Endothelin in the Microvasculature

Matthias Barton

University of Zurich School of Medicine, Medical Policlinic, Department of Medicine, University Hospital Zurich, Switzerland

Introduction

Vascular activity of a peptide secreted from endothelial cells was discovered in the mid-1980s. In 1988, Yanagisawa and collegues published the sequencences of the gene and the peptide, which belongs to a peptide family structurally related to vasoconstrictor snake venoms. According to their cellular origin, the peptides were named endothelins. The predominant isoform, endothelin-1, is the most potent vasoconstrictor known and also promotes cell growth throughout the cardiovascular system. Endothelin is synthesized from its inactive precursor, big-endothelin, by endothelin-converting enzymes and other peptidases, and in mammals signal transduction is mediated through activation of two G-protein-coupled endothelin receptors. The endothelin system is activated in conditions associated with vascular injury and disease. This chapter discusses the role of endo-thelin in the microvasculature for some of the clinically most prevalent entities, atherogenesis, inflammation, and cancer development.

Endothelins: Vasoactive Peptides

After the discovery of endothelium-derived relaxing factor, later identified as nitric oxide [1], the vascular activity of a peptide secreted from endothelial cells was described in the mid-1980s [1,2]. In 1988, the gene sequencence of a 21-amino acid protein was identified [3] and found to be structurally similar to snake venoms, the sarafatoxins. The protein was named endothelin based on its cellular origin, and it soon turned out that three functionally different iso-forms exist [4]. In the late 1990s, additional endothelin iso-forms consisting of 31 and 32 amino acids were discovered

[5, 6]. The predominant isoform of the endothelin peptide family, endothelin-11-21, is the most potent vasoconstrictor known in terms of duration of action and potency and also stimulates cell growth throughout the cardiovascular system. In the vasculature, endothelin is synthesized from its inactive precursor, big-endothelin, by endothelin-converting enzymes and other peptidases (Figure 1). In mammals, endothelin signaling is mediated through activation of two high-affinity, G-protein-coupled endothelin receptors [5, 6]. Endothelin ETA receptors predominantly promote vasoconstriction and growth, whereas activation of the ETB receptor—which is highly expressed in endothelial cells—is mainly coupled to the release of the vasodilators and growth inhibitors nitric oxide and prostacyclin [5]. ETA receptors preferably bind endothelin-1, whereas the ETB receptor binds all three isoforms with equal affinity.

Regulatory Role of Endothelin for Microvascular Function

Expression of endothelin receptors in the microvascula-ture differs from the expressional pattern observed in larger vessels. Specifically, the endothelin ETA receptor—which is rarely detected in macrovascular endothelial cells—is expressed in microvascular endothelium. Microvascular endothelial cells are also structurally different from endothelial cells in other parts of the vascular tree. They differ in terms of abundency of cytoskeleton stress fibers, metabolic activity, and number of tight junctions [7]. Also, microvascular endothelial cells contain more vesicles. These cell organelles play a central role for endothelin synthesis: Endothelin-converting enzyme-2 protein expression is localized to endothelial cell vesicles, structures that store

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Endothelin

Figure 1 Biosynthesis and multiple functions of endothelin-1(1-21) in the cardiovascular system. Preproendothelin-1 messenger RNA is translated into preproendothelin-1, a 203 amino acid peptide, which is further cleaved by furin convertases to the inactive precursor, big-endothelin-1(1-38). Several enzymes, including endothelin-converting enzymes (ECE), cleave big-endothelin-1 to the active 21-amino acid peptide, endothelin-1. ECE, endothelin-converting enzyme; SEP, secreted soluble endopeptidase; VSMC, vascular smooth muscle cell; ROS, reactive oxygen species; ETA, endothelin ETA receptor; ETB, endothelin ETB receptor. Reproduced from reference [5] with permission.

Figure 1 Biosynthesis and multiple functions of endothelin-1(1-21) in the cardiovascular system. Preproendothelin-1 messenger RNA is translated into preproendothelin-1, a 203 amino acid peptide, which is further cleaved by furin convertases to the inactive precursor, big-endothelin-1(1-38). Several enzymes, including endothelin-converting enzymes (ECE), cleave big-endothelin-1 to the active 21-amino acid peptide, endothelin-1. ECE, endothelin-converting enzyme; SEP, secreted soluble endopeptidase; VSMC, vascular smooth muscle cell; ROS, reactive oxygen species; ETA, endothelin ETA receptor; ETB, endothelin ETB receptor. Reproduced from reference [5] with permission.

and release mature endothelin-1 upon stimulation. In vitro work suggests that endothelin also has important functions for capillary formation under normal and pathological conditions. Furthermore, endothelin-1 leads to activation of cytokines in circulating blood cells and also in resident cells such as endothelial cells or vascular smooth muscle. This activation includes induction of interleukin-6, nuclear factor kappa B, and C-reactive protein, effects that in turn can be enhanced by other cytokines under certain conditions. Thus, inflammatory changes and subsequent increases in vascular permeability are key factors determining microvascular injury mediated by endogenous endothelin-1.

Endothelin and the Microvasculature in Disease

Carcinogenesis and Angiogenesis

Autocrine endothelin receptor signaling controls growth of different types of tumor cells, including ovarian cancer melanoma, Kaposi sarcoma, colon carcinoma, prostate cancer, and bone metastases of tumors [8]. In most cancer cells, growth responses involve activation of ETA receptors. In addition, angiogenesis provides an important means to increase blood supply of the tumor growth and metastasis.

Proliferation and wound healing of endothelial cells is mediated by ETB receptors. Therefore, endothelin-1 via ETB receptors may act as an angiogenic peptide, providing additional avenues for cancer cells to increase blood supply and disseminate. By controlling expression of vascular endothelial cell growth factor (VEGF) in an autocrine fashion, endothelin-1 also acts as an important inducer of angiogen-esis, thereby stimulating tumor cell proliferation. In consequence, endothelin-1 can be considered an essential factor for tumor growth, neovascularization, and metastasis. ETA receptor blockade has been shown to inhibit tumor growth directly and to exert antiangiogenic effects by inhibiting VEGF production and by acting on vascular smooth muscle cell and on microvascular channels lined by tumor cells (Figure 2). Moreover, because ETA receptors are expressed by microvascular endothelial cells, inhibiton of these receptors is likely to play a role for capillary formation and growth as well. It is therefore not surprising that targeting of endothelin receptors currently appears to be a promising new anticancer strategy.

Inflammation and Vascular Permeability

Hypoxia represents a strong stimulus inducing both transcription and protein synthesis of endothelin-1. Moreover,

Endothelium Endothelin

in Pntfnctwcfagy A AtoJflbofiarrr

Figure 2 Proposed Overview of the Possible Role of Endothelin Signaling in Tumor Angiogenesis. This process involves a series of linked signaling pathways that typically begins with upregulation of the synthesis of angiogenic factors, such as ET-1 and VEGF, that are released by tumor cells. ETA receptor (ETAR) activation by ET-1 could promote angiogenesis by increasing VEGF production through HIF-1-dependent mechanisms. ET-1 binds to specific ETB receptors (ETBR) on endothelial cells and to ETA receptors on pericytes/vascular smooth muscle cells or on tumor cells lining vessels (vasculogenic mimicry). ETA receptor blockade (ABT627) could exert antiangiogenic effects by inhibiting VEGF production and by acting on VSMC and on micro-vascular channels lined by tumor cells. Another cascade of signaling events is then initiated, which leads to the formation of new tumor vessels. Erk, extracellular signal-related kinase; ET-1, endothelin-1; Mek, mitogen-activated protein kinase kinase; Akt, protein kinase B; Hif, hypoxia-inducible factor-1; VEGF, vascular endothelial cell growth factor; VEGFR, vascular endothelial cell growth factor receptor. Reproduced from reference [8] with permission. (see color insert)

in Pntfnctwcfagy A AtoJflbofiarrr

Figure 2 Proposed Overview of the Possible Role of Endothelin Signaling in Tumor Angiogenesis. This process involves a series of linked signaling pathways that typically begins with upregulation of the synthesis of angiogenic factors, such as ET-1 and VEGF, that are released by tumor cells. ETA receptor (ETAR) activation by ET-1 could promote angiogenesis by increasing VEGF production through HIF-1-dependent mechanisms. ET-1 binds to specific ETB receptors (ETBR) on endothelial cells and to ETA receptors on pericytes/vascular smooth muscle cells or on tumor cells lining vessels (vasculogenic mimicry). ETA receptor blockade (ABT627) could exert antiangiogenic effects by inhibiting VEGF production and by acting on VSMC and on micro-vascular channels lined by tumor cells. Another cascade of signaling events is then initiated, which leads to the formation of new tumor vessels. Erk, extracellular signal-related kinase; ET-1, endothelin-1; Mek, mitogen-activated protein kinase kinase; Akt, protein kinase B; Hif, hypoxia-inducible factor-1; VEGF, vascular endothelial cell growth factor; VEGFR, vascular endothelial cell growth factor receptor. Reproduced from reference [8] with permission. (see color insert)

oxidative stress associated with reperfusion further increases endothelin synthesis. There is now evidence suggesting that endothelin-1 directly contributes to vascular inflammation and microvascular permeability [9, 10]. Although the latter function is usually attributed to mediators such as histamine or VEGF, endothelin may not only contribute to tissue edema but also induces rolling and adherence of leukocytes to the capillary wall [11] (Figure 3). This results in inflammation, blood hyperviscosity, and microcirculatory stasis, leading to a substantial decrease in microcircular O2 extraction and cell dysfunction. These changes can be prevented to a large extent by endothelin receptor blockers targeting the ETA receptor, a treatment that has been shown to substantially reduce microcirculatory dysfunction in the myocardium [10, 12], brain [11], and intestine. Activation of the microvascular endothelin system and its direct and indirect effects are therefore responsible for cell injury, particularly in conditions associated with ischemia.

Atherosclerosis and Hypercholesterolemia

Cardiovascular risk factors such as increases in blood pressure, increased plasma levels of cholesterol or glucose, or lack of female sex hormones—all of which accelerate the development of atherosclerosis—result in activation of the endothelin system in vitro and in vivo [5, 6]. Recently, several studies have demonstrated that changes in microvas-culature may importantly contribute to atherogenesis. Experimental evidence from John Cooke's laboratory suggests that enhancing angiogenesis may accelerate rather than limit the atherosclerotic disease process. Elegant studies by Lerman and coworkers [10, 13] have investigated some of the regulatory processes by which endothelin-dependent microvascular changes may contribute to early atherogenesis [13]. Even in the absence of structural injury, hypercholesterolemia, one of the cardinal risk factors for atherosclerosis, results in vascular inflammation, increased microvascular permeability [10], and adventitial neovascularization (Figure 5B). These alterations are associated with induction of VEGF expression (Figure 5D). Interestingly, increased microvascular permeability, adventitial neovascu-larization, as well as VEGF expression can be fully prevented by blocking ETA receptors [10, 13]. Similarly, prevention of myocardial inflammation, infarction, and fibrosis in atherosclerotic mice by endothelin ETA receptor blockade has been demonstrated [12], and beneficial effects on structural changes in mesenteric resistance vasculature of

Endothelin

Figure 3 Role of Endothelin for Leukocyte-Endothelium Interactions in Gerbil Pial Venules. Frequency of rolling (A) and firm adherent leukocytes at the vessel wall of pial venules (B) in the absence (O) or presence (•) of the ETA antagonist BQ-610 before and after global ischemia in gerbils. Arrow indicates time point of drug administration, bar indicates time of ischemia. *p < 0.05 versus control. From reference [11] with permission.

Figure 3 Role of Endothelin for Leukocyte-Endothelium Interactions in Gerbil Pial Venules. Frequency of rolling (A) and firm adherent leukocytes at the vessel wall of pial venules (B) in the absence (O) or presence (•) of the ETA antagonist BQ-610 before and after global ischemia in gerbils. Arrow indicates time point of drug administration, bar indicates time of ischemia. *p < 0.05 versus control. From reference [11] with permission.

Endothelin

Figure 4 Vascular Permeability Measured by FITC-albumin in Guinea Pig Intestinal Submucosal Microcirculation. Effects of histamine (HIST) or endothelin-1 (ET-1) in absence or presence of treatment with antagonists of the ETA receptor (BQ-123, BQ), platelet-activating factor (WEB-2086, WEB), or Hj histamine receptors (diphenhydramine, H1). * p < 0.05 versus CON; t p < 0.05 versus ET-1. From reference [9] with permission.

Figure 4 Vascular Permeability Measured by FITC-albumin in Guinea Pig Intestinal Submucosal Microcirculation. Effects of histamine (HIST) or endothelin-1 (ET-1) in absence or presence of treatment with antagonists of the ETA receptor (BQ-123, BQ), platelet-activating factor (WEB-2086, WEB), or Hj histamine receptors (diphenhydramine, H1). * p < 0.05 versus CON; t p < 0.05 versus ET-1. From reference [9] with permission.

Table I Effects of atherosclerosis and endothelin ETA receptor blockade with darusentan for 30 weeks on vascular structure in mesenteric resistance arteries in apolipoprotein E-deficient mice (apoE0) and wild-type controls (C57BL6/J) measured after treatement in isolated vessels in a arteriograph system in vitro under perfused and pressurized conditions.

Group

C57

C57 + Darusentan

apoE0

ApoE0 + Darusentan

Lumen diameter (|lm)

222 ± 4

235 ± 7

217 ± 5

220 ± 8

Intima-media thickness (|lm)

18.9 ± 0.2

18.3 ± 0.4

20.9 ± 0.7*

17.0 ± 0.4t

Intima-media CSA (x103 ||m2)

14.3 ± 0.2

13.9 ± 0.5

15.6 ± 0.6*

12.9 ± 0.5t

ApoE0, apolipoprotein E deficient; C57, wild-type control; darusentan, ETA receptor antagonist (Knoll LU135252); CSA, cross-sectional area; *P < 0.05 versus C57; tP < 0.05 versus apoE0. Modified from reference [15] with permission.

Image Vasa Vasorum

Figure 5A-C Effects of normal diet (A), high-cholesterol diet alone (B), or in combination with endothelin ETA receptor antagonist treatment (C) on formation and spatial distribution of coronary vasa vasorum in pigs. Concomitant endothelin blockade essentially prevented adventitial neovascularization. From reference [13] with permission.

Figure 5A-C Effects of normal diet (A), high-cholesterol diet alone (B), or in combination with endothelin ETA receptor antagonist treatment (C) on formation and spatial distribution of coronary vasa vasorum in pigs. Concomitant endothelin blockade essentially prevented adventitial neovascularization. From reference [13] with permission.

Endothelin Receptor And Vegf

Figure 5D Coronary artery wall expression of VEGF protein in pigs after 12weeks of high-cholesterol diet. Note that endothelin ETA receptor blockade (ET-A) fully prevented vascular upregulation of VEGF induced by hypercholesteremia. N, normal diet; HC, high-cholesterol diet; HC + ET-A, high-cholesterol diet and endothelin ETA receptor antagonist ABT-627. From reference [13] with permission.

Figure 5D Coronary artery wall expression of VEGF protein in pigs after 12weeks of high-cholesterol diet. Note that endothelin ETA receptor blockade (ET-A) fully prevented vascular upregulation of VEGF induced by hypercholesteremia. N, normal diet; HC, high-cholesterol diet; HC + ET-A, high-cholesterol diet and endothelin ETA receptor antagonist ABT-627. From reference [13] with permission.

mice with atherosclerosis have been reported (Table I). It is not known whether the microvascular changes described mechanistically translate into the inhibition of advanced human-like atherosclerotic lesions observed after ETA receptor blockade [14], but it is likely that anti-inflammatory and antiangiogenic effects of endothelin blockade play an essential role.

References

1. Furchgott, R. F., and Vanhoutte, P. M. (1989). Endothelium-derived relaxing and contracting factors. FASEB J. 3, 2007-2018.

2. Highsmith, R. F. (1992). From endotensin to endothelin: The discovery and characterization of an endothelial cell-derived constricting factor. In Endothelin (G. M. Rubanyi, ed.), pp. 1-192. New York: Oxford University Press.

3. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988). Anovel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-415.

4. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., and Masaki, T. (1989). The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Natl. Acad. Sci. USA 86, 2863-2867.

5. Barton, M., Traupe, T., and Haudenschild, C. C. (2003). Endothelin, hypercholesterolemia, and atherosclerosis. Coron. Artery Dis. 14, 477-490.

6. Lüscher, T., and Barton, M. (2000). Endothelins and endothelin receptor antagonists: Therapeutic considerations for a novel class of cardiovascular drugs. Circulation 102, 2434-2440.

7. Kuruvilla, L., and Kartha, C. C. (2003). Molecular mechanisms of endothelial regulation of cardiac function. Mol. Cell. Biochem. 253, 113-123.

8. Bagnato, A., and Spinella, F. (2002). Emerging role of endothelin-1 in tumor angiogenesis. Trends Endocrinol. Metab. 14, 44-50.

A thorough review on the paracrine and autocrine effects of endothe-lin in cancer development and angiogenesis.

9. King-VanVlack, C. E., Mewburn, J. D., Chapler, C. K., and MacDonald, P. H. (2003). Hemodynamic and proinflammatory actions of endothelin-1 in guinea pig small intestine submucosal microcirculation. Am. J. Physiol. Gastrointest. Liver Physiol. 284, G940-G948.

10. Bonetti, P. O., Best, P. J., Rodriguez-Porcel, M., Holmes, D. R., Lerman, L. O., and Lerman, A. (2003). Endothelin type A receptor antagonism restores myocardial perfusion response to adenosine in experimental hypercholesterolemia. Atherosclerosis 168, 367-373.

11. Lehmberg, J., Putz, C., Fürst, M., Beck, J., Baethmann, A., and Uhl, E. (2003). Impact of the endothelin-A receptor antagonist BQ 610 on microcirculation in global cerebral ischemia and reperfusion. Brain Res. 961, 277-286.

12. Caligiuri, G., Levy, B., Pernow, J., Thoren, P., and Hansson, G. K. (1999). Myocardial infarction mediated by endothelin receptor signaling in hypercholesterolemic mice. Proc. Natl. Acad. Sci. USA 96, 6920-6924.

Important work demonstrating a causal role of endothelin for endothe-lin receptors in myocardial inflammation and infarction in mice with advanced atherosclerosis.

13. Herrmann, J., Best, P. J., Ritman, E. L., Holmes, D. R., Lerman, L. O., and Lerman, A. (2002). Chronic endothelin receptor antagonism prevents vasa vasorum neovascularization in experimental hypercholes-terolemia. J. Am. Coll. Cardiol. 39, 1555-1561.

An interesting article demonstrating a role of endothelin for adventitial neovascularization during hypercholesterolemia in pigs.

14. Barton, M., Haudenschild, C. C., d'Uscio, L. V, Shaw, S., Munter, K., and Luscher, T. F. (1998). Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice. Proc. Natl. Acad. Sci. USA 95, 14367-14372.

15. d'Uscio, L. V., Barton, M., Shaw, S., and Lüscher, T. F. (2002). Chronic ET(A) receptor blockade prevents endothelial dysfunction of small arteries in apolipoprotein E-deficient mice. Cardiovasc Res. 53, 487-495.

Capsule Biography cian at the Department of Medicine and Director of Research of the Medical Policlinic at the University Hospital Zurich. The research in his laboratory focuses on atherogenesis, obesity, as well as vascular and renal biology. His research is supported by grants from the Swiss National Foundation (SNF), the Deutsche Forschungsgemeinschaft, and the University of Zürich.

Dr. Barton is Associate Professor of Cardiology at the University of Zürich School of Medicine, Switzerland. He is currently Attending Physi-

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Responses

  • Robin
    Are the families of caligiuri and Bagnato related?
    3 years ago

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