Vascular Endothelium Role in Health and Disease
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Vascular Endothelium Role in Health and Disease
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- 2. Objectives • Discuss the importance of the vascular endothelium in the overall functioning of the body • Explain the heterogeneity of EC’s as core property of the endothelium • Discuss the normal functions of the vascular endothelium • Describe the role of the vascular endothelium in the following disease processes: – Cardiovascular disease – Diabetes – CKD – Cancer – Sepsis
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- 6. MECHANISMS OOFF EECC HHEETTEERROOGGEENNEEIITTYY Aird W C Circulation Research. 2007;100:158-173 Copyright © American Heart Association, Inc. All rights reserved.
- 8. BARRIER AND TRANSDUCING FUNCTIONS
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- 18. SIGNALING MECHANISMS IN THE REGULATION OF ENDOTHELIAL PERMEABILITY
- 19. Effects of Signalling Molecules IInnccrreeaasseedd DDeeccrreeaasseedd MMiixxeedd PPeerrmmeeaabbiilliittyy PPeerrmmeeaabbiilliittyy IInnccrreeaasseedd IICC CCaallcciiuumm PPKKAA NNOO PPKKCC EEppaacc ccGGMMPP MMLLCCKK SSpphhiinnggoossiinnee--11-- pphhoossppaahhttee RRaacc--11 SSrrcc ffaammiillyy kkiinnaasseess SSmmaallll GGTTPPaasseess RRaacc--11 aanndd ccddcc4422 SSmmaallll GGTTPPaassee RRhhooAA YYuuaann && RRiiggoorr,,22001111
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- 33. VASCULAR ENDOTHELIUM AND DISEASE
- 35. Types of EC Activation • Type 1 – Transient – No de novo protein synthesis or gene upregulation – Loosening of tight junctions – Increasing permeability – Export Weibel-Palade bodies releasing vWF and P-selectin – Increased adhesion of leukocytes and platelets • Type 2 - Prolonged – Gene expression of pro-inflammatory cytokines and adhesion molecules
- 37. TNF EC Activation Lei Xiao et. al, 2013
- 38. EC Activation Lei Xiao et. al, 2013
- 41. MECHANISMS UNDERLYING ENDOTHELIAL DYSFUNCTION
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- 46. PUTATIVE MECHANISMS • Production of an EDCF that counteracts the normal dilator effect of NO • Reduced activity of NO synthase • Enhanced breakdown of NO after its generation • Presence of hemoglobin in subarachnoid hemorrhage – Inhibition of NO leading to vasospasm – Inactivation of endothelium-derived NO by generating superoxide anions CCoosseennttiinnoo eett ..aall,, 22000011
- 47. ENDOTHELIUM AND HYPERTENSION AND CAD
- 48. Increase in FBF above basal (b) induced by intra-arterial acetylcholine (left) and sodium nitroprusside (right) in FH+ (○; n=34) and FH− (•; n=30) subjects. Taddei SS eett aall.. CCiirrccuullaattiioonn.. 11999966;;9944::11229988--11330033 Copyright © American Heart Association, Inc. All rights reserved.
- 49. • Decreased availability of NO –Decreased production –Increased degradation • Overproduction of endothelin TThhuuiilllleezz && RRiicchhaarrdd,,22000055
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- 61. GGaallaavvaass eett..aall,, 22001133 CCAANNCCEERR CCEELLLLSS
- 66. Effect of Vasoactive Mediators on Splanchnic Circulation Sam et al, 1997
- 68. REFERENCES • 1. Sarah Y. Yuan and Robert R. Rigor. Regulation of Endothelial Barrier Function. Colloquium Series on Integrated Systems Physiology: from Molecule to Function to Disease. Morgan & Claypool Life Sciences Publishers, 2011 • 2. Michel Félétou. The Endothelium Part 1: Multiple Functions of the Endothelial Cells—Focus on Endothelium-Derived Vasoactive Mediators. Colloquium Series on Integrated Systems Physiology: from Molecule to Function to Disease. Morgan & Claypool Life Sciences Publishers, 2011 • 3. Carine Michiels. Endothelial Cell Functions. Journal of Cellular Physiology 196:430–443, 2003 • 4. Cines et al. Endothelial Cells in Physiology and in the Pathophysiology of Vascular Disorders. BLOOD. The Journal of The American Society of Hematology VOL 91, NO 10 MAY 15, 1998 • 5. William C. Aird. Endothelium in health and disease. Pharmacological Reports, 2008, 60, 139-142
- 69. • 6. William C. Aird. Phenotypic Heterogeneity of the Endothelium: I. Structure, Function, and Mechanisms. Circ Res. 2007;100:158-173. doi: 10.1161/01.RES.0000255691.76142.4a • 6. Lei Xiao, Yahan Liu and Nanping Wang. New paradigms in inflammatory signaling in vascular endothelial cells. Am J Physiol Heart Circ Physiol 306: H317–H325, 2014.First published November 27, 2013; doi:10.1152/ajpheart.00182.2013. • 7. Michel Fe´le´tou and Paul M. Vanhoutte. Endothelial dysfunction: a multifaceted disorder. Am J Physiol Heart Circ Physiol 291: H985–H1002, 2006. • 8. Joseph A Vita MD, Naomi M Hamburg MD. Does endothelial dysfunction contribute to the clinical status of patients with peripheral arterial disease? Can J Cardiol 2010;26(Suppl A):45A-50A. • 9. Cosentino et .al. Endothelial Dysfunction and Stroke. Journal of Cardiovascular Pharmacology. 38 (Suppl. 2):S75–S78 © 2001 • 10. Taddei et.al. Defective l-Arginine–Nitric Oxide Pathway in Offspring of Essential Hypertensive Patients. Circulation. 1996; 94: 1298-1303 doi: 10.1161/01.CIR.94.6.1298 • 11. Thuillez & Richard. Targeting endothelial dysfunction in hypertensive subjects. Journal of Human Hypertension (2005) 19, S21–S25 & 2005 Nature Publishing Group
- 70. • 12. Landmesser et. Al. Endothelial Function: A Critical Determinant in Atherosclerosis?. Circulation. 2004;109:II-27-II-33. doi: 10.1161/01.CIR.0000129501.88485.1f • 13. Jian Xu, PhD; Ming-Hui Zou. Molecular Insights and Therapeutic Targets for Diabetic Endothelial Dysfunction. Circulation. 2009;120:1266-1286. doi: 10.1161/CIRCULATIONAHA.108.835223 • 14. Taddei et al. Vitamin C Improves Endothelium-Dependent Vasodilation by Restoring Nitric Oxide Activity in Essential Hypertension. Circulation. 1998;97:2222-2229. doi: 10.1161/01.CIR.97.22.2222 • 15. Kajimoto et.al . Inhibition of eNOS phosphorylation mediates endothelial dysfunction in renal failure: new effect of asymmetric dimethylarginine. http://www.kidney-international.org. & 2012 International Society of Nephrology • 16. Judah Folkman, Angiogenesis in Cancer, Vascular, Rheumatoid and other diseases. Nature Medicine Vol.1 No.1 1995
- 71. • 17. Goon et.al. Circulating Endothelial Cells, Endothelial Progenitor Cells, and Endothelial Microparticles in Cancer. Neoplasia . Vol. 8, No. 2, February 2006, pp. 79 – 88 • 18. Galavas et.al. Angiogenesis-Related Pathways in the Pathogenesis of Ovarian Cancer. Int. J. Mol. Sci. 2013, 14, 15885-15909; doi:10.3390/ijms140815885 • 19. Demir et al. Sequential Steps During Vasculogenesis and Angiogenesis in the Very Early Human Placenta. Placenta (2006), 27, 535e539 doi:10.1016/j.placenta.2005.05.011 • 20. Roberts and Porter, Cellular and molecular mechanisms of endothelial dysfunction in diabetes. Diabetes and Vascular Disease Research 2013 10: 472 originally published online 3 September 2013. DOI: 10.1177/1479164113500680
Notes de l'éditeur
- How do we define the endothelium? - From an anatomical standpoint, the endothelium represents the inner cellular lining of the blood and lymphatic vessels. However, there are examples of vascular mimicry in which other cell types, eg, trophoblasts, form the inner lining of blood vessels. Many of the characteristic ultrastructural features of the endothelium, such as Weibel–Palade bodies or fenestrae, are not present in every EC. Developmentally, endothelium arises from mesoderm via the differentiation of hemangioblasts and/or angioblasts. However, other cell lineages may transdifferentiate into ECs, and ECs into other lineages. From a functional standpoint, the endothelium displays a remarkable ‘division of labor’. For example, endothelial cells that line the postcapillary venules are primarily responsible for mediating leukocyte trafficking, while arteriolar endothelial cells regulate vasomotor tone. In summary, each of the above definitions falls short of fully capturing the endothelium. -The ‘elusiveness’ of the endothelium reflects its marked heterogeneity in structure and function. - In the final analysis, the endothelium is best defined not by a single marker or function, but rather by its enormous behavioral repertoire. Phenotypic heterogeneity is not simply a descriptor, but rather is, in and of itself, a core property of the endothelium.
- Permeability -The endothelium is semipermeable; it must allow for regulated transport of fluids and solutes into and out of the blood. For purposes of discussion, permeability may be separated into 2 types: basal and inducible. -Under basal conditions, there is a continuous (although physiologically regulatable) flux of material between blood and underlying interstitium. Such activity takes place primarily in the capillaries, the major exchange vessels of the circulation. This may occur via the transcellular or the paracellular route. - The endothelium is capable of mediating inducible permeability in states of acute and chronic inflammation. The predominant site of inducible permeability is the postcapillary venule. According to the conventional view, agonists (eg, histamine, serotonin, bradykinin, substance P, and VEGF) induce EC retraction and the formation of intercellular gaps.66–68 Others have argued permeability-enhancing agents do not cause gap formation but result in increased transcellular vascular leakage of macromolecules via VVO-derived transendothelial pores. - Endothelium and permeability. A, Capillaries mediate constitutive (albeit physiologically regulatable) transfer of solutes and fluids between blood and underlying tissue. In continuous nonfenestrated endothelium, water and small solutes (molecular radius, 3 nm) pass between ECs, whereas larger solutes (depicted as back tracer) pass through ECs either via transendothelial channels or transcytosis, the latter process being mediated primarily by caveolae. Caveolae are particularly prevalent in capillaries of heart and skeletal muscle and rare in blood–brain barrier. -Compared with their nonfenestrated counterpart, continuous fenestrated endothelium demonstrates greater permeability to water and small solutes but similar reflection coefficients to albumin and larger macromolecules (the diaphragms of the fenestrae act as molecular filters). -Discontinuous endothelium is characterized by fenestrae (without diaphragms), gaps, and poorly organized basement membrane. These ECs contain many clathrin-coated pits, which play an important role in receptor-mediated endocytosis (although they may also take part in transcytosis). The endocytic pathway includes endosomal and lysosomal compartments (shown on the right). B, -In response to inflammation, postcapillary venules demonstrate increased permeability. Inducible transfer of water and solutes occurs between ECs (paracellular route) (I) and/or through ECs (transcellular route) (II). The paracellular route involves formation of gaps between ECs; the transcellular route involves VVO-mediated formation of transcellular pores. VVOs are enriched in the perijunctional regions of the cell. Red shapes in intercellular cleft represent tight junctional complexes.
- Mechanisms of EC heterogeneity. A, Hemangioblasts give rise to endothelial progenitor cells (angioblasts), which in turn differentiate into ECs of arteries, veins, and capillaries. Cell phenotypes are represented by color shades. Shown is the hypothetical relative role for microenvironment and epigenetics in mediating cell type–specific phenotypes. B, The role of the microenvironment in mediating nonheritable changes in EC phenotype is represented by receptor-mediated posttranslational modification of protein (eg, phosphorylation of a signal intermediate) and transcription factor–dependent induction of gene expression. Removal of the extracellular signal will result in eventual loss of translational/transcriptional effects, and residual effects will be “diluted out” with cell division. C, The role of epigenetics in mediating heritable changes in EC phenotype is represented by DNA methylation (•), histone methylation (CH3, •), and histone acetylation (red lines), which in turn negatively or positively influences gene expression. Methylation is regulated by a balance between methylases and demethylases, whereas acetylation of histones is mediated by a balance between histone acetyltransferases (HAT) and histone deacetylases (HDAC). Although epigenetic modifications are triggered by extracellular signals and are dynamically regulated, they may persist on removal of the signals, and are transmitted during mitosis. Epigenetics - functionally relevant changes to the genome that do not involve a change in the nucleotide sequence. Examples of mechanisms that produce such changes are DNA methylation and histone modification, each of which alters how genes are expressed without altering the underlying DNA sequence.
- The Main Functions of the endothelium may be classified into the following
- -The barrier function of exchange microvascular endothelium derives from the integrity of the endothelial structure, which maintains a low and selective permeability to fluid and solutes under normal physiological conditions. Blood fluid, solutes, and even circulating cells can cross the endothelium via two routes: through the cell body (transcellular), or between the cells (paracellular, or intercellular). Here, we discuss the ultrastructural basis and function of the transcellular pathway vs. paracellular pathway.
- -Transcytosis represents an important pathway of endothelial transcellular permeability to macromolecules. The mechanism involves vesicle-mediated endocytosis at the endothelial luminal membrane, vesicle-mediated transcytosis occurs when albumin binds to gp60 receptors on the endothelial cell surface followed by transcytosis across the cell, and exocytosis at the basolateral membrane. This process can be completed by individual vesicles capable of shuttling from the apical to basolateral membrane of an endothelial cell, or by clusters of interconnected vesiculo-vacuolar organelles (VVOs) that form channel-like structures 80–200 nm in diameter, spanning the cell interior. Endocytosis and exocytosis are mediated by caveolae, lipid raft microdomains that form “cave-like” invaginations in the plasma membrane. -Aquaporins are integral membrane proteins expressed in endothelial cells that permit the diffusive flux of water across the cell membrane.
- - Vesicle formation is triggered by Src kinase-mediated phosphorylation of caveolin-1 (cav-1) at the endothelial luminal membrane. Cav-1 subunits aggregate in lipid rafts and oligomerize to form caveolae. Other events, including albumin binding to the gp60 albumin receptor trigger vesicle endocytosis. Accessory proteins including dynamin and intersectin-2 are recruited to form the elongated neck of caveolae invaginations, which are then pinched off in response to Ras signaling to form enclosed vesicles. Vesicles remain docked to the inner surface of the cell membrane by vesicular (v)-SNARE binding to membrane-bound target (t)- SNARE, until docking is disrupted by the N-ethylmaleimide-sensitive factor (NSF). Undocked vesicles may attach to microtubules via ATP-driven motor molecules (kinesin/dynein) that facilitate vesicle movement across the cell interior. Trafficking vesicles may fuse with VVOs, or may dock (via SNAREs) at the basolateral membrane and undergo membrane fusion and exocytosis, releasing contents into the extravascular space. This entire process is transcytosis.
- Most endothelial cell–cell interfaces are fused together by intercellular junctions or pores that selectively allow water, macromolecules, and even blood cells to pass through. The structural and functional integrity of these junctions is a major determinant of paracellular permeability. - Two types of intercellular junctions have been characterized as the cell–cell adhesive barrier structures in the microvascular endothelium: the adherens junction (AJ) and tight junction (TJ).
- -Endothelial cells of the microvessel wall are joined together by intercellular junction proteins: adherens junctions (AJs), tight junctions (TJs) and/or gap junctions. -Barrier function in most vascular beds is provided by AJs. -Some specialized microvascular beds rely upon TJs for additional barrier function. -Endothelial cells are anchored to the basement membrane via focal adhesions. Focal adhesions and intercellular junctions are interconnected via cytoskeleton; Barrier function is dependent upon the stability and integrity of these three elements. - There are two additional structures related to endothelial cell–cell junctions that are not considered to be determinants of paracellular permeability. Gap junctions, formed of connexins, primarily facilitate signaling between endothelial cells, and do not directly contribute to barrier function. - The molecular structure of gap junctions is characterized by six units of connexin forming a channel that connects the cytosols of adjacent endothelial cells, allowing rapid propagation of signaling molecules (e.g., Ca2+) between the cells [242].
- Adherens junctions. Adherens junctions (AJs) are ubiquitous throughout the vasculature. The intercellular adhesion protein vascular endothelial (VE)-cadherin is principally responsible for barrier function and is believed to be the most important protein in forming the molecular basis, as well as regulating the function of AJs. VE– cadherin is a transmembrane receptor; its extracellular domain binds to the extracellular domain of another VE–cadherin expressed in the membrane of an adjacent endothelial cell. By forming a homotypic bond in this manner, VE–cadherin glues the neighboring cells together. The intermolecular binding of VE–cadherin extracellular domains is dependent upon extracellular calcium. Intracellularly, VE–cadherin is connected to the actin cytoskeleton via a family of catenins (α-, β-,γ-, and p120-catenins) VE–cadherin binds directly to β-catenin and γ-catenin, which in turn are connected to actin via binding to α-catenin VE–cadherin is also stabilized by binding to p120-catenin, though p120-catenin does not directly bind to actin. Rather, p120-catenin binds to protein kinases (Src kinases) and phosphatases, serving as a scaffold to bring these signaling molecules into proximity with adherens junction proteins for further interactions. The stability of the VE–cadherin–catenin–cytoskeleton complex is essential to the maintenance of endothelial barrier function Other junction proteins contribute to AJ structure, including Junction adhesion molecules ( JAM-A, -B and -C) and platelet-endothelial cell adhesion molecule (PECAM)-1. JAMs connect to the actin cytoskeleton via zona occludens (ZO)-1 and α-catenin, which may stabilize AJs. PECAM-1 facilitates cell–cell binding with circulating blood cells.
- Tight junctions. Tight junctions (TJs) are found in most vascular beds; however, TJs contribute to microvascular barrier function only in a few specialized tissues, including the brain, retina and testicles. Endothelial tight junctions are similar to adherens junctions, but are composed of interactions of tight junction proteins: occludin, claudins (3/5), and JAM-A Occludins and claudins are integral membrane proteins, each with four transmembrane domains and two extracellular loop domains. -The extracellular loop domains of occludin or claudins form homotypic binding with the extracellular domains of like molecules on neighboring endothelial cells. JAM-A, a member of the immunoglobin superfamily of proteins, is also present in tight junctions, though the role of JAM-A in tight junctions is not understood. Occludin, claudins, and JAM-A are connected to the actin cytoskeleton via zona occludens proteins (ZO-1, ZO-2) and α-catenin
- Focal adhesions are points of attachment between the endothelial basolateral membrane and the surrounding extracellular matrix (ECM) of the microvascular wall The major structural components of focal adhesions are transmembrane receptors called integrins Their intracellular domains interact with the cytoskeleton either directly or indirectly through the linker proteins paxillin, talin, vinculin, or α-actinin Their large extracellular domains bind to respective matrix proteins, such as fibronectin, vitronectin, collagen, fibrinogen, and Laminin - Integrin–matrix binding is essential to the establishment and stabilization of endothelial barriers. Altering integrin-binding properties reduces focal adhesion strength or causes cell detachment from the substratum
- How do focal adhesions help maintain the endothelial barrier function? The precise mechanisms are not completely understood. It appears that various physical and chemical signals can be sensed and coordinated at the cell–matrix focal contact sites where integrins play a central role in transmembrane crosstalk between the cells and extracellular matrix. It seems that focal adhesions are lipid raft domains containing scaffold proteins that bind to multiple intracellular signaling molecules. ECM–integrin interactions induce “outside-in” signaling events that may contribute to the maintenance of endothelial barrier integrity. Binding of RGD (Arg-Gly-Asp) proteins to integrins triggers intracellular signaling events (outside-in signaling) including activation and recruitment of kinases (e.g., Src kinase, focal adhesion kinase (FAK). While agonist-receptor binding triggers “inside-out” signaling events that modulate integrin–ECM adhesions which increases integrin extracellular binding affinity and induces integrin clustering at focal adhesions (inside-out signaling). Clustering increases the collective binding capacity (avidity) of integrins at focal adhesions, strengthening adhesion to the basement membrane. Integrin clustering is further stabilized by intracellular connections to the actin cytoskeleton (via α-actinin, vinculin, talin, paxillin, etc.) and serves to maintain barrier integrity. The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands.
- The endothelial cytoskeleton is composed of microtubules, intermediate filaments and actin filaments Polymeric components of the cytoskeleton: Actin microfilaments, microtubules and vimentin intermediate filaments, stabilize endothelial cell structure. Cell peripheral (cortical) actin filaments stabilize intercellular junctions and maintain focal adhesion integrity to support normal physiological endothelial barrier function. Microtubules and intermediate filaments provide additional support. Under inflammatory conditions, including RhoA activation, microtubules disassemble into α- and β- tubulin subunits, and actin filaments, reorganize into parallel and linear stress fiber bundles that span the cell interior, pathological characteristics of endothelial hyperpermeability.
- A summary of the signaling molecules that are well accepted as permeability regulators in microvascular endothelium. In general, the canonical pathway leading to increased permeability includes elevation of intracellular calcium and activation of protein kinase C (PKC), myosin light-chain kinase (MLCK), Src family kinases, and the small GTPase RhoA. Molecules that are considered barrier protectors (permeability-decreasing factors) include protein kinase A (PKA), Epac, sphingosine-1-phosphate (S1P), and the small GTPases Rac-1 and cdc42. Other molecules are known to alter barrier function but their effects (i.e., increase or decrease permeability) are controversial, including nitric oxide (NO), cyclic GMP, and Rac-1.
- The two faces of the endothelial cell. Like Janus, the Roman god of thresholds or portals, the endothelial cell has two faces. Strategically positioned at the interface of the blood and the tissues, the endothelial cell likewise must cast a gaze forward and backwards. The duality between the homeostatic properties (left) and the functions involved in host defences, tissue injury and many diseases (right) also evokes the two faces of Janus.
- Pivotal role of the endothelial cells in hemostasis, thrombosis and fibrinolysis. Upper panel: In normal conditions, endothelial cells show an anti-thrombotic phenotype Degradation of the aggregating agent ADP with ectonucleotidases; Prevention of primary hemostasis: i.e., platelet activation, adhesion and aggregation, by expressing 13-hydroxyoctadecadienoic acid (13-HODE), and releasing prostacyclin (PGI2), and NO], An anticoagulant phenotype [expression of the thrombin receptor thrombomodulin and the protein C/S system, which converts thrombin from a procoagulant to an anticoagulant enzyme; Expression of the tissue factor pathway inhibitor and the binding/activation of anti-thrombin to cell surface heparin-like molecules such as glycosaminoglycans (heparan sulfate)] and a pro-fibrinolytic phenotype [release of tissue-type plasminogen activator (t-PA) and possibly urokinase-type plasminogen activator (u-PA)]. Lower panel: Following vascular injury and under pathological conditions, the endothelium shifts to a pro-thrombotic/pro-coagulant phenotype, by releasing the von Willebrand factor (vWF), thromboxane A2 (TXA2) and platelet-activating factor (PAF), synthesized via the remodeling pathway, which play a major role in initial platelet recruitment and thrombus formation, by expressing the thrombin receptor protease-activated receptor-1 (PAR-1), which enhances the release of vWF and reinforces endothelial cell activation, for instance, by promoting the expression of adhesion molecules to the cell surface (ICAM-1, VCAM-1, E-selectin, P-selectin), and by producing plasminogen activator inhibitor type-I (PAI-1), which prevents fibrinolysis by inhibiting t-PA (and u-PA) activity. ADP: adenosine diphosphate; AMP: adenosine monophosphate; COX: cyclooxygenase; NOS: nitric oxide synthase; PLA2: phospholipase A; EC2.3.1.67: acetyl-CoA:2-lyso-PAF acetyltransferase.
- Release of tissue factor. Traumatized tissue releases a complex of several factors called tissue factor or tissue thromboplastin. This factor is composed especially of phospholipids from the membranes of the tissue plus a lipoprotein complex that functions mainly as a proteolytic enzyme. Activation of Factor X-role of Factor VII and tissue factor. The lipoprotein complex of tissue factor further complexes with blood coagulation Factor VII and, in the presence of calcium ions, acts enzymatically on Factor X to form activated Factor X (Xa). Effect of Xa to form prothrombin activator-role of Factor V. The activated Factor X combines immediately with tissue phospholipids that are part of tissue factors or with additional phospholipids released from platelets, as well as with Factor V to form the complex called prothrombin activator. Within a few seconds, in the presence of calcium ions (Ca++), this splits prothrombin to form thrombin, and the clotting process proceeds as already explained. At first, the Factor V in the prothrombin activator complex is inactive, but once clotting begins and thrombin begins to form, the proteolytic action of thrombin activates Factor V. This then becomes an additional strong accelerator of prothrombin activation. Thus, in the final prothrombin activator complex, activated Factor X is the actual protease that causes splitting of prothrombin to form thrombin; activated Factor V greatly accelerates this protease activity, and platelet phospholipids act as a vehicle that further accelerates the process. Note especially the positive feedback effect of thrombin, acting through Factor V, to accelerate the entire process once it begins.
- Blood trauma causes (1) activation of Factor XII and (2) release of platelet phospholipids. Trauma to the blood or exposure of the blood to vascular wall collagen alters two important clotting factors in the blood: Factor XII and the platelets. When Factor XII is disturbed, such as by coming into contact with collagen or with a wettable surface such as glass, it takes on a new molecular configuration that converts it into a proteolytic enzyme called "activated Factor XII." Simultaneously, the blood trauma also damages the platelets because of adherence to either collagen or a wettable surface (or by damage in other ways), and this releases platelet phospholipids that contain the lipoprotein called platelet factor 3, which also plays a role in subsequent clotting reactions. Activation of Factor XI. The activated Factor XII acts enzymatically on Factor XI to activate this factor as well, which is the second step in the intrinsic pathway. This reaction also requires HMW (high-molecular-weight) kininogen and is accelerated by prekallikrein. Activation of Factor IX by activated Factor XI. The activated Factor XI then acts enzymatically on Factor IX to activate this factor as well. Activation of Factor X-role of Factor VIII. The activated Factor IX, acting in concert with activated Factor VIII and with the platelet phospholipids and factor 3 from the traumatized platelets, activates Factor X. It is clear that when either Factor VIII or platelets are in short supply, this step is deficient. Factor VIII is the factor that is missing in a person who has classic hemophilia, for which reason it is called antihemophilic factor. Platelets are the clotting factor that is lacking in the bleeding disease called thrombocytopenia. Action of activated Factor X to form prothrombin activator-role of Factor V. This step in the intrinsic pathway is the same as the last step in the extrinsic pathway. That is, activated Factor X combines with Factor V and platelet or tissue phospholipids to form the complex called prothrombin activator. The prothrombin activator in turn initiates within seconds the cleavage of prothrombin to form thrombin, thereby setting into motion the final clotting process, as described earlier.
- NO is produced from the amino acid L-arginine by the enzymatic action of nitric oxide synthase (NOS). There are two endothelial forms of NOS: constitutive NOS (cNOS; type III) and inducible NOS (iNOS; type II). Co-factors for NOS include oxygen, NADPH, tetrahydrobiopterin and flavin adenine nucleotides. In addition to endothelial NOS, there is a neural NOS (nNOS; type I) that serves as a transmitter in the brain and in different nerves of the peripheral nervous system, such as non-adrenergic, non-cholinergic (NANC) autonomic nerves that innervate penile erectile tissues and other specialized tissues in the body to produce vasodilation. Under normal, basal conditions in blood vessels, NO is continually being produced by cNOS. The activity of cNOS is calcium- and calmodulin-dependent. There are two basic pathways for the stimulation of cNOS, both of which involve release of calcium ions from subsarcolemmal storage sites. First, shearing forces acting on the vascular endothelium generated by blood flow causes a release of calcium and subsequent cNOS activation. Therefore, increases in blood flow stimulate NO formation (flow-dependent NO formation). Second, endothelial receptors for a variety of ligands stimulate calcium release and subsequent NO production (receptor-stimulated NO formation). Included are receptors for acetylcholine, bradykinin, substance-P, adenosine, and many others vasoactive substances. In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980 he and others identified this substance as being NO. When NO forms, it has a half-life of only a few seconds, in large part because superoxide anion has a high affinity for NO (both molecules have an unpaired electron making them highly reactive). Therefore, superoxide anion reduces NO bioavailability. NO also avidly binds to the heme moiety of hemoglobin (in red blood cells) and the heme moiety of the enzyme guanylyl cyclase, which is found in vascular smooth muscle cells and most other cells of the body. Therefore, when NO is formed by vascular endothelium, it rapidly diffuses into the blood where it binds to hemoglobin and subsequently broken down. It also diffuses into the vascular smooth muscle cells adjacent to the endothelium where it binds to and activates guanylyl cyclase. This enzyme catalyzes the dephosphorylation of GTP to cGMP, which serves as a second messenger for many important cellular functions, particularly for signalling smooth muscle relaxation. Cyclic GMP induces smooth muscle relaxation by multiple mechanisms including increased intracellular cGMP, which inhibits calcium entry into the cell, and decreases intracellular calcium concentrations (click here for details) activates K+ channels, which leads to hyperpolarization and relaxation stimulates a cGMP-dependent protein kinase that activates myosin light chain phosphatase, the enzyme that dephosphorylates myosin light chains, which leads to smooth muscle relaxation.
- ET was soon defined as the most potent and long-lasting endogenous vasoconstrictive substance yet discovered [2–4]. Since then the ET system has been found to be involved in multiple physiologic functions related to the nervous, renal, cardiovascular, respiratory, gastrointestinal and endocrine systems. ET is produced predominantly by endothelial cells but it is also produced by leukocytes, macrophages, smooth muscle cells, cardiomyocytes and mesangial cells. Endothelial cells predominately produce ET-1 while ET-2 and ET-3 are also expressed in a wide variety of cell types. ET genes (preproET genes) code for a large precursor-protein mRNA (preproET mRNA). Different stimuli modulate the transcription of the preproET-1 gene. The translation of preproET-1 mRNA results in the formation of a 203-amino acid preproET-1 peptide, which is cleaved by a furin convertase [10] to the 38-amino acid peptide big ET-1. Big ET-1 is transformed to ET-11–21 through cleavage of the Trp21–Val22 bond by ET-converting enzyme-1 (ECE-1), which exists in four isoforms (a, b, c and d) [11] and by chymase and non ECE metalloproteases . In addition, chymase cleaves big ET-1 at the Tyr31–Gly32 bond, resulting in the formation of ET-11–31 [12]. The 31- residue ET-1 has vasoconstrictive effects but its exact physiologic and pathophysiologic roles are currently not clear. Promoters Hypoxia Ischemia Shear stress Inhibitors Nitric oxide Prostacyclin Atrial natriuretic peptides Estrogens All three ETs bind to two types of receptors named ETA and ETB: in the cardiovascular system, ETA-receptors are found in smooth muscle cells and cardiac myocytes, whereas ETB-receptors are localized on both endothelial cells and in smooth muscle cells. The binding of ET-1 to smooth muscle cells ETA- and ETB receptors activates phospholipase C, which leads to an increase of inositol triphosphate, diacylglycerol and intracellular calcium and, consequently, to long-lasting vasoconstriction [14]. The increase of diacylglycerol and calcium stimulates also protein kinase C, which mediates the mitogenic action of ET-1 [15]. On the other hand, the activation of endothelial ETB-receptors stimulates the release of NO and prostacyclin [16], prevents apoptosis [17], inhibits ECE-1 expression in endothelial cells and plays a minor role in endothelial-dependent vasodilatation [18]. ETB-receptors also mediate the pulmonary clearance of circulating ET-1 [19] and the reuptake of ET-1 by endothelial cells (Fig. 2). There is a cross talk between ETAand ETB-receptors apparently causing compensation when only one receptor is antagonized [20,21].
- The migration of leukocytes from the vascular system to sites of injury or pathogenic exposure is a key event in the process of inflammation. The entry of leukocytes into sites of injury or infection requires molecular mechanisms, which enable them to recognize such sites from within the vasculature and to establish contact with the endothelium in order to exit and migrate through the endothelium of post-capillary venules.
- - Recognition as well as contact formation is mediated by several adhesion molecules, which act in a sequential manner in coordination with regulatory mediators such as the chemokines (Fig. 3). Monocytes, lymphocytes, and neutrophils all migrate by these similar, sequence-dependent mechanisms but differ in their response to chemotactic and inflammatory signals, particularly in their qualitative and quantitative expression of adhesion molecules (Springer, 1994). -Capture and rolling. Both the capture or initial tethering and removal of leukocytes from the flowing blood, and their rolling along the vessel wall are due to the reversible binding of transmembrane glycoprotein adhesive molecules called selectins which are found both on the leukocytes and on endothelial cells (Ebnet and Vestweber, 1999; Wagner and Roth, 2000; Ley, 2002). In contrast to the vast majority of other adhesion molecules, the selectins mediate cell contact via binding to carbohydrate ligand structure and thus, they are lectins. L-selectin is found on most types of leukocytes (Finger et al., 1996), E-selectin is specific of endothelial cells (Bevilacqua et al., 1989) and P-selectin is found on endothelium and platelets (McEver et al., 1989). - A limited number of discrete glycoproteins, modified with certain oligosaccharides, for example, the sLeX motif, define the physiological ligands of the selectins. However, the exact nature of these ligands on cell surface is still unclear. L-selectin is a major carbohydrate- presenting ligand for E-selectin on human neutrophils identified in vitro but the actual in vivo ligand may be an unique mucin modified with N-linked sugars that has been characterized in murine cells and called E-selectin ligand-1 (Willmroth and Beaudet, 1999). L-selectin binds to at least three glycoproteins: Gly- CAM-1, CD-34, and MAdCAM-1 which are expressed on the endothelial cell surface, the most probable being a fucosylated variant of CD34 (Tedder et al., 1995; Varki, 1997). P-selectin interacts with its leukocyte counterpart, P-selectin glycoprotein ligand-1 (PSGL-1) which is, like CD34, modified with O-linked sialic acid and fucose (McEver and Cummings, 1997). - The process of margination, capture, and rolling, is a normal behavior of circulating neutrophils. Only after appropriate stimuli, do rolling leukocytes become firmly bound to endothelial cells. Leukocyte rolling is first mediated by L-selectin. Since L-selectin is constitutively expressed on leukocytes, the induced adherence in inflammatory situation requires induction of L-selectin ligand and/or of E- and P-selectins on the endothelial surface. E-selectin is transcriptionally induced by cytokines such as IL-1, TNF-a, or by LPS (Klein et al., 1995; Scholz et al., 1996). In contrast, P-selectin is stored in the membrane of storage granules, the Weibel– Palade bodies, in endothelial cells (McEver et al., 1989). Fusion of these granules with the plasma membrane is rapidly stimulated by proinflammatory mediators such as histamine and thrombin. In addition to the regulation of its transport to the cell surface, P-selectin is also inducible by cytokines. - L-selectin (constitutive rolling on endothelial cells) and P- and/or E-selectins (inflammatory mediator induced capture) work cooperatively to initiate leukocyte migration. The reversible selectin–ligand interaction allows time for leukocytes to associate with endothelial cells and to sense and respond to stimuli presented on the endothelial cell surface which stimulate them to adhere more firmly. Such signals are provided by chemokines or by other mediators like PAF. -
- Endothelium and leukocyte trafficking. A, According to the classical multistep paradigm for leukocyte recruitment (based on studies of skin, skeletal muscle, and mesentery), leukocytes (neutrophils are shown) undergo initial attachment, followed by rolling, firm adhesion, and transmigration through activated endothelium in postcapillary venules (labeled as venule). Rolling and adhesion are mediated by interactions between EC receptors (P- and E-selectin and ICAM-1) and leukocyte counter-receptors/ ligands (not shown). Leukocytes may transmigrate between or through ECs. The molecular details of transendothelial cell migration (also known as diapedesis or extravasation) are less well understood but appear to involve PECAM-1/CD31, junctional adhesion molecule-1 (JAM-1), and CD99. Activated ECs present chemokines that induce integrin activation on the surface rolling leukocytes (not shown). B, In the lung, the majority of leukocyte trafficking takes place in pulmonary capillaries. Leukocyte trafficking at this site is dependent on ICAM-1 but not E- or P-selectin. Other contributing mechanisms include reduced deformability of activated neutrophils and activation state of the endothelium. Note that leukocytes must cross 2 barriers (endothelium and epithelium) to reach the airspace. C, In the liver, leukocyte trafficking occurs primarily in sinusoids by an ICAM-1–dependent mechanism. Cytoplasmic projections of the neutrophil may reach through fenestrations and gaps into the space of Disse and thus sense and respond to signals from hepatocytes and Ito cells. D, In HEVs of mesenteric lymph nodes, lymphocytes constitutively roll on, adhere to and transmigrate across specialized cuboidal endothelium via distinct mechanisms that involve binding of lymphocyte L-selectin to EC PNAd and mucosal addressin cell adhesion molecule (MadCAM-1), and leukocyte integrins to ICAM-1, ICAM-2, and MadCAM-1. Not shown in the figure (but of importance in health and disease) is the transmigration of monocytes or lymphocytes in peripheral tissues, as well as leukocyte trafficking in large veins and arteries.
- -It is generally accepted that blood vessels develop via two subsequent processes. During vasculogenesis, formation of the earliest primitive capillaries is achieved by in situ differentiation of hemangiogenic stem cells that are derived from pluripotent mesenchymal cells. The resulting angioblastic cells give rise to endothelial precursor cells (Figure 1). Only thereafter, during angiogenesis new blood vessels derive from already existing vessels. - Physiological as well as pathological processes require vasculogenesis and angiogenesis for the same reason, blood supply. Different inducers and stimulators affect angiogenesis and vasculogenesis by directly or indirectly stimulating proliferation, differentiation and migration of endothelial or respective precursor cells.
- Putative molecular and morphological steps during vasculogenesis and angiogenesis in placental villi. Depicted on the left are the regulating cells and extracellular matrix components together with growth factors and receptors involved; on the right the steps of differentiation can be found. For details in branching and non-branching angiogenesis
- -Alterations of endothelial cells and the vasculature play a central role in the pathogenesis of a broad spectrum of the most dreadful of human diseases, as endothelial cells have the key function of participating in the maintenance of patent and functional capillaries. -When considering the role of the endothelium in disease, the two most common terms that are used are endothelial cell activation and endothelial cell dysfunction. -Pober and Gimbrone were the first to demonstrate that a well defined stimulus could induce the expression of an endothelial cell marker. (Pober JS, Gimbrone MAJr.: Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro: demonstration by monoclonal antibody binding and immunoprecipitation. Proc Natl Acad Sci USA, 1982, 79, 6641–6645.)
- Endothelial cell activation refers to the phenotypic response to an inflammatory stimulus and is not an all-or-none phenomenon. Rather, in keeping with the theme of heterogeneity, endothelial cells display a spectrum of responses. By contrast, endothelial cell dysfunction is by definition definition maladaptive. - The endothelial cell is not like a toggle (on-and off switch) rather is works more like a dimmer switch in which the phenotype displays a spectrum of responses.
- EC activation is commonly classified into type I and II responses. Type I activation loosen the EC junctions to increase the permeability and export Weibel-Palade bodies (exocytosis) to release the stored vonWillebrand factor (vWF) and P-selectin, initiating the endothelial interaction with leukocyte and platelets. It is a rapid but transient response independent of de novo gene expression. Type II activation provides a more sustained inflammatory response and invokes the gene expression of a variety of proinflammatory cytokines and adhesion molecules.
- Tumor necrosis factor (TNF)-alpha, produced primarily by activated macrophages and helper T-cells (38-40) is the most proximal cytokine mediator. It is detected in the serum within 20 minutes after an immune challenge (41). Its concentration peaks between 90 minutes and 2 h after endotoxin injection
- As a transcription factor, NF-B activates the transcription of the proinflammatory genes including TNF-, interleukin-1 (IL-1), interleukin-8 (IL-8), E-selectin, VCAM-1, and ICAM-1 by binding to the cognate cis-elements within the regulatory regions of these target genes (22, 69, 80, 98). TNF signaling pathways in endothelial cells (ECs). The binding of TNF with its receptor (TNFR1) causes the recruitment of TNFR-associated via death domain protein (TRADD), receptor-interacting serine/threonine-protein kinase 1 (RIPK1, also called as RIP), and TNFR-associated factor 2 (TRAF2). These molecules form the TNFR1 signalosome. TRAF2 catalyzes the ubiquitination (Ub) of RIP and recruits the NF-B essential modifier (NEMO)/IB kinase (IKK) complex, leading to the phosphorylation (P) of IB by IKK and the ensuing ubiquitination and degradation of IB in proteasome. The release of NF-B p65 and p50 into nucleus results in the transcriptional activation of the proinflammatory target genes such as ICAM-1, VCAM-1, and E-selectin to mediate leukocyte adhesion and transmigration. CIAP, cellular inhibitor of apoptosis proteins. The IκB kinase (IKK) is an enzyme complex that is involved in propagating the cellular response to inflammation.[1] The IκB kinase enzyme complex is part of the upstream NF-κB signal transduction cascade. The IκBα (inhibitor of kappa B) protein inactivates the NF-κB transcription factor by masking the nuclear localization signals (NLS) of NF-κB proteins and keeping them sequestered in an inactive state in the cytoplasm.[2][3][4] IKK specifically, phosphorylates the inhibitory IκBα protein.[5] This phosphorylation results in the dissociation of IκBα from NF-κB. NF-κB, which is now free migrates into the nucleus and activates the expression of at least 150 genes
- TLR activation in ECs. Upon activation by pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs), Toll-like receptor 4 (TLR4) dimerizes and associates with myeloid differentiation primary response gene 88 (MyD88), which recruits IL-1 receptor associated kinases (IRAKs) to TLR. IRAKs are activated by phosphorylation and then associate with TRAF6. The IRAK-1/TRAF6 complex dissociates from the TLR4 and associates with TGF-- activated kinase 1 (TAK1), TAK1-binding protein 1 (TAB1), and TAB2. TRAF6 in turn polyubiquitinates and facilitates the binding of TAK1 with IKK, leading to the activation of NF-B.
- The healthy endothelium not only arbitrates endothelium-dependent vasodilation, but also actively suppresses thrombosis, vascular inflammation, and hypertrophy. This schematic depicts differences between a healthy endothelium (A) and a dysfunctional one (B). A healthy endothelium displays a vasodilatory phenotype consisting of high levels of vasodilators such as: nitric oxide (NO) and prostacyclin (PGI2) and Low levels of reactive oxygen species (ROS) and uric acid. A healthy endothelium also has an anticoagulative phenotype consisting of low levels of plasminogen activator inhibitor 1 (PAI-1) von Willebrand factor (vWF) P-selectin. Very little inflammation may be present, as indicated by low levels of: soluble vascular cell adhesion molecule (sVCAM.), soluble intercellular adhesion molecule (sICAM), E-selectin, C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6). Finally, the population of endothelial progenitor cells (EPCs, indicative of vascular repair capacity) is high Whereas levels of endothelial microparticles (EMPs) and circulating endothelial cells (CECs), indicative of endothelial stress/damage, are low. In the case of a dysfunctional endothelium, the phenotypic characteristics include impaired vasodilation, increased oxidative stress/uric acid, lipid peroxide radical,Nitrotyrosine and Nitirc oxide, and a procoagulant and pro-inflammatory phenotype with decreased vascular repair capacity and increased numbers of EMPs and CECs. 6-keto PGF1α: 6 keto prostaglandin F1-alpha, a stable product of PGI2; ADMA; asymmetric dimethyl arginine, inhibitor of NO biosynthesis; EC: endothelial cell; NO2 −: nitrite ion, stable degradation product of NO; NO3 − nitrate ion, stable degradation product of NO; ONOO−: peroxynitrite, the product of superoxide-mediated inactivation of NO; VSMC: vascular smooth muscle cell; WBC: white blood cell. (Modified from Dylan Burger and Rhian MT 2012).
- Although endothelial dysfunction occurs in many different disease processes, oxidative stress can be identified as a common denominator (77, 78). Reactive oxygen species play a central role in vascular physiology and pathophysiology. NO,O2 , the hydroxyl radical (OH), H2O2, and peroxynitrite (ONOO) are produced in the vasculature under both normal and stress conditions such as inflammation or injury. O2 can be generated by different enzymes (e.g., NADPH oxidase, xanthine oxidase, cyclooxygenases, NO synthases, cytochrome P450 monooxygenases, and enzymes of the mitochondrial respiratory chain) in virtually all cell types, including vascular smooth muscle and endothelial cells.
- NO, O2 , the hydroxyl radical (OH), H2O2, and peroxynitrite (ONOO) are produced in the vasculature under both normal and stress conditions such as inflammation or injury. O2 can be generated by different enzymes (e.g., NADPH oxidase, xanthine oxidase, cyclooxygenases, NO synthases, cytochrome P450 monooxygenases, and enzymes of the mitochondrial respiratory chain) in virtually all cell types, including vascular smooth muscle and endothelial cells. Superoxide either spontaneously or enzymatically [through dismutation by superoxide dismutase (SOD)] is reduced to the uncharged H2O2. H2O2 in the presence of the enzyme catalase or glutathione peroxidase is then dismutated into water and oxygen. In the presence of transition metals (copper, iron) or superoxide anions, H2O2 generates, through the Fenton or Haber-Weiss reaction, respectively, the highly reactive hydroxyl radicals that can be scavenged by mannitol or dimethylthiourea. Hydroxyl radicals cause cell damage through the peroxidation of lipids and sulfhydryl groups. When NO and O2 are produced in close vicinity, they interact to form ONOO, a potent oxidant also capable of oxidizing sulfhydryl groups as well as nitrating and hydroxylating aromatic groups, including tyrosine, tryptophan, and guanine (305; Figs. 1 and 2).
- Endothelial dysfunction could contribute to lower extremity ischemia by impairing blood flow responses to ischemia, collateral formation and arterial remodelling, and by promoting vasospasm, thrombosis, plaque rupture and lesion progression.
- Under pathological conditions, the endothelium may also be ‘activated’ to express prothrombotic and proinflammatory factors, including tissue factor, adhesion molecules, chemotactic factors and cytokines. In general, cardiovascular risk factors are associated with an alteration of endothelial phenotype that promotes atherosclerosis. Potential links between endothelial dysfunction and the clinical expression of peripheral arterial disease (PAD). COX-2 Cyclooxygenase-2; CRP C-reactive protein; eNOS Endothelial nitric oxide synthase; FMD Flow-mediated dilation; ICAM-1 Intercellular adhesion molecule-1; IL-1β Interleukin-1-beta; MCP-1 Monocyte chemoattractant protein-1; NFκB Nuclear factor kappa B; ox-LDL Oxidized low-density lipoprotein; RH Reactive hyperemia; ROS Reactive oxygen species; TNF-α Tumor necrosis factor-alpha
- There is much evidence suggesting that endothelial dysfunction can play a role in the pathogenesis of ischemic stroke.
- Several lines of evidence suggest that formation of NO occurs in cerebral blood vessels under basal conditions both in vitro and in vivo. Inhibitors of NO synthase decrease basal levels of cyclic guanosine monophosphate (cGMP), and produce contraction of cerebral arteries (5). In addition to exerting a tonic dilator effect on the cerebral circulation, basal release of NO may protect cerebral endothelium by inhibiting aggregation of platelets and leukocytes. Impaired endothelium-dependent relaxation of cerebral blood vessels has been observed during chronic hypertension, diabetes, hypercholesterolemia, subarachnoid haemorrhage (6), ischaemia, and ageing. PUTATIVE MECHANISMS Production of an EDCF that counteracts the normal dilator effect of NO This EDCF appears to be a cyclooxygenase product of arachidonic acid metabolism that activates a prostaglandin H2/thromboxane A2 receptor. Reduced activity of NO synthase Enhanced breakdown of NO after its generation. Presence of hemoglobin in subarachnoid hemorrhage Inhibition of NO leading to vasospasm inactivate endothelium-derived NO by generating superoxide anions
- -Endothelial cell damage occurs in many vascular beds during hypertension. However, it is not clear whether hypertension is the cause or the result of this damage. - Supporting the notion that endothelial dysfunction is one of the causes of hypertension is the finding of impaired endothelial function in the normotensive offspring of patients with essential hypertension
- -The impaired endothelial function was described as a decreased vasodilating response to acetylcholine, an endothelium-dependent relaxant agent, in the forearm of normotensive offspring of essential hypertensive patients compared with normotensive subjects without familial histories of hypertension. -Because the response to sodium nitroprusside, a direct smooth muscle cell–dependent vasodilator, and calculated minimal FVR, an index of vessel wall structural changes, were not different in the same two groups of subjects, the present findings are consistent with the presence of impaired endothelium-dependent vasodilation in subjects with genetic predispositions to hypertension. -In humans, although not universally demonstrated, endothelium-dependent vasodilation to acetylcholine is reduced in essential hypertensive patients compared with normotensive control subjects suggesting that endothelial function can be impaired in human hypertension. -This abnormality seems to be caused by the simultaneous presence of a defect in the L-arginine–nitric oxide pathway and production of a cyclooxygenase-dependent EDCF.
- -Higher oxidative stress in some hypertensive states could be due to increased levels of angiotensin- II, which stimulates NADPH oxidase to generate ROS, thus causing vascular inflammation (60). By preventing these effects, ACEI’s and ARB’s improve vasorelaxation in hypertensive patients. - Increased oxidative stress in hypertensive states leads to decreased availability of NO Overproduction of endothelin-1 may play a role in hypertension
- Presumed mechanism of changes in the nitric oxide (NO) pathway in hypertension. (a) Decreased NO production. L-arginine deficiency, Changes in L-arginine transporter, Accumulation of endogenous endothelial NO synthase (eNOS) inhibitors (asymmetric dimethylarginine (ADMA) Cofactor deficiency (tetrahydrobiopterin (BH4) Decreased eNOS gene Decreased half-life of eNOS mRNA Changes in Gi proteins Changes in calcium-independent pathways of eNOS activation (tyrosine or serine phosphorylation) and Changes in the interactions between eNOS and caveolin−HSP90. (b) Increased NO degradation (oxidative stress). (10) uncoupling of eNOS, (11) NADPH (reduced nicotinamide adenine dinucleotide phosphate) expression and (12) increased xanthine oxidase. (Akt, protein kinase B; Ang II, angiotensin II; CaM, calmodulin; CAT, cationic amino-acid transporter; DDAH, dimethylarginine dimethylaminohydrolase; GTP, guanosine 5'-triphosphate; HSP, heat shock protein; PIP3K, phosphatidylinositol 3-kinase; THB, tetrahydrobiopterin; TNF, tumour necrosis factor ; Tyr K, tyrosine kinase.) -Given the mechanisms of increased inactivation of NO, this inactivation results from binding to various molecules like haemoglobin or albumin, but the decrease in NO bioavailability is mostly the consequence of its interaction with superoxide anions, a phenomenon that is probably of importance. in arterial hypertension
- The major cardiovascular risk factors increase vascular production of reactive oxygen species, which may reduce endothelial NO availability via at least 3 pathways: direct inactivation of NO by superoxide (O2 .), resulting in loss of the bioactivity of NO; reduced NO-synthase activity due to increased levels of ADMA, an endogenous NO-synthase inhibitor, resulting from redox-sensitive inhibition of DDAH; and eNOS “uncoupling,” due to increased oxidation of H4B. ONOO- indicates peroxynitrite.
- Potential mechanisms leading to increased vascular superoxide (O2.) production in patients with coronary disease. Coronary activities of NAD(P)Hoxidase and xanthine oxidase, both potent O2 .-producing enzymes, are increased in patients with coronary disease, as determined by electron spin resonance spectroscopy. In contrast, the coronary activity of ecSOD, a major vascular O2 .-degrading enzyme system, is markedly reduced in these patients. CAD indicates coronary artery disease.
- Patients with diabetes invariably show impaired endothelium-dependent vasodilation. This is partly due to the frequent association of the disease with other cardiovascular risk factors, including hypertension, obesity, and dyslipidemia. Moreover, diabetic as well as obese patients usually consume a high-calorie diet rich in macronutrients that per se is able to induce vascular abnormalities (77). Indeed, protein, lipid, and glucose loads are associated with a marked production of ROS (78); and high-fat meals, with impaired endothelium-dependent vasodilation (79). A crucial negative effect of such meals is particularly attributable to high levels of circulating free fatty acids, which are able to induce ROS production and impair endothelial function (80). Mechanisms leading to endothelial damage in diabetes, independent of the damage due to other cardiovascular risk factors, include insulin resistance, hyperglycemia, and low-grade systemic inflammation.
- Origin of endothelial dysfunction in diabetes mellitus. Endothelial dysfunction in diabetes can be induced solely by or by a combination of hyperglycemia fatty acids inflammation (4) insulin resistance. - Activation and effects of protein kinase C (PKC): High levels of free fatty acids and hyperglycaemia activate DAG, which in turn activates PKC. This increases expression of ET-1, VCAM, ICAM, NFκB and NADPH oxidase. NADPH oxidase activation results in decreased NO bioavailability. PKC may have a role in PI3-kinase pathway inhibition. Ruboxistaurin inhibits PKC activation. DAG: diacylglycerol; VCAM: vascular cell adhesion molecule; ICAM: intercellular adhesion molecule; NFκB: nuclear factor κB; ET-1: endothelin 1; NADPH: nicotinamide adenine dinucleotide phosphate; PI3-kinase, phosphoinositide-3 kinase. Prolonged exposure to hyperglycemia is now recognized as a major factor in the pathogenesis of diabetic complications, including atherosclerosis, mechanistically involving enhanced enzymatic and nonenzymatic protein/lipid glycosylation, protein kinase C activation, inflammation, and ROS production. Other factors including dyslipidemia, elevated FFAs, inflammation, and insulin resistance, can cause endothelial dysfunction. RNS indicates reactive nitrogen species; EDCF, endothelium-derived COX-dependent vasoconstricting factor; AGE, advanced glycation end products; and PKC, protein kinase C.
- Insulin resistance induces endothelial dysfunction in diabetes. In addition to crucial metabolic actions, insulin plays a critical role in the maintenance of physiological endothelial function through its ability to stimulate NO release via a cascade of signaling, initiated by binding to its cognate receptor (IR) expressed on endothelial cells. The cascade involves activation of the PI3K-Akt axis and downstream serine 1177 phosphorylation of eNOS for NO-dependent vasodilator actions or stimulation of the endothelial release of ET-1 for its vasoconstrictor effect. In insulin-resistance vessels, pathway-specific impairments in PI3K-dependent signaling decrease the expression and activity of eNOS, whereas compensatory secretion of insulin augment its mitogen-activated protein kinase pathways, which results in both the overexpression of adhesion molecules (vascular cell adhesion molecule-1 and E-selectin) and increased secretion of ET-1. Insulin-resistant endothelium becomes highly inflammatory, with impaired blood supply, which in turn worsens insulin resistance. SHC indicates Src[sarcoma] Homology domain C-terminal; PDK-1, phosphoinositide-dependent kinase-1; MEK, MAPK (mitogen-activated protein kinase)/ERK (extracellular signal-regulated kinase) kinase; MAPK, mitogen-activated protein kinase; VCAM, vascular cell adhesion molecule; .
- Asymmetric dimethylarginine (ADMA) is generated during the process of protein turnover and is actively degraded by the intracellular enzyme, dimethylarginine dimethylaminohydrolase (DDAH). ADMA is an endogenous inhibitor of all types of nitric oxide synthases (NOSs). It has long been thought that the NOS inhibition by ADMA is attributable to its competitive inhibition as an L-arginine analog. However, there is increasing evidence that ADMA may have additional effects that are independent of the competitive inhibition of NOS although the precise mechanisms are unknown.
- Proposed mechanism for endothelial dysfunction in chronic kidney disease (CKD). The increase in circulating asymmetric dimethylarginine (ADMA) causes endothelial dysfunction in CKD by reducing vascular endothelial nitric oxide synthase (eNOS) phosphorylation. Inhibition of the eNOS signaling pathway may be an additional mechanism accounting for the eNOS inhibition by ADMA in CKD, independently of competitive inhibition. DDAH, dimethylarginine dimethylaminohydrolase; NO, nitric oxide.
- Tumor angiogenesis is important for tumor progression. If vascular supply cannot be recruited, tumors remain dormant at a size of approximately 1mm3 which is the limit of diffusion for small molecules.
- The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is essential for tumor progression. Tumor-associated vessels promote tumor growth by providing oxygen and nutrients and favor tumor metastasis by facilitating tumor cell entry into the circulation. During development, VEGF induces differentiation and proliferation of endothelial cells from its progenitors (the hemangioblast and angioblast) to form a poorly differentiated primitive vascular plexus (vasculogenesis). Angiopoietin-1 (Ang-1) and other morphogens (e.g. Ephrins-Eph) induce remodeling of the vascular plexus into a hierarchically structured mature vascular system through endothelial cell sprouting, trimming differentiation and pericyte recruitment (angiogenesis). During tumor angiogenesis, angiopoietin-2 (Ang-2) destabilizes the vessel wall of mature vessels. Quiescent endothelial cells become sensitive to VEGF (or other angiogenic factors), proliferate and migrate to form new vessels. Bone marrow-derived endothelial cell progenitors are found in the peripheral blood and can recruit at sites of angiogenesis. If this vascular supply cannot be recruited, tumors, for the most part based upon preclinical models, should remain dormant at a size of approximately 1 mm3, which is the limit of diffusion for small molecules.
- VEGF is produced by cancer cells and relates to the metastatic potential of a number of different types of tumors, including ovarian cancer [82,83]. It is detected by immunostaining in most ovarian cancerous tissues and it is also an important facilitator of the creation of ascites in the latter stages of the disease [84,85]. VEGF alongside its receptors constitute the dominant pathway that regulates angiogenesis in ovarian cancer [39,86]. The role of the VEGF/VEGFR axis in ovarian cancer has been well documented due to pharmacological studies of agents that reduce the burden of women with the disease [83,87]. Intracellular signaling related to VEGF in ovarian cancer includes the elaboration of molecules such as JAK and STAT pathway components, PI-3 kinases, and MAP kinases [82]. More specifically, PI-3K has been shown to play an important role in angiogenesis with its expression correlating with VEGF upregulation [88], and an upregulation of the PI3K/Akt pathway is observed [89]. The activation of the JAK-STAT pathway has been correlated with upregulation of VEGF and intracellular signaling in angiogenesis, especially the upregulation of STAT3 and STAT5 [90]. MAP kinases are also involved in an interplay with VEGF levels [91]. For the initiation of signaling, an autocrine loop of VEGF/VEGFR has been indicated to be responsible [36,40]. Lately, there are other protein molecules that have been studied and shown to be involved in a signaling interplay with the VEGF/VEGFR complex, mainly VEGF/VEGFR2. These include the Src kinases, which increase vascular permeability [92], and phospholipase C that may interact with Erk/MAPK molecules enhancing the VEGF effect on vascular permeability and vessel formation [93]. VEGF plays an exceptional role in angiogenesis. It is involved in this process by mainly regulating new blood vessel growth [59]. It also promotes survival of immature vasculature before it turns into its mature form. VEGF was first discovered by Ferrara and colleagues [60] and was previously known as the vascular permeability factor due to its capacity of increasing vascular permeability [61]. There are seven member molecules that fall into this family of proteins, including VEGF A–E, and also the placental growth factor 1 and 2 (PIGF-1 and PIGF-2) [62,63]. VEGF-A multiple isoforms may be formed due to alternative mRNA splicing, with VEGF165 being the most prevalent VEGF isoform in a number of tumors [64–66]. Molecules of this family exert their effect via signaling through their tyrosine kinase receptor counterparts that are expressed normally on the surface of endothelial cells [62,64] and are termed vascular endothelial growth factor receptors (VEGFR). There are three isoforms of this type of receptors namely VEGFR1-3 [67–69]. VEGF-A binds preferentially to VEGFR1 and 2, VEGFB and PIGF-1 and PIGF-2 bind to VEGFR1, whereas VEGF-C and D bind preferentially to VEGFR3 [70,71]. The binding of the ligand onto the receptor induces a receptor dimerization that leads to intracellular signaling initiation. VEGF expression has been shown to be upregulated by factors such as IGF-1 and IL-6 [72,73]. The expression of VEGF may also be regulated by mutations in genes such as p53, ras, src, and vhl [74,75] Function-wise, VEGFR2 is the main receptor isoform through which VEGF, mainly VEGF-A, mediates its effects that are directly related to angiogenesis [76,77]. VEGFR-1 has a less defined role, although recent studies have shown that both VEGF and PIGF may bind onto the receptor, in pathological conditions such as tumors, and enhance angiogenesis effects [78]. Moreover, soluble VEGFR1 may even play a role in controlling VEGFR2 signaling as it can act as a decoy receptor molecule [79]. In turn, VEGFR3 plays a lesser role in angiogenesis, but it has been documented to play an important role in lymphangiogenesis upon binding of VEGF-C and VEGF-D [59,80,81]. The main VEGF isoform being important in angiogenesis is VEGF-A and it will be referred as VEGF from this point onwards.
- PDGF is an essential protein to pericyte recruitment, which is a critical aspect of blood vessel maturation. It has been shown that the activation of the PDGF Receptor (PDGFR) leads to upregulation of angiogenic events [94,95]. PDGF also interacts with VEGF and they either converge their signaling cascades or the PDGF pathway may be activated in response to resistance to VEGF inhibition [95,96]. The importance of PDGF in angiogenesis and in tumor progress is highlighted by the correlation of its expression with ovarian cancer patients’ prognosis [97]. Four isoforms of the PDGF molecule have been identified namely PDGF A-D [98,99]. As in the case of VEGF, there is specificity on which isoforms of PDGF bind specific corresponding receptor isoforms either PDGFR-α or PDGFR-β in order to exert their effects. In this case, PDGF A–C bind onto PDGFR-α, whereas PDGF-B and PDGF-D bind onto PDGFR-β [100]. As in the case of VEGF/VEGFR, an autocrine signaling mechanism, may be responsible for PDGF promoting angiogenesis and tumor growth [101]. Upon activation of the PDGF pathway, signaling occurs via the use of the PI3K/Akt complex pathway but there are also MAPK molecules involved alongside proteins of the Src family and Phospholipase C-γ [102]. Other molecules related to the PDGF signaling include the Ras protein [103], STAT proteins, and guanine-5'-triphosphate (GTP-ase) activating protein [104]. In the case of ovarian cancer, PDGF has been recorded in a large number of samples and a five to six-fold increase in the level of PDGF has been measured in ovarian cancer tumor cells when compared to cells of the normal ovarian epithelium [105–107]. PDGFR is expressed in ovarian carcinomas and it is also present in malignant ascites [108]. PDGF has been shown to interfere with the stroma formation and also act as a substrate for angiogenesis [109]. It has also been shown to act in concert with VEGF in order to promote new vessel formation and stabilize newly synthesized vessels [75,110,111], so PDGF molecules are key regulatory molecules in oncogenesis and angiogenesis, important in ovarian cancer. Some elements of the PDGF signaling pathways are shown in Figure 3.
- FGF signaling mechanism has originally been studied as a significant embryogenesis pathway [112] and it has since become an important research target when it comes to angiogenesis research in cancer [113,114]. There are over 20 FGF isoforms identified, namely 23, and five receptor molecules (FGFR) have also been described [115]. The receptor molecules pose great similarity in structure, including an extracellular immunoglobulin (Ig)-like domain and an intracellular tyrosine kinase domain [116,117]. These domains are conserved between the first four isoforms of the receptor but the fifth isoform (FGFR-5) lacks the intracellular tyrosine kinase domain [118,119]. Upon binding of the ligand onto the receptor, the receptor molecules dimerise, a process that leads to the initiation of the intracellular signaling cascade. In ovarian cancer, disruptions to the appropriate signaling cascade have been reported, such as alternative splicing events differentiating the ability of the receptor to bind ligands effectively, while mutation events have not been considered significant in altering the receptor’s function [119–121]. In ovarian cancer, differences in alternative splicing may confer sensitivity to the ligand [122]. Moreover, in the case of ovarian cancer, FGF may be secreted into malignant ascites alongside VEGF, therefore, it may be contributing to cancer progression and angiogenesis [123,124]. The expression of FGF may be associated with prognosis [125]. It has been shown that FGF may play a direct role in tumor cell proliferation in ovarian cancer [126,127], but may also play a role in angiogenesis acting alongside other pro-angiogenic factors such as VEGF [128,129]. The FGF signaling pathway involves the employment of downstream proteins such as MAPK proteins and proteins of the PI3K/Akt cascade [130]. Phospsholipase-c and IP3 cascades are also involved in the downstream signaling of FGF, whereas the FGF pathway may crosstalk with other pathways such as the Notch pathway [131]. A schematic overview of some elements of the FGF related pathways are shown in Figure 4.
- Angiogenin, also known as RNase 5, induces angiogenesis mainly through four pathways: its ribonuclease activity, basement membrane degradation, signaling transduction, and nuclear translocation. 1. Upon binding of ANG to actin, some of the ANG-actin complexs dissociate from the cell surface. Thereafter, this complex accelerates tissuetype plasminogen activator (tPA)-catalyzed generation of plasmin from plasminogen [14]. Therefore, through the formation of its actin complex, ANG promotes the degradation of basement membrane and extracellular matrix and thus allows endothelial cells to penetrate and migrate into the perivascular tissue [15], an essential feature of angiogenesis. 2. Although there is a lack of knowledge on ANG receptors, several pathways have been proposed to be activated by ANG stimulation. In response to ANG treatment, extracellular signal-related kinase1/2 (ERK1/2) [17] as well as protein kinase B/Akt [18] were activated in human umbilical vein endothelial (HUVE) cells, and phosphorylation of stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was observed in human umbilical artery smooth muscle (HuASM) cells [19] (Fig. 1). Activations of these signaling pathways by ANG are considered to be an important mechanism leading to cell proliferation and further angiogenesis. 3. Angiogenin undergoes nuclear translocation in endothelial cells and smooth muscle cells [19], which has also been shown to be necessary for ANG-induced angiogenesis. 4. RNase activity is necessary for the functions of ANG. Mutations of His13, Lys40, or His114, key amino acids for the RNase activity of ANG, greatly decrease its angiogenic activity in the chick embryo chorioallantoic membrane. 5. ANG takes part in cancer development by stimulating both angiogenesis and cancer cell proliferation
- In septic shock, systemic vasodilation and pooling of blood in the periphery leads to tissue hypoperfusion, even though cardiac output may be preserved or even increased early in the course. This is accompanied by widespread endothelial cell activation and injury, often leading to a hypercoagulable state that can manifest as DIC. In addition, septic shock is associated with changes in metabolism that directly suppress cellular function. The net effect of these abnormalities is hypoperfusion and dysfunction of multiple organs—culminating in the extraordinary morbidity and mortality associated with sepsis.
- Inflammatory mediators. Various microbial cell wall constituents engage receptors on neutrophils, mononuclear inflammatory cells, and endothelial cells, leading to cellular activation. Toll-like receptors (TLRs, Chapter 2 ) recognize microbial elements and trigger the responses that initiate sepsis. However, mice genetically deficient in TLRs still succumb to sepsis, [59,] [60] and it is believed that other pathways are probably also involved in the initiation of sepsis in humans (e.g., G-protein coupled receptors that detect bacterial peptides and nucleotide oligomerization domain proteins 1 and 2 [NOD1, NOD2]). [62] Upon activation, inflammatory cells produce TNF, IL-1, IFN-γ, IL-12, and IL-18, as well as other inflammatory mediators such as high mobility group box 1 protein (HMGB1). [62] Reactive oxygen species and lipid mediators such as prostaglandins and platelet activating factor (PAF) are also elaborated. These effector molecules activate endothelial cells (and other cell types) resulting in adhesion molecule expression, a procoagulant phenotype, and secondary waves of cytokine production. [61] The complement cascade is also activated by microbial components, both directly and through the proteolytic activity of plasmin ( Chapter 2 ), resulting in the production of anaphylotoxins (C3a, C5a), chemotactic fragments (C5a), and opsonins (C3b) that contribute to the pro-inflammatory state. [63] In addition, microbial components such as endotoxin can activate coagulation directly through factor XII and indirectly through altered endothelial function (discussed below). The systemic procoagulant state induced by sepsis not only leads to thrombosis, but also augments inflammation through effects mediated by protease-activated receptors (PARs) found on inflammatory cells. Endothelial cell activation and injury . Endothelial cell activation by microbial constituents or inflammatory mediators produced by leukocytes has three major sequelae: (1) thrombosis; (2) increased vascular permeability; and (3) vasodilation. The derangement in coagulation is sufficient to produce the fearsome complication of DIC in up to half of septic patients. [60] Sepsis alters the expression of many factors so as to favor coagulation. Pro-inflammatory cytokines result in increased tissue factor production by endothelial cells (and monocytes as well), while at the same time reining in fibrinolysis by increasing PAI-1 expression (see Fig. 4-6B and Fig. 4-8 ). The production of other endothelial anti-coagulant factors, such as tissue factor pathway inhibitor, thrombomodulin, and protein C (see Fig. 4-6 and Fig. 4-8 ), are diminished. [60,] [61,] [64] The procoagulant tendency is further exacerbated by decreased blood flow at the level of small vessels, producing stasis and diminishing the washout of activated coagulation factors. Acting in concert, these effects promote the deposition of fibrin-rich thrombi in small vessels, often throughout the body, which also contributes to the hypoperfusion of tissues. [60] In full-blown DIC, the consumption of coagulation factors and platelets is so great that deficiencies of these factors appear, leading to concomitant bleeding and hemorrhage ( Chapter 14 ). The increase in vascular permeability leads to exudation of fluid into the interstitium, causing edema and an increase in interstitial fluid pressure that may further impede blood flow into tissues, particularly following resuscitation of the patient with intravenous fluids. The endothelium also increases its expression of inducible nitric oxide synthetase and the production of nitric oxide (NO). These alterations, along with increases in vasoactive inflammatory mediators (e.g., C3a, C5a, and PAF), cause the systemic relaxation of vascular smooth muscle, leading to hypotension and diminished tissue perfusion. - Metabolic abnormalities. Septic patients exhibit insulin resistance and hyperglycemia. Cytokines such as TNF and IL-1, stress-induced hormones (such as glucagon, growth hormone, and glucocorticoids), and catecholamines all drive gluconeogenesis. At the same time, the pro-inflammatory cytokines suppress insulin release while simultaneously promoting insulin resistance in the liver and other tissues, likely by impairing the surface expression of GLUT-4, [65] a glucose transporter. Hyperglycemia decreases neutrophil function—thereby suppressing bactericidal activity—and causes increased adhesion molecule expression on endothelial cells. [65] Although sepsis is initially associated with an acute surge in glucocorticoid production, this phase is frequently followed by adrenal insufficiency and a functional deficit of glucocorticoids. This may stem from depression of the synthetic capacity of intact adrenal glands or frank adrenal necrosis due to DIC (Waterhouse-Friderichsen syndrome, Chapter 24 ). - counter-regulatory immunosuppressive mechanisms, which may involve both innate and adaptive immunity. [59] [60] [61] Proposed mechanisms for the immune suppression include a shift from pro-inflammatory (TH1) to anti-inflammatory (TH2) cytokines ( Chapter 6 ), production of anti-inflammatory mediators (e.g., soluble TNF receptor, IL-1 receptor antagonist, and IL-10), lymphocyte apoptosis, the immunosuppressive effects of apoptotic cells, and the induction of cellular anergy. [59] [60] [61] It is still debated whether immunosuppressive mediators are deleterious or protective in sepsis. [59] • Organ dysfunction. Systemic hypotension, interstitial edema, and small vessel thrombosis all decrease the delivery of oxygen and nutrients to the tissues, which fail to properly utilize those nutrients that are delivered due to changes in cellular metabolism. High levels of cytokines and secondary mediators may diminish myocardial contractility and cardiac output, and increased vascular permeability and endothelial injury can lead to the adult respiratory distress syndrome ( Chapter 15 ). Ultimately, these factors may conspire to cause the failure of multiple organs, particularly the kidneys, liver, lungs, and heart, culminating in death.
- Endothelins (ET), potent vasoconstrictors also produced by vascular endothelium, have recently gained acceptance as important mediators of vascular tone, particularly in sepsis. They are 21-amino acid peptides consisting of ET-1, ET-2, ET-3, and vasoactive intestinal contractor (VIC). ET-1 gene expression is up regulated by endotoxin or LPS in human macrophages (109). Cytokines have also been shown to affect ET gene expression (109). Produced from the cleavage of 203-213 amino-acid proteins called preproendothelins into precursors called big endothelins (big ET-1, big ET- 2, big ET-3), their final conversion is dependent on metalo-endoproteases called endothelial converting enzymes (Figure 5) generating ET-1, ET-2, or ET-3. Three isoenzymes (ECE-1a, ECE-1b, and ECE-2) have been isolated to date. ET’s binding directly to their specific receptor results in alterations in vascular tone. -Progression of sepsis in our rat model may occur at least in two phases. Phase 1 (early phase, 0-12 h after induction of sepsis), when both plasma ET and NO showed an increase in response to induction of sepsis and phase 2 (late phase, 12-48 h after induction of sepsis), when plasma ET levels returns to basal levels while NO remained elevated. Thus, these two potent vasoactive agents have a divergent time course that is likely to be related to the different mechanisms of control of the vascular tone during sepsis.