BIOLOGICAL ACTIONS OF ENDOTHELIUM

Presenter : Dr. AnuPriya J

SCHEME

• Introduction• History • Structure • Biological actions of endothelium

-Transport functions-Vascular tone-Host defence-Growth factors-Haemostasis-Angiogenesis

• Role of endothelium in disease

INTRODUCTION

• Endothelial cells are mesodermal in origin

• Forms an interface between circulating blood or lymph in the lumen and the rest of the vessel wall.

• Also has various important biological functions.

• Key determinants of health and disease in blood vessels.

HISTORY

• 17th century – William Harvey – circulation of blood

• 19th century – new view of the circulatory system – tissues and cells

• Friedrich von Recklinghausen – Virchow’s assistant – credited with establishing a method for staining lines of cell junctions with silver

• Endothelium – first described by Virchow in capillaries as a simple membrane with flattened nuclei

• Swiss Anatomist Wilhelm His – introduced the term endothelium.

HISTORY

• Waldeyer – suggested restricting the term to those cells that make up the innermost layer of blood and lymph vessels & the posterior lining of the cornea.

• 20th century textbooks – Gray’s anatomy, A.A. Bohm and Colleagues’ textbook of Histology and other books.

• Newer advances – implications of technology – microscopy and cell culture.

• ENDOTHELIUM IN ACTION – NOT JUST A COVERING.

STRUCTURE

• The cells that form the endothelium are called endothelial cells.

• Endothelial cells in direct contact with blood are called vascular endothelial cells, whereas those in direct contact with lymph are known as lymphatic endothelial cells.

• The epithelial lining of the vascular system

• Almost always simple squamous epithelium with some exceptions

STRUCTURE

• Endothelial cells are very flat, have a central nucleus, are about 1-2 µm thick and 10-20 µm in diameter.

• The cells are typically flat and elongate, with their long axes oriented parallel to the direction of blood flow in the artery.

• Nuclei of endothelial cells are also elongated in the direction of blood flow.

STRUCTURE

• The cytoplasm is relatively simple with few organelles, mostly concentrated in the perinuclear zone.

• The most obvious feature is the concentration of small vesicles (pinocytotic vesicles)

• Weibel-Palade bodies : Rod like cytoplasmic inclusions –electron dense structures which contain Von WillebrandFactor(vWf)

• Endothelial cells are connected by adherens, tight and gap junctions.

• Fenestrated – glomeruli, capillaries of endocrine glands

• Sinusoidal – liver sinusoids.

Biological actions

• Maintenance of extracellular matrix

• Transport function

• Pro and antithrombotic

• Defense mechanism

• Vascular tone

• Regulation of cell growth

• Angiogenesis

Maintenance of extracellular matrix

Synthesis of

• Basal lamina – type IV collagen, laminin

• Glycocalyx – proteoglycans

Transport function

• Maintenance of selective permeability barrier

• Simple diffusion – oxygen, carbon dioxide.

• Active transport – glucose, amino acids, electrolytes.

• Pinocytosis – water, small molecules, soluble proteins.

• Receptor mediated endocytosis (clathrin dependent process)

– LDL, transferrin, growth factors, antibodies, MHC complexes.

Regulation of immune responses

• Secretion of interleukins (IL-1,IL-6,IL-8)

• Expression of MHC II molecules

• Cell adhesion molecules and their receptors expressed on the endothelial surface.

• Leucocyte rolling

• Smooth surface of endothelium - glycocalyx, a proteoglycancoat

• Negative charge on the surface of endothelial cells –glycosaminoglycans - mainly heparan sulfate

• Anticoagulants and antithrombotic substances present on intact endothelial surface.

• Prothrombotic substances released from damaged endothelium.

Pro and antithrombotic

Pro and antithrombotic

Secretion of

• Anticoagulants – thrombomodulin, tissue factor pathway inhibitor & others

• Antithrombogenic agents – prostacyclin, tissue plasminogenactivator, antithrombin III, heparin

• Prothrombogenic agents (released after damage to the cells) – tissue thromboplastin, von willebrand factor, plasminogenactivator inhibitor.

Regulation of cell growth

Secretion of

• Growth stimulating factors – PDGF, GM-CSF,

G-CSF, M-CSF,FGF

• Growth inhibiting factors – Heparin, TGF-β

Vascular tone

Secretion of

• Vasoconstrictors – endothelin, angiotensinconverting enzyme.

• Vasodilators – endothelium derived relaxing factor/nitric oxide, prostacyclin

Nitric oxide

Nitric oxide

o Nitric oxide synthase – 3 isoforms (NOS 1, NOS 2, NOS 3).

o NOS 3 – present in endothelial cells.

Functions

• Vasodilation

• Inhibits platelet aggregation

• Inhibits transcription of adhesion molecules

• Inhibits vascular smooth muscle proliferation

Stimulus

• Shear stress, Inflammation ,acetylcholine, substance P, bradykinin, serotonin, endothelin, thrombin

Inactivated by hemoglobin

Action lasts for 3-6 s

Prostacyclin - actions synergistic to NO

Carbon monoxide – acts in the same manner as NO

Endothelin

• Endothelial cells produce endothelin-1 (ET1)

• Potent vasoconstrictor

• Secretion – regulated via transcription of ET1 gene

Preprohormone

Big endothelin 1

ECE

Endothelin 1

• ECE – Endothelin converting enzyme

Endothelin

Endothelin

• ETA receptor – Specific to ET-1

– Found in many tissues

– Mediates vasoconstriction produced by ET-1

– Acts via cAMP

• ETB receptor – responds to all three endothelins

– coupled to Gi

– Mediates vasodilation

• Vasculogenesis – formation of new blood vessels denovo – embryonic life – angioblasts from mesoderm

• Angiogenesis – formation of new blood vessels from pre-existing blood vessels - Stimulants : pregnancy, hypoxia, inflammation, trauma, tumors

Angiogenesis

Angiogenesis

• Facilitated by vascular endothelial growth factor(VEGF).

• Also by Angiogenin, FGF,α5β1 integrin

• VEGF is produced by many cell types including tumorcells, macrophages, platelets, keratinocytes and renal mesangial cells.

• Release stimulated by HIF produced by hypoxic cells.

• Regulation of vascular cell growth in the placenta, wound healing, tissue repair and tumor growth.

APPLIED ASPECTS

• Animal experiment – inhibit NOS – prompt rise in BP

This suggests – tonic release of NO – necessary to maintain BP

• Nitroglycerin & other vasodilators – stimulate guanylyl cyclase - treat angina

APPLIED ASPECTS

ASPIRIN

- Irreversible inhibition of cyclooxygenase

- Reduction of both TXA2 and PGI2

- PGI2 – produced by endothelial cells

- TXA2 – produced by platelets

- Endothelial cells produce new COX in a matter of hours

- Platelets cannot manufacture the enzyme COX – the level rises only after new platelets enter the circulation (4-8 days)

- Therefore, aspirin – reduces clot formation

- Prevent MI, Unstable angina, TIA, Stroke

APPLIED ASPECTS

• Topical VEGF, PDGF and FGF help in wound healing.

• Inhibitors of VEGF, PDGF and FGF → treat cancer -prevent angiogenesis - stop or slow the growth or

spread of tumors.

• Colony stimulating factors injected - hemopoiesis

Atherosclerosis

• Damaged endothelium – free radicals

• Free radicals + LDL – Oxidized LDL

• Oxidized LDL + macrophages – foam cell

• Nitric oxide – reduced

Endothelium in cardiovascular disease

• Secretion of endothelin is increased by a variety of stimulants including hypoxia, catecholamines and angiotensin II.

• Activity of nitric oxide is blunted.

• Plasma concentration of endothelin is elevated and the levels correlate with hemodynamic disturbance.

• The major source of circulating endothelin in heart failure is the pulmonary vascular bed.

Endothelium in cardiovascular disease

Endothelium in cardiovascular disease

• Endothelin-1 is a potent vasoconstrictor and mitogen that binds to endothelin A and B receptors in the pulmonary vasculature.

• Acute intravenous administration of endothelin antagonists improved hemodynamics in patients with heart failure.

• Oral endothelin antagonists are being developed.

Smoking

• Nicotine - opens up the intercellular junctions and allow large molecules to pass through the wall.

• Such toxins can potentiate degenerative changes in blood vessels and lead to vascular disease.

Clinical Assessment of Endothelial Function

• Endothelial function can be assessed invasively using acetylcholine, which induces endothelium-dependent dilation and smooth muscle–mediated constriction.

• In healthy coronary arteries, endothelium-dependent dilation predominates. In the presence of endothelial damage, vasoconstriction predominates.

• The coronary artery diameter is compared by quantitative angiography before and after infusion of acetylcholine.

• Non invasive method - High-resolution ultrasound to measure the brachial artery diameter in response to reactive hyperemia.

• Close correlation between endothelial dysfunction in the forearm and coronary endothelial dysfunction.

• Endothelial function correlates inversely with serum

C-reactive protein (CRP).

• Endothelial cell activation leads to increased expression of inflammatory cytokines and adhesion molecules

• E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and P-selectin.

Clinical Assessment of Endothelial Function

• VENDY’S ENDOTHELIAL FUNCTION MEASUREMENT VIDEO

References

• Ganong's Review of Medical Physiology, 24th Edition

• Guyton and Hall Textbook of Medical Physiology, 12th Edition

• Histology: A Text and Atlas Michael H. Ross, 4th Edition

• Kumar and Clark's Clinical Medicine, 8th Edition

• Robbins and Cotran Pathologic basis of disease, 7th Edition

• Best & Taylor's Physiological Basis Of Medical Practice, 13/ E.

• Internet references

You are only as old as your endothelium-- Paul VanHoutte, Mayo Clinic (1983 )

• http://circ.ahajournals.org/content/115/10/1285.full

•http://circ.ahajournals.org/content/1 09/23_suppl_1/III-27.full

Atherosclerosis

• An active role in supplying nutrients to the subendothelial structures.

(note : tunica intima and media – blood supply from blood in vessel lumen

tunica adventitia – has its own blood vessels –k/a vasa vasorum in artery)

• Nitric oxide prevents oxidative modification of low-density lipoprotein (LDL) cholesterol.9 Oxidation of LDL has been proposed as a major mechanism of the atherosclerotic process;10furthermore, plasma and macrophage content of oxidized LDL in coronary plaques correlate with severity of acute coronary syndrome.11 Conversely, impaired production or activity of NO leads to events or actions that promote atherosclerosis, such as vasoconstriction, platelet aggregation, smooth muscle cell proliferation and migration, leukocyte adhesion, and oxidative stress.12 Oxidized LDL cholesterol increases synthesis of caveolin-1, which inhibits production of NO by inactivating eNOS.2 Oxidative stress can also interfere with the production and activity of NO by a number of mechanisms that are independent of LDL. For example, the free radical superoxide anion rapidly inactivates NO and destroys tetrahydrobiopterin, a cofactor required for NO synthesis.13

• The blood–brain barrier (BBB) is a highly selective permeability barrier that separates the circulating blood from the brainextracellular fluid (BECF) in the central nervous system (CNS). The blood–brain barrier is formed by capillary endothelial cells, which are connected by tight junctions with an extremely high electrical resistivity of at least 0.1 Ω⋅m. The blood–brain barrier allows the passage of water, some gases, and lipid soluble molecules by passive diffusion, as well as the selective transport of molecules such as glucose and amino acids that are crucial to neural function. On the other hand, the blood–brain barrier may prevent the entry of lipophilic, potential neurotoxins by way of an active transport mechanism mediated by P-glycoprotein. Astrocytes are necessary to create the blood–brain barrier. A small number of regions in the brain, including the circumventricular organs (CVOs), do not have a blood–brain barrier.

• The blood–brain barrier occurs along all capillaries and consists of tight junctions around the capillaries that do not exist in normal circulation.[1] Endothelial cells restrict the diffusion of microscopic objects (e.g., bacteria) and large or hydrophilic molecules into thecerebrospinalfluid (CSF), while allowing the diffusion of small hydrophobic molecules (O2, CO2, hormones).[2] Cells of the barrier actively transport metabolic products such as glucose across the barrier with specific proteins.[citation needed] This barrier also includes a thick basement membrane and astrocytic endfeet.[3]

Guyton

• in Formation of New Blood Vessels .Three of those that have been best characterized are vascular endothelial growth factor (VEGF), fibroblast growth factor, and angiogenin, each of which has been isolated from tissues that have inadequate blood supply.

• Presumably, it is deficiency of tissue oxygen or other nutrients, or both, that leads to formation of the vascular growth factors (also called "angiogenic factors").

• Essentially all the angiogenic factors promote new vessel growth in the same way. They cause new vessels to sprout from other small vessels. The first step is dissolution of the basement membrane of the endothelial cells at the point of sprouting. This is followed by rapid reproduction of new endothelial cells that stream outward through the vessel wall in extended cords directed toward the source of the angiogenic factor. The cells in each cord continue to divide and rapidly fold over into a tube. Next, the tube connects with another tube budding from another donor vessel (another arteriole or venule) and forms a capillary loop through which blood begins to flow. If the flow is great enough, smooth muscle cells eventually invade the wall, so some of the new vessels eventually grow to be new arterioles or venules or perhaps even larger vessels. Thus, angiogenesis explains the manner in which metabolic factors in local tissues can cause growth of new vessels.

Guyton

• Certain other substances, such as some steroid hormones, have exactly the opposite effect on small blood vessels, occasionally even causing dissolution of vascular cells and disappearance of vessels. Therefore, blood vessels can also be made to disappear when not needed. Peptides produced in the tissues can also block the growth of new blood vessels. For example, angiostatin, a fragment of the protein plasminogen, is a naturally occurring inhibitor of angiogenesis. Endostatin is another antiangiogenic peptide that is derived from the breakdown of collagen type XVII. Although the precise physiological functions of these antiangiogenic substances are still unknown, there is great interest in their potential use in arresting blood vessel growth in cancerous tumors and therefore preventing the large increases in blood flow needed to sustain the nutrient supply of rapidly growing tumors. Vascularity Is Determined by Maximum Blood Flow Need, Not by Average Need An especially valuable characteristic of long-term vascular control is that vascularity is determined mainly by the maximum level of blood flow need rather than by average need. For instance, during heavy exercise the need for whole body blood flow often increases to six to eight times the resting blood flow. This great excess of flow may not be required for more than a few minutes each day. Nevertheless, even this short need can cause enough VEGF to be formed by the muscles to increase their vascularity as required. Were it not for this capability, every time that a person attempted heavy exercise, the muscles would fail to receive the required nutrients, especially the required oxygen, so that the muscles simply would fail to contract. However, after extra vascularity does develop, the extra blood vessels normally remain mainly vasoconstricted, opening to allow extra flow only when appropriate local stimuli such as oxygen lack, nerve vasodilatory stimuli, or other stimuli call forth the required extra flow.

• Tm interaction with vasculature switch to angiogenicphenotype, enabling tumor progression

Angiogenic Cascade

- Endothelial receptor binding /

activation

- Formation of angiogenic mother

vessels

- Morphogenesis of mother vessels

- Basement membrane dissolution

- Endothelial cell proliferation

- Endothelial cell migration

- Vascular tube formation

- Arterial-venous differentiation

- Vascular stabilization

Guyton

• When blood flows through the arteries and arterioles, this causes shear stress on the endothelial cells because of viscous drag of the blood against the vascular walls. This stress contorts the endothelial cells in the direction of flow and causes significant increase in the release of NO. The NO then relaxes the blood vessels. This is fortunate because the local metabolic mechanisms for controlling tissue blood flow dilate mainly the very small arteries and arterioles in each tissue. Yet, when blood flow through a microvascular portion of the circulation increases, this secondarily stimulates the release of NO from larger vessels due to increased flow and shear stress in these vessels. The released NO increases the diameters of the larger upstream blood vessels whenever microvascular blood flow increases downstream. Without such a response, the effectiveness of local blood flow control would be decreased because a significant part of the resistance to blood flow is in the upstream small arteries. NO synthesis and release from endothelial cells are also stimulated by some vasoconstrictors, such as angiotensin II, which bind to specific receptors on endothelial cells. The increased NO release protects against excessive vasoconstriction.

CORNEAL ENDOTHELIUM

• Misnomer here • a simple squamous or low cuboidal monolayer of mitochondria-rich cells

responsible for regulating fluid and solute transport between the aqueous and corneal stromal compartments. (The term endothelium is a misnomer here. The corneal endothelium is bathed by aqueous humour, not by blood or lymph, and has a very different origin, function, and appearance from vascular endothelia.) Unlike the corneal epithelium the cells of the endothelium do not regenerate. Instead, they stretch to compensate for dead cells which reduces the overall cell density of the endothelium and has an impact on fluid regulation. If the endothelium can no longer maintain a proper fluid balance, stromal swelling due to excess fluids and subsequent loss of transparency will occur.

• Not derived from mesoderm• Derived from neural crest

Why blood doesn’t clot in an intact blood vessel?

• ADPase enzyme on the membrane degrades ADP

• Tissue Factor Pathway Inhibitor on the membrane

• Thrombomodulin functions as a cofactor in the thrombin-induced activation of protein C in the anticoagulant pathway

• Prostacyclin synthesis

• Inactivate vasoactive substances

Pro and antithrombotic

• The foundational model of anatomy makes a distinction between endothelial cells and epithelial cells on the basis of which tissues they develop from, and states that the presence of vimentin rather than keratin filaments separate these from epithelial cells.[4] Many considered the endothelium a specialized epithelial tissue.[5]