Autoantibodies Targeting AT1- and ETA-Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor.

Scleroderma renal crisis (SRC) is an acute life-threatening manifestation of systemic sclerosis (SSc) caused by obliterative vasculopathy and thrombotic microangiopathy. Evidence suggests a pathogenic role of immunoglobulin G (IgG) targeting G-protein coupled receptors (GPCR). We therefore dissected SRC-associated vascular obliteration and investigated the specific effects of patient-derived IgG directed against angiotensin II type 1 (AT1R) and endothelin-1 type A receptors (ETAR) on downstream signaling events and endothelial cell proliferation. SRC-IgG triggered endothelial cell proliferation via activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent activation of the E26 transformation-specific-1 transcription factor (Ets-1). Either AT1R or ETAR receptor inhibitors/shRNA abrogated endothelial proliferation, confirming receptor activation and Ets-1 signaling involvement. Binding of Ets-1 to the tissue factor (TF) promoter exclusively induced TF. In addition, TF inhibition prevented endothelial cell proliferation. Thus, our data revealed a thus far unknown link between SRC-IgG-induced intracellular signaling, endothelial cell proliferation and active coagulation in the context of obliterative vasculopathy and SRC. Patients' autoantibodies and their molecular effectors represent new therapeutic targets to address severe vascular complications in SSc.

Intrinsic Deregulation of Vascular Smooth Muscle and Myofibroblast Differentiation in Mesenchymal Stromal Cells from Patients with Systemic Sclerosis.

INTRODUCTION: Obliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast differentiation in response to SSc-microenvironment defining growth factors and determined responsible mechanisms. METHODS: We studied responses of multipotent MSCs from SSc-patients (SSc-MSCs) and healthy controls (H-MSCs) to long-term exposure to CTGF, b-FGF, PDGF-BB or TGF-ß1. Differentiation towards VSMC and myofibroblast lineages was analyzed on phenotypic, biochemical, and functional levels. Intracellular signaling studies included analysis of TGF-ß receptor regulation, SMAD, AKT, ERK1/2 and autocrine loops. RESULTS: VSMC differentiation towards both, contractile and synthetic VSMC phenotypes in response to CTGF and b-FGF was disturbed in SSc-MSCs. H-MSCs and SSc-MSCs responded equally to PDGF-BB with prototypic fibroblastic differentiation. TGF-ß1 initiated myofibroblast differentiation in both cell types, yet with striking phenotypic and functional differences: In relation to H-MSC-derived myofibroblasts induced by TGF-ß1, those obtained from SSc-MSCs expressed more contractile proteins, migrated towards TGF-ß1, had low proliferative capacity, and secreted higher amounts of collagen paralleled by reduced MMP expression. Higher levels of TGF-ß receptor 1 and enhanced canonical and noncanonical TGF-ß signaling in SSc-MSCs accompanied aberrant differentiation response of SSc-MSCs in comparison to H-MSCs. CONCLUSIONS: Deregulated VSMC differentiation with a shift towards myofibroblast differentiation expands the concept of disturbed endogenous regenerative capacity of MSCs from SSc patients. Disease related intrinsic hyperresponsiveness to TGF-ß1 with increased collagen production may represent one responsible mechanism. Better understanding of repair barriers and harnessing beneficial differentiation processes in MSCs could widen options of autologous MSC application in SSc patients.

The proto-oncogene c-Fos transcriptionally regulates VEGF production during peritoneal inflammation.

Vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1) are key mediators of adverse peritoneal membrane remodeling in peritoneal dialysis eventually leading to ultrafiltration failure. Both are pleiotropic growth factors with cell type-dependent regulation of expression and biological effects. Here we studied regulation of TGF-ß1-induced VEGF expression in human peritoneal mesothelial cells in the absence or presence of proinflammatory stimuli, tumor necrosis factor-α (TNF-α) or interleukin-1ß (IL-1ß). Quiescent human peritoneal mesothelial cells secreted only trace amounts of VEGF. Stimulation with TGF-ß1 resulted in time- and dose-dependent increases in VEGF mRNA expression and protein release. TNF-α and IL-1ß alone had minimal effects but acted in synergy with TGF-ß1. Combined stimulation led to induction of transcription factor c-Fos and activation of the VEGF promoter region with high-affinity binding sites for c-Fos. Inhibition of c-Fos by small interfering RNA interference or by pharmacological blockade with SR-11302 decreased VEGF promoter activity and downregulated its expression and release. Exposure of human peritoneal mesothelial cells to dialysate effluent containing increased levels of TGF-ß1, TNF-α, and IL-1ß obtained during peritonitis resulted in a dose-dependent VEGF induction that was significantly attenuated by SR-11302. Thus, dialysate TGF-ß1, IL-1ß, and TNF-α act through c-Fos to synergistically upregulate VEGF production in peritoneal mesothelium and may represent an important regulatory link between inflammation and angiogenesis in the peritoneal membrane.

Cytoprotective Actions of FTY720 Modulate Severe Preservation Reperfusion Injury in Rat Renal Transplants.

BACKGROUND: Fingolimod (FTY720) is a potent agonist of sphingosine 1 phosphate receptors and thereby interferes with lymphocyte trafficking. We previously showed that FTY720 protects from mild preservation reperfusion injury induced by 4 hr of cold ischemia. The purpose of this study was to explore the role of FTY720 in ischemic injury and regeneration using a clinically relevant rat renal transplant model with 24 hr of cold ischemia. METHODS: Donor kidneys were cold stored in the University of Wisconsin solution for 24 hr before transplantation into bilaterally nephrectomized syngeneic recipients (n=6 per group), which received 0.5 mg/kg/d FTY720 or vehicle through oral gavage. Grafts were harvested 2 or 7 days posttransplantation. Renal tissue was examined histologically, stained for apoptosis, proliferation, inflammatory cell infiltrates, and studied for transforming growth factor-beta, and tumor necrosis factor-alpha expression. Rat proximal tubular cells were incubated with 0.1 to 30 micromol/L of phosphorylated FTY720 to test for in vitro cytopathic effects. RESULTS: FTY720 induced peripheral lymphopenia and significantly reduced intragraft CD3 and ED1 infiltrates. Acute tubular damage scores and graft function were not influenced by FTY720. Tubular apoptosis was significantly reduced, whereas the number of proliferating cell nuclear antigen-positive tubular cells were markedly increased. FTY720 attenuated renal tumor necrosis factor-alpha and transforming growth factor-beta expression. In vitro, pharmacologic concentrations up to 1 micromol/L of phosphorylated FTY720 did not affect tubular cell viability. CONCLUSION: FTY720 confers tubular epithelial protection in the presence of severe preservation reperfusion injury. Beneficial effects may in part be due to reduction in cell-mediated immune mechanisms. Furthermore, FTY720 could be helpful in patients with delayed graft function.