Thrombospondin 1 in hypoxia-conditioned media blocks the growth of human microvascular endothelial cells and is increased in systemic sclerosis tissues

Abstract

BackgroundSystemic sclerosis (SSc) is a chronic inflammatory autoimmune disease characterised by vascular dysfunction and damage, excess collagen deposition and subsequent organ manifestations. Vasculopathy is an early feature of the disease which leads to a chronic hypoxic environment in the tissues. Paradoxically, there is a lack of angiogenesis. We hypothesised that this may in part be due to a nonphysiological, overriding upregulation in antiangiogenic factors produced by the hypoxic tissues. We considered thrombospondin 1 (TSP-1) as a candidate antiangiogenic factor.ResultsConditioned media from human microvascular endothelial cells cultured in both normoxic and hypoxic environments were able to block endothelial cell proliferation, with the latter environment having a more profound effect. Filtration to remove > 100-kDa proteins or heparin-binding proteins from the conditioned media eliminated their antiproliferative effect. TSP-1 was expressed in high concentrations in the hypoxic media, as was vascular endothelial growth factor (VEGF). Depletion of TSP-1 from the media by immunoprecipitation reduced the antiproliferative effect. We then show that, in a dose-dependent fashion, recombinant TSP-1 blocks the proliferation of endothelial cells. Immunohistochemistry of skin biopsy material revealed that TSP-1 expression was significantly higher throughout the skin of patients with SSc compared with healthy controls.ConclusionsDespite the environment of chronic tissue hypoxia in SSc, there is a paradoxical absence of angiogenesis. This is thought to be due in part to aberrant expression of antiangiogenic factors, including TSP-1. We have demonstrated that TSP-1 is released in high concentrations by hypoxic endothelial cells. The conditioned media from these cells is able to block proliferation and induce apoptosis in microvascular endothelial cells, an effect that is reduced when TSP-1 is immunoprecipitated out. Further, we have shown that recombinant TSP-1 is able to block proliferation and induce apoptosis at concentrations consistent with those found in the plasma of patients with SSc and that its effect occurs in the presence of elevated VEGF levels. Taken together, these data are consistent with a model wherein injured microvascular cells in SSc fail to repair because of dysregulated induction of TSP-1 in the hypoxic tissues.

Heart angiotensin II-induced cardiomyocyte hypertrophy suppresses coronary angiogenesis and progresses diabetic cardiomyopathy

“…Accordingly, TSP-1 upregulated in the extensively hypertrophied cardiomyocytes of SDT rat LVs at 16 wk could both induce capillary endothelial cell apoptosis and suppress capillary endothelial cell proliferation via the inhibition of VEGFstimulated VEGFR2 phosphorylation and thereby overcome the stimulatory effect of VEGF on coronary angiogenesis, leading to coronary capillary loss. These findings are compatible with those in a recent study (28) showing that in cultured human microvascular endothelial cells, despite the induction of VEGF in hypoxia-conditioned media, the increased levels of TSP-1 were able to override the protective effects of VEGF on their survival and proliferation. Taking these previous reports together with our findings, the signals from the hypertrophied cardiomyocytes under chronic hypoxia to inhibit coronary angiogenesis differ markedly between the above-mentioned models (37,45) and our own, and we suggest an important role for TSP-1 as an antiangiogenic factor in the diabetic heart of SDT rats.…”

Age-Associated Induction of Cell Membrane CD47 Limits Basal and Temperature-Induced Changes in Cutaneous Blood Flow

“…Loss of cutaneous blood flow, associated tissue necrosis and poor wound healing are known consequences of age and certain vasculopathies 27,28,45 . We have reported that skeletal muscle blood flow is maintained in old TSP1- and CD47-null mice compared to old wild type animals 21 .…”

“…The secreted matricellular protein thrombospondin-1 (TSP1) is unregulated in several disease states that are associated with loss of SkBF and impaired wound healing including diabetes, scleroderma 27 and systemic sclerosis 28 , and has recently been postulated to account for the loss of cutaneous blood flow in these individuals 29 . In pre-clinical models of cutaneous wound healing TSP1 antisense oligomers delayed wound healing 30 and overexpression of TSP1 in the skin of mice greatly slowed wound closure and wound-associated angiogenesis 31 .…”

Cytokines in the immunopathology of systemic sclerosis

“…In normal physiological circumstances, tissue hypoxia and angiogenic drive attempt to recover tissue perfusion and endothelial cell function. In SSc patients, the structural changes in the microvessels are irreversible culminating in the loss of capillaries with paradoxical absence of angiogenesis [55]. The imbalance between pro-angiogenic (e.g.…”