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The endothelial biology of sickle cell disease
REVIEW ARTICLE The endothelial biology of sickle cell disease ROBERT P. HEBBEL and GREGORY M. VERCELLOTTI MINNEAPOLIS, MINNESOTA
Abbreviations: G P
= g l y c o p r o t e i n ; I C A M = i n t e r c e l l u l a r a d h e s i o n m o l e c u l e ; IL = interleukin; M V E C = m i c r o v a s c u l a r e n d o t h e l i a l cell; INF = i n t e r f e r o n ; RBC = r e d b l o o d cell; TNF = t u m o r necrosis f a c t o r ; TSP = t h r o m b o s p o n d i n ; V C A M = v a s c u l a r cell a d h e s i o n m o l e c u l e ; W B C = w h i t e b l o o d cell; v W f = v o n W i l l e b r a n d f a c t o r
he vascular wall comprises the pathobiologic focal point of many disease states, a fact that derives from the myriad biologic roles played by the endothelial cell. This cell resides at the interfacial barrier between the blood space and tissues, providing both spatially defining physical confinement and an "inert" surface for blood contact. Yet it displays a host of adhesion molecules that potentially promote interaction with blood cells. It contributes separately to the physiology of both hemostasis and inflammation, although it inexorably links them by residing at the interface of these two critical homeostatic systems,1'2 both responding to and participating in each. It exists in a delicate balance between its numerous anticoagulant properties and potential procoagulant capabilities.2-4 It helps control vascular tone by liberation of either vasodilatory or vasoconstricting factors.5 And most importantly, the endothelial cell provides a responsive surface 1'6-9 that is modulated by an extraordinary array of biologic modifiers: thrombin, histamine, interleukins, hypoxia, endotoxin, oxidants, TNF, INF, and so on. These substances (and states) induce the endothelial cell to participate in vascular pathobiology by modulating its extensive repertoire of surface-dependent functions. The phenomenal respon-
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From the Department of Medicine, University of Minnesota Medical School. Submittedfor publicationSept. 13, 1996;acceptedSept. 26, 1996. Reprint requests: Robert P. Hebbel, MD, Department of Medicine, Box 480 UMHC, Universityof MinnesotaMedicalSchool, Harvard St. at E. River Rd., Minneapolis,MN 55455. J Lab Clin Med 1997;129:288-93. Copyright© 1997by Mosby-YearBook,Inc. 0022-2143/97 $5.00 + 0 5/1/78721 288
siveness of endothelial functional phenotype is most dramatically illustrated by this cell's hemostatic and adhesive functions. ROLE IN HEMOSTASIS
The endothelial cell exerts numerous physiologically important influences on hemostasis2-4 that can be influenced by biologic modifiers. For example, thrombin not only is the most potent physiologic activator of platelets but also is an excellent stimulant of various endothelial changes, including the release of vWf, prostacyclin, nitric oxide, plasminogen activators, and platelet-activating factor; the expression of tissue factor and adhesion molecules such as P-selectin; the promotion of endotheliumdependent vasoconstriction; and the activation of thrombomodulin. Numerous other modulators of endothelial cell hemostatic function have been described and are summarized, albeit not all-inclusively, in Table I. ROLE IN ADHESION BIOLOGY
Nature likewise has provided the endothelial surface with a host of receptors and other structures that promote the attachment of blood cells, with or without the involvement of adhesogenic plasma proteins (Table II). Integrins. These comprise heterodimeric associations of multiple ~ and [3 chains, x° These receptors tend to exhibit overlapping binding specificities, many of which depend on a ligand RGD sequence. The [31 integrins tend to bind fibronectin or laminin or collagen (or a combination). Of these, endothelial cells express several fibronectin receptors (at least ~s[31 and o~v[~l). Broad binding capability is exhibited by the [33 integrins. Of these, endothelial
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T a b l e I. Endothelial cell h e m o s t a t i c functions Anticoagulant functions
Procoagulant functions
Antithrombotic Sulfated proteoglycans (1' by INF; 4, by TNF, I_-1) Thrombomodulin (1' by THR; ,t, by TNF, IL-1, _PS) Fibrinolytic Plasminogen activators (1' by THR; $ by IL-1) Vasodilatation Prostacyclin ( 1' by THR; IL-1) Nitric oxide ( 1' by IL-1, THR, LPS; $ by OX, HYP)
Prothrombotic vWf release ( I' by THR) Tissue factor expression ( 1` by THR, LPS, IL-1, TNF) Antifibrinolytic Plasminogen activator inhibitor 1 ( 1` by THR, LPS, IL-1) Vasoconstriction Endothelin ( 1' by THR, HYP) PDGF ( 1' by THR, HYP, TGFq3) Platelet activating factor ( 1' by THR) Proinflammatory Adhesion molecules ( 1' by THR, LPS, IL-1, OX, INF-) Chemokine production ( 1" by TNF, LPS, IL-1) Platelet activation Platelet activating factor ( 1' by THR, IL-1)
Antiinflammatory Leukocyte adhesion inhibitor ( 1' by TNF, LPS, IL-1) Platelet inhibition Prostacyclin ( 1' by THR, IL-1) Nitric oxide ( 1' by IL-1, THR, LPS; 4, by OX, F-YP) HYP, Hypoxia;LPS, endotoxin;OX, oxidation; THR,thrombin.
T a b l e II. Endothelial cell a d h e s i v e functions Surface adhesion receptor
Integrins 0~5131 ~vl~l %133 Ig superfamily ICAM-1,2 VCAM* PECAM Leucine-rich receptors GP1 b Selectins P-selectin E-selectin* Unique CD36 (GplV)
Ligand
FN FN VN, FN, FB, TSP, vWf
Influences on receptor expresslon
i' by IL-1 I" by IL-1; $ by INF plus TNF
WBC CD11 b/18 WBC 0~41~1 PECAM
1' by INF, IL-1, TNF, LPS 1' IL-1, TNF, OX
vWf
1' by TNF (augmented by INF)
sLx/WBC sLx/WBC
1` by THR, HIS, LTC 1' by TNF, LPS, IL-1 (augmented by INF)
TSP, COL_
1' by IFN~/
FN, Fibronectin;VN, vitronectin; FB, Fibrinogen;COL, collagen;HIS, histamine;LTC, leukotriene04; s/x, (sialyI-Lewis-x);others as in footnote to Table I.
*Net constitutivelyexpressedand only detectableon stimulated endothelium.
cells express the vitronectin recepor (%~3), which potentially provides for the binding of vitronectin, fibrinogen, vWf, TSP, and fibronectin) °'u Expression of avf33 and %~1 on endothelial cells is increased by IL-loL,12 while INF--/plus TNF depresses %133 expression even while preserving %131.13 The immunoglobulin superfamily. These receptors 14 are represented on endothelial cells by constitutive expression of ICAM-1,2 and by inducible expression of VCAM. ICAM participates in WBC-endothelial cell interactions, and its expression is increased by INF--y/IL-lodTNF-odlipopolysaccharide. 15 VCAM expression is induced by endothelial activation with IL-1 or TNF and possibly by H202 or lysolipids.14 Endothelium also expresses platelet endothelial cell
adhesion molecule, which is probably a homotypic adhesion molecule important in neutrophil transmigration. Seleetins. These are single-chain receptors, the ligand for which appears to be carbohydrates like sialyl-Lewis-x.16 On endothelium, P-selectin (also called GMP140) is brought to the surface from Weibel-Palade bodies by the same stimuli (e.g., thrombin, histamine, leukotriene C4) 1 that raise cytosolic [Ca] and lead to vWf release. E-selectin is not expressed on quiescent endothelium, only on cells activated by TNF or IL-1, an effect potentiated by INF--/. Selectins mediate the adhesion of neutrophils and monocytes to endothelium.
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GPIV (CD36). This is a unique glycosylated receptor 17 that is expressed on microvascular endothelium (MVECs) but not large vessel endothelium is and on platelets/megakaryocytes, monocytes, and hematopoietic progenitors. CD36 expression is increased by INF-'y. 18 It is a receptor for TSP, collagen, and oxidized low-density lipoprotein. The leucine-rich GP family. This is represented on endothelium by GPIb, 19 which contributes to functional vWf binding. 2° Endothelial expression of GPIb is increased by TNF-a, an effect greatly amplified by INF-y. 19 Endothelial-endothelial adhesion proteins. Endothelial-endothelial adhesion proteins include occludin at tight junctions; connexins at gap junctions; and cadherins at adherence junctions. 21 These additional adhesion molecules are important, because they maintain endothelial layer integrity and barrier function. However, they have no known or hypothesized role in blood cell-endothelial cell interaction.
ENDOTHELIAL CELL HETEROGENEITY
The existence of endothelial heterogeneity probably is a significant factor in pathobiology involving the endothelium. Although deduced largely from the study of cultured cells, it has been accepted as a valid concept that endothelial cells exhibit heterogeneity with respect to hemostatic functions. 22-a4 It also is likely to be relevant to adhesive interactions. For example, it may be noted that MVECs show a more brisk ICAM-1 response to cytokines than do human umbilical vein endothelial cells, x5 As noted above, CD36 is expressed on MVECs but not on large vessel endothelium. Its expression therefore provides a useful marker of endothelial cells as being microvascular in origin. Despite its probable fundamental physiologic importance, very little is known about organ-to-organ or temporal endothelial heterogeneity in situ. PARTICIPATION OF ENDOTHELIUM IN SICKLE CELL DISEASE
Remarkably, many endothelial functions could, in theory, participate in the vascular pathobiology of sickle disease. The following sections provide examples. Mechanical factors. The space-defining function of the endothelium provides the interfacial surface on which any mechanical obstruction to blood flow must first impact. Thus it is the interaction of sickled red cells with the vessel wall that actually enforces deoxygenation-induced vascular obstruction. WBC adhesion molecules. The responsiveness of endothelium to inflammatory stimuli is most well de-
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fined for those that elicit the expression of adhesion molecules that promote WBC-endothelium interaction. Host defense appears to depend not only on WBC responsiveness to inflammatory signaling but also on the endothelial cell's responsiveness. Thus the expression of selectins and ICAMs on the endothelial surface is a critical determinant of the ability of WBCs to marginate and roll along endothelium and to firmly attach, respectively. 1°'16 The possibility of WBC-endothelium adhesion in the patient with sickle cell disease has at least two fundamental implications. First, ample precedent exists in other vascular diseases for this consequence of the inflammatory state resulting in endothelial damage. 25 Various lines of evidence have identified the probable occurrence of endothelial damage in patients with sickle cell disease (summarized below). Second, the polymorphonuclear leukocyte is larger and far stiffer (i.e., less deformable) than the red cell. Thus its attachment to endothelium, particularly in the smallest vessels of limiting diameter, would impede passage of red cells, something that predictably causes greatly increased risk for red cell sickling. In this regard, it is notable that WBC count correlates with mortality in patients with sickle cell disease. 26 Hemostasis. Patients with sickle cell disease seem to be in a near-constant state of hemostatic perturbation or activation, a notion supported by multiple lines of evidence. 27 However, it cannot be discerned from the available data whether the endothelium has a net procoagulant or net anticoagulant impact on sickle blood. In any case, the very fact that endothelium responds to thrombin in multiple ways (as noted above) suggests that the excess thrombin generation of the patient with sickle cell disease 2s is likely to have an impact on disease pathobiology. Interestingly, patients with sickle cell disease are reported to have antiphospholipid antibodies with high frequency, 29 something that requires further investigation given the reported ability of such antibodies arising in the context of lupus anticoagulants to inhibit endothelial thrombomodulin 3° or prostacyclin release. 31 Vascular tone. The ability of the vascular endothelium to participate in the regulation of vascular tone is undisputed. For example, the endothelium-derived vasoconstrictor endothelin is induced by thrombin, transforming growth factor-t3, shear stress, hypoxia, and attachment of sickled red cells; it is inhibited by oxygen and nitric oxide. The vasodilating substance nitric oxide, on the other hand, is induced by shear stress, acetylcholine, bradykinin, and IL-1-TNF; it is inhibited by hypoxia. 32 Whether the endothelium's vasodilatory or vaso-
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constricting effects dominate, or are alternately expressed, in sickle disease is not established. Levels of endothelin are reported to be elevated in patients with sickle cell disease, 33'34 and apparent abnormalities of vascular tone have been identified in the form of oscillatory microvascular flow. 35 In this disease, where avoidance of vascular obstruction depends critically on unimpeded passage of RBCs, the tone-regulating function of the endothelium certainly must be a relevant factor. It has been reported that the attachment of sickled red cells greatly stimulates endothelin transcription in cultured endothelium, 36 which is consistent with the finding that adherent sickle RBCs inhibit vasorelaxation. 37 RBC adhesion. The postulated role of RBC-endothelial cell interaction as a trigger of sickle vasoocclusion 38 indicates that various endotheliurn-dependent factors relevant to this interesting cell-cell interaction may have a significant impact on pathobiology. Among the varied mechanisms for RBC adhesion to endothelium are several related to specific adhesion molecules noted above. For example, %[33 and CD36 have been implicated as mechanisms of TSP-mediated sickle RBC binding to endotheliurn. 39 VCAM is of particular interest because interaction between endothelial VCAM and 0~4[~1 o n sickle reticulocytes seems to be one major mechanism of RBC-endothelium interaction, and it is potentiated by TNF. 40 Similarly, cytokine effects may provide inducible vWf binding capacity relevant to vWf-mediated adhesion of sickle reticulocytes to endothelium, an additional major mechanism underlying RBC-endothelium interaction. 41 ,~dso, oxidatively damaged, 42 hypoxia-stressed, 43 or virally infected 44'45 endothelia provide a more adhesive substrate for sickle RBCs. Other largely preliminary studies suggest that endothelium stimulated by platelet-activating factor or IL-1 also supports greater sickle RBC adhesion. 3s Endothelial cell heterogeneity. This too is predicted to have a significant impact on sickle disease pathobiology. Of particular relevance is the example of a TSP receptor, CD36, that is hypothesized to mediate adhesion of sickle RBCs to endothelium. This molecule is expressed on microvascular, but not on large vessel, endothelial cells, is In this regard it is remarkable that the adhesion-promoting effect of vWf is much lower when microvascular endothelial cells are used than when large vessel endothelial cells are used, while the adhesion potentiating effect of unfractionated plasma is dramatically greater for microvascular than for large vessel endothelium. 45 This undoubtedly reflects the receptor complement
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on different endothelia and suggests that plasma substances other than vWf are more important for RBC interaction with small vessel endothelium. Additionally, the endothelial cell surface can obviously vary in conjunction with concurrent illness to the extent that endothelial surface modifiers are at play. Endothelial cell responsiveness. The probable relevance of all of these participants in sickle vascular pathobiology is heightened by the very responsiveness of the vascular endothelium to biologic modifiers. The assessment of blood levels of potential biologic modifiers in sickle disease is in its infancy, but already enough is known to predict participation of endothelial cell modulation in this disease. These influence the whole spectrum of endothelial cell functions outlined above as being of direct relevance to sickle disease. Significantly, elevated levels of endotoxin, 46 IL-1, 47 and TNF 4s all have been reported in patients with sickle cell disease; there is an apparent excess of thrombin generation as well. 28 Additionally, the very nature of this vascular disease predicts repetitive molestation of the endothelium by hypoxia and possibly by oxidative stress. Endothelial damage in sickle disease. The hypothesis that endothelial injury occurs and contributes to sickle vasoocclusion has been discussed but is poorly documented. Histopathologic changes suggestive of endothelial injury have been observed in splenic and cerebral vasculatures of patients with sickle cell disease. 48-51 Thromboses in these patients tend to occur at sites of underlying intimal hyperplasia, 49-51 implying that this vascular lesion is derived from gross disturbances of endothelial cell function. An abnormal presence of circulating endothelial surface molecules (ICAM-1, VCAM-1, E-selectin) has been reported in the plasma of patients with sickle cell disease, although it is unknown whether these are secreted and physiologic or represent an injury response. 52 The adhesion of sickle RBCs to endothelium is reported to stimulate a prostacyclin response 53'54 and to impair DNA synthetic ability. 55 Finally, the blood of patients with sickle cell disease, particularly the blood of those in acute painful crisis, contains increased numbers of circulating endothelial cells.56-58 In aggregate, these lines of evidence strongly suggest that the endothelium can be, and is, variably perturbed in the patient with sickle cell disease. THE NEED TO UNDERSTAND ENDOTHEL]UM IN SITU
Nothing is really known about the status of endothelium in vivo. Considering the importance of the endothelial cell in vascular biology, it is particularly
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remarkable that so little is known about it in sickle disease, in which there is ample reason to believe that it influences pathophysiology. A single article has suggested that retinal capillaries in patients with sickle cell disease exhibit greatly increased ICAM-1 expression, 59 which suggests prior stimulation by cytokines and the possibility of concomitant VCAM and GPIb expression. Proof that sickle disease is, in its pathobiology, a disease of endothelial biology will have to await development of techniques to assess the status of the endothelium in situ. Possibly the study of circulating endothelial cells may afford this opportunity. 58 REFERENCES
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16. Lasky LA. Selectins: interpreters of cell-specific carbohydrate information during inflammation. Science 1992;258: 964-9. 17. Greenwalt DE, Lipsky RH, Ockenhouse CF, Ikeda H, Tandon NN, Jamieson GA. Membrane glycoprotein CD36: a review of its roles in adherence, signal transduction, and transfusion medicine. Blood 1992;80:1105-15. 18. Swerlick RA, Lee KH, Wick TM, Lawley TJ. Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro. J Immunol 1992;148:78-83. 19. Konkle BA, Shapiro SS, Asch AS, Nachman RL. Cytokineenhanced expression of glycoprotein Iba in human endothelium. J Biol Chem 1990;265:19833-8. 20. Asch AS, Adelman B, Fujimoto M, Nachman RL. Identification and isolation of a platelet GPIb-like protein in human umbilical vein endothelial cells and bovine aortic smooth muscle cells. J Clin Invest 1988;81:1600-7. 21. Dejana E, Corada M, Lampugnani MG. Endothelial cell-tocell junctions. FASEB J 1995;9:910-8. 22. Speiser W, Anders E, Preissner KT, Wagner O, MiJllerBerghans G. Differences in coagulant and fibrinolytic activities of cultured human endothelial cells derived from omental tissue microvessels and umbilical veins. Blood 1987;69: 964-7. 23. Dupuy E, Bikfalvi A, Rendu F, Levy Tolendano S, Tobelem G. Thrombin mitogenic responses and protein phosphorylation are different in cultured human endothelial cells derived from large and microvessels. Exp Cell Res 1989;185:363-72. 24. Zwaginga J J, de Boer HC, IJsseldik MJW, Kerkhof A, Muller-Berghaus G, Gruhlichhenn J, et al. Thrombogenicity of vascular cells. Comparison between endothelial cells isolated from different sources and smooth muscle cells and fibroblasts. Arteriosclerosis 1990;10:437-48. 25. Varani J, Ward PA. Mechanisms of neutrophil-dependent and neutrophil-independent endothelial cell injury. Biological Signals 1994;3:1-14. 26. Platt OS, Thorington BD, Rosse WF, Milner PF, Castro O, Steinberg MH, et al. Mortality in sickle cell disease: life expectancy and risk factors for early death. N Engl J Med 1994;330:1639-44. 27. Francis RB Jr, Hebbel RP. Hemostasis. In: Embury S, Hebbel RP, Mohandas N, Steinberg MH, editors. Sickle cell disease: basic principles and clinical practice. New York: Raven Press, 1994:299-310. 28. Kurantsin-Mills J, Ofosu FA, Sara TK, Siegel RS, Lessin LS. Plasma factor VII and thrombin-antithrombin III levels indicate increased tissue factor activity in sickle cell patients. Br J Haematol 1992;81:539-44. 29. Kucuk O, Gilman-Sachs A, Beaman K, Lis LJ, Westerman MP. Antiphospholipid antibodies in sickle cell disease. Am J Hematol 1993;42:380-3. 30. Cariou R, Tobelem G, Bellucci S, Soria J, Soria C, Maclouf J, et al. Effect of lupus anticoagulant on antithrombogenic properties of endothelial cells--inhibition of thrombomodulin-dependent protein C activation. Thromb Haemost 1988; 60:54-8. 31. Schorer AE, Wickham NWR, Watson KV. Lupus anticoagulant induces a selective defect in thrombin-mediated endothelial prostacyclin release and platelet aggregation. Br J Haematol 1993;71:399-407. 32. Failer DV. Vascular modulation. In: Embury S, Hebbel RP, Mohandas N, Steinberg MH, editors. Sickle cell disease:
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basic principles and clinical practice. New York: Raven Press, 1994:235-46. Longenecker G, Culberson D, Beyers B, Manci E, Mankad V: Plasma endothelin (ET) levels are elevated in sickle cell disease (SCD)[abstract]. Blood 1991;78(suppl):414a. Rybicki AC, Musto S, Swinson GI, Benjamin LJ, Nagel RL, Schwartz RS. Increased levels of endothelin-1 in plasma of sickle cell anemia patients [abstract]. Blood 1991;78(suppl): 202a. Rodgers GP, Scheehter AN, Noguchi CT, Klein HG, Nienhuis AW, Bonnet RF. Periodic microcirculatory flow in patients with sickle-cell disease. N Engl J Med 1984;311:1534-8. Phelan M, Perrine SP, Brauer M, Faller DV. Sickle erythrocytes, after sickling, regulate the expression of the endothelin-1 gene and protein in human endothelial cells in culture. J Clin Invest 1995;96:1145-51. Mosseri M, Bartlett-Pandite AN, Wenc K, Isner MH, Weinstein R. Inhibition of endothelium-dependent vasorelaxation by sickle erythrocytes. Am Heart J 1993;126:338-46. Hebbel RP, Mohandas N. Sickle cell adherence. In: Embury S, Hebbel RP, Mohandas N, Steinberg MH, editc,rs. Sickle cell disease: basic principles and clinical practice. New York: Raven Press, 1994:217-30. Sugihara K, Sugihara T, Mohands N, Hebbel RF'. Thrombospondin mediates adherence of CD36 + sickle reticulocytes to endothelial cells. Blood 1992;80:2634-42. Swerlick RA, Eckman JR, Kumar A, Jeitler M, Wick TM. ag[31-integrin expression on sickle reticulocytes: vascular cell adhesion molecule-l-dependent binding to endothelium. Blood 1993;82:1891-9. Wick TM, Moake JL, Udden MM, Eskln SG, Sears DA, McIntire LV. Unusually large von Willebrand factor multimers increase adhesion of sickle erythrocytes to human endothelial cells under controlled flow. J Clin Invest 1987;80: 905-10. Hebbel RP, Moldow CF, Steinberg MH. Modulation of erythrocyte-endothelial interactions and the vasocc.lusive severity of sickling disorders. Blood 1981;58:947-52. Dampler CD, Stuart MJ. Hypoxia enhances red blood cell (RBC)-endothelial cell (EC) adherence. Role of 15-hydroxyeicosatetraenoic acid (15-HETE) in this interaction. Clin Res 1993;41:163A. Hebbe! RP, Visser MR, Goodman JL, Jacob HS, Vercellotti GM. Potentiated adherence of sickle erythrocytes to endothelium infected by virus. J Clin Invest 1987;80:1503-6. Brittain HA, Eckman JR, Wick TM. Sickle erythrocyte adherence to large vessel and microvascular endothelium under
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Abbreviations: G P
= g l y c o p r o t e i n ; I C A M = i n t e r c e l l u l a r a d h e s i o n m o l e c u l e ; IL = interleukin; M V E C = m i c r o v a s c u l a r e n d o t h e l i a l cell; INF = i n t e r f e r o n ; RBC = r e d b l o o d cell; TNF = t u m o r necrosis f a c t o r ; TSP = t h r o m b o s p o n d i n ; V C A M = v a s c u l a r cell a d h e s i o n m o l e c u l e ; W B C = w h i t e b l o o d cell; v W f = v o n W i l l e b r a n d f a c t o r
he vascular wall comprises the pathobiologic focal point of many disease states, a fact that derives from the myriad biologic roles played by the endothelial cell. This cell resides at the interfacial barrier between the blood space and tissues, providing both spatially defining physical confinement and an "inert" surface for blood contact. Yet it displays a host of adhesion molecules that potentially promote interaction with blood cells. It contributes separately to the physiology of both hemostasis and inflammation, although it inexorably links them by residing at the interface of these two critical homeostatic systems,1'2 both responding to and participating in each. It exists in a delicate balance between its numerous anticoagulant properties and potential procoagulant capabilities.2-4 It helps control vascular tone by liberation of either vasodilatory or vasoconstricting factors.5 And most importantly, the endothelial cell provides a responsive surface 1'6-9 that is modulated by an extraordinary array of biologic modifiers: thrombin, histamine, interleukins, hypoxia, endotoxin, oxidants, TNF, INF, and so on. These substances (and states) induce the endothelial cell to participate in vascular pathobiology by modulating its extensive repertoire of surface-dependent functions. The phenomenal respon-
T
From the Department of Medicine, University of Minnesota Medical School. Submittedfor publicationSept. 13, 1996;acceptedSept. 26, 1996. Reprint requests: Robert P. Hebbel, MD, Department of Medicine, Box 480 UMHC, Universityof MinnesotaMedicalSchool, Harvard St. at E. River Rd., Minneapolis,MN 55455. J Lab Clin Med 1997;129:288-93. Copyright© 1997by Mosby-YearBook,Inc. 0022-2143/97 $5.00 + 0 5/1/78721 288
siveness of endothelial functional phenotype is most dramatically illustrated by this cell's hemostatic and adhesive functions. ROLE IN HEMOSTASIS
The endothelial cell exerts numerous physiologically important influences on hemostasis2-4 that can be influenced by biologic modifiers. For example, thrombin not only is the most potent physiologic activator of platelets but also is an excellent stimulant of various endothelial changes, including the release of vWf, prostacyclin, nitric oxide, plasminogen activators, and platelet-activating factor; the expression of tissue factor and adhesion molecules such as P-selectin; the promotion of endotheliumdependent vasoconstriction; and the activation of thrombomodulin. Numerous other modulators of endothelial cell hemostatic function have been described and are summarized, albeit not all-inclusively, in Table I. ROLE IN ADHESION BIOLOGY
Nature likewise has provided the endothelial surface with a host of receptors and other structures that promote the attachment of blood cells, with or without the involvement of adhesogenic plasma proteins (Table II). Integrins. These comprise heterodimeric associations of multiple ~ and [3 chains, x° These receptors tend to exhibit overlapping binding specificities, many of which depend on a ligand RGD sequence. The [31 integrins tend to bind fibronectin or laminin or collagen (or a combination). Of these, endothelial cells express several fibronectin receptors (at least ~s[31 and o~v[~l). Broad binding capability is exhibited by the [33 integrins. Of these, endothelial
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T a b l e I. Endothelial cell h e m o s t a t i c functions Anticoagulant functions
Procoagulant functions
Antithrombotic Sulfated proteoglycans (1' by INF; 4, by TNF, I_-1) Thrombomodulin (1' by THR; ,t, by TNF, IL-1, _PS) Fibrinolytic Plasminogen activators (1' by THR; $ by IL-1) Vasodilatation Prostacyclin ( 1' by THR; IL-1) Nitric oxide ( 1' by IL-1, THR, LPS; $ by OX, HYP)
Prothrombotic vWf release ( I' by THR) Tissue factor expression ( 1` by THR, LPS, IL-1, TNF) Antifibrinolytic Plasminogen activator inhibitor 1 ( 1` by THR, LPS, IL-1) Vasoconstriction Endothelin ( 1' by THR, HYP) PDGF ( 1' by THR, HYP, TGFq3) Platelet activating factor ( 1' by THR) Proinflammatory Adhesion molecules ( 1' by THR, LPS, IL-1, OX, INF-) Chemokine production ( 1" by TNF, LPS, IL-1) Platelet activation Platelet activating factor ( 1' by THR, IL-1)
Antiinflammatory Leukocyte adhesion inhibitor ( 1' by TNF, LPS, IL-1) Platelet inhibition Prostacyclin ( 1' by THR, IL-1) Nitric oxide ( 1' by IL-1, THR, LPS; 4, by OX, F-YP) HYP, Hypoxia;LPS, endotoxin;OX, oxidation; THR,thrombin.
T a b l e II. Endothelial cell a d h e s i v e functions Surface adhesion receptor
Integrins 0~5131 ~vl~l %133 Ig superfamily ICAM-1,2 VCAM* PECAM Leucine-rich receptors GP1 b Selectins P-selectin E-selectin* Unique CD36 (GplV)
Ligand
FN FN VN, FN, FB, TSP, vWf
Influences on receptor expresslon
i' by IL-1 I" by IL-1; $ by INF plus TNF
WBC CD11 b/18 WBC 0~41~1 PECAM
1' by INF, IL-1, TNF, LPS 1' IL-1, TNF, OX
vWf
1' by TNF (augmented by INF)
sLx/WBC sLx/WBC
1` by THR, HIS, LTC 1' by TNF, LPS, IL-1 (augmented by INF)
TSP, COL_
1' by IFN~/
FN, Fibronectin;VN, vitronectin; FB, Fibrinogen;COL, collagen;HIS, histamine;LTC, leukotriene04; s/x, (sialyI-Lewis-x);others as in footnote to Table I.
*Net constitutivelyexpressedand only detectableon stimulated endothelium.
cells express the vitronectin recepor (%~3), which potentially provides for the binding of vitronectin, fibrinogen, vWf, TSP, and fibronectin) °'u Expression of avf33 and %~1 on endothelial cells is increased by IL-loL,12 while INF--/plus TNF depresses %133 expression even while preserving %131.13 The immunoglobulin superfamily. These receptors 14 are represented on endothelial cells by constitutive expression of ICAM-1,2 and by inducible expression of VCAM. ICAM participates in WBC-endothelial cell interactions, and its expression is increased by INF--y/IL-lodTNF-odlipopolysaccharide. 15 VCAM expression is induced by endothelial activation with IL-1 or TNF and possibly by H202 or lysolipids.14 Endothelium also expresses platelet endothelial cell
adhesion molecule, which is probably a homotypic adhesion molecule important in neutrophil transmigration. Seleetins. These are single-chain receptors, the ligand for which appears to be carbohydrates like sialyl-Lewis-x.16 On endothelium, P-selectin (also called GMP140) is brought to the surface from Weibel-Palade bodies by the same stimuli (e.g., thrombin, histamine, leukotriene C4) 1 that raise cytosolic [Ca] and lead to vWf release. E-selectin is not expressed on quiescent endothelium, only on cells activated by TNF or IL-1, an effect potentiated by INF--/. Selectins mediate the adhesion of neutrophils and monocytes to endothelium.
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GPIV (CD36). This is a unique glycosylated receptor 17 that is expressed on microvascular endothelium (MVECs) but not large vessel endothelium is and on platelets/megakaryocytes, monocytes, and hematopoietic progenitors. CD36 expression is increased by INF-'y. 18 It is a receptor for TSP, collagen, and oxidized low-density lipoprotein. The leucine-rich GP family. This is represented on endothelium by GPIb, 19 which contributes to functional vWf binding. 2° Endothelial expression of GPIb is increased by TNF-a, an effect greatly amplified by INF-y. 19 Endothelial-endothelial adhesion proteins. Endothelial-endothelial adhesion proteins include occludin at tight junctions; connexins at gap junctions; and cadherins at adherence junctions. 21 These additional adhesion molecules are important, because they maintain endothelial layer integrity and barrier function. However, they have no known or hypothesized role in blood cell-endothelial cell interaction.
ENDOTHELIAL CELL HETEROGENEITY
The existence of endothelial heterogeneity probably is a significant factor in pathobiology involving the endothelium. Although deduced largely from the study of cultured cells, it has been accepted as a valid concept that endothelial cells exhibit heterogeneity with respect to hemostatic functions. 22-a4 It also is likely to be relevant to adhesive interactions. For example, it may be noted that MVECs show a more brisk ICAM-1 response to cytokines than do human umbilical vein endothelial cells, x5 As noted above, CD36 is expressed on MVECs but not on large vessel endothelium. Its expression therefore provides a useful marker of endothelial cells as being microvascular in origin. Despite its probable fundamental physiologic importance, very little is known about organ-to-organ or temporal endothelial heterogeneity in situ. PARTICIPATION OF ENDOTHELIUM IN SICKLE CELL DISEASE
Remarkably, many endothelial functions could, in theory, participate in the vascular pathobiology of sickle disease. The following sections provide examples. Mechanical factors. The space-defining function of the endothelium provides the interfacial surface on which any mechanical obstruction to blood flow must first impact. Thus it is the interaction of sickled red cells with the vessel wall that actually enforces deoxygenation-induced vascular obstruction. WBC adhesion molecules. The responsiveness of endothelium to inflammatory stimuli is most well de-
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fined for those that elicit the expression of adhesion molecules that promote WBC-endothelium interaction. Host defense appears to depend not only on WBC responsiveness to inflammatory signaling but also on the endothelial cell's responsiveness. Thus the expression of selectins and ICAMs on the endothelial surface is a critical determinant of the ability of WBCs to marginate and roll along endothelium and to firmly attach, respectively. 1°'16 The possibility of WBC-endothelium adhesion in the patient with sickle cell disease has at least two fundamental implications. First, ample precedent exists in other vascular diseases for this consequence of the inflammatory state resulting in endothelial damage. 25 Various lines of evidence have identified the probable occurrence of endothelial damage in patients with sickle cell disease (summarized below). Second, the polymorphonuclear leukocyte is larger and far stiffer (i.e., less deformable) than the red cell. Thus its attachment to endothelium, particularly in the smallest vessels of limiting diameter, would impede passage of red cells, something that predictably causes greatly increased risk for red cell sickling. In this regard, it is notable that WBC count correlates with mortality in patients with sickle cell disease. 26 Hemostasis. Patients with sickle cell disease seem to be in a near-constant state of hemostatic perturbation or activation, a notion supported by multiple lines of evidence. 27 However, it cannot be discerned from the available data whether the endothelium has a net procoagulant or net anticoagulant impact on sickle blood. In any case, the very fact that endothelium responds to thrombin in multiple ways (as noted above) suggests that the excess thrombin generation of the patient with sickle cell disease 2s is likely to have an impact on disease pathobiology. Interestingly, patients with sickle cell disease are reported to have antiphospholipid antibodies with high frequency, 29 something that requires further investigation given the reported ability of such antibodies arising in the context of lupus anticoagulants to inhibit endothelial thrombomodulin 3° or prostacyclin release. 31 Vascular tone. The ability of the vascular endothelium to participate in the regulation of vascular tone is undisputed. For example, the endothelium-derived vasoconstrictor endothelin is induced by thrombin, transforming growth factor-t3, shear stress, hypoxia, and attachment of sickled red cells; it is inhibited by oxygen and nitric oxide. The vasodilating substance nitric oxide, on the other hand, is induced by shear stress, acetylcholine, bradykinin, and IL-1-TNF; it is inhibited by hypoxia. 32 Whether the endothelium's vasodilatory or vaso-
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constricting effects dominate, or are alternately expressed, in sickle disease is not established. Levels of endothelin are reported to be elevated in patients with sickle cell disease, 33'34 and apparent abnormalities of vascular tone have been identified in the form of oscillatory microvascular flow. 35 In this disease, where avoidance of vascular obstruction depends critically on unimpeded passage of RBCs, the tone-regulating function of the endothelium certainly must be a relevant factor. It has been reported that the attachment of sickled red cells greatly stimulates endothelin transcription in cultured endothelium, 36 which is consistent with the finding that adherent sickle RBCs inhibit vasorelaxation. 37 RBC adhesion. The postulated role of RBC-endothelial cell interaction as a trigger of sickle vasoocclusion 38 indicates that various endotheliurn-dependent factors relevant to this interesting cell-cell interaction may have a significant impact on pathobiology. Among the varied mechanisms for RBC adhesion to endothelium are several related to specific adhesion molecules noted above. For example, %[33 and CD36 have been implicated as mechanisms of TSP-mediated sickle RBC binding to endotheliurn. 39 VCAM is of particular interest because interaction between endothelial VCAM and 0~4[~1 o n sickle reticulocytes seems to be one major mechanism of RBC-endothelium interaction, and it is potentiated by TNF. 40 Similarly, cytokine effects may provide inducible vWf binding capacity relevant to vWf-mediated adhesion of sickle reticulocytes to endothelium, an additional major mechanism underlying RBC-endothelium interaction. 41 ,~dso, oxidatively damaged, 42 hypoxia-stressed, 43 or virally infected 44'45 endothelia provide a more adhesive substrate for sickle RBCs. Other largely preliminary studies suggest that endothelium stimulated by platelet-activating factor or IL-1 also supports greater sickle RBC adhesion. 3s Endothelial cell heterogeneity. This too is predicted to have a significant impact on sickle disease pathobiology. Of particular relevance is the example of a TSP receptor, CD36, that is hypothesized to mediate adhesion of sickle RBCs to endothelium. This molecule is expressed on microvascular, but not on large vessel, endothelial cells, is In this regard it is remarkable that the adhesion-promoting effect of vWf is much lower when microvascular endothelial cells are used than when large vessel endothelial cells are used, while the adhesion potentiating effect of unfractionated plasma is dramatically greater for microvascular than for large vessel endothelium. 45 This undoubtedly reflects the receptor complement
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on different endothelia and suggests that plasma substances other than vWf are more important for RBC interaction with small vessel endothelium. Additionally, the endothelial cell surface can obviously vary in conjunction with concurrent illness to the extent that endothelial surface modifiers are at play. Endothelial cell responsiveness. The probable relevance of all of these participants in sickle vascular pathobiology is heightened by the very responsiveness of the vascular endothelium to biologic modifiers. The assessment of blood levels of potential biologic modifiers in sickle disease is in its infancy, but already enough is known to predict participation of endothelial cell modulation in this disease. These influence the whole spectrum of endothelial cell functions outlined above as being of direct relevance to sickle disease. Significantly, elevated levels of endotoxin, 46 IL-1, 47 and TNF 4s all have been reported in patients with sickle cell disease; there is an apparent excess of thrombin generation as well. 28 Additionally, the very nature of this vascular disease predicts repetitive molestation of the endothelium by hypoxia and possibly by oxidative stress. Endothelial damage in sickle disease. The hypothesis that endothelial injury occurs and contributes to sickle vasoocclusion has been discussed but is poorly documented. Histopathologic changes suggestive of endothelial injury have been observed in splenic and cerebral vasculatures of patients with sickle cell disease. 48-51 Thromboses in these patients tend to occur at sites of underlying intimal hyperplasia, 49-51 implying that this vascular lesion is derived from gross disturbances of endothelial cell function. An abnormal presence of circulating endothelial surface molecules (ICAM-1, VCAM-1, E-selectin) has been reported in the plasma of patients with sickle cell disease, although it is unknown whether these are secreted and physiologic or represent an injury response. 52 The adhesion of sickle RBCs to endothelium is reported to stimulate a prostacyclin response 53'54 and to impair DNA synthetic ability. 55 Finally, the blood of patients with sickle cell disease, particularly the blood of those in acute painful crisis, contains increased numbers of circulating endothelial cells.56-58 In aggregate, these lines of evidence strongly suggest that the endothelium can be, and is, variably perturbed in the patient with sickle cell disease. THE NEED TO UNDERSTAND ENDOTHEL]UM IN SITU
Nothing is really known about the status of endothelium in vivo. Considering the importance of the endothelial cell in vascular biology, it is particularly
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remarkable that so little is known about it in sickle disease, in which there is ample reason to believe that it influences pathophysiology. A single article has suggested that retinal capillaries in patients with sickle cell disease exhibit greatly increased ICAM-1 expression, 59 which suggests prior stimulation by cytokines and the possibility of concomitant VCAM and GPIb expression. Proof that sickle disease is, in its pathobiology, a disease of endothelial biology will have to await development of techniques to assess the status of the endothelium in situ. Possibly the study of circulating endothelial cells may afford this opportunity. 58 REFERENCES
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