| Tissue engineering blood vessels(TEBV) is one of the most important substitutes for vascular diseases, such as atherosclerosis and vascular restenosis. Blood vessels are mainly composed of endothelial cells (EC) and smooth muscle cells (SMC). SMC are not terminally differentiated, and possess the ability to switch between a contractile (differentiated) and a synthetic (dedifferentiated) phenotype with changing local environmental cues. SMC within adult blood vessels proliferate at an extremely low rate, exhibit very low synthetic activity, and express a unique repertoire of contractile proteins for the cell's contractile function. The differentiation state of the SMCs in TEBV is very important in determining if the SMCs will be able to respond to various vasoactive mediators. Normal in vivo blood vessels contain differentiated SMCs that contract and relax in response to vasoactive molecules, thus controlling the blood flow through the vessel. EC lining the lumen of blood vessels have many important functions, such as vasodilation, platelet coagulation, clot formation, immune response, water and solute permeability. Many researches on EC and SMC interactions within TEBV showed that the co-culture of EC/SMC is crucial to better understand EC/SMC cross-talk and model vascular healing. More differentiated contractile SMC are desired in TEBV, so that the vessel will be able to fully respond to vasoactive mediators in the blood or released by the EC. Thus, we try to investigate SMC phenotype switch within EC/SMC co-culture system, and confirm the phenotype of SMC treated with the PI3K inhibitor Wortmannin.Mononuclear cells were isolated from fresh rat peripheral blood by density gradient centrifugation, induced and cultured in EGM-2-MV kit with PDGF-BB. EC were isolated from thoracic aortas of rat after removing endothelial cell, by using tissue section culturing. The cells were serially passaged and expanded in humidified incubator at 37℃with 5% CO2. EC and SMC were assessed to confirm their cell phenotype by phase contrast light microscopic and immunofluorescent microscopic. The floating gel co-culture model consisted of a subconfluent EC (1×104cells/well) attaching on the downside floating gel, when oppositely culturing a single layer of SMC (1×104cells/well) on cell culture plate. The expression of CRBP-1, Smemb, SM-MHC, and Smoothelin-B was analyzed by RT-PCR; and immunofluorescence was analyzed by Leica TCS SP2 confocal microscope and Image-pro Plus software.The results showed that isolated EC kept their cobblestone-like morphology and positively stained by vWF, an endothelial cell specific marker. The SMC displayed the spindle shape and specific "hill and valley" morphology and positively stained by smooth cell marker a-SMA. In the floating gel co-culture system, the expression of CRBP-1 and Smemb, the most stringent markers for the dedifferentiated phenotype of SMC isolated from rat peripheral blood, increased by 1.6-fold and 1.9-fold compared with SMC cultured alone at the 48h time point, by 1.9-fold and 2.4-fold at 72h. Then the trend of the gene expression reversed. The expression of CRBP-1 and Smemb at 96h is lower than 72h, while the differentiated phenotype marker Smoothelin-B is higher than 72h. There wasn't a statistically difference between the SMC from rat peripheral blood and those from thoracic aortas(P>0.05). In the monoculture group, Wortmannin inhibited SMC proliferation with dose-dependence. Wortmannin also down regulated the expression of CRBP-1.Our results showed that at first EC increased the synthetic capacity of SMC, then at 96h EC help to switch SMC from a synthetic (dedifferentiated) phenotype to a contractile (differentiated) phenotype. Wortmannin inhibited SMC proliferation and dowm-regulated the expression of CRBP-1 in the monoculture group. |
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