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ABSTRACT: A proper cavernous endothelial cell culture system would be advantageous for the study of the pathophysiologic mechanisms involved in endothelial dysfunction and erectile dysfunction (ED). To establish a nonenzymatic technique, which we termed the "Matrigel-based sprouting endothelial cell culture system," for the isolation of mouse cavernous endothelial cells (MCECs) and an in vitro model that mimics in vivo situation for diabetes-induced ED. For primary MCEC culture, mouse cavernous tissue was implanted into Matrigel and sprouting cells from the tissue were subcultivated. To establish an in vitro model for diabetes-induced ED, the primary cultured MCECs were exposed to a normal-glucose (5 mmoL) or a high-glucose (30 mmoL) condition for 48 hours. The purity of isolated cells was determined by fluorescence-activated cell sorting analysis. MCECs incubated under the normal- or the high-glucose condition were used for Western blot, cyclic guanosine monophosphate (cGMP) quantification, and in vitro angiogenesis assay. We could consistently isolate high-purity MCECs (about 97%) with the Matrigel-based sprouting endothelial cell culture system. MCECs were subcultured up to the fifth passage and no significant changes were noted in endothelial cell morphology or purity. The phosphorylation of Akt and eNOS and the cGMP concentration were significantly lower in MCECs exposed to high glucose than in those exposed to normal glucose. MCECs exposed to the normal-glucose condition formed well-organized capillary-like structures, whereas derangements in tube formation were noted in MCECs exposed to high glucose. The protein expression of transforming growth factor-β1 (TGF-β1) and phospho-Smad2 was significantly increased by exposure to high glucose. The Matrigel-based sprouting endothelial cell culture system is a simple, technically feasible, and reproducible technique for isolating pure cavernous endothelial cells in mice. An in vitro model for diabetic ED will be a valuable tool for evaluating the angiogenic potential of novel endogenous or synthetic modulators.