions bind multiprotein activator complexes including Sp1, Sp3, and Ets1 transcription activator complexes including Sp1, Sp3, and Ets1 transcription factors, among others. factors, among others.

Transgenic eNOS promoter-reporter mice faithfully recapitulate Transgenic eNOS promoter-reporter mice faithfully recapitulate expression of the native eNOS gene expression of the native eNOS gene

Demonstration of Epigenetic Demonstration of Epigenetic Regulation of eNOSRegulation of eNOS

In expressing cultured human endothelial cells, the eNOS promoter directs eNOS In expressing cultured human endothelial cells, the eNOS promoter directs eNOS expression at the endogenous locus, as well as reporter expression from episomal expression at the endogenous locus, as well as reporter expression from episomal eNOS promoter-reporter constructs. eNOS promoter-reporter constructs.

ChIP experiments demonstrated enrichment of the transcription factors Sp1, Sp3, ChIP experiments demonstrated enrichment of the transcription factors Sp1, Sp3, and Ets1 at the endogenous eNOS proximal promoter as well as recruitment of RNA and Ets1 at the endogenous eNOS proximal promoter as well as recruitment of RNA polymerase II. polymerase II.

In nonexpressing cultured human vascular smooth muscle cells, eNOS is not In nonexpressing cultured human vascular smooth muscle cells, eNOS is not expressed at the endogenous locus. expressed at the endogenous locus.

However, episomal eNOS promoter-reporter constructs demonstrated robust However, episomal eNOS promoter-reporter constructs demonstrated robust activity, similar to transient transfection of episomal constructs into human activity, similar to transient transfection of episomal constructs into human endothelial cells. endothelial cells.

ChIP experiments demonstrated no enrichment of Ets1, Sp1, and Sp3 at the ChIP experiments demonstrated no enrichment of Ets1, Sp1, and Sp3 at the proximal promoter of the endogenous eNOS gene despite similar global levels of proximal promoter of the endogenous eNOS gene despite similar global levels of these transcription factors in endothelial and vascular smooth muscle cells by these transcription factors in endothelial and vascular smooth muscle cells by Western blotting. Western blotting.

RNA polymerase II was not recruited to the eNOS proximal promoter. RNA polymerase II was not recruited to the eNOS proximal promoter. Episomal constructs demonstrated robust promoter activity in a majority of Episomal constructs demonstrated robust promoter activity in a majority of

nonexpressing cell types. nonexpressing cell types. These results from transient transfection experiments were in stark contrast to the These results from transient transfection experiments were in stark contrast to the

endothelial cell-restricted expression of stably integrated promoter-reporter endothelial cell-restricted expression of stably integrated promoter-reporter constructs in transgenic eNOS promoter-reporter mice.constructs in transgenic eNOS promoter-reporter mice.

Data demonstrated that nonexpressing cell types possess the requisite Data demonstrated that nonexpressing cell types possess the requisite transcriptional machinery to direct eNOS expression. transcriptional machinery to direct eNOS expression.

These data suggested that epigenetic, chromatin-based pathways may be relevant These data suggested that epigenetic, chromatin-based pathways may be relevant in the cell-specific expression of the eNOS gene 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 The Role of DNA Methylation in eNOSeNOS

Using Southern hybridization with methylation-sensitive Using Southern hybridization with methylation-sensitive isoschizomer mapping and nucleotide-resolution bisulfite genomic isoschizomer mapping and nucleotide-resolution bisulfite genomic sequencing, a differentially methylated region (DMR) was sequencing, a differentially methylated region (DMR) was demonstrated in the native eNOS proximal promoter (−361/+3) in demonstrated in the native eNOS proximal promoter (−361/+3) in expressing (endothelial) and nonexpressing cell types.expressing (endothelial) and nonexpressing cell types.

Genomic DNA isolated from endothelial cells was unmethylated or Genomic DNA isolated from endothelial cells was unmethylated or lightly methylated, whereas genomic DNA isolated from lightly methylated, whereas genomic DNA isolated from nonendothelial cells was heavily methylated at the eNOS proximal nonendothelial cells was heavily methylated at the eNOS proximal promoter. promoter.

Importantly DNA methylation was determined to be symmetrical Importantly DNA methylation was determined to be symmetrical (occurring on both sense and antisense strands) and restricted to (occurring on both sense and antisense strands) and restricted to CpG dinucleotides. CpG dinucleotides.

Methylation further upstream (−4912/−4587) in a region Methylation further upstream (−4912/−4587) in a region corresponding to an enhancer or further downstream in a CpG corresponding to an enhancer or further downstream in a CpG island located at the 3′-end of the gene failed to demonstrate island located at the 3′-end of the gene failed to demonstrate differential methylation in expressing and nonexpressing cell differential methylation in expressing and nonexpressing cell types. types.

The eNOS proximal promoter DMR was confirmed in vivo by The eNOS proximal promoter DMR was confirmed in vivo by performing bisulfite genomic sequencing of the eNOS proximal performing bisulfite genomic sequencing of the eNOS proximal promoter in endothelial and vascular smooth muscle cells isolated promoter in endothelial and vascular smooth muscle cells isolated by laser-capture microdissection from the murine aorta (a majority by laser-capture microdissection from the murine aorta (a majority of CpG sites is conserved between mouse and man). of CpG sites is conserved between mouse and man).

In Vivo Differential Regulation of In Vivo Differential Regulation of eNOSeNOS

To explore the functional relevance of the eNOS proximal promoter DMR, To explore the functional relevance of the eNOS proximal promoter DMR, chromatin immunoprecipitation (ChIP) combined with quantitative real-chromatin immunoprecipitation (ChIP) combined with quantitative real-time PCR was performed to assess the binding of relevant transfactors to time PCR was performed to assess the binding of relevant transfactors to the native eNOS proximal promoter in human endothelial and vascular the native eNOS proximal promoter in human endothelial and vascular smooth muscle cells.smooth muscle cells.

These experiments demonstrated preferential recruitment of Sp1, Sp3, These experiments demonstrated preferential recruitment of Sp1, Sp3, and Ets1 transcription factors to the eNOS proximal promoter in and Ets1 transcription factors to the eNOS proximal promoter in endothelial cells despite the presence of these factors in vascular smooth endothelial cells despite the presence of these factors in vascular smooth muscle cells, as determined by Western blotting. muscle cells, as determined by Western blotting.

Consistent with these results, the transcriptional machinery (RNA Consistent with these results, the transcriptional machinery (RNA polymerase II) was also preferentially bound to the eNOS proximal polymerase II) was also preferentially bound to the eNOS proximal promoter in endothelial cells. promoter in endothelial cells.

MeCP2, a methyl-CpG-binding protein associated with transcriptional MeCP2, a methyl-CpG-binding protein associated with transcriptional repression, was preferentially recruited to the eNOS proximal promoter in repression, was preferentially recruited to the eNOS proximal promoter in nonexpressing vascular smooth muscle cells.nonexpressing vascular smooth muscle cells.

Treatment of nonexpressing cell types with 5-azacytidine, a DNMT Treatment of nonexpressing cell types with 5-azacytidine, a DNMT inhibitor, demethylated the eNOS promoter in various nonendothelial cell inhibitor, demethylated the eNOS promoter in various nonendothelial cell types and increased expression of the eNOS mRNA.types and increased expression of the eNOS mRNA.

Several groups have previously demonstrated DMRs in the proximal Several groups have previously demonstrated DMRs in the proximal promoters of cell-restricted genes, for example, human maspin (promoters of cell-restricted genes, for example, human maspin (SERPINB5SERPINB5) ) and erythropoietin (and erythropoietin (EPOEPO). ).

Human eNOS represents the first example of a constitutively expressed Human eNOS represents the first example of a constitutively expressed gene in the vascular endothelium whose cell-restricted pattern of gene in the vascular endothelium whose cell-restricted pattern of expression is determined, at least in part, by DNA methylation pathways.expression is determined, at least in part, by DNA methylation pathways.

Vascular Endothelium Vascular Endothelium ProteinsProteins

Restricted expression of eNOS steady-state Restricted expression of eNOS steady-state RNA and protein, respectively, to the RNA and protein, respectively, to the endothelium, especially large- and medium-endothelium, especially large- and medium-sized arteries.sized arteries.

von Willebrand factor (VWF), vascular-von Willebrand factor (VWF), vascular-endothelial cadherin (VE-cadherin) (CDH5), endothelial cadherin (VE-cadherin) (CDH5), intercellular adhesion molecule-2 (ICAM-2), the intercellular adhesion molecule-2 (ICAM-2), the angiopoietin receptors (TIE1 and TIE2), and angiopoietin receptors (TIE1 and TIE2), and the vascular endothelial growth factor (VEGF) the vascular endothelial growth factor (VEGF) receptors (FLT-1/VEGFR1 and FLK-1/VEGFR2).receptors (FLT-1/VEGFR1 and FLK-1/VEGFR2).

Endothelial NOS (eNOS)Endothelial NOS (eNOS) In mammals, the production of nitric oxide is catalyzed by 3 isoforms of nitric 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: oxide synthase (NOS) encoded by separate genes on 3 different chromosomes: neuronal NOS (neuronal NOS (NOS1NOS1), inducible NOS (iNOS) (), inducible NOS (iNOS) (NOS2NOS2), and endothelial NOS ), and endothelial NOS (eNOS) ((eNOS) (NOS3NOS3). ).

These NOS isoforms differ in their regulation and cell-specific distribution. These NOS isoforms differ in their regulation and cell-specific distribution. The latter isoform, eNOS, is constitutively expressed and responsible for the The latter isoform, eNOS, is constitutively expressed and responsible for the

majority of nitric oxide produced by the vascular endothelium and, therefore, majority of nitric oxide produced by the vascular endothelium and, therefore, represents the dominant source of bioactive endothelium-derived relaxing represents the dominant source of bioactive endothelium-derived relaxing factor. factor.

Here, nitric oxide plays important antithrombotic and antiatherogenic roles Here, nitric oxide plays important antithrombotic and antiatherogenic roles characterized by the inhibition of platelet aggregation, leukocyte–endothelium characterized by the inhibition of platelet aggregation, leukocyte–endothelium adhesion, and vascular smooth muscle cell proliferation.adhesion, and vascular smooth muscle cell proliferation.

Its fundamental role in cardiovascular physiology is underscored by the Its fundamental role in cardiovascular physiology is underscored by the phenotype of eNOS-null mice. phenotype of eNOS-null mice.

These eNOS-deficient animals demonstrate systemic and pulmonary These eNOS-deficient animals demonstrate systemic and pulmonary hypertension, abnormal vascular remodeling, defective angiogenesis, hypertension, abnormal vascular remodeling, defective angiogenesis, pathological healing in response to vascular injury, and impaired mobilization pathological healing in response to vascular injury, and impaired mobilization of stem and progenitor cells.of stem and progenitor cells.

In human disease, eNOS deficiency has been documented in the lungs of In human disease, eNOS deficiency has been documented in the lungs of patients with pulmonary hypertension and in the neointimal covering of patients with pulmonary hypertension and in the neointimal covering of advanced atheromatous plaques. advanced atheromatous plaques.

Given its prominent role as a signaling molecule in the cardiovascular system, Given its prominent role as a signaling molecule in the cardiovascular system, much attention has been focused on deciphering the regulation of eNOS both much attention has been focused on deciphering the regulation of eNOS both in vitro and in vivo. in vitro and in vivo.

Important transcriptional, posttranscriptional, and posttranslational Important transcriptional, posttranscriptional, and posttranslational mechanisms have been defined.mechanisms have been defined.

Genetic and Epigenetic Genetic and Epigenetic Cardiovascular DiseaseCardiovascular Disease

Heritability estimates have been calculated Heritability estimates have been calculated for common cardiovascular diseases and for common cardiovascular diseases and range from 30% to 50%.range from 30% to 50%.

The recent characterization of evolutionarily The recent characterization of evolutionarily conserved, epigenetic mechanisms has conserved, epigenetic mechanisms has offered a fundamentally new paradigm for offered a fundamentally new paradigm for understanding mammalian gene regulation. understanding mammalian gene regulation.

Although best characterized in cancer and Although best characterized in cancer and developmental biology, these mechanisms developmental biology, these mechanisms provide the molecular substrate for the provide the molecular substrate for the improved understanding of complex, non-improved understanding of complex, non-Mendelian diseases including common Mendelian diseases including common diseases of the human vascular system. diseases of the human vascular system.

DNA Methylation at iNOS DNA Methylation at iNOS PromoterPromoter

iNOS is a cytokine-inducible gene whose expression is implicated in a iNOS is a cytokine-inducible gene whose expression is implicated in a number of human diseases, in particular, chronic inflammatory conditions, number of human diseases, in particular, chronic inflammatory conditions, e.g., iNOS mRNA and protein in the neointima of atherosclerotic human e.g., iNOS mRNA and protein in the neointima of atherosclerotic human blood vessels. blood vessels.

Consistent with a role for promoter DNA methylation in transcriptional Consistent with a role for promoter DNA methylation in transcriptional repression, heavy methylation of CpG dinucleotides in the human iNOS repression, heavy methylation of CpG dinucleotides in the human iNOS proximal promoter of nonresponsive human endothelial cells was proximal promoter of nonresponsive human endothelial cells was documented. documented.

In contrast, the highly homologous mouse iNOS promoter in responsive In contrast, the highly homologous mouse iNOS promoter in responsive cell types was only lightly methylated. cell types was only lightly methylated.

DNA methylation was symmetrical and restricted to CpG dinucleotides. DNA methylation was symmetrical and restricted to CpG dinucleotides. The percentage of methylation of CpG dinucleotides in the core iNOS The percentage of methylation of CpG dinucleotides in the core iNOS

promoter was well correlated with gene expression, as determined by promoter was well correlated with gene expression, as determined by quantitative real-time RT-PCR. quantitative real-time RT-PCR.

The functional relevance of these findings was demonstrated by treating The functional relevance of these findings was demonstrated by treating human cells in culture with 5-azacytidine, a pharmacological DNMT human cells in culture with 5-azacytidine, a pharmacological DNMT inhibitor. inhibitor.

In minimally responsive DLD-1 cells, the iNOS promoter was completely In minimally responsive DLD-1 cells, the iNOS promoter was completely demethylated by treatment with 5-azacytidine and associated with increased demethylated by treatment with 5-azacytidine and associated with increased iNOS steady-state mRNA after cytokine stimulation. iNOS steady-state mRNA after cytokine stimulation.

In contrast, the iNOS promoter in human endothelial cells remained In contrast, the iNOS promoter in human endothelial cells remained hypermethylated and refractory to cytokine stimulation, suggesting additional hypermethylated and refractory to cytokine stimulation, suggesting additional layers of epigenetic control. layers of epigenetic control.

Taken together, these data demonstrate a prominent role for DNA Taken together, these data demonstrate a prominent role for DNA methylation in the transcriptional silencing of the human iNOS promoter in methylation in the transcriptional silencing of the human iNOS promoter in nonresponsive human endothelial cells in culture.nonresponsive human endothelial cells in culture.

Histone posttranslational modifications Histone posttranslational modifications contribute to the transcriptional silencing of iNOS contribute to the transcriptional silencing of iNOS

in cultured human cellsin cultured human cells Using ChIP combined with real-time PCR, differential recruitment of the methyl-Using ChIP combined with real-time PCR, differential recruitment of the methyl-

binding domain protein MeCP2 to the heavily methylated iNOS promoter in human binding domain protein MeCP2 to the heavily methylated iNOS promoter in human endothelial cells but not to the highly cytokine-inducible endothelial cells but not to the highly cytokine-inducible VCAM-1VCAM-1 promoter were promoter were detected. detected.

Because MeCP2 can mediate transcriptional silencing by recruiting corepressor Because MeCP2 can mediate transcriptional silencing by recruiting corepressor complexes with associated HDAC and H3K9 methyltransferase activities, the complexes with associated HDAC and H3K9 methyltransferase activities, the corresponding histone posttranslational modifications at the iNOS and corresponding histone posttranslational modifications at the iNOS and VCAM-1VCAM-1 core promoters in a variety of cell types before and after cytokine stimulation were core promoters in a variety of cell types before and after cytokine stimulation were analyzed. analyzed.

Cytokine stimulation was associated with increased recruitment of RNA Cytokine stimulation was associated with increased recruitment of RNA polymerase II at the polymerase II at the VCAM-1VCAM-1 promoter, but not at the iNOS promoter, in human promoter, but not at the iNOS promoter, in human endothelial cells. endothelial cells.

Importantly, di- and trimethylated H3K9 marks were enriched at the iNOS proximal Importantly, di- and trimethylated H3K9 marks were enriched at the iNOS proximal promoter before and after cytokine stimulation in human endothelial cells. promoter before and after cytokine stimulation in human endothelial cells.

These repressive marks were not detected at the cytokine-inducible These repressive marks were not detected at the cytokine-inducible VCAM-1VCAM-1 promoter. promoter.

Similar results have been reported for E-selectin, another cytokine-inducible Similar results have been reported for E-selectin, another cytokine-inducible endothelial gene.endothelial gene.

These data establish a role for epigenetic pathways, in particular, DNA and histone 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 H3K9 methylation, in the transcriptional silencing of the human iNOS gene in cultured human endothelial cells.cultured human endothelial cells.

It is tempting to speculate that dysregulation of these epigenetic pathways in It is tempting to speculate that dysregulation of these epigenetic pathways in disease leads to aberrant iNOS expression in endothelial cells, for example, as disease leads to aberrant iNOS expression in endothelial cells, for example, as observed in human atherosclerosis. observed in human atherosclerosis.

Epigenetic Pathways in Vascular Epigenetic Pathways in Vascular Development and Endothelial Development and Endothelial

DifferentiationDifferentiation Epigenetic pathways have received increasing attention in embryonic Epigenetic pathways have received increasing attention in embryonic

development and cellular differentiation.development and cellular differentiation. Global and endothelial cell-specific knockout of a class II HDAC, Global and endothelial cell-specific knockout of a class II HDAC,

HDAC7, is embryonic lethal and required for the development of a HDAC7, is embryonic lethal and required for the development of a normal vasculature.normal vasculature.

Specifically, these mice demonstrated defects in endothelial–Specifically, these mice demonstrated defects in endothelial–endothelial and endothelial–smooth muscle cell contacts, resulting in endothelial and endothelial–smooth muscle cell contacts, resulting in vascular dilatation and rupture. vascular dilatation and rupture.

This phenotype is the result of a dysregulated MMP10/TIMP1 axis, This phenotype is the result of a dysregulated MMP10/TIMP1 axis, resulting in pathological degradation of the extracellular matrix. resulting in pathological degradation of the extracellular matrix.

In addition, HDAC activity has been shown by multiple independent In addition, HDAC activity has been shown by multiple independent laboratories to be critical for endothelial differentiation of embryonic laboratories to be critical for endothelial differentiation of embryonic stem cells and adult endothelial progenitor cells.stem cells and adult endothelial progenitor cells.

HDAC3 activity appears to be particularly relevant.HDAC3 activity appears to be particularly relevant. HDAC3 and HDAC7 are specifically implicated in endothelial HDAC3 and HDAC7 are specifically implicated in endothelial

development and differentiation, colocalize in vivo and are development and differentiation, colocalize in vivo and are constituents of the same multiprotein, corepressor complexes, SMRT constituents of the same multiprotein, corepressor complexes, SMRT and N-CoR.and N-CoR.

Whether HDAC activity is also required for maintenance of the mature Whether HDAC activity is also required for maintenance of the mature endothelial phenotype is presently not known. endothelial phenotype is presently not known.

Hint of Vascular GenesHint of Vascular Genes

The histone methyltransferase WHSC1 The histone methyltransferase WHSC1 interacts with an important cardiac interacts with an important cardiac transcription factor, Nkx2-5, to regulate transcription factor, Nkx2-5, to regulate the normal development of the heart.the normal development of the heart.

Jarid2, also known as Jumonji, is an integral Jarid2, also known as Jumonji, is an integral component of the Polycomb repressor component of the Polycomb repressor complex, which deposits repressive complex, which deposits repressive histone marks. Jarid2 has long been known histone marks. Jarid2 has long been known to function in heart development, but its to function in heart development, but its mechanism of action was unknown. mechanism of action was unknown.

Epigenetic Pathways in Epigenetic Pathways in Postnatal AngiogenesisPostnatal Angiogenesis

In the mature vascular system, HDACs regulate postnatal angiogenesis in In the mature vascular system, HDACs regulate postnatal angiogenesis in response to various pathological cues. response to various pathological cues.

The utility of pharmacological HDAC inhibitors as cancer chemotherapeutics is The utility of pharmacological HDAC inhibitors as cancer chemotherapeutics is related, in part, to their potent antiangiogenic activity.related, in part, to their potent antiangiogenic activity.

Similar findings were reported in VEGF-induced, hypoxia-induced, and other Similar findings were reported in VEGF-induced, hypoxia-induced, and other models of postnatal angiogenesis and endothelial cell migration.models of postnatal angiogenesis and endothelial cell migration.

HDAC1 has been implicated in hypoxia-induced angiogenesis.HDAC1 has been implicated in hypoxia-induced angiogenesis. Specific knockdown of HDAC7 using an siRNA strategy inhibited cell migration Specific knockdown of HDAC7 using an siRNA strategy inhibited cell migration

and angiogenesis in mature primary endothelial cells in culture.and angiogenesis in mature primary endothelial cells in culture. Potente et al demonstrated a fundamental role for SIRT1, a class III HDAC, in Potente et al demonstrated a fundamental role for SIRT1, a class III HDAC, in

angiogenic signaling both in vitro and in vivo.angiogenic signaling both in vitro and in vivo. The mechanistic basis for the requirement of HDAC activity in the angiogenic The mechanistic basis for the requirement of HDAC activity in the angiogenic

response is presently not clear and likely involves the regulation of the response is presently not clear and likely involves the regulation of the acetylation status of various transcription factors, such as the forkhead acetylation status of various transcription factors, such as the forkhead transcription factor, Foxo1, and other proteins, including eNOS itself.transcription factor, Foxo1, and other proteins, including eNOS itself.

It remains to be defined whether global or locus-specific histone modifications It remains to be defined whether global or locus-specific histone modifications important in the chromatin-based control of gene expression are also important in the chromatin-based control of gene expression are also relevant. relevant.

Perhaps not surprisingly other epigenetic pathways are beginning to emerge Perhaps not surprisingly other epigenetic pathways are beginning to emerge as important regulators of postnatal angiogenesis and include DNA as important regulators of postnatal angiogenesis and include DNA methylation, histone methylation, and the RNA interference machinery methylation, histone methylation, and the RNA interference machinery

Epigenetic Pathways in Inflammation Epigenetic Pathways in Inflammation and the Endothelial Response to and the Endothelial Response to

Blood FlowBlood Flow Hyperacetylation of histones H3 and H4 in the 5′-regulatory regions of genes is strongly Hyperacetylation of histones H3 and H4 in the 5′-regulatory regions of genes is strongly

associated with transcriptional activation. associated with transcriptional activation. Treatment with HDAC inhibitors induce gene expression at sensitive promoters. Treatment with HDAC inhibitors induce gene expression at sensitive promoters. HDAC inhibitors have emerged as an important new class of potent antiinflammatory agents in HDAC inhibitors have emerged as an important new class of potent antiinflammatory agents in

a number of cell types, including endothelial cells. a number of cell types, including endothelial cells. HDAC inhibitors have shown early promise in the treatment of a growing number of chronic 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, inflammatory diseases such as inflammatory bowel disease, systemic lupus erythematosus, and rheumatoid arthritis.and rheumatoid arthritis.

To date, the mechanism of action remains unclear but may involve modulation of NF-κB To date, the mechanism of action remains unclear but may involve modulation of NF-κB transcriptional activity.transcriptional activity.

In human cultured endothelial cells, HDAC inhibitors inhibited TNF-α–induced monocyte In human cultured endothelial cells, HDAC inhibitors inhibited TNF-α–induced monocyte adhesion in vitro and in vivo via suppression of the adhesion in vitro and in vivo via suppression of the VCAM-1VCAM-1 gene. gene.

Interestingly, other cytokine-inducible genes, Interestingly, other cytokine-inducible genes, ICAM-1ICAM-1 and E-selectin, were not suppressed, and E-selectin, were not suppressed, suggesting a direct effect on the suggesting a direct effect on the VCAM-1VCAM-1 promoter as opposed to a general inhibition of the promoter as opposed to a general inhibition of the NF-κB signaling pathway. NF-κB signaling pathway.

Similar results have recently been reported for the repression of cytokine-induced tissue factor 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.in human endothelial cells by a variety of structurally distinct HDAC inhibitors.

What accounts for this unique promoter specificity is presently not known. What accounts for this unique promoter specificity is presently not known. The recent demonstration that the administration of the pharmacological HDAC inhibitor, TSA, The recent demonstration that the administration of the pharmacological HDAC inhibitor, TSA,

to atherosclerosis-prone, to atherosclerosis-prone, LdlrLdlr−/− mice exacerbates neointimal lesions emphasizes the need −/− mice exacerbates neointimal lesions emphasizes the need for a better understanding of epigenetic pathways in animal models of atherosclerosis.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 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. physical forces of blood flow, in particular, laminar shear stress.

Pronounced changes in global and locus-specific histone posttranslational modifications in Pronounced changes in global and locus-specific histone posttranslational modifications in cultured human endothelial cells in response to laminar shear stress.cultured human endothelial cells in response to laminar shear stress.

It is interesting to speculate that epigenetic pathways, in part, determine the susceptibility of It is interesting to speculate that epigenetic pathways, in part, determine the susceptibility of different regions of the vascular system to atherosclerosis. different regions of the vascular system to atherosclerosis.

Relative reduction of eNOS transcription in atherosclerosis prone regions of the mouse aorta.Relative reduction of eNOS transcription in atherosclerosis prone regions of the mouse aorta.

Epigenetic mechanisms in Epigenetic mechanisms in diabetic vascular diabetic vascular

complicationscomplications Increasing evidence suggests that epigenetic factors play a Increasing evidence suggests that epigenetic factors play a

key role in the complex interplay between genes and the key role in the complex interplay between genes and the environment. environment.

Actions of major pathological mediators of diabetes and its Actions of major pathological mediators of diabetes and its complications such as hyperglycaemia, oxidant stress, and complications such as hyperglycaemia, oxidant stress, and inflammatory factors can lead to dysregulated epigenetic inflammatory factors can lead to dysregulated epigenetic mechanisms that affect chromatin structure and gene mechanisms that affect chromatin structure and gene expression. expression.

Furthermore, persistence of this altered state of the Furthermore, persistence of this altered state of the epigenome may be the underlying mechanism contributing to epigenome may be the underlying mechanism contributing to a ‘metabolic memory’ that results in chronic inflammation and a ‘metabolic memory’ that results in chronic inflammation and vascular dysfunction in diabetes even after achieving vascular dysfunction in diabetes even after achieving glycaemic control. glycaemic control.

Further examination of epigenetic mechanisms by also taking Further examination of epigenetic mechanisms by also taking advantage of recently developed next-generation sequencing advantage of recently developed next-generation sequencing technologies can provide novel insights into the pathology of technologies can provide novel insights into the pathology of diabetes and its complications in vasculature. diabetes and its complications in vasculature.

DNA methylation: relation to DNA methylation: relation to diabetes and vascular diabetes and vascular

complicationscomplications Regulation of Regulation of AgoutiAgouti gene expression by DNA methylation  gene expression by DNA methylation

plays an important role in the development of obesity and plays an important role in the development of obesity and diabetes in mice diabetes in mice

The islet dysfunction and development of diabetes in rats is The islet dysfunction and development of diabetes in rats is associated with epigenetic silencing via promoter DNA associated with epigenetic silencing via promoter DNA methylation of Pdx1, a key transcription factor that methylation of Pdx1, a key transcription factor that regulates β-cell differentiation and insulin gene expression regulates β-cell differentiation and insulin gene expression

Peroxisome proliferator-activated receptor-γ co-activator 1α Peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) regulates insulin production in pancreatic β-cells. (PGC-1α) regulates insulin production in pancreatic β-cells.

Studies with T2D animals showed that DNA Studies with T2D animals showed that DNA hypermethylation at the promoter of its hypermethylation at the promoter of its gene gene PPARGC1APPARGC1A reduces PGC-1α expression and inhibits  reduces PGC-1α expression and inhibits insulin production insulin production

Methylation and Cardiovascular Methylation and Cardiovascular DiseasesDiseases

The role of DNA methylation in the pathogenesis of cardiovascular diseases The role of DNA methylation in the pathogenesis of cardiovascular diseases (CVDs) is not completely understood. (CVDs) is not completely understood.

Atherosclerosis was associated with global hypomethylation in SMCs of Atherosclerosis was associated with global hypomethylation in SMCs of atherosclerotic lesions from humans, and animal models such as high-fat atherosclerotic lesions from humans, and animal models such as high-fat diet-fed ApoE null mice and balloon-injured rabbits.diet-fed ApoE null mice and balloon-injured rabbits.

Furthermore, altered DNA methylation of several candidate genes linked with Furthermore, altered DNA methylation of several candidate genes linked with atherosclerosis was identified in both VSMCs and ECs, and in mouse models. atherosclerosis was identified in both VSMCs and ECs, and in mouse models.

These include hypoxia-inducible factor-1α, c-fos, p53 and oestrogen receptor, These include hypoxia-inducible factor-1α, c-fos, p53 and oestrogen receptor, growth factors, arachidonic acid-metabolizing enzymes (15-lipoxygenase), growth factors, arachidonic acid-metabolizing enzymes (15-lipoxygenase), vasodilator endothelial nitric oxide synthase, and matrix metalloproteinases.vasodilator endothelial nitric oxide synthase, and matrix metalloproteinases.

Alterations in genomic DNA methylation were also demonstrated in Alterations in genomic DNA methylation were also demonstrated in leucocytes derived from ApoE null mice preceding the development of leucocytes derived from ApoE null mice preceding the development of atherosclerosis.atherosclerosis.

Other CVD risk factors such as hyperhomocysteinaemia, Other CVD risk factors such as hyperhomocysteinaemia, hypercholesterolaemia, and inflammation have also been implicated in DNA hypercholesterolaemia, and inflammation have also been implicated in DNA methylation changes associated with atherosclerosis.methylation changes associated with atherosclerosis.

Altered global DNA methylation was noted in peripheral blood monocytes of Altered global DNA methylation was noted in peripheral blood monocytes of patients with increased risk for CVDs.patients with increased risk for CVDs.

Risk for CVDs and diabetes increases with age, and ageing is associated with Risk for CVDs and diabetes increases with age, and ageing is associated with hypomethylation of genomic DNA. hypomethylation of genomic DNA.

miRNAs in vascular miRNAs in vascular complicationscomplications

The 22-nucleotide small non-coding miRNAs play important roles in The 22-nucleotide small non-coding miRNAs play important roles in diverse biological processes and disease conditions such as cancer, diverse biological processes and disease conditions such as cancer, diabetes, DN, cardiogenesis, angiogenesis, and vascular development diabetes, DN, cardiogenesis, angiogenesis, and vascular development by post-transcriptional mechanisms.by post-transcriptional mechanisms.

Interaction of mature miRNAs with specific binding sites in the 3′ Interaction of mature miRNAs with specific binding sites in the 3′ untranslated regions of target mRNAs in the RNA-induced silencing untranslated regions of target mRNAs in the RNA-induced silencing complex leads to either mRNA degradation or inhibition of translation.complex leads to either mRNA degradation or inhibition of translation.

Each miRNA can regulate multiple targets including signal transduction Each miRNA can regulate multiple targets including signal transduction components, transcription and epigenetic factors, and provide another components, transcription and epigenetic factors, and provide another level of epigenetic mechanism to fine tune gene regulation in response level of epigenetic mechanism to fine tune gene regulation in response to environmental stimuli.to environmental stimuli.

Evidence shows that miRNAs can affect the function of both ECs and Evidence shows that miRNAs can affect the function of both ECs and VSMCs relevant to vascular diseases.VSMCs relevant to vascular diseases.

miRNAs are implicated in phenotypic switching, proliferation, migration, miRNAs are implicated in phenotypic switching, proliferation, migration, and neointimal thickening in VSMCs, as well as capillary formation, and neointimal thickening in VSMCs, as well as capillary formation, migration, senescence, expression of adhesion molecules, and migration, senescence, expression of adhesion molecules, and angiogenic growth and transcription factors in ECs.angiogenic growth and transcription factors in ECs.

In monocytes and macrophages, miRNAs regulate inflammation, In monocytes and macrophages, miRNAs regulate inflammation, response to oxidized lipids, oxidative stress, immune function, response to oxidized lipids, oxidative stress, immune function, cholesterol homeostasis, and differentiation. cholesterol homeostasis, and differentiation.

Use of Vascular Use of Vascular Endothelial Growth Endothelial Growth Factor (VEGF) as a Factor (VEGF) as a Treatment for End Treatment for End

Stage Coronary Artery Stage Coronary Artery Disease (CAD)Disease (CAD)

AngiogenesisAngiogenesis TThe formation of new blood vessels from existing he formation of new blood vessels from existing microvesselsmicrovessels

VEGF cVEGF contributes to the preservation of ischemic ontributes to the preservation of ischemic tissue and myocardial pump function after tissue and myocardial pump function after myocardial infarctionmyocardial infarction

VEGF is iVEGF is important in:mportant in: Embryogenesis (called vasculogenesis)Embryogenesis (called vasculogenesis) Wound healing Wound healing Tumor growth and metastasizationTumor growth and metastasization Rheumatoid arthritisRheumatoid arthritis Ischemic heart diseaseIschemic heart disease Ischemic peripheral vascular diseaseIschemic peripheral vascular disease

VEGF is involved in VEGF is involved in AngiogenesisAngiogenesis

Inducing AngiogenesisInducing Angiogenesis

Need a stimulusNeed a stimulusHypoxic tissue, Ischemic tissue, Mechanically damaged Hypoxic tissue, Ischemic tissue, Mechanically damaged tissuetissue

Need expression of angiogenic molecules to Need expression of angiogenic molecules to initiate angiogenesisinitiate angiogenesisVEGF, FGF, TGFVEGF, FGF, TGF, PDGF, PDGFNeed angiogenesis to occurNeed angiogenesis to occur1. Proliferation and migration of endothelial cells from the 1. Proliferation and migration of endothelial cells from the microvasculaturemicrovasculature2. Controlled expression of proteolytic enzymes2. Controlled expression of proteolytic enzymes3. Breakdown and reassembly of extracellular matrix3. Breakdown and reassembly of extracellular matrix4. Morphogenic process of endothelial tube formation4. Morphogenic process of endothelial tube formationMechanism of Angiogenesis not completely knownMechanism of Angiogenesis not completely known

VEGF VEGF (vascular endothelial growth factor)(vascular endothelial growth factor) Specific for only endothelial cellsSpecific for only endothelial cells

May inhibit smooth muscle growth…reduce restenosisMay inhibit smooth muscle growth…reduce restenosis FGF FGF (fibroblast growth factor)(fibroblast growth factor) Associated with tumor angiogenesisAssociated with tumor angiogenesis Can stimulate growth in other cells besides endothelial cellsCan stimulate growth in other cells besides endothelial cells Not as specific as VEGFNot as specific as VEGF

TGF-TGF- (transforming growth factor ß)(transforming growth factor ß) Indirect angiogenesis effectIndirect angiogenesis effect Possibly induces VEGF expression (Protein Kinase C pathway)Possibly induces VEGF expression (Protein Kinase C pathway)

PDGF PDGF (platelet derived growth factor)(platelet derived growth factor) Not well characterized in angiogenesisNot well characterized in angiogenesis

Why use VEGF to Promote Why use VEGF to Promote Angiogenesis?Angiogenesis?

Other VEGF CharacteristicsOther VEGF Characteristics

VEGF expressed by Macrophages, fibroblasts, VEGF expressed by Macrophages, fibroblasts, smooth muscle cells, endothelial cells (all are smooth muscle cells, endothelial cells (all are present in the heart)present in the heart)

Action is direct because of the exclusive Action is direct because of the exclusive specificity for receptors (flt-1 and flk-1) specificity for receptors (flt-1 and flk-1)

Receptors only found on endothelial cells Receptors only found on endothelial cells

Causes activation of many other genes involved Causes activation of many other genes involved in angiogenic responsein angiogenic response

How to Deliver VEGFHow to Deliver VEGFProtein TherapyProtein TherapyDirect injection of proteinDirect injection of protein Time delay deliveryTime delay delivery Local intercoronary bolusLocal intercoronary bolusGene TherapyGene TherapyAdenovirus vectorAdenovirus vector

Excellent specificity for endothelial cellsExcellent specificity for endothelial cells Extended expression of VEGExtended expression of VEGFFDirect gene transferDirect gene transfer

Involves direct injection of eukaryotic plasmid DNA Involves direct injection of eukaryotic plasmid DNA containing VEGF cDNA containing VEGF cDNA

Should VEGF administration prove effective, it is likely that Should VEGF administration prove effective, it is likely that VEGF/VEGF DNA will be delivered on a catheter platformVEGF/VEGF DNA will be delivered on a catheter platform

Case StudiesCase Studies

Injection of naked VEGF cDNA contained Injection of naked VEGF cDNA contained in an Eukaryotic Expression Vectorin an Eukaryotic Expression Vector

Jeffery Isner Jeffery Isner et al.et al. St. Elizabeth’s Medical St. Elizabeth’s Medical CenterCenter

Phase I clinical trial…designed to assess Phase I clinical trial…designed to assess safety and bioactivity of treatment methodssafety and bioactivity of treatment methods

Limited sample…only 5 patients involvedLimited sample…only 5 patients involved Prior Bypass and/or angioplastyPrior Bypass and/or angioplasty Class 3-4 AnginaClass 3-4 Angina No longer respond to additional treatmentNo longer respond to additional treatment

Animal Data:Animal Data:

Charles Mack Charles Mack et al. et al. New York Hospital- New York Hospital-Cornell Medical CenterCornell Medical Center

Administration VEGF gene through Administration VEGF gene through Adenovirus mediated gene therapyAdenovirus mediated gene therapy

Model:Model: Pig with a constrictor band around circumflex artery to Pig with a constrictor band around circumflex artery to

induce myocardial infarction and ischemiainduce myocardial infarction and ischemia Eventually results in complete occlusion of circumflex Eventually results in complete occlusion of circumflex

arteryartery

Vector:Vector: Adenovirus vector in E1a-, partial E1b-, and partial E3- Adenovirus vector in E1a-, partial E1b-, and partial E3-

mutations (makes them replication deficient)mutations (makes them replication deficient) Adenovirus used because of the natural selectivity for Adenovirus used because of the natural selectivity for

endothelial cellsendothelial cells Minimal inflammation detected in animals 4 weeks post Minimal inflammation detected in animals 4 weeks post

therapy therapy In vivo conformation of expression confirmed by ELISA 3 In vivo conformation of expression confirmed by ELISA 3

days after injection days after injection

ResultsResults

Treatment Resulted in significantly reduced Treatment Resulted in significantly reduced ischemic area (area of oxygen starved tissue) ischemic area (area of oxygen starved tissue) and and

Ischemic maximum (severity of ischemia) in Ischemic maximum (severity of ischemia) in treated animals treated animals

Strength of heartbeat returned in treated Strength of heartbeat returned in treated animals more than untreated animalsanimals more than untreated animals

More vessels visible angiographically in More vessels visible angiographically in treated animals vs. untreated animals treated animals vs. untreated animals

Treated animals seemed to route around the Treated animals seemed to route around the occlusion as demonstrated by the filling of occlusion as demonstrated by the filling of branching arteriebranching arteriess

Why it works?Why it works?

VEGF stimulates growth of “collateral”VEGF stimulates growth of “collateral” vessels?vessels?