Endothelium

Question 1

Cell differentiation, heterogeneity and dysfunction

Answer 1

• Lines all blood and lymphatic vessels recognised as large endocrine gland, control vascular function by responding to hormones, neurotransmitters, and vasoactive factors which affect vasomotion, thrombosis, platelet aggregation, and inflammation
• Majority of cells are present in microvasculature and capillaries, small distribution of endothelial cells in aorta, arteries, veins, and vena cava, but large distribution in arterioles, capillaries, and venules
• Cells in arteries and veins = continuous and thicker, capillaries = fenestrated and thinner to allow exchange of metabolites and gases
• Cell culture study function: migration/permeability/proliferation/survival/ angiogenesis
• Endothelial markers + haematopoietic precursor cells define stages of differentiation
• Phenotypic heterogeneity = endothelium conforms to needs of underlying tissues in body and allows for adaptation to microenvironments
• Endothelial cell heterogeneity: through epigenetic changes (by cell division) or by local environment cues (these must be maintained)
• Local environment determines cell phenotype, influences receptor signalling and gene expression, epigenetic changes more resistant to microenvironment changes
• Shift in EC function to ↓ vasodilation and more pro-inflammatory & pro-thrombotic state, due to ↓ bioavailability of NO and ↓ vasodilation, blood vessels may become damaged and leaky with loss of EC
• Cardiovascular disease: dysfunction is evident before presentation of obstructive atherosclerotic lesions in both conduit and resistance coronary vessel, magnitude of ED ↑ with accumulation of CVD risk factors in peripheral conduit vessels
• Endothelial function = good prognostic marker of future cardiac events in patients with CVD, but administration of L-arginine can ↑ NO bioavailability and improve endothelial function in patients with CVD risk factors
• Medications control CVD risk factors: anti-hypertensives or statins have beneficial effects on endothelial function through ↓ oxidative stress and lipid accumulation
• Inflammatory disease: patients with inflammatory disorders e.g rheumatoid arthritis, Bechet’s disease and IBD have increased risk of developing CVD
• Anti-inflammatory medications: improve endothelial function in resistance + conduit vessels, supports role of inflammation, predictor of CVD in inflammatory diseases

Question 2

Permeability/barrier function

Answer 2

• Respond to environmental changes, produce different changes to alter change in environment (they are sensors and effectors)
• Endothelial barrier = main barrier of escape for substance from blood to tissues
• Barrier is selective: specialised in different regions of body based on function
• Non-fenestrated continuous endothelium: forms majority of vascular tree: arteries, veins, capillaries of the brain, skin, and heart
• Endothelium basement membrane: polarised cell with distinct expression of receptors on luminal/apical and abluminal/basal sides
• Endothelial abluminal membrane resides on basement membrane, associated with ECM (collagen, fibronectin, and laminin)
• Endothelial intercellular junctions: tight on arterial side, looser on post capillary venules
• Tight & adherens junctions (VE-Cadherin) form main barrier to paracellular transport
• Junctional proteins + integrins connected to EC cytoskeleton
• Continuous endothelium = paracellular transport of H2O + small solutes pass between endothelial cells, tight junctions = transcytosis → passage of larger solutes
• Caveolae: smooth membrane invaginations and vesicles, high density in capillary endothelial cells
• Transendothelial channels: pores open between caveolae = passage through endothelium
• Fenestrated continuous endothelium: glomerular endothelium, vessels in endocrine & exocrine glands, gastric and intestinal mucosa
• Fenestrae have a diaphragm which may increase size selectivity of a pore
• Fenestrations permit greater trans-endothelial transport of fluids and small solutes
• Discontinuous/ sinusoidal endothelium = present in liver + bone marrow, small endothelial cells which are clear colloids and soluble waste macromolecules from circulation, basement membrane is formed poorly with gaps
• High endocytic activity in the clathrin-coated pits which are receptor mediated and allow for fluid phase endocytosis
• Vesiculo-vacuolar organelles: major route of fluid + solute transport across endothelium in inflammatory situation, form transcellular channels when connected, predominantly at post-capillary venules

Question 3

Nitric oxide and vascular tone

Answer 3

• Vascular tone = degree of constriction experienced by a blood vessel relative to its maximally dilated state, all arterial and venous vessels under basal conditions exhibit a degree of smooth muscle contract that determines diameter and tone of vessel
• Endothelium releases vasoactive factors: vasodilatory (NO, prostacyclin (PGI2) endothelium derived hyperpolarising factor (EDHF)) or vasoconstrictive (thromboxane (TXA2), endothelin-1 (ET-1), or H2O2)
• NO: endothelium-dependent vasodilator of underlying smooth muscle, role in maintenance of basal vasodilator tone of blood vessels
• NO formed under influence of enzyme nitric oxide synthase in presence of BH4, which converts L-arginine to NO and its 3 isoforms:
  1. Neuronal isoform (nNOS) produces NO to act as neuronal messenger regulates synaptic neurotransmitter release, long term potentiation, coordination between neuronal activity and blood flow, and is main modulation
  2. Macrophage/ inducible isoform (iNOS): only expressed in cells exposed to inflammatory mediators or other injurious stimuli that activate macrophages
  3. Endothelial NOS (eNOS) produces NO in vasculature, involved in regulation of vascular tone, inhibition of SMC proliferation, and inhibition of platelet aggregation
• Inactive eNOS: bound to protein caveolin, located in small invaginations (caveolae), when intracellular levels of Ca2+ ↑ , eNOS detaches from caveolin and is activated (NO agonists influence detachments of eNOS by releasing Ca2+ from ER)
• Intracellular Ca2+ stores depletion → signal sent to membrane receptors → open Ca2+ allowing extracellular Ca2+ into cell (store-operated Ca2+)
• Ca2+ attaches to protein calmodulin in cytoplasm → undergoes structural changes → bind to eNOS to convert to L-arginine to NO (NO production is Ca2+ level dependent)
• Activity of NO in vasculature: freely diffusible gas, acts as a signalling molecule, has short half-life, involved in local activity (activity limited by circulating haemoglobin α)
• Prevents thrombosis (inhibits platelet adhesion to vessels & activation), anti-inflammatory (inhibits leukocyte adhesion & migration), antioxidant (inhibits smooth muscle cell proliferation & migration) and athreoprotective
• Shear stress initiates eNOS phosphorylation by actions of protein kinase B, shear stress results from increased blood flow in vessel, can ↑ NO production by eNOS phosphorylation, or stimulating endothelial cell receptors by allowing transfer of blood-borne agonists to attach to endothelial cell receptors, ↑ intracellular Ca2+
• Shear stress activates specialised Ca2+ activated K+ channels on endothelial cell surface → K+ efflux and Ca2+ influx (contribution of Ca2+ and eNOS phosphorylation to NO production is dependent on duration of shear stress)
• Shear stress = most potent physiological NO production mediator, endothelium turns shear stress into vasorelaxation response by production of NO ↑ blood flow
• Relaxation of vascular smooth muscle (vSMC): synthesised NO diffuses across endothelial cell to adjacent smooth muscle, binds to enzyme soluble guanylyl cyclase
• Activated enzyme ↑ conversion rate of GTP to cGMP, ↓ smooth muscle tension
• cGMP: ↓ Ca2+ release from sarcoplasmic reticulum in smooth muscle, and helps restore Ca2+ to SR, both actions ↓ contraction of smooth muscle cells
• Flow-mediated dilation: ↑ blood flow through artery → vasodilation on principal that ↑ blood flow = shear forces on endothelium → stimulates endothelial cells to release NO
• Reduced vasodilation following ↑ shear forces is representative of impaired NO bioavailability, FMD = good surrogate marker of NO bioavailability
• Endothelium-derived relaxing factor: Ach-induced vasodilation does not occur when endothelium is removed means endothelium produces a factor allowing for a relaxation response (relaxing agent reduces hypertension)