| In recent years, the gap junction protein connexin43 has been shown to play a pivotal role in wound healing process. If downregulate the expression of Cx43 at skin wound site after wounding immediately,it can markedly improve the rate and quality of wound healing. There are three kinds of repair cells in skin wound healing, keratinocytes, fibroblasts and vascular endothelial cells. Many studies about the mechanism of how Cx43 downregulation improve the wound healing showed that Cx43 downregulation can increase the rate of migration and proliferation of fibroblasts and keratinocytes, also improve the differentiation capacity of keratinocytes.However, the effects of Cx43 downregulation on endothelial cells are still unclear. As we has known, vascular endothelial cell participate in multi-step process of wound healing. During inflammatory phase, leukocytes need to transmigrate vascular endothelial cells reaching wound site to cause inflammatory response. At the stage of proliferation, vascular endothelial cells influence angiogenesis which is a central feature of wound repair and can provide oxygen and nutrients necessary to sustain cell metabolism. Based on these, we choose endothelial cells as the research object to analyze effects of Cx43 downregulation on endothelial cell functions.Human umbilical vein endothelial cell(HUVEC) is the mostly used model for endothelial cell research. What’s more, it has a good reactivity with a variety of factors, including inflammatory factors. Hence, this study choose HUVECs as the research object.Small interference RNA(si RNA) is usually used to downregulate proteins expression. We designed three si RNA sequences(Cx43 si RNA 1,Cx43 si RNA 2,Cx43 si RNA 3) to reduce the expression of Cx43 in HUVECs according Cx43 gene sequence. The results of Western blot showed that the maximum silence effects was reached when cells were treated 24 h and continue to 72 h, and Cx43 si RNA 1 was the most effective si RNA sequence when HUVECs were treated by Cx43 si RNA 1 24 h, the expression of connexin 43 reduced by ~80%. Hence, here we chose Cx43 si RNA 1 treating HUVECs 24 h to investigate the effects of connexin43 downregulation on intercellular communication, viability, proliferation, migration and angiogenic activity of HUVECs. The results showed that the intercellular communication was decreased by ~65%. The viability, proliferation, migration and angiogenic activity of HUVECs were decreased significantly, compared with the normal cells.Inflammation process is a necessary process for skin wound healing. Inflammation response needs leukocytes adhering to endothelial cells and transmigrating across blood vessel wall. Hence, we used connexin43 si RNA 1 to reduce the expression of connexin43 in HUVECs to investigate the effects of connexin43 downregulation on adhesion between monocyte U937 and HUVEC, and the ability of U937 transmigrating HUVECs layer. TNF-α can stimulate HUVECs to stay in inflammation state. We used TNF-α to stimulate HUVECs to simulate inflammation in vivo. The results of Calcein-AM labeling U937 and rose bengal staining showed that TNF-α indeed stimulated HUVECs to present inflammation state, and downregulation of connexin43 expression could decrease the ability of adhesion between U937 and HUVEC and the ability of transmigration HUVECs layer.To study the molecular mechanism, we analyzed the expression of cell adhension molecules that play an important role in inflammation response. The results of Western blot and rt-PCR showed that the expression of ICAM-1、VCAM-1 was reduced because of Cx43 downregulation, but CD44 had no change.In conclusion, downregulation of connexin43 expression can significantly decrease the viability, proliferation, migration and angiogenic activity of HUVECs, and decrease the ability of adhesion between U937 and HUVEC, the ability of U937 transmigration HUVECs layer, and the expression of ICAM-1、VCAM-1 to reduce the intensity of the inflammatory response, and hence, further to improve the rate and quality of wound healing. |
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