Block II Learning Objectives

Lecture XVIIIntroduction to Part II - Presentation of a Case StudyTerminologyTermDefinition

Hereditary Hemorrhagic Telangiectasia (HHT)Genetic (autosomal dominant) disorder that leads to abnormal blood vessel formation in the skin, mucous membranes, and often in organs such as the lungs and brain. This can cause nosebleeds, GI bleeding.

AnemiaCommon blood disorder attributed to a general decrease in number of erythrocytes or decrease in hemoglobin.

Focal Vascular LesionAbnormality near the surface of epidermal tissue attributed from vascular origins, particularly through dilation of blood vessels.

EpistaxisCommon occurrence of nosebleeds, where blood drains from the nostril, attributed to rupture of the blood vessels in the nose.

TelangiectasiasSmall, dilated blood vessels near the surface of the skin or mucous membranes.

Arteriovenous MalformationAbnormal connection between veins and arteries, typically from congenital origins.

NidusLarge, fragile tangle/accumulation of arteries and veins.

Autosomal DominantThis refers to the inheritance pattern of a disease. If it is autosomal dominant, then only one abnormal gene from a parent is necessary to attain the disorder.

HaploinsufficiencyWhen an organism only has one functional copy of the gene, with the other inactivated, which can cause abnormal presentations such as in disease-states.

Incomplete PenetranceProportion of patients carrying the genotype that do not express the phenotype.

Variable ExpressivityVariations in phenotype carrying a particular genotype. It is analogous to the severity of a condition in clinical medicine.

Lecture ObjectivesIntroduction to Block II and the case study.Block II surrounds the elements of signal transduction pathways that alter gene expression. This strategy is mainly the way to target genes. This can occur in elements of development and can be utilized to our advantage in stem cell research. In short, signal affects expression.

The case study involved in this block is the medically unique tale of a patient named Sarah. She is a 32-year old martial arts instructor in South Arizona, married with kids. She has experienced elements of fatigue (tired and sluggish). She initially attributed it to stress until she observes blood in her stool. Sarahs doctor does a history and physical, with no indication of colon cancer in the family. A fecal occult blood test confirms blood in her stool, and Sarah is then scheduled for a colonoscopy. Her doctor finds numerous small red spots on her skin, distributed mostly in her mouth and on the top of her hands. They increased in number, as she got older. She also gets small lesions on the inside of her mouth or tongue from time to time. Sarah also tells her doctor she had a history of nosebleeds, even though her sister does not get nosebleeds. Her mom also had a history of nose bleeds, and that one of her sons had a lot of nosebleeds. The colonoscopy revealed vascular lesions (known as telangiectasias) in her colon, and laboratory test revealed anemia in her blood. This led to the diagnosis of hereditary hemorrhagic telangiectasia, and DNA testing later revealed that she has specifically HHT Type II.Learn about the presentation, diagnosis, complications, and inheritance pattern of HHT.Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder found in ~1 in 5-8000 people, with 1.2 million people affected. It is a multisystem vascular disorder consisting of focal vascular lesions (telangiectasias or arteriovenous malformations). It is an autosomal dominant disease, with the bad gene inhibits the good gene, and so the bad protein is created so that it inhibits the desired effects from normal proteins. It exhibits haploinsufficiency in which the on good copy is not enough for normal effects. The disease has marked intrafamilial variation in terms of both penetrance and variable expressivity.

The presentation is typically observed in childhood but the symptoms can be aggravated with age, with patients exhibiting only nosebleeds and telangiectasia. Of the patient population, approximately a third of the patients exhibit chronic anemia and GI bleeding. However, a majority can have typically silent arteriovenous malformations in the pulmonary, hepatic, cerebral, and spinal circulations. Complications can involve not only anemia due to chronic GI and epistaxis, but also in the larger arteriovenous malformations in lungs, brain, liver, and GI tract (such as in stroke, hemorrhage, and brain infection). More rare complications tend to include congestive high output heart failure from the decrease in capillary bed size.

Diagnosis involves three of the following criteria: (1) spontaneous and recurrent epistaxis, (2) multiple telangiectasias at characteristic sites, (3) visceral vascular lesions (gastrointestinal telangiectasias and proven arterioveous malformations on lung, liver, brain, or spine), (4) family history of HHT (first-degree relative)

Treatment and management involves strategies to: (1) stop or prevent excessive bleeding, (2) increase clotting ability, (3) replenish lost blood, (4) monitor for more serious complications, and (5) procedures to block off local sites where there is a blood vessel defect.Know the underlying structural disorder in HHT. Distinguish between Telangiectasias and Arteriovenous Malformations.The underlying disorder in HHT is attributed to the abnormal vascular architecture. There are two types of vascular abnormalities: telangiectasis and arteriovenous malformations (AVMs). Telangiectasis are focal dilatations of microvessels. There is a decrease in the number of capillaries, and are convoluted in larger lesions in later stages. This implies that there is really a direct connect between arteries and veins, and thus does not allow appropriate capillary exchange. They are common on skin and mucous membranes. Arteriovenous malformations are essentially direct connections between larger arteries and veins. These are typically larger than telangiectasis, up to several centimeters. At this point , there is a decrease (if not a lack) of capillaries, and can be present as a nidus (essentially a big, fragile tangle of arteries and veins). AVMs can particular develop in distinct parts of the vasculature to limit bypasses in normal circulation. Think about what we might learn by examining this case.From observing the pathogenic mechanisms underlying the vascular malformations in HHT, we can learn about how the genetic mutations can overall affect signaling patterns in the body and disrupt normal physiological processes. In HHT, one of the physiological consequences is essentially excessive bleeding and poor capillary exchange.

Lecture XVIIICardiovascular System: Organization, Functions, PropertiesTerminologyTermDefinition

Transport and ExchangeMajor function of the cardiovascular system, involving movement of a media (particularly blood) through a circulatory system to allow for tissue exchange of nutrients and metabolic waste.

Closed versus Open CirculationClosed Circulation: Blood never leaves the network of arteries, veins and capillaries, with oxygen and nutrients diffusing into interstitial fluid. Allows for diffusion of nutrients at low pressure states.Open Circulation: Blood leaves network of arteries, veins, and capillaries in the form of hemolymph, which provides no distinction between blood and interstial fluid. Allows for movement at higher pressure.

William HarveyEnglish physician who first described the systemic circulation and properties of blood in a closed circulation.

Pulmonary CirculationPortion of the cardiovascular system carrying oxygen-depleted blood away from the heart to the lungs, to bring oxygenated blood to the heart.

Systemic CirculationPortion of the cardiovascular system where oxygenated blood is carried to the various organs (excluding lungs) and brings oxygen-depleted blood to the heart.

In Series

Type of organization consisting of consecutive elements, such as A B.

In Parallel

Type of organization consisting of one attending to multiple events, such as A B + C + D (simultaneously).

ArteryBlood vessels that carry blood away from the heart.

VeinBlood vessels that carry blood towards the heart.

MicrocirculationSmall vessels in the vasculature that are embedded in organs and are the main element in the distribution of nutrients from blood to tissue.

ArterioleBlood vessel in microcirculation that moves out of the artery and into the capillaries. They are also the primary sites of vascular resistance, where the greatest change in blood pressure and velocity of blood flow occurs.

VenuleBlood vessel in microcirculation that return blood from the capillary to the larger blood vessels.

CapillarySmallest blood vessels and are involved in exchange of nutrients and materials, and connect arterioles to venules.

AtriumChamber of the heart involved in receiving blood. The right atrium receives blood rom the superior or inferior vena cava, while the left atrium receives blood from the pulmonary veins.

VentricleLarge chamber that collect and expel blood from the atrium towards either lungs (right ventricle) or to the body (left ventricle). The left ventricle is typically bigger than the right ventricle.

MyocardiumMuscular layer of the heart involved in contraction and pumping of blood, consisting of cardiac muscle.

Atrioventricular ValvesValves of the heart between the atria and ventricle, known as the mitral valve (left) and the tricuspid (right).

Cardiac OutputVolume of blood being pumped by the heart, by a left or right ventricle within one minute. It is the multiplication product of stroke volume and heart rate.

Total Cross-Sectional AreaTotal cross sectional area of all blood vessels of a particular type. Capillaries (through their numbers) have the largest cross sectional area despite their small size.

Velocity of Blood FlowA value equal to the total volume flow divided by the cross-sectional area:

Volumetric Flow RateVolume of fluid that passes through a given surface per unit time.

Blood PressurePressure exerted by circulating blood upon the walls of blood vessels.

Arteriovenous MalformationAbnormal connection between veins and arteries, typically from congenital origins.

TelangiectasiaSmall, dilated blood vessels near the surface of the skin or mucous membranes.

Lecture ObjectivesKnow the functions of the CV system.The cardiovascular system has the following major functions: (1) transport and exchange of nutrients, (2) fluid balance to regulate intracellular and extracellular volume of body cells, (3) dissipation of excess heat, (4) as a buffer system to stabilize pH. The ultimate goal of this is to maintain homeostasis, or a relatively constant environment around cells.Learn how the CV system is organized.The cardiovascular system is organized in a closed circulation, in which blood is contained within vessels (of varying size and thickness) at all times. The components of the human cardiovascular system are: (1) the heart, (2) blood vessels, and (3) blood. The heart is the muscular pump that provides the driving force of the movement of blood. The blood vessels are ultimately the plumbing of the heart, directing movement and keeping the blood confined. Finally, the blood is the tissue involved in carrying materials.

On a larger scale, it is organized into two circulations: (1) the pulmonary circulation (involving the right side of the heart) and (2) the system circulation (involving the left side of the heart). The systemic circulation is typically in series with the pulmonary circulation, while the other organ systems are supplied with blood in parallel by the systemic circulation. It can also be organized by function. Lungs represent a site of gas exchange. The heart is the pump. The arteries are the distributing tubes. The capillaries are the large networks of very thin tubes where exchange occurs at tissues. Veins are the collecting tubes.Trace the flow of blood through the CV system.The flow of blood through the cardiovascular system is typically in the following manner. Blood flows into the right atrium from the superior and inferior vena cava and consequently flow into the right ventricle. From the right ventricle, blood will then flow (via the pulmonary arteries) to the lungs, where they will be replenished with oxygen while simultaneously releasing carbon dioxide. The newly oxygenated blood will then flow into the left atrium via the pulmonary veins. It will then flow into the left ventricle, and then be pumped into the aorta and thus into the systemic circulation.Know about cardiac output and its distribution and how it can change.Cardiac output is the volume of blood pumped by each ventricle per minute (approximately 5 liters/minute). The cardiac output from right to left ventricles is typically the same (at 5 liters/minute as it is a closed circulatory system). If not the same, it may imply backup of flow. The distribution of the cardiac output to the different organ systems is rather unequal, determined by function, size, and metabolism. Some changes in cardiac output are rather normal, especially in states of exercise, where the metabolic demands (by neurological excitation) require greater flow of blood.

Learn how CV parameters change at different levels of the circulation. Think about how these might change in HHT.The cardiovascular parameters can change at different levels of the circulation. In terms of total cross-sectional area, the capillaries (collectively) have the greatest cross-secitonal area, because of their numbers. The total cross-sectional area is the key determinant of the velocity of blood flow. Typically, as the cross sectional area increases the velocity of flow decreases (at constant blood flow rate). This is important because we can also tell that the pressure is the lowest at the capillaries, while highest at the aorta. The mean arterial pressure remains constant through larger arteries and then drops at the arterioles and troughs at the venules and veins. This can change in hereditary hemorrhagic telangiectasia (where direct connections allow paths of lower resistance) by making venous pressure increase. People with hereditary hemorrhagic telangiectasia can have arteriovenous malformations that spur a shunt (a low resistance shortcut) between the arteries and veins. The problem is that the veins are not as structurally stable in comparison to the muscular arteries, and can easily hemorrhage because of such relatively extreme pressures by such bypasses. Patients with this same disorder can develop a shunt in the hepatic circulation, with the chronic high cardiac output that can spur cardiac failure.

Lecture XIXBlood Vessel Structure and FunctionTerminologyTermDefinition

Tunica InternaMost interior layer of the blood vessel, which includes endothelial cells and connective tissue.

Tunica MediaMedial layer of the cell, containing vascular smooth muscle cells in loose connective tissue and elastic fibers.

Tunica ExternaMost external layer of the blood vessel, which is essentially a connective tissue sheath, also known as an adventitia.

Vascular EndotheliumThin layer of cells that line the interior surface of blood vessels, with the individual cells called endothelial cells.

Vascular Smooth MuscleSmooth muscle found within, and composing the majority of the wall of blood vessels, particularly the arteries and arterioles.

Basement MembraneSheet of fibers that underlie the epithelium, which line cavities and surfaces such the endothelium.

ElastinProtein in elastic connective tissue to allow consistency in shape after expansion or contraction.

Collagen fibersMajor component of connective tissue that allows maintenance in shape. This is seen in greater amounts in venules and veins.

ArteriesBlood vessels that carry blood away from the heart, exhibiting more smooth muscle in comparison to veins.

Pressure ReservoirsType of reservoir in which pressure, not volume, is the main element regulated. This is seen in arteries, where the mean arterial pressure is kept constant.

Pulsatile Blood PressureQuantity of pressure required creating the feeling of a pulse.

Systolic PressureMaximal blood pressure, or the pressure exhibited in contraction.

Diastolic PressureMinimal blood pressure, or the pressure exhibited in relaxation.

Mean Arterial Blood PressureAverage arterial pressure during a single cardiac cycle, or the average blood pressure in an individual. Calculated as the product as the sum of the product of cardiac output and stroke volume and central venous pressure [.

VeinsBlood vessels that carry blood towards the heart, exhibiting more collagen in comparison to arteries.

Volume ReservoirsType of reservoir where volume, not pressure, is under regulation. Veins are considered volume reservoirs, retaining the majority of blood volume.

Distribution of Blood VolumeDistribution of blood is where volume changes to different parts of the body despite the constant pressure gradient.

Venous ValvesMain force involved in allowing blood to flow towards the heart unidirectionally.

MicrocirculationSmaller vessels in vasculature involved in distribution of blood within tissues.

ArteriolesBlood vessel in microcirculation that moves out of the artery and into the capillaries. They are also the primary sites of vascular resistance, where the greatest change in blood pressure and velocity of blood flow occurs.

VenulesBlood vessel in microcirculation that return blood from the capillary to the larger blood vessels.

Volumetric Flow RateVolume of fluid that passes through a given surface per unit time. It is proportional to the pressure gradient across the vessel and is inversely related to the resistance to blood flow: .

Pressure GradientDifference in pressure along a blood vessel.

Resistance to FlowDespite a constant pressure, the major variable involved in altering volume and distribution of blood. It determines how much blood flow goes through a particular blood vessel, and also determines how much flow goes into a particular organ.

Poisseuilles LawPhysical law that provides a pressure drops along a blood vessel. Calculated by: , where r is radius, P is pressure, is viscosity, and L is length.

Smooth Muscle Cell ContractilityAbility of the smooth muscle to relax or contract independent of volume.

Primary Resistance VesselsVessels involved in altering blood volume despite constant pressure. Arterioles are the primary resistance vessels.

VasoconstrictionNarrowing of blood vessels from contraction of smooth muscles, found particularly in larger arteries and arterioles.

VasodilationThe widening of blood vessels from relaxation of smooth muscles.

CapillariesSmallest blood vessels and are involved in exchange of nutrients and materials, and connect arterioles to venules.

Lecture ObjectivesLearn about the anatomy of blood vessels.The anatomy of blood vessels is distinctly different among the arteries and veins. Remember that their structure is reflective upon function, but as blood vessels (excluding the capillaries), they have similar parts. The blood vessel contains three parts: the tunica interna, the tunica media, and the tunica externa. The tunica interna is the innermost layer of the blood vessel, containing the endothelium and connective tissue. All blood vessels are lined with the endothelial cells, which function in clot prevention, cell signaling, and capillary exchange. The tunica media is the smooth muscle layer, containing the smooth muscle in loose connective tissue with elastin. Elastin is important in allow the recoil from the larger volume of blood flow. The tunica externa (also known as the adventitia) represents a connective tissue sheath that encapsulated the blood vessel.

Though arteries and veins are classified under blood vessels, they have observable differences. Arteries have a larger amount of smooth muscle and elastin, while veins tend to have greater amounts of collagen fibers. Thus, compensate in order to establish unidirectional flow, the veins are accommodated with valves made from endothelial tissue. The exclusion of capillaries is simply because they are one cell thick and are the main exchange points between the blood and tissue.

See how the anatomy of different types of blood vessels reflects function.The anatomy is reflective upon function. Arteries typically move blood away from the heart, while veins move blood towards the heart. Their function can be represented structurally in the fact that arteries have larger amounts of elastin and vascular smooth muscle lining this type of blood vessel. This allows the artery to propagate pressure waves from the heart (allowing transient increases in blood), and allow it to recoil back from expansion. Thus, the arteries are treated as the pressure reservoirs of the circulatory system, keeping the mean arterial pressure constant and allow even pressure distribution to the system.

Meanwhile, veins display a different structure for their function. They are involved in carrying tissues back to the heart. They have thinner walls compared to arteries and less elastic tissue and smooth muscle, and are typically adapted for lower pressures. To facilitate venous return to the heart in a unidirectional fashion, they have a series of valves. Thus, they are typically considered to be volume reservoirs, as they are the major storage of blood volume (containing approximately 64% of volume distributed).

Know the functions of endothelial cells and smooth muscle cells.The endothelial cells of the body are involved in (1) provision of a smooth interface, (2) secretion of cellular signals, and (3) in the capillaries, provide a penetrable barrier for exchange.

Smooth muscle cells in the vasculature can contract or relax depending on what kinds of signals they receive. Arterioles are the major resistance vessels. A change in arterioles will cause a change in flow. The largest drop in pressure occurs at the arterioles, and arterioles control the flow of blood amid the constant pressure. They typically act by vasoconstriction and vasodilation. Vasoconstriction occurs by increased contraction of smooth muscle, where there is increases in resistance and eventual decrease in flow. Vasodilation occurs when there I a decreased contraction of smooth muscle, where there is a decrease in resistance and an increase in flow. Resistance is what determines how much flow goes into each organ.

Local and extrinsic factors can affect the activity of the vascular smooth muscle. It can be affected by (1) autonomic nervous system activity, such as in state of physical activity, (2) local factors, or metabolites, released by the nearby tissue, giving a metabolic control of blood flow, (3) local changes in blood flow, showing myogenic control, and (4) signals released by the vascular endothelium, such as nitric oxide and endothelin.

Know the different parameters that affect blood flow through a blood vessel.There are several general parameters that can affect blood flow through a blood pressure, as Poiseuilles Law can describe. We do know that pressure and resistance are the two major variables that can affect flow, but there are others. The radius is inversely proportional to resistance, such that an increase in the radius can cause a decrease in resistance. However resistance is directly proportional to the viscosity and length, so that increasing either viscosity or length can cause an increase in resistance.

However, we should also examine the role of blood pressure. It is not dependent on the absolute values of pressure along the blood vessel, but more of the difference between the pressures along the blood vessel, which is known as a pressure gradient. In physiological states, however, we should remember that there is going to be change in blood vessel structure as the absolute pressures increase.Think about how the above relate to the case study. What changes occur with HHT?In hereditary, hemorrhagic telangiectasia, we should remember that the arteriovenous malformations allow a low resistance bypass into the vein. Higher pressures in the draining veins at focal vascular lesions in HHT can lead to consequential structural changes in the veins. However, veins are particularly thinner relative to arteries, and the compensatory measures are inadequate. If the pressure is too high and the structural compensations are insufficient, it can lead to strokes, aneurysm, and hemorrhage due to such structural insufficiency. The laminar flow is disrupted in the arteriovenous malformation and spur turbulent flow. Such turbulent flow can cause structural changes and greater proliferation to accommodate such changes in flow.

Lecture XXTransport and Exchange in the MicrocirculationTerminologyTermDefinition

MicrocirculationSmaller vessels in vasculature involved in distribution of blood within tissues.

ArteriolesBlood vessel in microcirculation that moves out of the artery and into the capillaries. They are also the primary sites of vascular resistance, where the greatest change in blood pressure and velocity of blood flow occurs.

MetarteriolesShort vessel that links arterioles to capillaries, which contain smooth muscle cells a short distance apart that forms a precapillary sphincter that surrounds entrance to capillary bed.

Precapillary sphinctersBand of smooth muscle that adjusts flow into capillary.

VenulesBlood vessel in microcirculation that return blood from the capillary to the larger blood vessels.

CapillariesSmallest blood vessels and are involved in exchange of nutrients and materials, and connect arterioles to venules. Capillaries are suited as a site of exchange because of the minimal diffusion distance, maximal surface area, and the maximal time for exchange.

Capillary RecruitmentIncrease in the number of perfused capillaries in response to stimuli.

Continuous Capillaries

Type of capillary that has an uninterrupted lining in the endothelial cells, to allow water and ions to diffuse with two types of tight junctions: (1) one with many transport vesicles such as those in skeletal muscle, and (2) those with few vesicles, such as in the central nervous system.

Fenestrated Capillaries

Type of capillary with pores in the endothelial cells that allow small molecules and small proteins to diffuse, found in kidneys and exocrine glands.

Sinusoidal Capillaries

Type of capillary with larger openings in the endothelium due to a discontinuous basal lamina, which allow cells and larger proteins to enter. These are found in bone marrow, lymph nodes, liver, spleen and adrenal gland.

DiffusionMovement of particles from areas of higher concentration to areas of low concentration.

Ficks LawMathematical relationship in diffusion: , where C is concentration, P is permeability of membrane to substance, A is surface area of membrane, MW is the molecular weight of substance, and X is distance.

Concentration GradientGradual difference in the concentration of solutes in a solution between two regions. Rate of diffusion depends on the concentration gradient.

Lipid-Soluble SubstancesNonpolar substances that can dissolve in fats thus can pass through the phospholipid bilayer.

Small, Water-Soluble SubstancesPolar or charged solutes that need to be dissolved in water thus will diffuse across certain pores or gaps in cells.

Exchangeable ProteinsProteins that are shuttled across the membrane by a general vesicular mechanism of transport, such as endocytosis and exocytosis.

Plasma ProteinsProteins that are generally too large to pass through the water-filled pores thus remain in the plasma.

EndocytosisCells absorb molecules through engulfment.

ExocytosisCells direct contents of solutes out of the cell membrane via secretory vesicles.

Secondary PolycythemiaType of increase in erythrocytes either by natural or artificial causes, with a known underlying cause.

Interstitial FluidFluid that bathes and surround cells of tissue, and is the major component of extracellular fluid.

Plasma FluidLiquid component of blood where blood cells in whole blood are suspended.

Intracellular FluidFluid found inside cells, which is also known as cytosol or cytoplasm.

Bulk FlowMovement of a fluid driven by pressure.

UltrafiltrationType of membrane filtration involving hydrostatic pressure pushing against a semipermeable membrane. In this case, there is a push for fluid out of the capillary. Typically, ultrafiltration is larger than reabsorption by 3 L.

ReabsorptionType of membran filtration in which there is a uptake of fluid into the capillary.

LymphaticsOrgan system involved in filtration of lymph and reuptake of fluid that is not reabsorbed into the capillary. Consist of blind-ended capillaries that overlap the capillary system to transport fluid back into the venous system.

Net Fluid Exchange PressureContributor of bulk flow which is the difference in the outward and inward pressures, such is also influenced by derivatives of the outward and inward pressure, such as capillary pressure (PC), interstitial fluid pressure (PIF), plasma colloid osmotic pressure (P), and the interstial fluid colloid osmotic pressure (IF). Thus: .

Capillary PressureOutward force for fluid exchange.

Interstitial Fluid Exchange PressureInward force for fluid exchange.

Plasma Colloid Osmotic PressureInward Force for fluid exchange.

Interstitial Fluid Colloid Osmotic PressureOutward force for fluid exchange.

Starlings LawMathematical illustration of hydrostatic and oncotic forces to determine amount of fluid movement from blood to tissue. Calculated as: . Exchange pressure determines direction of movement, while both exchange pressure, and permeability will determine magnitude of the movement.

Lecture ObjectivesKnow the organization, anatomy, and function of the microcirculation.We need to remember that microcirculation is the site of exchange of solutes and fluid. The microcirculation consists of three general anatomical parts: the arteriole, the capillaries, and the venule. On the side of the arterioles, there are smooth muscle cells in areas such as the metarteriole and the precapillary sphincter that allow regulation of flow through the capillaries. It should also be remembered that the capillary is one-cell thick to allow a site of exchange. Learn how the exchange of solutes occurs in capillaries.The exchange of solutes between the plasma and tissue cells occurs via a mechanism known as diffusion in the capillaries. Diffusion is the movement of solutes from areas of high concentration to areas of low concentration. One can calculate the net rate of diffusion Q by Ficks Law: , where C is concentration gradient, P is permeability of membrane to substance, A is surface area of membrane, MW is the molecular weight of substance, and X is distance. Capillries are well suited as a site of exchange because of the minimal diffusion distances (low X), maximal surface area (large A), and a low flow rate (giving it a lot of time for the necessary exchange to occur, because flow is inversely proportional to cross-sectional area). However, at any particular moment, not all the capillaries are open. In skeletal muscle, only 10% of the capillaries are open. In more physical states, such as in exercise, metabolites diffuse in the precapillary sphincters and allow blood to flow into the capillaries due to dilation (relaxing) of the sphincters and arterioles. The major strategy of this is to maintain the concentration gradient. Capillary recruitment is also self-regulatory, because decreases in physical activity will cause a decrease in flow and removal of the vasodilating metabolites and cause the precapillary sphincter and arterioles to contract.

In the capillary, the exchange of solutes is known as transcapillary exchange, in which the solutes will move based on the intrinsic diffusion characteristics. Lipid-soluble substances, such as O2 and CO2, will diffuse across the membrane because they are chemically nonpolar and are able to dissolve in lipids. Other substances, particularly those that are water-soluble and small, need to be dissolved in water first, but can diffuse across gaps in cells. Remember, the rate of diffusion is dependent on the concentration gradient, size of the solute, and the polarity of the solute. With this, there are also three different types of capillaries, the (1) continuous (which is the most common) and found in skeletal muscle, skin, the (2) fenetrated, which is found in kidneys and exocrine glands for filtration purposes, and (3) sinusoidal, found in the liver, spleen, where there is necessary movement of cells and larger proteins. The capillary pore sizes vary in different tissues. In general, permeability to small solutes is greater than permeability to large molecules. However, the capillary permeability can be altered under certain conditions, such as histamine (an inflammatory response chemical released by mast cells that increases the size of the capillary pores). For larger molecules, such as proteins, diffusion is not the optimal mechanism, so vesicular mechanisms such as endocytosis and exocytosis are utilized to shuttle proteins to the interior or the exterior of the cell. So, we can summarize the factors of transcapillary exchange of small solutes in the following diagram:

Know how exchange of fluid occurs in capillaries and learn about Starlings Law.The movement of solutes occurs through the interstitial fluid, which serves as a passive intermediary. Interstitial fluid is the majority of extracellular fluid (approximately 80%) and serves a homeostatic role of the cell. Intracellular fluid constitutes the majority of fluid, but plasma fluid and interstitial fluid play a role in providing the optimal environment for cells. The exchange of fluid between capillaries and interstitial fluid typically occurs via bulk flow. In bulk flow, there are two movements: (1) ultrafiltration, in which the fluid moves out of the capillaries, and (2) reabsorption, in which the capillaries take up the fluid. Ultrafiltration is typically greater than reabsorption by approximately 3 liters a day, but the fluid is returned to the plasma by the lymphatic system, which transports the excess fluid (known as lymph) back into the venous system. Bulk flow is influenced by net fluid exchange pressure, which is an outward and inward pressure difference. Four forces determine them: capillary pressure (outward), interstitial fluid pressure (inward), plasma colloid osmotic pressure (inward), and interstitial fluid colloid osmotic pressure (outward). Thus, we can calculate the net fluid exchange pressure: . From there, we can determine the fluid movement between blood and tissue, which is known as Starlings Law/Equation, which is: . From there, we can draw two conclusions: (1) the pressure difference determines the direction of fluid movement, and (2) either fluid permeability coefficient, capillary surface area, or pressure difference will determine the magnitude of the fluid movement. Discuss how the above relate to the case study.In hereditary hemorrhagic telangiectasia, the transfer of small solutes can be locally affected at the site of the vascular lesions. Thus, the decrease in number of capillaries can spur less exchange occurring at the local areas. If there is a pulmonary arteriovenous malformation in a patient, individuals can develop secondary polycythemia, which is essentially an elevated hematocrit. This response is mainly to compensate for the low oxygen concentration in the blood. Thus, the adrenal glands secrete erythropoietin to increase carrying of oxygen to the tissues in response to the presence of the pulmonary arteriovenous malformation. In terms of pressure, one can relate the low-resistance bypass as not beneficial because they do not retain the properties that allow for optimal exchange (which is the complete opposite of a capillary). Thus there is little diffusion across the membrane in the between the capillary and lung or between the capillary and tissues. This is why patients with HHT often will present with fatigue and secondary polycythemia.

Lecture XXIVascular DevelopmentTerminologyTermDefinition

Cell DifferentiationProcess by which a less specialized cell becomes more specialized in a multicellular organism.

Cell ProliferationThe growth or production of cell by multiplication of parts

Cell MovementMovement of the cell in response to

Cell InteractionDirect interactions between cells that play a role in development and function of multicellular organisms.

Inductive InteractionInteraction between two groups of cells in which a signal passed from one group of cells causes the other group of cells to change their developmental state (or fate).

Gene ExpressionProcess by which information from a gene is used to synthesize a protein or gene product.

VasculogenesisDifferentiation of angioblasts into endothelial cells and their assembly into a primary vascular plexus. Formation of the vascular network in some organs occurs mainly by vasculogenesis.

AngiogenesisThe process by which new vessels form by sprouting or splitting of pre-existing vessels. In other organ systems, angiogenesis may be more prominent.

AngioblastsDifferentiated cell from the mesoderm formed from hemangioblasts.

MesodermPrimary germ cell layer that yields the cardiovascular system. In response to BMP, they form hemangioblast, which eventually forms parts of the vasculature.

Yolk SacMembranous sac attached to the embryo, providing nourishment to the embryo. Around the 4th week of embryonic development, the functioning vasculature is present in the yolk sac, and angioblasts develop in the blood islands.

Blood IslandsStructures in the developing embryo that can yield different parts of the circulatory system, derived from plexuses formed from angioblasts.

Primary Capillary PlexusInitial site of vascular remodeling. It is formed by vasculogenesis.

Embryonic versus Extra-Embryonic VasculatureEmbryonic: Involving angioblasts to form tubes that yield mature vessels.Extra-Embryonic: Involving angioblasts developing blood islands, which form a primary plexus to yield a mature vessel.

Vascular Endothelial Cell Growth Factor (VEGF)Signaling protein produced to stimulate vasculogenesis and angiogenesis.

Receptor Tyrosine KinaseHigh-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones. Play a critical role in binding of VEGF for angiogenesis and vasculogenesis.

TransphosphorylationMovement of one phosphate group of one molecule to another molecule. VEGF causes receptor dimerization and transphophorylation of tyrosine residues.

AngiogenesisProcess by which new vessels form by sprouting or splitting of preexisting vessels.

Sprouting AngiogenesisForm of angiogenesis in which tip cells (designated endothelial cells) extend into the extracellular matrix creating vessels.

Non-Sprouting (Intussusceptive) AngiogenesisForm of angiogenesis in which the capillary wall extends into lumen to split the vessel into two.

Activation PhasePhase in which vascular permeability and degradation of basement membrane allowing endothelial cells to migrate into the matrix to proliferate.

Resolution PhaseEndothelial cells discontinue growth and migration and allow reformation of basement membrane.

Basement MembraneSheet of fibers that underlie the endothelium of the vasculature.

Mesenchymal CellsMultipotent stem cells that can differentiate into different cells. In the presence of activated TGF-, mesenchymal cells differentiate into mural cells, which consequently differentiate into vascular smooth muscle or pericytes.

Tip CellsDesignated endothelial cells that are the point men of the activation phase. Specific inductive signaling occurs to regulation formation and migration of tip cells, particularly VEGF-A and Notch signaling.

Platelet-Derived Growth Factor (PDGF)One of many growth factos involved in regulation of cell growth and division.

Mural CellsVascular smooth mucle cells or pericytes that are involved in the formation of normal vasculature, which respond to VEGF.

Transforming Growth Factor (TGF-)Protein that controls proliferation, differentiation in of endothelial cells. Important in vessel maturation and balances proliferation and differentiation.

Postnatal AngiogenesisAngiogenesis that occurs after the birth and later development of the human, which can occur in states of hypoxia and trauma.

Lecture ObjectivesKnow some general principles of development.The general principles of development typically involve starting simple and simply refining. Vascular development involves a series of maturation and refinement, and recruitment of other cells for the associations, leading to stabilization and maturation. The remodeling and maturation then allows formation of vasculature, with tissue-specific accommodations. This process occurs in the embryo. The angioblasts, the precursor cells, become defined to move from vasculature without forming intermediate primary plexus. There are two major phases of vascular development: (1) vasculogenesis and (2) angiogenesis. Vasculogenesis is the formation of blood cells from undifferentiated cells to tube. Angiogenesis involves formation of vessels from pre-existing vessels, which can involve splitting of pre-existing vessels.

Vascular development involves the differentiation, proliferation, and migration of cells that will form the blood vessel tubes (endothelial cells). Within this process, there is a formation of a primitive network of tubes of endothelial cells known as the primary capillary plexus. Recruitment and differentiation of mural cells will yield smooth muscle cells or pericytes. From this, sprouting, branching, intussusception and remodeling occurs to form a more mature network, with eventual further maturation of blood vessels to reflect specific functions of vessel types.

These processes are mediated by cellular signaling. Multicellular animals have proteins (that are typically conserved) that are involved in the mediation of cellular interactions and signal transduction. The inductive signals are important to spur orderly differences from initially identical cells. Signals are typically involved in gene expression, meaning that the genes are in control of the developmental process, sometimes repeatedly.Learn about the process of vasculogenesis during embryonic development.Vasculogenesis is considered the first phase in vascular development. Endothelial cells are derived from precursor cells, known as angioblasts, that originate from the mesoderm. Remember that the mesoderm gives rise to the cardiovascular system. By approximately the fourth week of embryonic development, there is a functioning vasculature, and nutrients are from the placenta as well as the yolk sac. Angioblasts typicaly develop from the blood islands of the yolk sac. From this, a primary capillary plexus in the yolk sac is formed by vasculogenesis and then goes through remodeling. The larger vessel is giving rise to a smaller vessel. On top of this, the angioblasts arise in the embryo proper as well as migrate out of the mesoderm in somites and form vessels in various parts of the body, including part of the aorta. They will eventually spur formation of blood vessels. However, there is such a thing as too much of a good thing in this case. Obviously, having not enough angioblasts can spur poor formation of the cardiovascular system, but having too much can compromise the embryo due to nutrient depletion and excessive remodeling. Thus, the embryo must regulate the formation of angioblasts.

One factor that is important in vasculogenesis is known as vascular endothelial cell growth factor (VEGF). They promote angioblast formation and proliferation and allow formation of blood vessels by inductive interaction.

Inductive interaction is a stepwise interaction between two groups of cells in which a signal passed from one group of cells causes the other group of cells to change their evelopmental state (or fate). VEGF binds to a receptor tyrosine kinase, in which the binding of VEGF to the extracellular domain causes protein changes to spur a signaling cascade and desired effect. Though there are different types of VEGFs and associated receptors, there are different effects present that are dependent on the receptor (not on the factor). The receptor dictates the effect of the binding, NOT the factor. VEGF can promote vasculogenesis and angiogenesis, but also lymphangiogenesis (formation of lymphatic vessels). VEGF causes the receptor dimerization and transphosphorylation of tyrosine residues, similar to growth factors such as EGF, and causes attraction of proteins to the signaling complex as well as recruitment of different enzymes for the signaling pathway. Different effects can occur depending on the signaling pathway by VEGF. VEGF can ensure (1) cell survival, (2) gene expression and cellular proliferation, (3) cellular proliferation and vasopermability, which ultimately leads to (4) vasculogenesis and angiogenesis.

Learn about the process of angiogenesis during embryonic development and postnatal angiogenesis.Angiogenesis, as stated before, is the growth of blood vessels from preexisting vessels. The second phase of vascular development has two types: (1) non-sprouting (or intussusceptive) angiogenesis and (2) sprouting angiogenesis. They typically can either sprout from the primary plexus or be non-sprouting. Intussusceptive angiogenesis typically presents with a formation of a blood vessel pillar that splits off into two different vessels. The holes will eventually connect with each other to produce a much finer network. In some body regions (such as the limbs), the blood vessels develop during embryogenesis mainly by angiogenesis not by vasculogenesis.

Sprouting angiogenesis involves two phases: (1) Activation and (2) Resolution. Activation involves the preexisting tube to sprout out and form tip cells. In the Activation Phase, cells loosen connections and degrade the extracellular matrix to allow for proliferation and migration from tube. In this phase, vascular permeability increases, degradation of the basement membrane is permitted to allow endothelial cell migration and proliferation. Tip cells are the endothelial point men that move in the Activation Phase. The specific inductive signaling occurs to regulate formation and migration of tip cells, involving VEGF-A signaling and the Notch signaling pathway. VEGF-A is the signal that designates the endothelial cell to be a tip cell and sprout. The Notch Signaling Pathway is simply a preventive tool involving lateral inhibition of neighboring endothelial cells to prevent unnecessary sprouting in proximity of the designated tip cell. The Resolution Phase involves reformation of the extracellular matrix as well as recruitment and differentiation of mural cells to become endothelial tubes. Endothelial cells halt migration and proliferation, the cell-cell junctions are tightened, and reformation of the basement membrane occurs, allowing mesenchymal cells to be recruited to differentiate into mural cells. At this time, the pericytes (which are sparsely covered) involves the recruitment of mesenchymal cells to migrate and associate with the newly developed blood vessels. Recruitment and differentiation of mural cells to the endothelial tubes involves PDGF- secretion from the tip cells to recruit mesenchymal cells to migrate and contact endothelial cells. The cell-cell contact results in activation of latent transforming growth factor (TGF-) in the extracellular matrix. The activated TGF- causes mesenchymal cells ot differentiate into the mural cells (either vascular smooth muscle cells or pericytes). TGF- signaling is needed for formation of vascular smooth muscle cells, and also occurs in endothelial cells (in a paracrine fashion) to control balance between proliferation/migration and differentiation. Other signaling molecules are also involved in the development of new blood vessels, and their numbers can be altered depending on the body organ involved.

Postnatally, angiogenesis can also occur, such as in states of wounding or hypoxia. In states of wounding or vascular trauma, the factors are present and can allow proliferation of these factors and spur a much finer network. In states of hypoxia, VEGF is also present. Tissue cells produce a factor known as hypoxia inducible factor that is sensitive to O2 levels in the blood. Normally, HIF is inhibited due to high oxygen levels, but can be turned on in low oxygen states and allow capillary sprouting through increased secretion of VEGF.

This leads to this case study. Local hypoxia can affect the progression of arteriovenous malformation. The hypoxia is not from the decreased concentrations of oxygen in the blood, but the poor diffusion of oxygen into the tissues from a low-resistance bypass. Thus, there is increased production of VEGF and proliferation of vascular endothelial cells. However, they promote the formation of vessels called torturous vessels that worsen the arteriovenous malformation due to contortion and capillary bed destruction.Look at a few selected signaling systems (VEGF, PDGF, TGF-) that are important for vasculogenesis and angiogenesis.Growth FactorAcronymRole in Vasculogenesis and Angiogenesis

Vascular Endothelial Cell Growth FactorVEGFImportant in vasculogenesis. It binds to receptor tyrosine kinase, causing vasculogenesis, angiogenesis, or lymphangiogenesis depending on te receptor. Important in (1) cell survival, (2) cell proliferation and vasopermeability, (3) gene expression, and ultimately (4) vasculogenesis and angiogenesis.

Platelet-Derived Growth FactorPDGFSecreted by the tip cells, allow recruitment of mesenchymal cells to migrate and contact endothelial cells. Like VEGF, it acts through receptor tyrosine kinase.

Transforming Growth Factor, Type TGF-Important in vessel maturation, and allow mesenchymal cells to differentiated into mural cells.

Hypoxia-Inducible FactorHIFAllow postnatal angiogenesis in response to hypoxic states.

Lecture XXIITGF- Ligand Superfamily: Properties, Cell Biology, Physiological EffectsTerminologyTermDefinition

HHT TypesThere are five major types of HHT depending on the problem area: HHT Type I: Involves a mutation in the endoglin (ENG) gene, which is a type III TGF receptor family co-receptor. HHT Type II: Involves a mutation in the activin receptor-like kinase (ALK-1) gene, which is a Type I TGF superfamily receptor. HHT Type III: Mutation on a locus on chromosome V. HHT Type IV: Mutation on a locus of chromosome VII. Juvenile Polyposis/HHT Syndrome: Mutations in the gene for SMAD4, a transcription factor.

Activin Receptor-Like Kinase 1 (ALK-1)Receptor in the TGF Signaling Pathway that allows increases in the endothelial cell proliferation and migration.

EndoglinGlycoprotein on surface of cells that participates in the TGF signaling pathway.

TGF- SuperfamilyGroup of structurally related proteins involved in the TGF signaling pathway. They are involved in embryogenesis, cell differentiation, cell cycle arrest, and apoptosis.

Transforming Growth FactorsPolypeptide growth factors that are involved in cellular proliferation and differentiation, but have structural and functional differences.

Transformed CellsCells that have been altered to a cancer-like state due to the direct uptake, incorporation, and expression of exogenous genetic material.

Soft Agar Colony Growth AssayType of assay in which cell colonies are grown independent of attachment to a surface an essentially suspended in agar.

Anchorage-Independent GrowthA type of growth in which cells are not suspended in a solid environment (to allow determination of growth in certain factors by not being attached).

Autocrine SignalingType of signaling in which cell secretes a messenger that binds to receptors on the same cell

Paracrine SignalingType of signaling in which cell secretes a messenger that binds to receptors on a neighboring cell.

Endocrine SignalingType of signaling in which cell secretes a messenger into the bloodstream which binds to receptors on a distant cell.

ApoptosisProcess of programmed cell death by certain biochemical events.

Proteinase InhibitorsType of inhibitor involved in the repression of extracellular matrix degradation.

Extracellular MatrixPart of tissue that provides structural support to cells, consisting of the interstitial matrix and the basement membrane. It provides support, segregation, and regulation of intercellular communication, maintaining stability amid the cells dynamic behavior.

MetalloproteinasesGroup of proteinases that are classified by the most prominent functional group on their active site. They are proteolytic enzymes involving a metal in their catalytic enzyme.

2D CultureCulturing cells in flat plastic petri dishes (where the cells adhere to the surface), which allows for a 2-dimensional environment for the cells.

3D CultureCulturing cells in a suspension to provide a more physiologically relevant enevironment for the cells, without attachment.

ActivinsDimer that is involved in cellular proliferation, differentiation, apoptosis, metabolism, homeostasis, immune response, wound repair, and endocrine function.

BMPsType of growth factors that are interact with receptors on the cell surfafe that are involved in growth and differentiation.

AgonistChemical that binds to receptor of a cell that triggers a response by the cell, typically mimicking a naturally occurring susbstance.

AntagonistType of chemical that blocks a biological response when binding to a receptor, dampening cellular response.

Dorsal-Ventral PatterningType of patterning that involves compartmentalization the embryo to account for certain features at the dorsal-ventral locations.

Left-Right AsymmetryNo symmetry at the left and right side of the body.

N-terminal Signal PeptideThe n-terminus is the first part of the protein that emerges from the ribosome in translation. It has signal peptide sequences that spurs delivery to the proper organelle. The peptide is removed at the destination by a signal peptidase.

Latency Associated PeptideProtein derived rom N-terminal region of the TGF that interacts with the TGF homodimer to for a complex.

Small Latent ComplexComplex consisting of the N-terminal region of the TGF gene product and the TGF homodimer.

Large Latent ComplexLarger complex secreted in the extracellular matrix that consists of latent TGF- binding to the Small Latent Complex.

TGF- ActivationActivation of latent TGF to an active state by signaling pathways or various other factors.

Lecture ObjectivesLearn about the molecular genetics of HHT.There are several types of HHT, which can be determined via laboratory testing involving DNA markers. The mutations in the Endoglin and ALK1 are the most common cause of hereditary hemorrhagic telangiectasia. These mutations can cause alterations in splicing at the donor and receptor sites and spur a loss of function due to their scattered distribution of the key sites. Most mutations are a loss of function, possibly haploinsufficiency from a having at least one bad-coding copy in the genotype.

TypeMutation

IEndoglin (ENG) Gene

IIActivin Receptor-Like Kinase 1 (ALK-1) Gene

IIICandidate Locus on Chromosome 5

IVCandidate Locus on Chromosome 7

Juvenile Polyposis/HHT SyndromeMutations in gene for SMAD4 (transcription factor)

Learn about the discovery of TGF-.The genes that are mutated in HHT are part of the TGF- signaling pathway, in which binding to the receptor causes a signal transduction that can alter gene expression. And remember that latent TGF needs to be activated to spur the vascular development. This signaling pathway is one of many that can influence gene expression.

TGF (known as Transforming Growth Factor-) was discovered about 30 years ago around the late 1970s. It was discovered when researchers began testing the hypothesis that some tumor viruses could transform cells into a cancer-like state by inducing cells to secrete molecules that acted on the same cells, causing transformation via autocrine control. These factors were partially purified which could cause normal fibroblasts to form progressively growing colonies in soft agar with reversible effects. After isolation and purification by column chromatography and various other testing, they found two proteins that are involved: TGF- and TGF-. TGF- is structurally related to epidermal growth factor and was the factor involved in the growth. However, TGF- was the protein that had the growth-inhibitory effect, among several other effects. When grown in a 2-dimensional culture (meaning that the cells attached to the flat petri dish), there is actually an inhibition of proliferation and migration of cells. However, in the three-dimensional soft agar culture, there is the formation of tube-like structures.Learn about the diverse functions of the TGF- superfamily.The TGF- superfamily is involved in many functions:1. Cell growth (inhibition)2. Cell differentiation (promotion)3. Apoptosis (induction)4. Embryonic Development5. Wound Healing6. Vascular Remodeling7. Roles in immunity, fibrosis, cancer, heart disease, diabetes, and Marfan Syndrome

Scientists determining other functions eventually converged upon the finding of TGF-. It contributes to vascular development in the following ways:1. Regulation in the proliferation, differentiation, and migration of endothelial cells and mural cells.2. Vascular remodeling and vessel maturation3. Stimulation in the production of extracellular matrix proteins and proteinase inhibitors (repressing matrix degradation)Know the general properties of TGF- superfamily ligands.This family of proteins is often context dependent, in which the response is dependent on: (1) ligand and receptors, (2) other signals, (3) cell type, (4) signal intensity, (5) signal duration. However, experimentally with endothelial cells, there is a negative feedback mechanism associated with the context-dependent response. Low levels of TGF are associated with an enhancement of proliferation and migration of cells, while high levels of the same protein can inhibit the effect. One way to regulate the levels is through binding affinity of the receptor.

The TGF-s are often associated with embryogenesis, cell differentiation, cell cycle arrest, and apoptosis. The properties within the TGF- superfamily are similar. They are a secreted protein containing an N-terminal signal peptide (a zip code that ensures that the protein goes to the specific organelle). The C-terminal region consists of approximately 112 amino acids that become the mature TGF that is the active ligand (as a dimer). The middle portion of the TGF- encodes the latency-associated peptide. All three of the forms of TGF- are initially released from the cell in a latent/inactive form, and need to be activated to achieve an effect. There are several advantages of having the latent form:1. Inactive complex provides an opportunity for intricate regulation. Activation pathways may be cell or tissue specific.2. Differences in the LAPs provide different TGF complexes that are selective to specific stimuli.3. Signaling can be locally activated by proteolysis or by mechanical strain.Know about the process of TGF- activation from a latent state.TGF- activation involves the latency-associated protein combining with the active TGF- (in various catalysts) to form the small latent complex. This complex can make further associations, by attaching the small latent complex with latent TGF- binding protein via a disulfide bond, forming the Large Latent Complex. This Large Latent Complex can then covalently link with the extracellular matrix. The mature ligand dimerizes to form the active molecule. From there, there are several mechanisms to fully activate TGF-:1. Degradation of ECM proteins (fibrillin or LTBP) by proteases.2. Cleavage, dissociation, or conformational change in LAP.3. Integrin-dependent activation (by activating metalloproteases or by a mechanical strain-dependent form).Learn about the role of antagonists of TGF- superfamily ligands.The antagonists of the TGF- signaling can function to establish complex spatial patterns of functional activity because there is regulation. Antagonists are important to establish the dorsal-ventral patterning, such as in Noggin. Antagonists are also important for establishing left-right asymmetry. The inhibitors can be expressed in interesting patterns. The protein involved is Lefty, a TGF- superfamily member that inhibits Nodal signaling (another TGF- family member), helping to restrict Nodal expression to the left side of the embryo to push for molecular asymmetry.

Lecture XXIIITGF- Signaling: Receptors and CoreceptorsTerminologyTermDefinition

Receptor Serine/Threonine KinaseType of kinase enzyme that phosphorylates the OH group of serine or threonine, playing a role in regulation of cell proliferation, apoptosis, differentiation, and embryonic development. TGF

Catalytic ReceptorTransmembrane receptor where the binding of an extracellular ligand causes intracellular enzymatic activity.

Type I ReceptorsProtein receptor in heteromeric complex which is the main switch involved in the signal transduction.

Type II ReceptorsProtein receptor in heteromeric complex which can remain active, but needs a allosteric, conformational change in order to fully activate signal transduction.

HomodimerMacromolecular complex formed by two identical molecular subunits.

HeterodimerMacromolecular complex formed by two non-identical molecular subunits.

Constitutively ActiveType of activity in which the molecule is constant and active, but is not in appropriate conformation to phosphorylate another receptor.

Induced Proximity ModelLigand binding brings proteins together in the membrane and this close proximity allows for signaling to occur.

GS RegionRegulatory site for TGF- type I receptor. It needs to be phosphorylated by TGF- type II receptor for it to occur.

SMAD ProteinIntracellular proteins that transduce extracellular signals from TGF- ligands to the nucleus to activate downstream gene transcription. Exist in three classes: (1) Receptor-regulated, (2) Common-mediator, and (3) Antagonistic

TranscriptionProduction of a complementary RNA copy (mRNA) from a sequence of DNA.

PhosphorylationAddition of a phosphate group to a protein or another organic molecule, activating or deactivating protein enzymes.

SMAD SignalingSignaling involves transduction of extracellular signaling to the nucleus. They form a trimer or two receptor-regulated SMADs and one co-SMAD to create a transcription factor to regulate expression of certain genes.

Non-SMAD SignalingConveyance of non-SMAD signaling proteins such as p38 or MAPK, with no use of SMAD proteins.

EndoglinType I membrane glycoprotein on cell surfaces that is part of the TGF- receptor complex.

Co-Receptor (Accessory Receptor)Cell surface receptor that binds a signaling molecule in addition to a primary receptor to facilitate ligand recognition and initiate signaling.

EndocytosisProcess by which cells absorb molecules through engulfment.

Clathrin-coated PitInvolved in clathrin-mediated endocytosis, which is formed from the inward budding of plasma membrane vesicles to allow internalization of the molecules.

Lipid-RaftOrganization of glycosphingolipids and protein receptors to compartmentalize cellular processes by serving as centers for assembly of signaling molecules.

Transcription FactorsProtein that binds to specific DNA sequences to regulate transcription.

Lecture ObjectivesLearn about the different types of receptors and co-receptors in the TGF- superfamily.TypeExamples

ITRI (ALK5), ActRIB (ALK4), ALK 7, ALK1, ALK2, ALK3, ALK6

IITRII, ActRII, ActRIIB, BMPRII, MISRII

Generally there are two types of receptors: Type I and II. In the signaling pathway, be sure to remember that Type II must be activated in order for activation of Type II to occur. They are quite structurally similar, and can only be differentiated by peptide mapping. They form homodimers and heterodimers. The number of ligands very much exceeds the number of receptors, so there can be convergence on signaling, particularly to a receptor. Consequently, there are combinatorial interactions, or diverse responses by small numbers of receptors and communication. The significance is simply because there is one way for cells to generate the capacity for diverse responses derived from a small number of ligands and receptors (with TGF- superfamily responding to a specific the TGF- receptor). It also allows for crosstalking between different signals. However, cells need to respond appropriate to the signal.Know how signal transduction occurs through the receptors.The general signaling pathway to understand is that it is activated by an extracellular ligand. The receptors involved in TGF- Signaling typically involved Receptor Serine/Threonine Kinases, which are consequently involved serine/threonine phosphorylation, which has intrinsic catalytic activity. The pathway generally involves several steps:1. TGF- brings together two Type I Receptors with Type II Receptors.2. Type II Receptors phosphorylate and activate type I receptors.3. The R-SMAD proteins bind to Type I Receptors to be in complex with SARA.4. Type I receptor phosphorylates R-SMAD, promoting dissociation from the kinase and SARA.5. The product binds to a Co-SMAD.6. SMAD hetero-oligomer enters the nucleus.7. The SMADs associate with other DNA-binding proteins to activate or inhibit transcription of specific genes.

The TGF- dimer binding results in a heterotetrameric complex of receptor subunits. The receptos essentially cluster to form heterotetrameric complex, consisting of 4 receptors and 2 ligand molecules [4+2]. A jigsaw arrangement can explain the specificity of the TGF- superfamily to the TGF- receptor. It is simply a stepwise, allosteric, cooperative mechanism. When the Type II receptor binds to TGF, it allows a conformation that allows the Type I receptor to fit, yielding the TGF- receptor complex. Essentially, the Type I receptor binds much more favorable in the presence of another factor (Type II), because the receptor affinity is higher for Type I after Type II enters. This is not necessary for other proteins such as BMP, which binding and activation of the complex can occur simultaneously. The system is to bring the Type I and II receptors for an intimate embrace. The complex puts the cytoplasmic kinase domains in a catalytically favorable orientation. This system is also associated with the induced proximity model, in which the ligand binding causes proteins to move closer together in the membrane and this close proximity causes the signaling to occur. The phosphorylation of the GS region of the TGF- 1 receptor activates the kinase. The GS region is the regulatory factor that interferes with the kinase. The GS region will move out of the way when the serines of the Type I receptor are phosphorylated. Type II does not have the GS, but Type I needs to move the GS region to cause the desired activation. Phosphorylation allow allows the type I receptor to phosphorylate SMAD proteins, and the phosphorylation of the SMAD proteins allow SMADs to accumulate in the nucleus. Phosphorylation is used both to activate Type I receptors and SMAD proteins, and thus one can conclude that phosphorylation is the mechanism to activate or deactive proteins or recruit proteins to a complex.

Endoglin has been noted as a co-receptor that is abundant in the vascular endothelial cells, mainly to potentiate signaling through the Type I and Type II receptors, but signaling can occur without the co-receptor. Endoglins function still remains a clinical mystery, but co-receptors play a role in endocytosis (especially in clathrin-mediated uptake) of TGF- receptors, and the signaling may be ongoing.Know the difference between SMAD and non-SMAD signaling via the TGF- pathway.TGF- signaling typically involves SMAD-mediated responses, but non-SMAD responses have also been noted. Some receptors act in other ways without utilizing SMAD, and was discovered through observation of rapid effects that were not involving SMAD proteins. This may allow for a bypass mechanism to allow for a desired effect in the event of faulty SMAD signaling.Discuss the parameters that determine what kind of response is elicited in cells during signaling.Now this leads to another question: what determines what kind of response will be elicited by TGF- superfamily ligands in any particular cell? Experiments and studies have lead to these factors:1. Ligands2. Receptors and Co-Receptors3. SMADs4. Cell type-specific cooperating transcription factors

Lecture XXIVTranscriptional Regulation in PhysiologyTerminologyTermDefinition

Central Dogma of Molecular BiologyFramework of comprehsnion in the transfer of information between DNA, RNA, and protein among living organisms, in which DNA can yield RNA, which then yields protein. However, this does not happen in the reverse, generating a one-way flow of information.

Differential ExpressionDifferences among cell expression even though the cells have the same DNA, mainly because they express different parts of the DNA.

Messenger RNA (mRNA)Molecule of RNA that encodes information for the protein product.

Ribosomal RNA (rRNA)RNA component of the ribosome, providing the mechanism for decoding mRNA into amino acids and interacts with tRNAs during translation.

Transfer RNA (tRNA)Adaptor molecule composed of RNA utilized in bridging genetic code in mRNA.

Small, noncoding RNAsShort ribonucleic acid molecule in eukaryotes that are regulatory involved in post-transcriptional regulation.

Transcription UnitStretch of DNA transcribed into an RNA molecule to allow for eventual translation to protein.

Transcriptional ControlRegulation of gene expression by controlling number of RNA transcripts of a region of DNA. Major regulatory mechanism of protein synthesis.

Acute RegulationProcesses that need to be regulated in the range of seconds to minutes involving changes in protein activity, brecause these changes can occur rapidly.

Long-Term RegulationProcesses that are regulated over a longer time period typically involving transcriptional regulation.

RNA Polymerases (I, II, III)Enzyme that produces RNA. There are three types: RNA Polymerase I: Involved in the transcription of DNA to rRNA RNA Polymerase II: Involved in the transcription of DNA to mRNA or snRNA. RNA Polymerase III: Involved in the transcription of DNA to tRN, 5S rRNA, or 7S RNA of signal recognition particle.

Transcription CycleProcess by which the DNA transcribes an RNA product. Contains three phases: (1) Initiation, (2) Elongation, and (3) Termination

Initiation PhasePhase of transcription where promoters designate area and allow binding of RNA polymerase to DNA promoter.

Elongation PhasePhase of transcription where DNA template strands allows for the synthesis of an RNA copy.

Termination PhasePhase of transcription consisting of the halting of RNA synthesis and release of mRNA.

Gene PromoterRegion of DNA that facilitates transcription of a gene.

TATA BoxDNA sequence found in promoter region of genes. Part of the promoter sequence, it is the binding site of general transcription factors and involved in transcription.

Initiator ElementDNA sequence element that overlaps a transcription start site and allow determination of start site location in a promoter and enhancing strength of a promoter with a TATA box.

Basal PromoterPromoter elements that can direct low levels of transcription, but are insufficient for full expression of a gene.

Preinitiation ComplexLarge complex of proteins necessary for transcription of protein-coding genes.

General Transcription FactorsTranscription factors involved in the transcription of class II genes to mRNA templates.

(TATA Box Binding Protein ) TBPGeneral transcription factor that binds to TATA box.

Closed ComplexComplex state of a protein where the aqueous environment is isolated with the non-aqueous.

Open ComplexComplex state of a protein where the protein opens, allowing for aqueous and non-aqueous environment to interact. In this case, the open complex allows the transcription start site to be unpaired and allows exposure to the template strand to allow nucleotides to join.

Conformational ChangeChange in the proteins structure in response to the environment or other factors.

Promoter Proximal ElementsShort regions of DNa involved for constitutive expression, or regulated expression.

Enhancer ElementsShort region of DNA that can be bound with proteins to enhance transcription levels of genes, which work by increasing the rate of initiation from a basal promoter.

Gene-Specific Transcription FactorsDiverse protein involved in gene regulation that are specific to a seuqnece of DNA.

Chromatin StructureCombination of DNA nad proteins that make up the contents of the nucleus of a cell. Its function is to package DNA to a smaller volume to fit into the cell and to strengthen DNA to allow cell division and control gene expression and DNA replication.

5 to 3 DirectionChemical orientation of a strand of nucleic acid, with the start designated as 5 and the end as 3.

Pausing and EditingProcess in elongation phase consisting of halting transcription to allow replacement or removal of nucleic acid.

mRNA CappingWith the help of enzymes, a process in which a cap is added to the 5 end of the nascent transcript, allowing protection against degradation and to promote translation.

Poly-A TailString of adenines added to the 3 end of the transcript to designate end of sequence.

mRNA ExportRelease of mRNA from nucleus through nuclear pore complex.

Nuclear Pore ComplexPore in the nucleus in which the nascent strand of mRNA is release into to arrive at the cytoplasm.

Lecture ObjectivesKnow the difference between transcriptional and non-transcriptional regulation of gene expression.In order to understand transcriptional and non-transcriptional regulation of gene expression, we need to first understand the elements of transcription. Transcription is simply the generation of mRNA from DNA, which (in eukaryotes) occurs in the nucleus. It is one of the processes within the Central Dogma, in which DNA synthesizes RNA, and RNA consequently produces a protein. This one-way flow of information is key to the biological processes at a unicellular and multicellular level. From this, researchers eventually discovered mRNA, tRNA, rRNA, miRNA, and snRNA. All cells have the same genes (as ingrained in the form of DNA), but the cells are specialized due to the expression of different sets of genes.

Expression of genes is different in each type of cell. Some are on all the time (constitutively), or only on at certain times. It can be also expressed in a cell-type specific manner. At times, some are expressed at high levels, while other are expressed at lower levels, and even some are expressed at higher levels only when specific cell surface receptors or cytoplasmic receptors are activated by ligand binding.

Now, to discuss the genetic material, there are two major types of genetic material: DNA and RNA. DNA is a double helix with deoxyribose sugars consisting of one side with a strand of nucleotides while the other has a strand of the complementary sequence, according to Watson-Crick base pairing. RNA is a single-stranded molecule with a ribose sugar and contains uracil instead of the thymine in DNA. There are several types of RNA, but only one type of DNA. The types of RNA are listed at the table below:Type (Abbreviation)Description% Of RNA

Messenger RNA (mRNA)Major encoding element for proteins.< 10

Ribosomal RNA (rRNA)Major component of ribosomes.75

Small Stable RNAsEncompass RNAs that are mainly adaptor proteins. Transfer RNAs (tRNAs) are involved in protein synthesis. Small Nuclear RNAs (snRNAs) are involved in RNA splicing, regulation of transcription15

Small noncoding (ncRNAs) or microRNAs (miRNA)Regulatory RNAs involved in controlling gene expression.Small.

The strand of DNA that is being transcribed is typically referred to as the transcription unit. After transcription occurs, the mRNA is produced, but undergoes editing and splicing (to remove introns will keeping the exons at certain splice sites) to yield the mature mRNA. Transcriptional control is especially crucial to prevent expression of possible mutations. Transcriptional regulation is altering the rate of gene expression by altering the rate of transcription. Non-transcriptional regulation is associated with the rate of gene expression by altering the protein activity, typically after mRNA has been synthesized. For example, non-SMAD mediated responses dont involve changes at the level of transcription, so it can be considered a non-transcription regulation.Learn about acute responses versus longer-term responses.Acute regulation typically encompass processes that need to be regulated in the range of seconds to minutes that involve changes in the protein activity, because these changes that can occur rapidly. Altering the environment of the protein to chemical phosphorylation of isomerization can affect the proteins activity. Long-term regulation involves processes that are regulated over a longer time period, typically involving transcriptional regulation. There are several advantages of this. One, it is energetically efficient, only producing a protein when it is needed. Second, it allows the cell to switch expression of one type of gene to another, particularly during development or differentiation. Finally, it can also do large-scale expression of a whole network of genes involved in a particular pathway or process.Know the basics about the basal transcription machinery. Learn about TFIID, TBP, and TATA boxes.In order to understand the machinery, it is necessary to comprehend the various parts involved. Recall that RNA polymerase is involved in the synthesis of RNA from DNA. However, there are three types of RNA. RNA polymerase I is involved in the synthesis rRNA, and RNA polymerase III is involved in the production tRNA, 5S rRNA, and 7S RNA of signal recognition particle. RNA Polymerase II is the enzyme of interest for transcription, which yields mRNAs and some snRNAs. RNA Polymerase II is a large multiprotein complex with a repeptive carboxyl-terminal domain. The conserved portions of the residues are located in the inner surfaces of the enzymes. The structure of the enzyme contains a cleft for the DNA and a site for nucleotides to enter, as well as a channel for newly synthesized RNA to exit.Know the transcription cycle.The transcription cycle is a process creating RNA from DNA. It contains three steps: (1) Initiation, (2) Elongation, and (3) Termination. Among these steps (which are all regulated), initiation is the most regulated step. Prior to Initiation, a pre-initiation complex must be formed because RNA polymerase II on its own cannot initiate transcription from promoters. Consequently, factors, known as general transcription factors, are required to form a complex. For RNA Pol II, there are more than 20 proteins. Though there are other factos involved in this complex, the first one to go in is transcription factor IID (TFIID). The binding of TBP to the TATA box leads to a pronounced bend in the DNA and recruitment of other factors.

Initiation of transcription (the Initiation Phase) involves a shift of the RNA Polymerase/DNA Complex from a closed complex to an open complex conformation. This conformation change in the polymerase causes a region of DNA around the transcription start site to become unpaired, allowing exposure of the template strand. Consequently the first ribonucleotides are joined. At this point, several sequences, such as promoters, proximal promoter elements, and enhancers, are also add to spur effects on the rate of initiation from the basal promoter.

The Elongation Phase involves the clearance of promoter that needs to occur before synthesis begins. The transcript is extended in a 5 to 3 direction at a rate of approximately 30-100 nucleotides per second by the following chemical reaction: . During this process, pausing and editing may occur. As the new (or nascent) transcript emerges, a special cap structure is added on to the 5 end. This cap help to protect against degradation and later will promote translation of the message in the cytoplasm. Termination of transcription (the Termination Phase) involves RNA Polymerase II reaching the end of the gene at the 3 end. Once this occurs, the RNA gets cleaved at the polyadenylation site and a poly-A tail is added to the transcript. The polymerase may continue transcribing for a while but soon falls off. After splicing occurs, the mature mRNAs are exported from the nucleus through the nuclear pore complex.Be aware of the role of promoters, proximal promoter elements, and enhancers.Promoters are the sum of DNA sequences necessary for transcription initiation. Other elements include a TATA box and initiator element, which is found near the start site of transcription, and consequently part of the basal promoter. Basal promoter elements can direct low levels of transcription, but are insufficient for full expression of a gene, so (as a result) promoters also contain promoter proximal elements that are needed for constitutive expression or regulated expression. Another group of sequences, known as enhancers, typically increase the rate of initiation from a basal promoter. They can be distant from the start site, and exhibit flexibility (allowing itself to work in different positions or orientations), and contain clusters of regulatory elements. Such regulation can have an effect on gene-specific transcription factors as well as chromatin structure. The following table summarizes promoters, proximal promoter elements, and enhancers.

SequenceDiagramDescription

PromoterLoosely defined as the sum of DNA sequences necessary for transcription initiation. TATA box and initiator element are found near start of transcription as part of a basal promoter.

Proximal Promoter ElementsNeeded for constitutive/regulated expression.

EnhancersFlexible part of the sequence that can be far from the start site that increase the rate of initiation from a basal promoter. They can contain clusters of regulatory elements.

Lecture XXVStrategies for Transcriptional Regulation in EukaryotesTerminologyTermDefinition

Promoter proximal elementsProximal sequence upstream of the gene that tends to contain primary regulatory elements.

Enhancer ElementsElements that increase the rate of initiation from a basal promoter, which can be distant from start site and contain clusters of regulatory elements.

Insulator ElementsGenetic boundary eleent that is an enhancer-blocking element or a barrier against condensed chromatin proteins spreading onto active chromatin.

Locus Control RegionsRegions defined by their ability to enhance the expression of linked genes.

Gene Specific Transcription FactorsBinding sites for proteins allow differential control of gene transcription.

Major GrooveType of groove seen in DNA structure containing the nitrogen and oxygen atoms of the base pairs pointing inward toward the helical axis. It is more dependent on base composition and may be the site for protein recognition of specific DNA sequences or regions.

Hydrogen BondingAttractive interaction of a hydrogen atom with an electronegative atom.

Combinatorial ControlComplex regulatory regions (in enhancers and promoters) are constructed from different combinations of simple regulatory molecules.

SynergyTwo or more elements functioning together to produce a result not independent obtainable.

CooperativityBehavior observed in enzymes and receptors that have multiple binding sites where the affinity of the binding sites for a ligand is increased or decreased as a consequence of binding of the ligand to the receptor.

Mediator ComplexMultiprotein complex that functions as a transcriptional coactivator.

Lambda RepressorSwitch in the lifecycle of a bacteriophage responsible for maintenance of lambda phage. It binds to operator associated with the RNA polymerase promoter to prevent RNA polymerase from initiating transcription, and cannot enter the lytic cycle.

DNA Binding DomainIndependently folded protein domain that contains at least one motif that recognizes DNA.

Activation DomainProtein domains involved in the formation and stability of the preinitiation complex, activating transcription.

RepressorsDNA-binding protein that regulates expression of one or more genes by binding to the operator and blocking attachment of RNA polymerase to the promoter, blocking transcription.

CompetitionMethod of utilizing transcription factors as repressors by competitive DNA binding.

MaskingMethod of utilizing transcription factors as repressors by masking the activation surface.

De Novo SynthesisSynthesis of complex molecules from simple molcules.

CoactivatorProtein that increases gene expression by binding to an activator containing the DNA binding domain. Coactivators cannot bind DNA by itself.

Histone AcetyltransferaseEnzymes that acetylate conserved lysine amino acids on histone proteins.

CorepressorSubstance that inhibits the expression of genes, by indirect means (interaction with repressor proteins that in turn bind to the promoter)

Gene SilencingThe deactivation of a gene by a mechanism other than genetic modification, meaning that it would be expressed under normal circumstances.

DNA MethylationAddition of a methyl group to the 5 position of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring on DNA.

Histone MethylationModification of certain amino acids in a histone protein by addition of one, two, or three methyl groups.

Genetic ProgramPhysiological change brought about by a temporal pattern of activation of a particular subset of genes.

Network MotifsConnectivity patterns that occur more often in comparison to random networks.

Lecture ObjectivesKnow the different types of DNA regulatory elements that control transcription of a gene.Regulatory ElementIllustrationFunctionCharacteristics

Promoter ProximalProximal sequence upstream of the gene that tends to contain primary regulatory elements.Can be within 300 base pairs upstream or downstream of the basal promoters.

EnhancerElements that increase the rate of initiation from a basal promoter, which can be distant from start site and contain clusters of regulatory elements.Can be distances away from the start site. They will work in different positions or orientations. They also contain clusters of regulatory elements.

InsulatorGenetic boundary element that is an enhancer-blocking element or a barrier against condensed chromatin proteins spreading onto active chromatin.Insulators are involved in the protections of regions of a chromosome from the effects of neighboring regions. They suppress activation of one gene, so that the enhancer only affects a specific gene.

Locus Control RegionsRegions defined by their ability to enhance the expression of linked genes.Locus control regions are short regions of DNA rich in binding sites for transcription regulators, which create open chromatin promoting the expression of nearby genes. These regions can be important in regulating the expression of a cluster of nearby genes, so that they are expressed in the correct order during development or in the current location in the embryo. All these regulatory elements contain binding sites for proteins called gene-specific transcription factors.

It is important to remember that all cells have the sam