Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling

Rafiee Parvaneh, Heidemann Jan, Ogawa Hitoshi, Johnson, Fisher, Li, Otterson, Binion

Description

The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on non-immune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a long-term clinical complication and significant cause of mortality in solid-organ transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsA's effect on four in vitro measures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX-2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2004
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Cell Communication and Signaling

BioMedCentral

Open Access Research Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling 1,2 3 3 2 Parvaneh Rafiee* , Jan Heidemann , Hitoshi Ogawa , Nathan A Johnson , 3 1 1,4 1 Pamela J Fisher , Mona S Li , Mary F Otterson , Christopher P Johnson and 3 David G Binion

1 2 Address: Department of Surgery, Medical College of Wisconsin, Milwaukee, WI, 53226, USA, Children's Hospital of Wisconsin, Milwaukee, WI, 3 4 53226, USA, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI, 53226, USA and Zablocki Veterans Affairs Medical Center, Milwaukee, WI, 53295, USA

Email: Parvaneh Rafiee*  [e-mail]; Jan Heidemann  [e-mail]; Hitoshi Ogawa  [e-mail]; Nathan A Johnson  [e-mail]; Pamela J Fisher  [e-mail]; Mona S Li  [e-mail]; Mary F Otterson  [e-mail]; Christopher P Johnson  [e-mail]; David G Binion  [e-mail] * Corresponding author

Published: 02 June 2004 Received: 12 January 2004 Accepted: 02 June 2004 Cell Communication and Signaling2004,2:3 This article is available from: http://www.biosignaling.com/content/2/1/3 © 2004 Rafiee et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

Abstract The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on nonimmune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a longterm clinical complication and significant cause of mortality in solidorgan transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsA's effect on fourin vitromeasures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection.

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Cell Communication and Signaling2004,2

Background The calcineurin inhibitor cyclosporin A (CsA) is a potent immunosuppressive agent that has formed the pharmaco logic cornerstone of solid organ transplantation. CsA pre vents the activation of lymphokine genes essential for T cell proliferation by disrupting calciumdependent signal transduction pathways in leukocytes [1]. Although phar macologic studies of CsA have focused primarily on T cell responses, there is emerging evidence that this agent may exert potent effects on blood vessels, promoting arterial hypertension, inducing longterm vascular dysfunction, and contributing to obliterative vasculopathy in chronic transplant rejection [25]. At the present time, chronic rejection with its associated vasculopathy, is the major cause of late allograft dysfunction, including patients with intestinal transplants [6,7].

In solidorgan transplantation, the vascular endothelium has received attention because of its unique role as the interface between the donor graft and the host's circulat ing immune cells, and as a focus of acute rejection [8,9]. More recent investigation has demonstrated that the endothelium plays a central role in chronic rejection, where inappropriate activation of endothelial cells results in obliterative vasculopathy and accelerated posttrans plant atherosclerosis [10], a major cause of morbidity and mortality in solid organ transplant recipients. Activation of graft endothelium in chronic rejection may result from host/graft immunologic attack, as well as dysfunction associated with transplant immunosuppression [5]. In transplantation of the small bowel, microvascular dys function may contribute to more significant problems with both acute and chronic rejection in these patients. Indeed, small bowel transplantation has been one of the more problematic clinical areas in the realm of solid organ grafts, where patients require increased immuno suppressive regimens and have had overall, less successful clinical outcomes [1113].

The growth of new microvessels, or angiogenesis, is now appreciated to be a critical biologic process involved in tis sue homeostasis. Angiogenesis is initiated by local activa tion of genes encoding diffusable angiogenic factors, or by the release of vascular growth factors which subsequently act on local microvasular cell populations, as well as by a decrease in local angiostatic factors, including interferon beta [14]. Angiogenesis involves an orchestrated sequence of steps which include endothelial activation, stress fiber assembly, fibrinolysis, proteolytic degradation of the basement membrane and the extracellular matrix, migra tion, proliferation and neovascularization [15]. One of the major angiogenic growth factors is the vascular endothelial growth factor (VEGF), which selectively induces activation, migration, proliferation and tube for mation in endothelial cellsin vitro. VEGF is a 34–42 kDa

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glycoprotein which exerts its biological effects on endothelial cells through its two major tyrosine kinase receptors, VEGFR1/Flt1 (fetal liver kinase1) and VEGFR2/Flk1/KDR (kinase insert domain containing receptor). By binding to these receptors, VEGF activates various signaling cascades, including the mitogen acti vated protein kinase family (ERK1/2, p38 MAPK and SAPK/JNK) and phosphoinositol3kinase (PI3 kinase) [16,17]. A downstream result of these signaling events is the expression of COX2, which plays an integral role in VEGF induced angiogenesis [1820]. Finally, investigation has demonstrated a pivotal role for the transcription fac tor nuclear factor activated in Tcells (NFAT) in the ang iogenic signaling of VEGF in human umbilical vein endothelial cells (HUVEC) [21], which is inhibited by CsA. Thus, there is a potential role for CsA in blocking angiogenesis in microvascular cell populations, poten tially through its effect on NFAT.

An integrated analysis of the effect of CsA on the multiple stages of the angiogenic process in human organ specific microvascular endothelial cells has not been performed to date. Studies evaluating the effect of CsA on human umbilical vein endothelial cells [22] may not accurately reflect microvascular events, and are further complicated by their wide functional variability in tissue culture, caus ing discrepant results reported by different laboratories [23]. In addition, it is also now appreciated that human microvascular endothelial cells derived from differenti ated, organ specific vascular beds have been shown to dif fer significantly from HUVEC in their responsiveness to cytokines, the expression profile of antigens, and the elab oration of secretory products [2427]. We investigated the mechanisms of VEGFinduced endothelial cell signaling and angiogenesis in HIMEC, an organ specific microvas cular cell population, and the effect of CsA on fourin vitro components of angiogenesis, including stress fiber assem bly, migration, proliferation/growth and tube formation.

Results VEGF, but not TNF α /LPS, enhances growth of HIMEC We have previously shown that VEGF is a potent stimulus forin vitrogrowth and proliferation of HIMEC [28,29]. Additional studies have demonstrated thatin vitroactiva tion of HIMEC with TNFα/LPS will result in inflamma tory activation, expression of cell adhesion molecules and increased leukocyte binding which is mediated by activa tion of NFκB and mitogen activated protein kinases [30]. Because MAPK activation has been linked to proliferation of various cell types, including endothelial cells, we per formedin vitrogrowth studies to assess the effect of VEGF and TNFα/LPS on HIMEC. HIMEC monolayers stimu lated with VEGF for 24 hr resulted in a significant increase in cell number, while TNFα/LPS had essentially no effect and was similar to unstimulated cell growth (Figure 1). As

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