Endothelium and endotheliopathy









  • Because of their location, endothelial cells have the ability to interact with blood components, such as flow, soluble factors, and other cells. Endothelial cells integrate these signals into a cohesive regulation of vascular responses.



  • The endothelium controls the vascular tone of the underlying smooth muscle cells through the production of vasodilator and vasoconstrictor mediators.



  • Endothelial cell activation in response to inflammation changes endothelial cellular physiology and alters vascular function.



  • A large number of endothelial cell–active molecules are potential biomarkers for the early diagnosis of sepsis.



Until recently, scientists and clinicians considered the endothelium, the cell layer that lines the blood vessels, as an inert barrier separating the various components of blood and the surrounding tissues. The vascular endothelium is now recognized as a highly specialized and metabolically active organ performing a number of critical physiologic, immunologic, and synthetic functions. These functions include regulation of vascular permeability, fluid and solute exchange between the blood and interstitial space, vascular tone, cell adhesion, homeostasis, and vasculogenesis.


The normal vascular endothelium is only one cell layer thick, separating the blood and vascular smooth muscle. The endothelium responds to physical and biochemical stimuli by releasing regulatory substances affecting vascular tone and growth, thrombosis and thrombolysis, and platelet and leukocyte interactions with the endothelium. Because normal endothelial function plays a central role in vascular homeostasis, it is logical to conclude that endothelial dysfunction contributes to disease states characterized by vasomotor dysfunction, abnormal thrombosis, or abnormal vascular proliferation.


The endothelium lies between the lumen and vascular smooth muscle, where it is uniquely positioned to “sense” changes in hemodynamic forces or blood-borne signals by membrane receptor mechanisms. The endothelial cells can respond to physical and chemical stimuli by synthesis or release of a variety of vasoactive and thromboregulatory molecules and growth factors.


The vascular endothelium possesses numerous enzymes, receptors, and transduction molecules, and interacts with other vessel wall constituents and circulating blood cells. In addition to these universal functions, the endothelium may have organ-specific roles that are differentiated for various parts of the body, such as gas exchange in the lungs, control of myocardial function in the heart, or phagocytosis in the liver and spleen.


Normal endothelial function


Endothelial cell heterogeneity


Many vascular diseases appear to be restricted to specific vascular beds. For example, thrombotic events are often localized to single vessels. It is also common for certain vasculitides to specifically affect certain arteries, veins, or capillaries or to affect certain organs. Tumor cells often metastasize more commonly within particular vascular beds. The basis for this variability in vascular disease is poorly understood but may be explained by the heterogeneity of endothelial cells. There has been a greater understanding of how endothelial cell heterogeneity may contribute both to the maintenance of organ-specific function and to the development of disorders restricted to specific vascular beds.


The cell biology of capillary endothelium from different vascular beds may explain differences in tissue function. For example, the brain microcirculation is lined by endothelial cells connected by tight junctions that maintain the blood-brain barrier. By contrast, sinusoids found in the liver, spleen, and bone marrow are lined by endothelial cells that allow transcellular trafficking between intercellular gaps. Similarly, fenestrated endothelial cells found in the intestinal villi, endocrine glands, and kidneys facilitate selective permeability, which is required for efficient absorption, secretion, and filtering.


Another example of endothelial cell heterogeneity lies in the expression of cell surface receptors involved in cell-to-cell signaling and cell trafficking. For example, in the mouse, lung-specific endothelial cell adhesion molecules are exclusively expressed by pulmonary postcapillary endothelial cells and some splenic venules. Similarly, specific mucosal cell adhesion molecules are expressed primarily on endothelial venules in the Peyer patches of the small intestine. , Tumor cells may show clear preferential adhesion to the endothelium of specific organs paralleling their in vivo metastatic propensities.


Endothelial progenitor cells


A significant amount of literature has shown that maintenance and repair of vasculature in ischemic diseases may be at least partially mediated through recruitment of endothelial progenitor cells (EPCs) from the bone marrow to areas of vascular injury.


An EPC is a specific subtype of hematopoietic stem cell that has been isolated from circulating mononuclear cells, bone marrow, and cord blood. EPCs migrate from the bone marrow to the peripheral circulation, where they contribute to vascular repair. , When injected into animal models of ischemia, EPCs are incorporated into sites of neovascularization , , and have contributed to improved outcomes in patients with ischemic vascular disorders. In addition, there has been accumulating evidence for the function of EPCs in critical illnesses such as sepsis.


Recruitment of EPCs to areas of endothelial and vascular damage may have prognostic implications and could be associated with clinical outcome. The pathophysiologic changes associated with critical illness—notably, sepsis and sepsis-related organ dysfunction—may lead to apoptosis and necrosis of endothelial cells from the vasculature and recruitment of EPCs from the bone marrow.


In various models of vascular injury and organ dysfunction, only a few studies have emerged regarding EPCs in particular as a therapeutic strategy. Transplanted EPCs have been shown to improve survival of mice following liver injury. Infusion of EPCs also restored blood flow in a mouse model of hind limb ischemia. A prospective randomized trial compared the effects of EPC transplantation in patients with idiopathic pulmonary arterial hypertension versus conventional therapy and showed that after 12 weeks, patients who had received EPCs had a significant improvement in their 6-minute walk test, mean pulmonary artery pressure, pulmonary vascular resistance, and cardiac output.


Coagulation and fibrinolysis


A normal physiologic function of the endothelium is to provide an antithrombotic surface inhibiting platelet adhesion and clotting, facilitating normal blood flow. Under pathophysiologic conditions, the endothelium transforms into a prothrombotic surface. A dynamic equilibrium exists between both states that permits a rapid response to an insult and a rapid recovery.


Anticoagulant mechanisms


The endothelium has anticoagulant, antiplatelet, and fibrinolytic properties. Endothelial cells are the major sites for anticoagulant reactions involving thrombin. Thrombin plays a key role in coagulation, including the activation of platelets, activation of several coagulation enzymes and cofactors, and stimulation of procoagulation pathways on the endothelial cell surface. In the normal state, there is little thrombin enzyme activity. The surrounding endothelial cell matrix contains heparin sulfate and related glycosaminoglycans that activate antithrombin III. In addition, the subendothelial cell matrix contains dermatan sulfate, which promotes the antithrombin activity of heparin cofactor II. Furthermore, microvascular endothelial cells release a tissue factor pathway inhibitor that inhibits the factor VIIa/tissue factor complex and further contributes to anticoagulation ( Fig. 25.1 ).




• Fig. 25.1


Endothelium control of the coagulation cascade. An inflammatory stimulus upregulates the interaction of tissue factor (TF) with factor VII, which generates activated factor VII (factor VIIa). The TF–factor VIIa complex then leads to the conversion of factor X to factor Xa. The interaction of factors Xa and Va results in the conversion of prothrombin to thrombin and the conversion of fibrinogen to fibrin. Three key anticoagulant pathways can inhibit this process. Protein C is activated through its interaction with cell-surface thrombomodulin and inhibits the activities of factors Va and VIIIa. Antithrombin blocks the activation of multiple factors, including factor X and thrombin. Tissue factor pathway inhibitor interferes directly with the tissue factor–factor VIIa complex.


Thrombin activity is also modulated by endothelial cell synthesis of thrombomodulin. , The binding of thrombin to thrombomodulin facilitates the enzyme’s activation of the anticoagulant protein C. Activated protein C (APC) activity is enhanced by cofactor C, also called protein S, which is synthesized by endothelial cells as well as by other cells (see Fig. 25.1 ). APC inhibits factor Va and factor VIIIa. Thrombomodulin (TM) also inhibits prothrombinase activity indirectly by binding factor Xa ( Fig. 25.2 ). Protein C has a special receptor on the endothelial cells: endothelial cell protein C receptor (EPCR). EPCR augments protein C activation approximately 20-fold in vivo by binding protein C and presenting it to the thrombin-TM activation complex. Both EPCR and TM can be found in plasma as soluble proteins. APC retains its ability to bind EPCR; this complex appears to be involved in some of the cellular signaling mechanisms that downregulate inflammatory cytokine formation (tumor necrosis factor [TNF], interleukin-6). In addition, platelet adhesion to endothelial cells is markedly inhibited by endothelium-derived prostacyclin. The interactions between platelets and endothelium regulate platelet function, coagulation cascades, and local vascular tone.




• Fig. 25.2


The interaction of the protein C system with the endothelium: Thrombin bound to thrombomodulin (TM) modifies protein C bound to the endothelial protein C (Prot C) receptor on the cell surface to generate activated protein C (APC). APC acts as a natural anticoagulant by inactivating activated factors V (fVa) and VIII (fVIIIa), modulating inflammation by downregulating the synthesis of proinflammatory cytokines, leukocyte adherence, and apoptosis and enhancing fibrinolysis by inhibiting thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor type-1 (PAI-1). C4Bbp, C4b binding protein (binds protein S); + PS , in the presence of protein S; sEPCR, soluble endothelial cell protein C receptor; sTM, soluble thrombomodulin.

(Modified from Hazelzet J. Pathophysiology of pediatric sepsis. In: Nadel S. Infectious Diseases in the Pediatric Intensive Care Unit . London: Springer; 2008.)


Microvascular endothelial cells may secrete tissue plasminogen activator (t-PA), the powerful thrombolytic agent in frequent clinical use for treatment of coronary thrombotic occlusion. t-PA release is stimulated in vivo by norepinephrine, vasopressin, or stasis within the vessel lumen. Thrombin may also stimulate t-PA release, providing a further endothelium-mediated safeguard against uncontrolled coagulation.


Procoagulant mechanisms


The expression and release of tissue factor is the pivotal step in transforming the endothelium from an anticoagulant to a procoagulant surface. , Tissue factor accelerates factor VIIa-dependent activation of factors X and IX (see Fig. 25.1 ). The synthesis of tissue factor is induced by a number of agonists—including thrombin, endotoxin, several cytokines, shear stress, hypoxia, oxidized lipoproteins, and other endothelial insults. Once endothelial cells expressing tissue factor are exposed to plasma, prothrombinase activity is generated and fibrin is formed on the surface of the cells. Tissue factor can also be found in plasma as a soluble protein. Its role there is not well understood, but it probably plays a role in the initiation of coagulation.


Endothelium-derived vasodilators


The important role that the endothelium plays in controlling vascular tone has only recently been appreciated. Clinicians and researchers have come to appreciate that the endothelium controls underlying smooth muscle tone in response to certain pharmacologic and physiologic stimuli. This response involves a number of luminal membrane receptors and complex intracellular pathways and the synthesis and release of a variety of relaxing and constricting substances.


Nitric oxide


Furchgott and Zawadzki first postulated the existence of an endothelium-derived relaxing factor (EDRF) in 1980, when they noticed that the presence of endothelium was essential for rabbit aortic rings to relax in response to acetylcholine. Later, it was determined that the biologic effects of EDRF are mediated by nitric oxide (NO).


NO is generated from the conversion of L -arginine to NO and L -citrulline by the enzyme nitric oxide synthase (NOS). There are two general forms of NOS: constitutive and inducible. In the unstimulated state, NO is continuously produced by constitutive NO synthase (cNOS). The activity of cNOS is modulated by calcium that is released from endoplasmic stores in response to the activation of certain receptors. Substances such as acetylcholine, bradykinin, histamine, insulin, and substance P stimulate NO production through this mechanism. Similarly, shearing forces acting on the endothelium are another important mechanism regulating the release of NO. The inducible form of NOS (iNOS) is not calcium dependent but instead is stimulated by the actions of cytokines (e.g., TNF-α, interleukins) or bacterial endotoxins (e.g., lipopolysaccharide). iNOS occurs over several hours and results in NO production that may be more than a thousandfold greater than that produced by cNOS. This is an important mechanism in the pathogenesis of inflammation ( Fig. 25.3 ). Inhibition of NOS using competitive analogs of L -arginine drastically reduces endothelium-dependent relaxation in vitro, particularly in large conduit arteries, thereby evoking vasoconstriction. Chronic treatment of animals with NOS inhibitors or suppression of the cNOS gene is reported to induce hypertension.




• Fig. 25.3


Nitric oxide (NO) is generated from L -arginine ( L -arg) by the action of nitric oxide synthase (NOS). In the resting state, constitutive NOS (cNOS) is modulated by intracellular Ca 2+ and calmodulin (CaM). Stored Ca 2+ is released in response to vasoactive agents (e.g., acetylcholine and bradykinin) and other external stimuli, such as shearing forces. Activation of endothelial cells by cytokines and endotoxins increases expression of inducible NOS (iNOS). Citrulline (Cit) is a byproduct of NO production. NO has a half-life of only a few seconds in vivo and quickly diffuses to surrounding cells, such as smooth muscle cells. NO stimulates the production of the intracellular mediator cyclic GMP (cGMP). Increased cGMP activates a series of cGMP-dependent protein kinases (cGMP-PKs) and cGMP-dependent phosphatases (cGMP-Ps).


Once NO is formed by an endothelial cell, it readily diffuses out of the cell and into adjacent smooth muscle cells where it binds and activates the soluble form of guanylyl cyclase, resulting in the production of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. cGMP, in turn, activates a number of cGMP-modulated enzymes (see Fig. 25.3 ). Increased cGMP activates a kinase that subsequently leads to the inhibition of calcium influx into the smooth muscle cell and decreased calcium-calmodulin stimulation of myosin light chain kinase. This, in turn, decreases the phosphorylation of myosin light chains, decreasing smooth muscle tension development and causing vasodilation. There is also some evidence that increases in cGMP can lead to myosin light chain dephosphorylation by activating the phosphatase. In addition, cGMP-dependent protein kinase phosphorylates potassium ion (K + ) channels to induce hyperpolarization, thereby inhibiting vasoconstriction. , Interestingly, NO inhibition of platelet aggregation is also related to the increase in cGMP. Drugs that inhibit the breakdown of cGMP, such as inhibitors of cGMP-dependent phosphodiesterase (e.g., sildenafil), potentiate the effects of NO-mediated actions on the target cell. Therefore, NO contributes to the balance between vasodilator and vasoconstrictor influences that determine vascular tone.


Prostacyclin


Another major endothelium-derived vasodilator is the prostaglandin prostacyclin (PGI 2 ), a derivative of arachidonic acid synthesized through the action of the enzyme cyclooxygenase. Endothelium cells are capable of producing a variety of vasoactive substances that are products of arachidonic acid metabolism. Among these are prostaglandins, PGI 2 , leukotrienes, and thromboxanes. These substances act as either vasodilators or vasoconstrictors, among their other biological activities. PGI 2 is a potent vasodilator and is active in both the pulmonary and systemic circulations. In addition to its vasodilatory effects, prostacyclin also has antithrombotic and antiplatelet activity. Its release may be stimulated by bradykinin and adenine nucleotides. Like NO, it is chemically unstable, with a short half-life. However, unlike NO, PGI 2 activity in arterial beds depends on its ability to bind to specific receptors in vascular smooth muscle. Therefore, its vasodilator activity is determined by the expression of such receptors. PGI 2 receptors are coupled to adenylate cyclase to elevate cyclic adenosine monophosphate (cAMP) levels in vascular smooth muscle. The increase in cAMP results in (1) stimulation of adenosine triphosphate (ATP)-sensitive K + channels, leading to hyperpolarization of the cell membrane and inhibition of the development of contraction; and (2) increased efflux of calcium ions (Ca 2+ ) from the smooth muscle cell and inhibition of the contractile machinery.


In addition, PGI 2 facilitates the release of NO by endothelial cells, and NO potentiates the action of PGI 2 in vascular smooth muscle. Interestingly, NO may also potentiate the effects of prostacyclin. The NO-mediated increase in cGMP in smooth muscle cells inhibits a phosphodiesterase that breaks down cAMP and therefore indirectly prolongs the half-life of the second messenger of PGI 2 .


Endothelium-derived hyperpolarizing factor


Endothelium stimulation by acetylcholine also produces hyperpolarization of the underlying smooth muscle and thereby induces vasorelaxation. This process is not mediated by NO but is instead mediated by another endothelium-derived factor. This factor increases K + -channel conductance in smooth muscle cells, resulting in smooth muscle cell relaxation. The resulting vasodilation is not inhibited by NG-methyl-L-arginine (L-NMMA), the specific antagonist of NO, but rather is inhibited by ouabain, a sodium-potassium adenosine triphosphatase inhibitor. In addition, in most medium- to resistance-sized arteries, electrophysiologic studies have established that endothelium-dependent hyperpolarization of vascular smooth muscle is resistant to the combined inhibition of both NOs and cyclooxygenases. Accordingly, a component of the endothelium-dependent relaxation in these arteries is mediated by a substance different from NO and PGI 2 . This component of endothelium-dependent vasodilation has been attributed to an as yet unidentified diffusible endothelium-derived hyperpolarizing factor (EDHF).


Of significant clinical importance is the fact that the EDHF-mediated effect increases as the arterial diameter decreases, such as in resistance arteries. EDHF likely plays a significant role in the regulation of peripheral vascular resistance and local hemodynamics. Unfortunately, in the absence of selective inhibitors of the EDHF pathway, it is not possible to evaluate the relevance of EDHF in vivo.


Endothelium-derived vasoconstrictors


Endothelins (endothelium-derived contracting factors)


Endothelin is a 21-amino-acid peptide and is one of the most potent vasoconstrictors identified to date. Endothelial cells synthesize the prohormone big endothelin and express endothelin-converting enzymes to generate endothelin. There are three isoforms of endothelin, but only one (ET-1) has been shown to be released from human endothelial cells. ET-1 is synthesized in the endothelial cells. Its release is mediated by a variety of stimuli. ET-1 release is stimulated by angiotensin II, antidiuretic hormone, thrombin, cytokines, reactive oxygen species, and shearing forces acting on the vascular endothelium. ET-1 release is inhibited by NO as well as by PGI 2 and atrial natriuretic peptide. ,


ET-1 has a short half-life, suggesting that, similarly to NO, ET-1 is mainly a locally active vasoregulator. Once released by endothelial cells, ET-1 binds to a membrane receptor (ET A ) found on adjacent vascular smooth muscle cells. This binding leads to calcium mobilization and smooth muscle contraction. The ET A receptor is coupled with a G-protein linked to phospholipase-C, resulting in the formation of IP 3 . Interestingly, ET-1 can also bind to an ET B receptor located on the vascular endothelium, which stimulates the formation of NO by the endothelium. This release of NO appears to modulate the ET A receptor–mediated contraction of the vascular smooth muscle. Its physiologic role includes maintenance of basal vascular resistance, and it is present in healthy subjects in low concentrations.


Reactive oxygen species


Endothelial cells secrete oxygen-derived free radicals and hydrogen peroxide in response to shear stress and endothelial agonists such as bradykinin. Such reactive oxygen species are reported to inactivate NO, resulting in vasoconstriction. Reactive oxygen species may also facilitate the mobilization of cytosolic Ca 2+ in vascular smooth muscle cells and promote Ca 2+ sensitization of the contractile elements. Under conditions of hyperoxia, endothelium-derived superoxide anion may combine with NO with diffusion-limited kinetics to generate peroxynitrite, negating NO-mediated vasodilation, an effect inhibited by superoxide dismutase, which metabolizes superoxide anion to hydrogen peroxide.


Vasoconstrictor prostaglandins


The metabolism of arachidonic acid by cyclooxygenase in endothelial cells may lead to the secretion of precursors of thromboxanes and leukotrienes. These prostaglandins act on receptors in vascular smooth muscle to induce vasoconstriction. PGI 2 , however, is the major endothelial metabolite of arachidonic acid that is generated through the cyclooxygenase pathway. Thus, under normal circumstances, the influence of the small amounts of vasoconstrictor prostanoids released by endothelial cells is masked by the production of PGI 2 , NO, and EDHF. ,


Endothelium and blood cell interactions


In addition to the interactions of the endothelium with blood coagulation factors, endothelial cells also express cell-surface molecules known as the endothelial glycocalyx (EG). Over the past decade, we have learned about the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The glycocalyx is a gel-like layer enriched with carbohydrates. Its dimensions vary according to the type of vasculature, ranging from 0.2 μm to more than 2 μm. The EG consists of various types of glycosaminoglycans covalently attached to plasma membrane–bound core proteoglycans. Heparan sulfate comprises 50% to 90% of endothelial glycosaminoglycans. The remainder is a mixture of hyaluronic acid, dermatan, keratan, and chondroitin sulfates. In healthy vessels, the EG determines vascular permeability, attenuates blood cell–vessel wall interactions, mediates shear stress sensing, enables balanced signaling, and fulfills a vasculoprotective role. When the EG is disrupted or modified, these properties are lost ( Fig. 25.4 ). Evidence is emerging that damage to the glycocalyx plays a pivotal role in several vascular pathologies.


Jun 26, 2021 | Posted by in CRITICAL CARE | Comments Off on Endothelium and endotheliopathy
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