Background MVECs damage is a central event in the pathogenesis of SSc leading to capillaries rarefaction and consequent ischemia. VEGF is the best characterized angiogenic growth factor; its increase after ischemia is a timed controlled event, acting only in the first stages of new blood vessels formation when it regulates ECs sprouting and proliferation. During SSc, VEGF lacks this tight control and is strongly up-regulated over time both at local and systemic levels. Caveolae, specialized plasma membrane microdomains, abundant in ECs, and caveolin-1 (cav-1), their principal residual protein, compartmentalize VEGF-induced signaling toward angiogenesis.
Objectives Here we aimed to prove whether caveolae/cav-1 act as platform for organizing and compartmentalizing VEGF/VEGFR2 signaling in MVECs during SSc. In fact an abnormal cav-1 expression in microvascular endothelium, may contribute to angiogenic defective response during the disease.
Methods After ethical approval skin was collected from 10 patients with SSc and used for immunofluorescence (IF), qPCR and MVECs isolation. Normal skin was obtained from healthy donors (HC) who underwent surgery for trauma. Cav-1 detection and its co-localization with VEGFR-2 was perfomed on skin sections and MVECS isolated from SSc patients and compared to HC by IF, immunoprecipitation and Western Blot (WB). qPCR analysis was used to assess mRNA cav-1 expression level both in SSc skin and MVECs. To determine whether VEGF treatment could affect cav-1/VEGFR2 colocalization in MVECs during SSC, all the above was assessed after culturing these cells in the presence of VEGF.
Results We found that VEGFR-2 colocalizes with cav-1 in blood vessels within SSc skin, as already observed in HC skin. However semi-quantitative analysis of IF cav-1 expression level revealed lower expression in SSc MVECS with respect to HC skin. This result was confirmed by qPCR, showing cav-1 was down-regulated in MVECs isolated from SSc patients when compared to HC cells. When we assessed cav-1/VEGFR.2 co-localization within MVECS, we observed that VEGFR-2 was localized in the caveolae directly bound by cav-1. This observation was further confirmed by cav-1 immunoprecipitation and WB for VEGFR-2 in SSc MVECs, without differences from HC. Interestingly, after culturing these cells in the presence of VEGF, we found a disruption of cav-1/VEGFR-2 co-localization both in HC and SSc MVECs, without cav-1 mRNA levels re-modulation
Conclusions During SSc cav-1 expression down-regulation within MVECs, could possibly explain the dysfunctional, ineffective VEGF angiogenic signaling. This mechanism might maintain the redundancy of deregulated VEGF abundance in a vicious loop, strongly contributing to not compensative angiogenic response during SSc.
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Disclosure of Interest None Declared