Microvascular pericytes have detrimental effects on tubulo-interstitial fibrosis via PMT. However, therapeutic strategies that might interfere with this process are currently lacking. We aimed to investigate the possible effects of Everolimus (EVE) in reversing PMT. Human pericytes were stimulated with pro-fibrotic factors such as TGFβ (10 ng/ml) and C5a (10^-7 M) at different time points. By immunofluorescence analysis we found that TGFβ and C5a down-regulated PDGFRβ (%Bas: 15.22±3.63; C5a: 3.66±2.35; TGFβ: 2.08±1.04, p<0.05) and induced αSMA remodeling fibers (p<0.05), indicating the occurrence of PMT. When EVE (5 ng/mL) was added 24h after TGFβ stimulation, we found a significant abrogation of PMT with decreased Collagen I production (qPCR CollagenI 2^-DDCT : TGFβ 24h + EVE 24h: 5.47±1.71 vs TGFβ 24h: 9.81±1.33). In addition, 4h pre-treatment with EVE significantly hampered TGFβ-induced Collagen I mRNA (CollagenI 2^-DDCT : EVE 4h + TGFβ 24h: 3.02±0.41 vs TGFβ 24h). EVE was also capable to induce significant ANGPT1 mRNA expression, a pivotal factor involved in vascular stabilization (ANGPT1 2^-DDCT: EVE24h: 8,4±1.42 vs Basal 1.2±0.72). We then performed functional Matrigel assays, where endothelial cells were co-coltured with Pericytes to test tube formation. C5a and TGFβ induced vascular rarefaction with limited tube formation. In presence of EVE, we found the development of capillary-like structure and inhibition of C5a-induced vessels rarefaction (p<0.05). Our data suggest that mTOR signaling pathways might be pivotal in regulating PMT. EVE might play a role in preventing pericyte activation thereby hampering the development of graft fibrosis.

CITATION INFORMATION: Castellano G, Franzin R, Stasi A, Divella C, Gesualdo L. Everolimus Abrogates C5a and TGFβ Mediated Perycite-to-Myofibroblast Transition (PMT) Inhibiting Vascular Rarefaction. Am J Transplant. 2016;16 (suppl 3).

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