Vascular endothelium: recent advances

ARTICLE

Auteur(s) : Krystyna Anna PASYK¹, Barbara Anna JAKOBCZAK²

¹ Institute of Gerontology, The University of Michigan, 300 North Ingalls St., Ann Arbor, Michigan 48109-2007, USA
² Department of Chemistry, University of Michigan, 930 N. University St., Ann Arbor, Michigan 48109-1055, USA

Article accepted on 11/05/2004

Our knowledge of vascular endothelium (VE) is relatively new. Rudolf Virchow, in the 19^th century postulated a passive role of the VE in transferring nutrients from the bloodstream to tissues, and functioning as an insulation barrier, separating platelets from the thrombogenic subendothelial connective tissue [1]. Major advances have been made during the last two decades, and VE started to be considered a dynamic “widespread organ” with a crucial homeostatic role in regulation of vascular tone, thrombogenesis, and vascular remodeling. VE has vital functions in the body, with numerous secretory properties, and is actively involved in multiple physiological and pathological processes [2].

Morphology

VE is formed of a monolayer of endothelial cells (ECs) that line the circulatory system. The VE may be continuous, fenestrated, or discontinuous. In human dermis, the blood vessels are lined by continuous and fenestrated endothelium. The major ultrastructural differences between dermal VE and other organs are greater thickness of ECs in dermal vasculature and the presence of large bundles of microfilaments in their cytoplasm. In the dermal arterial vasculature, nuclei of ECs are elongated, giving a smooth luminal surface, but in venous vessels they are round, indented, and bulge into the lumen. ECs have three surfaces: cohesive, adhesive, and luminal. The cohesive surface adjoins ECs to one another and facilitates transport processes. This surface consists of specialized intercellular junctions: gap junctions, tight junctions, adherent junctions, and syndesmos [3]. Myoendothelial junctions are involved in intercellular exchange of information. The adhesive surface of ECs adheres to the basal lamina. The luminal side of the VE consists of molecules and specific binding proteins that regulate trafficking of circulating blood cells. Microvilli and microfolds have been found in some pathological conditions [4]. Their role is not clear. The pinocytotic vesicles are involved in molecular transport and caveolae, in endothelial vesicular trafficking and signal transduction [5]. The Weibel-Palade bodies are EC cytoplasmic granules. They secrete von Willebrand factor (vWF), glycoprotein, GMP-140, P-selectin, IL-8, α1,3 fucosyltransferase VI, and tetraspanin CD63/lamp3 molecules. However, the nature and function of crystalloids (Fig. 1) identified within the cytoplasm of ECs, in normal skin and some diseases, are unknown [6]. The blood vessel ECs (BECs) are supported externally by basal lamina; lymphatic ECs (LECs) by fine collagenous and reticular fibrils only.

Physiology

The VE is a dispersed “organ” that synthesizes several peptides and biologically active molecules taking part in vascular tone regulation, vessel contraction and permeability. ECs have a role in regulation of blood pressure and blood flow by releasing both vasodilatators (nitric oxide [NO], endothelium-derived hyperpolarizing factor [EDHF], prostacyclin [PGI2]), and vasoconstrictions (endothelin-1 [ET], endoperoxides, thromboxane A2, superoxide anions, platelet-activating factor [PAF]) [7, 8]. VE is a source of arachidonate metabolites, tissue factors, and numerous cytokines, GMP-140, fibronectin, collagen IV and VIII, and proteoglycans [9]. Laminin 8 and 10, involved during wound angiogenesis, are produced by dermal VE [10]. The VE synthesizes basic fibroblast growth factor (bFGF), endothelial cell derived growth factors (ECDGF), platelet-derived growth factor (PDGF), granulocyte-macrophage colony-stimulating factor (GMCSF), heparin-like growth inhibitor, growth factors and a mitogenic factor which regulate the growth of vascular smooth muscle cells. It was recently discovered that Nox 4 is abundant in ECs and may act as a catalytic component [11].
A main function of VE is to sustain thromboresistance through producing factors involved in blood clotting: vWF, vW Ag II, plasminogen activator, factor V, heparin-like molecules, thrombospondin, thrombomodulin, protein C, and protein S [12]. Antithrombotic EC properties are maintained by the balance between plasminogen activators (PAs) and PA inhibitors (PAIs) [13]. Mediation and control of transendothelial exchange of materials between the plasma and the interstitial fluid (including gas exchange in the lungs) are also principal VE functions. Several other functions of VE are mediated through membrane receptors (IL-1, IL-4, IL-8, G- and GM-CSF, IFN gamma, MIP-2, PDGF, VEGF, urokinase, OP4) [2, 14]. Receptors for steroid hormones, neurotransmitters, and polypeptides provides evidence for the potential hormone responsiveness of the VE [15]. Glycoproteins on the EC surface are receptors for: fibrinogen, vWF, and fibronectin. The EC receptors: NPY, VP and P2X are under investigation. Angiopoietins and two receptors (VEGFR-1, VEGFR-2) expressed by ECs, are important during embryonic development and in angiogenesis [16]. Angiopoietins are the major ligands of the endothelial-specific receptor Tie2. Upregulation of angiopoietins and Tie2 receptor is associated with the microvascular proliferation in psoriasis [17]. Vitronectin receptor αvβ3 probably also plays a role in angiogenesis [2]. Recently lymphangiogenic growth factors and their receptors have been discovered. These molecules are involved in tumor-induced lymphangiogenesis and lymphatic dissemination of tumor cells [18].
Apoptotic EC death is crucial during fetal development and is important in cellular homeostasis. Balance of apoptosis and EC proliferation is a major factor in angiogenesis [19]. Several molecules synthesized by ECs regulate apoptosis and angiogenesis. Some hormones and endocrine peptides, mediated by EC receptors, are able to regulate capillary formation and regression of vasculature. Control of angiogenesis in tumor growth inhibition, tumor invasion and metastasis is a recent research topic.

Pathology

VE is involved in numerous pathological processes: inflammation, tissue repair, tumor growth and metastasis, transplant rejection, atherogenesis, immunity, etc. Under normal conditions VE has anticoagulant and non-thrombogenic properties, but after injurious stimuli, the antithrombotic surface of ECs may start to be prothrombotic and antifibrinolytic and promote blood coagulation. This occurs during high hydrodynamic shear stress or at sites of inflammation, and may initiate disseminated intravascular coagulation, atherosclerosis, and immune vasculitis. Injury of VE can arise from mechanical or thermal trauma, hemodynamic stress, hypercholesterolemia, oxygen free radicals, chemical agents such as homocysteine, nicotine, immune/inflammatory mediators, bacterial and viral infections, and bacterial toxins [20]. Herpes simplex virus type I, influenza, or cytomegalovirus induce Fc and C3 receptors on VE. These receptors are attachment sites for immune complexes and may initiate cell injury. ECs, after exposition to bacterial endotoxins, IL-1 or TNF, may induce the synthesis and expression of a procoagulant molecule on EC surface and alter EC function [21]. Alloantibodies produced after transplantation may also injure VE.
There are three types of EC responses to injury.
1. The immediate transient response: After mild injury response begins immediately and terms 15 to 30 minutes causing increased vascular permeability (edema, allergic wheal).
2. The immediate - sustained response or the immediate – prolonged reaction: This occurs after severe injury and is associated with necrosis of ECs. Leakage starts immediately after injury and may last for several days (burn).
3. The delayed – prolonged response: Vascular leakage takes place after direct injury of ECs caused by mild-to-moderate thermal exposure, X-ray or UV irradiation, contact with bacterial toxins, and in delayed type IV hypersensitivity reactions. Leakage is delayed and lasts for several hours or days. Morphologic changes in VE after injurious stimuli have been observed in light and electron microscopy. Altered plump ECs, containing increased number of organelles, were called “activated cells” almost four decades ago [22]. The nature of the activation was unknown at that time. Alteration of ECs after various stimuli may cause either fluctuations in normal cellular activity or initiation of new EC functions and formation of new molecules which take part in coagulation, inflammation, and immunity [23]. The term “endothelial activation” was introduced for this stimulating process [24]. Quiescent ECs cannot interact with circulating leukocytes, but upon signal-induced changes, they become responsible for local recruitment of leukocytes and T cells. After EC activation, T cells as well as leukocytes are tethered by selectins to the luminal EC surface and are propelled in the direction of blood flow, which rolls them along the EC surface. The adherence and transmigration of white cells through VE are mediated by β₂ integrins and ICAM-1 [21].
VE may respond to cytokines in three ways: 1. ECs may be injured by cytokine binding to EC receptors. This takes place during transplant rejection. 2. ECs may migrate, proliferate, and form new capillaries (angiogenesis). 3. Activated ECs may perform new functions. Usually EC activation is beneficial, especially in host defense, because it can cause development of cell-mediated immune reactions. Activation of ECs, however, can cause their dysfunction and can lead to pathological processes (vascular leak syndrome, vasculitis in Kawasaki disease). Proliferation of ECs may result from infection, immunologic disturbances, trauma, or other etiologies and is observed in: bacilliary angiomatosis, verruga peruana (Carrion’s disease), papillary endothelial hyperplasia, benign (reactive) angioendotheliomatosis, ALHE, and some hemangiomas. Inflammation-induced angiogenesis is the main site of leukocyte-EC interaction which causes inflammatory infiltrates in giant cell arteritis. Vascular adhesion molecules (E-selectin, ICAM-1, VCAM-1) play a role in inflammatory skin diseases. In pathological conditions presence of these molecules is detected along the dermal capillary loop, which leads T cells and leukocytes to transmigration through the VE [25]. Hypoxia stimulates angiogenesis and enhances VEGF mRNA [26, 27]. Increased oxygen tension causes inhibition of EC proliferation and decreases the synthesis of a major surface protein (PECAM-1) and type IV collagen [28]. Potent angiogenesis inhibitors are: endostatin, thrombospondin-1, fragments of basal lamina collagens type IV, XV, and XVIII, rhuMAb-VEGF, constatin, tumstatin, and edible berry [29-31].
VE is exposed to circulating antibodies, antigens, and immune complexes, and for this reason, VE may regulate the transport of these agents into the tissues. In this way, VE is involved in initiation of immune reactions and plays a pivotal role in immunological diseases [32]. Cutaneous VE strongly express class I and class II major histocompatibility complex (MHC) molecules. The ability to activate memory T cells allows differentiation of ECs from other cells, which also express class I and class II MHC molecules, but cannot activate memory T cells. ECs can costimulate memory and naïve T cells which are mediated by LFA-3 (CD58) [33].
Apoptosis is induced not only by various stimuli, but also by several molecules (caspases, Bcl-2-like proteins, mitochondrial factors, FasL, stress-activated protein kinases, sphingomyelinases, etc) [34]. Some anti-EC antibodies activate ECs, others trigger apoptosis. Staphylococcus aureus induces rapid apoptosis of ECs and causes a break of the endothelial barrier, permitting invasion of the bacterium to the organs [35]. Conditions such as high concentration of homocysteine-thiolactone and reoxygenation after hypoxia, initiate EC apoptosis. Similarly, hyperbaric oxygenation of hematopoietic cells enhances apoptosis by increasing the intracellular accumulation of H₂O₂ [36]. Acidosis may protect ECs from apoptosis and inhibit their proangiogenic tendency. Dysregulation of the apoptotic process may be associated with cancer, AIDS, neurodegeneration, heart disease, autoimmunity, and allergy [37]. Apoptosis of ECs plays an important role in development of atherosclerosis. Further research studies on proapoptotic markers (Bax, Bak), antiapoptotic markers (Bcl-2, p53), and the proliferation marker (ki-67) may help early detection of dysregulation of apoptosis [38, 39]. It has recently been discovered that proteosome selective inhibitor (PSI) inhibits growth of tumors not only by induction of apoptosis, but also by inhibition of angiogenesis [40].

Vascular endothelial cell markers

Immunohistochemical or immunofluorescence techniques currently are commonly used to localize EC markers. Molecular markers of VE are still in the growing phase. The basal lamina of small blood vessels is visible in the presence of antibodies against the basal lamina components [9]. However EC of lymphatics give a negative reaction in immunologic staining because the basal lamina is absent. In electron microscopy Weibel-Palade bodies are recognized as morphologic EC markers.
The earliest markers used for identification of ECs were: angiotensin converting enzyme (ACE), vWF, and Ulex europaeus lectin [2, 41]. Monoclonal antibodies (B721, E431) recognize EC surface antigens, and together with other known EC markers (F8rAg, Ia, HCL-1) they are used in the study of Kaposi’s sarcoma in patients with AIDS. Monoclonal antibodies (E92, OKM5, HCL-1) react with BECs, but not with LECs [42]. Monoclonal antibodies to ICAM-1 are excellent markers of the VE. Staining with anti-CD34 may differentiate Kaposi’s sarcoma from hemosiderotic dermatofibromas. Similarly, tumor cells in tufted angioma can be detected with this technique [41].
Monoclonal antibody CD31 (PECAM-1) is also a reliable VE marker, but CD36 is present on VE as well as on monocytes and platelets [9, 43]. A new monoclonal antibody (S-Endo-1), which recognizes CD146, allows detection of ciculating ECs in thrombotic, infectious or immunologic disorders and is a useful marker for vascular wall injury [44]. Other EC markers are cytokeratin 8, 18, 19, vimentin, EN7/44, EN4, 1F10, BMA120, ELAM-1, sVCAM-1, sTM, E-selectin, and P-selectin [45]. E-selectin and VEGF are present in proliferating ECs in hemangiomas [9, 46]. The bFGF is positive in the proliferative and only in the early involuting phases of hemangioma [9]. Glut-1 is recently used for distinguishing hemangiomas from vascular malformations [47, 48]. Other EC markers are: alkaline phosphatase (ALPase), adenylate and guanylate cyclase (AC and GC), VEGFR-1, VEGFR-2, VEGFR-3, fms tyrosine kinase 4 gene (Flt-4), 5’-nucleotidase (5’Nase), PAL-E, beta-chemokine receptor D6, and VE-cadherin or cadherin-5 [2, 49, 50, 51]. A number of these markers are expressed with variable sensitivity on VE, whereas others are also present in different cells. VEGFR-3, Prox-1, Lyve-1, and podoplanin are specific markers for LECs [52-55]. Fli-1 is a novel nuclear marker of ECs of vascular tumors [56]. Integrin αvβ3 is a marker for activated and proliferating ECs [57, 58]. Fibronectin isoform (B-FN), and CD105 (endoglin) are markers of angiogenesis [59]. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TiMPs), as well as VEGF and bFGF are also markers for new vessel formation [60]. Endomucin is a new marker identified on VE of each tissue [61]. Guanylate-binding protein-1 (GPB-1), also a new molecular marker, identifies inflammatory cytokines-activated ECs in psoriasis, adverse drug reaction, and Kaposi’s sarcoma [62]. Anionic phospholipids are expressed on ECs in tumor vasculature, apoptotic, necrotic, injured, activated and malignant cells, and for this reason they can be used in the future, as receptors for vascular target substances during cancer treatment [63]. Recent studies demonstrated that antimouse VEGFR-2 antibodies may be used as antiangiogenic or vascular targeting agents in tumor therapy [64].

Conclusion

This brief and rather fragmentary review of VE morphology, physiology, and pathology, which focused on ECs in the skin, shows how these cells are pluripotent and have vital functions. Stimulated ECs may respond by vasodilatation and increased permeability, production of cytokines and cell mediators, activation, converting adherent leukocytes into mobile cells migrating to the inflammatory sites, antigen presentation, angiogenesis, and apoptosis. Currently much has been discovered about this dynamic organ, through genetic manipulation to engineer ECs. The number of discoveries of new EC functions, their biosynthetic and metabolic activities, as well as the markers expressed by VE is still growing. Studies on limitation or promotion of angiogenesis and apoptosis will be promising in the progress of opening new horizons for future therapy of various diseases. n

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