Experimental Study On The Effects Of Integrin-linked Kinase On Angiogenesis In Hypertrophic Scar In Vitro

BackgroundScar is a product of replacing normal skin with fibrous tissue after injury. Scar formation contains an important mechanism——scar angiogenesis. Aberrant angiogesis may be a critical factor to the abnormal scar formation. In recent years, the role of angiogenesis in scar formation has been a hot spot in studying mechanism of scar formation.Integrin-linked kinase (ILK) is a newly discovered serine / threonine kinase, which can mediate connections between integrants and ECM in order to achieve transmission of extracellular signals into inside cells. ILK can serve as a bridge between integrin and growth factors / receptor signaling pathways, and participates in the synthesis and secretion of various growth factors. ILK is wildly distributed in mammal's animals and related to a variety of diseases. Currently, there are many researches about ILK in tumor formation and tissue fibrosis, and some researches also reported that ILK may play a role in angiogenesis. However, there is not a report about ILK effect on scar angiogenesis yet. The purpose of this study is mainly to explore the mechanism of ILK in angiogenesis of scar in vitro.The effects of ILK on proliferation and cell cycle of vascular endothelial cells, which were isolated from human scar and cultured in vitro, were observed. Then, the effects of ILK on synthesis and secretion of VEGF from fibroblasts and regulation for expression of KDR mRNA and Flt-1 mRNA in vascular endothelial cells from scar were observed. Last, the role of hypoxia in the regulation for ILK expression in endothelial cells from scars was explored. This study is to prove the role of ILK in angiogenesis of hypertrophic scar from cell proliferation, environmental influence and growth factor receptor signaling pathway to investigate the mechanism of ILK in scar angiogenesis.PARTⅠStudy of the effect of ILK on proliferation and cell cycle of microvascular endothelial cell in hypertrophic scarObjective To explore the effect of proliferation and cell cycle of hypertrophic scar microvascular endothelial cells by interfering ILK gene expression with siRNA. Methods Eight patients' hypertrophic scar were isolated and cultured into microvascular endothelial cells (HSMECs) in vitro. Then the cells were divided into 4 groups:①the control group,②Interferin group,③ILK con-siRNA group and,④ILK siRNA group. ILK siRNA was transfected into the HSMEC by InterferinTM. First, we observed the ILK protein expression after siRNA were transfected into HSMECs by Western blot detection.XTT and flow cytometry was used to observe the effect of ILK siRNA on HSMECs proliferation and cell cycle respectively.Result The results of Western blot showed that the expression of ILK protein was inhibited by ILK siRNA in HSMECs. XTT results showed that the cellular proliferation rate was the lowest one among them ((F= 164.154, P=0.000)). Cell cycle analysis by flow cytometry indicated that the ratio of G1 phase was higher in ILK siRNA group than those in other groups (P<0.05), the ratios of S phase was lower in ILK siRNA group than those in other groups (P<0.05).Conclusion ILK siRNA can interfere with the expression of ILK at protein level in HSMECs, result in inhibition of HSMECs proliferation, and arrest the cell cycle progress toward S phase from G1 phase. ILK may be a critical regulator to promote the proliferation and cell cycle in HSMECs.PART II Study on the influence of integrin-linked kinase to VEGF and its receptors in hypertrophic scarObjective To explore the regulating role of integrin-linked kinase (ILK) on secretion of VEGF in human scar fibroblasts (hFBs) and expression of KDR mRNA and Flt-1 mRNA in scar vascular endothelial cells (HSMECs)Methods The hFBs and HSMECs were isolated from human hypertrophic scar and cultured in vitro. Then, these two kinds of cells were divided into three groups respectively:①The control group,②Empty plasmid group, and③ILK cDNA plasmid transfected group. First, the expressions of ILK and VEGF were detected with immunocytochemistry in the transfected hFBs. Then, the level of VEGF mRNA and its protein expressions were investigated by Real-time PCR(RT-PCR) and Western blot. Finally, the secretions of VEGF in supernatant of culture medium for fibroblasts were detected by ELISA. The expressions of Flt-1 mRNA and KDR mRNA were also investigated with Real-time PCR in HSMECs, which were divided into groups according to the method mentioned above. Results The immunocytochemistry showed that the ILK and VEGF staining enhanced after ILK cDNA transfection; RT-PCR and Western blot showed that the levels of VEGF mRNA and its protein were significantly higher in ILK cDNA transfected group than those in other groups (P<0.05). ELISA showed that the protein of VEGF was higher remarkably in ILK cDNA transfected group comparing with those of other two groups (P<0.05). The levels of Flt-1 mRNA and KDR mRNA in ILK cDNA transfected group were significantly higher than those in control group and empty plasmid group (P<0.05).Conclusion ILK could regulate the effect of VEGF of hFBs in mRNA and protein levels, and promotes VEGF secretion from fibroblasts. It could also up-regulate the expression of Flt-1 mRNA and KDR mRNA in HSMECs at the same time. ILK may promote angiogenesis in hypertrophic scar by means of improving binding between VEGF and its receptor.PARTⅢStudy of hypoxia regulating growth of microvascular endothelial cell and expression of intergrin-linked kinaseObjective To explore the effect of chemical hypoxia induced by CoCl2 on growth of microvascular endothelial cell and its ILK expression in hypertrophic scar.Methods Eight patients' hypertrophic scar were isolated and cultured into HSMECs in vitro. Chemical hypoxia model was established in vitro by CoCl2. Scar microvascular endothelial cells were stimulated with different concentrations of CoCl2. Growth of cell was detected with XTT assay. The expression of ILK and HIF-la mRNA were detected with RT-PCR. The protein levels of ILK were detected with 60μmol/L of CoCl2 at different time points by Western blot.Result XTT showed that cells stimulated by 60μmol/L of CoCl2 were the highest in proliferation. With the increase of concentration, the cells showed the inhibitory responses. RT-PCR showed that the expressions of ILK mRNA were promoted in mild hypoxia, and inhibited in aberrant hypoxia. The change of rule is same with the cell proliferation change. Western blot analysis revealed that ILK protein expressions were normal on the zero hour (normoxic condition), decreased on the 1st hour, and increased gradually from 2nd hour to 24th hour.Conclusion Hypoxia environment may play a critical role on change of ILK expression in scar microvascular endothelial cells. The cell growth and the expressions of ILK could be promoted in mild hypoxia, and exhibited suppression in aberrant hypoxia. ILK may affect the growth of microvascular endothelial cell by means of hypoxia signals to promote angiogenesis in scar.