Endothelial cell (EC) proliferation is characteristic of cell-mediated immune responses and it occurs early in association with alloimmune targeting of transplants. In human renal allografts, EC proliferation (by Ki67+ staining) has been shown to precede and predict chronic rejection. Furthermore, using rat and mouse cardiac allograft models, we found that interruption blockade with angiogenesis inhibitors attenuates the long-term development of chronic rejection/fibrosis. Here, we wished to test the hypothesis that EC proliferation drives chronic allograft fibrosis through EndMT. Primary cultures of human umbilical vein endothelial cells (HUVEC), that underwent either <20, 40-60, or 80-120 cellular divisions, were treated with TGFΒ1 (10ng/ml), TGFΒ2 (2.5ng/ml), and/or TNFΑ (100U/ml) alone for 7-10 days. EndMT was evaluated by FACS (N-cadherin + CD31) and qPCR (N-cadherin, ΑSMA, and Snail mRNA expression). We initially found that EC with <20 or 40-60 cellular divisions failed to undergo EndMT in response to TGFΒ1 or TGFΒ2. Yet, TNFΑ alone or TNFΑ+TGFΒ1 caused a small, but significant, degree of EndMT in these cells by FACS (8.7±4.2% and 10.2±1.4%, respectively, vs 2.1±0.3% in untreated controls; n≥3, P<0.05) and by qPCR (∼2.7-fold and ∼3.8-fold increase, respectively, in N-cadherin, ΑSMA, and Snail mRNA expression; n≥3, P<0.04). Interestingly, in EC with >80 doublings, TGFΒ1 or TGFΒ2, in combination with TNFΑ, led to marked EndMT by FACS (19.6±4.3% and 49.3±7.0%, respectively; n=4, P<0.02) and by qPCR (N-cadherin, ΑSMA, and Snail mRNA expression increased ∼3.6-fold vs untreated controls; n≥4, P<0.05). Finally, we evaluated whether hypoxia plays a role in EndMT and found that reducing pO2 (from 21 to 10%) for 7-10 days induced EndMT in 9.7±2.3% of EC with <20 divisions vs 1.7±0.5% of EC in normoxia (p<0.03, n=6). Following exposure to TGFΒ1+TNFΑ, hypoxia augmented EndMT by FACS, especially in EC with >80 doublings (34.8±6.3%, n=6, P<0.02). Collectively, these findings strongly suggest that proliferating EC are more susceptible to undergoing EndMT when exposed to inflammatory cytokines and hypoxia, factors well established to be present in a rejecting allograft. Thus, monitoring EC doublings within grafts may identify risk for the development of chronic rejection and provides further evidence that anti-angiogenic therapeutics may prevent chronic allograft fibrosis through reduced susceptibility to EndMT.

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