Endothelial Cell Notes

Oedema Formation

  • Capillary Hydrostatic Pressure: An increase in capillary hydrostatic pressure increases the gap between hydrostatic pressure in the capillary and interstitium, favoring fluid filtration.
  • Oncotic Oedema: Decreasing the gradient between protein in the vessel and protein in the tissue encourages fluid filtration.
    • Etiologies include conditions that decrease capillary oncotic pressure or increase interstitial oncotic pressure, such as liver disease or protein loss.
  • Permeability Oedema: A leaky vessel promotes fluid filtration.
    • Inflammation increases the filtration constant, leading to permeability oedema.
  • Lymphoedema: Impaired lymph drainage results in fluid accumulation in the interstitial space, increasing interstitial hydrostatic pressure.

The Endothelium

  • Lines all blood and lymphatic vessels.

  • Consists of 1−6×10131-6 \times 10^{13} cells lining over 60,000 miles of blood vessels.

  • Originally thought to be a simple semi-permeable barrier between blood and tissues.

  • Now recognized as a large endocrine gland, approximately 1 Kg in size, similar to the liver.

  • Majority of endothelial cells are located within microvasculature/capillaries.

  • Distribution of EC in the Human Vasculature:

    • Velocity of blood in blood vessels is inversely proportional to cross-sectional area.
    • Cross-sectional area increases, velocity decreases.
    • Blood flow is slowest in capillaries, allowing time for gas and nutrient exchange.
    • Total cross-sectional area of capillaries is greater than that of arteries or any other part of the circulatory system.

    *Illustration:
    * Aorta: high velocity, low cross-sectional area
    * Capillaries: low velocity, high cross-sectional area
    * Vena Cava

Endothelial Cell Culture

  • Allows study of endothelial function in isolation, including:
    • Migration
    • Permeability
    • Proliferation
    • Survival
    • Angiogenesis (tube formation and sprouting assays in extracellular matrix gels)
    • Tube Formation Assay: ECs plated on Matrigel for 24 hours.
    • Sprouting Assay: ECs growing on beads embedded in Matrigel after 72 hours.

Developmental Origin of Endothelium

  • Sequential development from mesoderm through the hemangioblast to the hemogenic endothelium and hematopoietic progenitors.
  • Markers of endothelial & haematopoietic precursor cells define stages of differentiation, such as vasculogenesis.
    *Hemogenic endothelium is a special subset of endothelial cells scattered within blood vessels that can differentiate into haematopoietic cells.

Blood Vessel Generation (Angiogenesis) from Existing BVs

  • ECs synthesize a tube of endothelial cells (ECs).
  • Capillaries consist of ECs surrounded by a basement membrane and a sparse layer of pericytes.
    • They form the main site of nutrient exchange between blood and tissue due to their wall structure and large surface-area-to-volume ratio.
    • Capillary endothelial layer can be continuous (muscle), fenestrated (kidney, endocrine glands), or discontinuous (liver sinusoids).
    • Endothelia of the blood-brain barrier or blood-retina barrier have tight junctions, making them impermeable to various molecules.
  • Arterioles and venules have increased coverage of mural cells compared to capillaries.
    • Precapillary arterioles are invested with vascular smooth muscle cells (SMCs).
    • Extravasation of macromolecules and cells from the bloodstream typically occurs from postcapillary venules.
  • Walls of larger vessels consist of three specialized layers:
    • Intima: endothelial cells
    • Media: SMCs
    • Adventitia: fibroblasts, matrix, and elastic laminae
    • The adventitial layer has its own blood supply (vasa vasorum).
    • SMCs and elastic laminae control vessel tone and diameter.
    • Arterio-venous shunts divert blood away from the capillary bed when necessary.
  • Lymphatic capillaries lack pericytes.
    • Larger (collecting) lymphatic vessels have basement membranes and valves for unidirectional lymph flow.
    • Lymphatic endothelial cells connect to surrounding connective tissue via anchoring filaments.

Phenotypic Heterogeneity

  • Allows endothelium to conform to the diverse needs of underlying tissues throughout the body.
  • Allows adaptation to diverse microenvironments.

Artery vs. Vein Endothelium

  • Artery:
    • ECs aligned in the direction of undisturbed flow
    • Long and narrow cells
    • Continuous endothelium with many tight junctions
    • No valves
    • Specific markers: Ephrin B2, DII4, ALK1, EPAS-1, Hey 1/2, Depp, NRP1
  • Vein:
    • Continuous endothelium
    • Shorter, wider cells
    • Not aligned in the direction of blood flow
    • Possess valves
    • Specific markers: EphB4, NRP2, COUP-TFII
  • Post-capillary venule:
    *Caveolae in their areas.
    *VVOs in thick portions

Mechanisms of EC Heterogeneity

  1. Hemangioblasts differentiate into endothelial progenitor cells (angioblasts), which then become ECs of arteries, veins, and capillaries.
    • Cell phenotypes are influenced by microenvironment and epigenetics.
  2. The microenvironment mediates nonheritable changes in EC phenotype via receptor-mediated posttranslational modification of protein and transcription factor-dependent induction of gene expression.
    • Removal of extracellular signals leads to loss of translational/transcriptional effects.
  3. Epigenetics mediate heritable changes in EC phenotype through DNA methylation, histone methylation, and histone acetylation.
    • These modifications influence gene expression and can persist even after signal removal.

Endothelial Functions

  1. Regulates vascular homeostasis.
  2. Acts as sensor and effector.
  3. Barrier function/permeability.
    • Main barrier to the escape of substances from blood to tissues.
    • Selective specialization in different regions of the body based on function.
    • Acts as sieve.
    • Non-fenestrated continuous endothelium forms the majority of the vascular tree in arteries, veins, and capillaries of the brain, skin, and heart.

Endothelial Basement Membrane

  • Polarized cells with distinct expression of receptors on luminal/apical & abluminal/basal sides.
  • Endothelial abluminal membrane resides on a basement membrane and is associated with extracellular matrix (ECM) - collagen, fibronectin, laminin.
  • Significant differences found in BM based on location and physical properties of vessel.
  • Vascular BM is composed of an intricate meshwork of pores and fibers.

Endothelial Intracellular Junctions

  • Tighter on the arterial side, looser on post-capillary venules.
  • Tight and adherens junctions form the main barrier to paracellular transport.

Tight junctions

  • ESAM
  • JAM1,-2,-3
  • Claudins (Cldn):
    • Cldin3,-5,-12
  • Occludins (Ocln)
    • ZO-1

Adherens junctions

  • VE-Cadherin
  • p120
  • Actin

Gap junctions

  • Connexins
    • connexon

How tight junctions work

  • Tight junction molecules such as occludin (Ocln) and claudins (Cldn) have four transmembrane domains and can copolymerize heterophilically or homophilically with each other. Tight junction molecules such as endothelial cell-specific adhesion molecule (ESAM) and junction adhesion molecules (JAMs) are immunoglobulin-like molecules that only bind homophilically.
  • The adaptor protein zonula occludens (ZO) couples the tight junctions to the actin cytoskeleton.

How adherens junctions work

  • Adherens junctions are composed of VE-cadherin and a number of partnering compounds, including a- and ß-catenin (a,ß) plakoglobulin (plako) and p120. a-Catenin connects the adherens junctions to the cytoskeleton.

How gap junctions work

  • Gap junctions consist of hemi-channels (connexons) that are formed by six identical or different connexins.

Paracellular Transport

  • Continuous endothelium allows water & small solutes (<3 nm molecular radius) to pass between ECs.
  • Continuous Non-fenestrated paracellular transport: Allows passage of larger solutes, e.g. albumin

Transcellular Transport

  • Transcytosis: Transcellular caveolae-mediated route of albumin transport

Caveolae smooth membrane invaginations & vesicles - highest density in capillary EC

Continuous Fenestrated Endothelium

  • Occurs in locations characterized by increased filtration or transendothelial transport, such as capillaries of exocrine and endocrine glands, gastric and intestinal mucosa, choroid plexus, glomeruli, and a subpopulation of renal tubules.
  • The presence of fenestrae in continuous endothelium is associated with increased permeability of fluids and small solutes, but not macromolecules.

Discontinuous/Sinusoidal Endothelium

  • Found in liver sinusoids & bone marrow.
  • Large fenestrations and poorly formed basement membrane with gaps.
  • Small ECs clear colloids & soluble waste macromolecules from the circulation
  • Has Sinusoidal fenestrae / Gaps.
  • High endocytic activity in clathrin-coated pits - receptor-mediated (scavenger pathways-e.g. uptake of LDL) and fluid phase endocytosis

Vesiculo-vacuolar Organelles (VVO)

  • A major route for the transport of fluids & solutes across the endothelium, particularly in inflammatory situations.
  • Form transcellular channels when they connect, predominantly at post-capillary venules.

Blood Flow Regulation/Permeability

  • Regulation of vascular tone is determined by endothelial-derived mediators.
  • Vasodilation:
    • Nitric oxide (NO) / Endothelial - derived relaxing factor (EDRF)
    • Prostacyclin (PGI2)
    • Endothelium-derived hyperpolarising factor (EDHF), CO, H2S
  • Vasoconstriction:
    • Endothelin-1 (ET-1)
    • Thromboxane A2
      *H2O2, superoxide anion (O2)
  • Acetylcholine-induced vasodilatation does not occur when the endothelium is removed; the endothelium produces "EDRF" (nitric oxide).

Nitric Oxide (NO) Synthesis

  • Mammalian nitric oxide (NO) synthesis is catalyzed by three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS).
  • L-arginine is the substrate for all three isoforms of NOS.
    *Following their incorporation into proteins, L-arginine residues that lie within certain sequences can be methylated by protein arginine methyltransferases. Proteolysis of arginine-methylated proteins releases free methylarginines into the cytosol.
  • The asymmetrically methylated arginines (w-N,N -asymmetric dimethylarginine (ADMA) and N -monomethyl-L-arginine (L-NMMA)) are competitive inhibitors of all NOS isoforms.
  • Asymmetric methylarginines are predominantly removed via their metabolism, which is catalyzed by dimethylarginine dimethylaminohydrolase (DDAH) enzymes and, to a lesser extent, by renal excretion. DDAH enzymes may exert physiological effects via NOS-independent pathways, such as regulation of vascular endothelial growth factor (VEGF) expression. EDRF, endothelium-derived relaxing factor; SMC, smooth muscle cell.
  • BH4BH4 = tetrahydrobiopterin is an important co-factor that maintains eNOS function/NO production

Three isoforms of NOS and their distribution

  • nNOS (NOSI): neurotransmission
    *Coordination between neuronal activity and blood flow
    *Pain modulation
  • eNOS (NOS III): cardiovascular EDRF
    *Regulation of vascular tone
    *Inhibition of SMC proliferation
    *Inhibition of platelet aggregation
  • iNOS (NOS II): inflammation and host defence
    *Cytoxicity against bacteria, viruses and other micro-organisms

Activity of NO on Vasculature

  • Freely diffusible gas that acts as a signalling molecule.
  • Very short half-life (6−30s6-30 s) = local activity
  • Activity in blood limited by circulating haemoglobin.
  • Prevents thrombosis - Inhibits platelet adhesion to vessels & activation
  • Anti-inflammatory - inhibits leukocyte adhesion & migration
  • Antioxidant
  • Inhibits smooth muscle cell proliferation & migration
  • Atheroprotective: enhanced vascular relaxation; inhibition of platelet activation and aggregation, apoptosis and endothelial-dependent monocyte adhesion.

Shear Stress

  • The most potent physiological mediator of NO production; a stress applied parallel to a face of a material.
  • Endothelium transduces shear stress into a vasorelaxation response via production of NO, increasing blood flow.
  • Induced eNOS expression & activity = increased NO production.

Relaxation of Vascular Smooth Muscle (vSMC) Mechanism:

  1. Activates guanylate cyclase
  2. Reduces [Ca2+][Ca^{2+}] and cGMP phosphodiesterase activity
  3. Activates PKG → limits activation of myosin-light chain kinase (MLCK) essential for myosin-actin cross bridge formation

Other EC Derived Vasodilators

  • Prostacyclin

Illustration

SGC, AC, Vascular smooth muscle cell, TNFa, HIS, CaM, PKG

Key Points on Nitric Oxide (NO)

  • Nature and Role:
    • NO is a ubiquitous, cell-permeable intracellular messenger. *Crucial for maintaining vascular endothelial barrier homeostasis: *vasodilatory *anti-coagulative *anti-proliferative *anti-inflammatory
      • Physiological Functions:
        *Participates in vasodilation and modulation of blood flow
        *Regulates endothelial cell function.
        *vascular endothelial dysfunction
        *vasoconstrictor
        *pro-coagulative
        *proliferative
        *pro-inflammatory
        *risk of cardiovascular disease with age
    • Synthesis:
      *Produced from L-arginine by nitric oxide synthases (NOS).
    • Mechanism of Action:
      *Acts through the activation of guanylate cyclase
      *Increases levels of cyclic GMP (cGMP), vital for signaling
    • Importance:
      *Maintains the integrity of the endothelial barrier
      *Regulates vascular permeability, contributing to overall vascular health

Endothelium and Homeostasis

  • Endothelium Provides a non-thrombogenic surface to maintain blood flow
    *Inhibits the activation of coagulation factors

Key points about endothelium and coagulation

  • Procoagulant Activity:
    • Endothelial cells (ECS) exhibit procoagulant activity when damaged.
    • Key mechanisms include:
      • Induction of tissue factor (thromboplastin) in response to injury or pro-inflammatory cytokines.
      • Increased expression of plasminogen activator inhibitor (PAI-1).
      • Release of von Willebrand factor (vWF) from Weibel-Palade bodies.
  • Anticoagulant Functions:
    • ECs maintain blood in a fluid state and promote limited clot formation to prevent excessive bleeding.
    • Key anticoagulant molecules produced by ECs include:
      • Tissue factor pathway inhibitor (TFPI).
      • Heparan, thrombomodulin, endothelial protein C receptor (EPCR).
      • Tissue-type plasminogen activator (t-PA), ecto-ADPase, prostacyclin, and nitric oxide.
  • Molecular Distribution:
    • Anticoagulant and procoagulant molecules are unevenly distributed throughout the vasculature, indicating localized regulation of coagulation processes.
  • Overall Balance:
    • The endothelium plays a crucial role in balancing coagulation and anticoagulation to maintain vascular integrity and prevent pathological clot formation.

Other Functions

  1. Leukocyte recruitment
  2. Hormone trafficking