Effect Of Cobalt Chloride-induced Hypoxia On Expression Of Ang-1 In Pericytes

BackgroundWound healing is the process that lots of healing related cells coordinate with each other,play their respective biological functions,and rely on the interaction between cells,extracellular matrix and cytokines and growth factors to repair the damage caused by various factors to the body.Angiogenesis is an essential factor in wound healing.Intact blood vessels can provide sufficient nutrients,oxygen and bioactive substances for wounds,and can promptly remove metabolic wastes.The endothelial cells connect with each other to form a blood vessel and form a microvascular barrier.The microvascular barrier allows the free passage of small molecules to exchange material,but it restricts the free passage of macromolecules.When the blood vessels injured,the function of the microvascular barrier is damaged.At this time,the large molecules are free to pass through the blood vessels,causing vascular leakage.Vascular leakage is common in the early stage of trauma.Any factors that cause destruction of blood vessels can cause vascular leakage and cause edema in wound.The wound sustained abnormal leakage is a sign of poor wound healing,commonly in chronic wounds.Therefore,inhibiting the microvascular leakage of the wound and regulating the microvascular permeability are of important clinical significance to wound healing.Pericyte is a component of blood vessels,which can regulate the permeability of blood vessels by covering the interstitial space of the vascular endothelium and paracrine.It is an indispensable cell for regulating angiogenesis,vascular remodeling and vascular permeability.Pericyte play an regulatory role by covering the gaps in vascular endothelial cells,and can also regulated indirectly by paracrine Ang-1.Ang-1 is a cytokine secreted by the pericytes.By combining the receptor Tie-2of vascular endothelial cells,it plays an important role in maintaining the integrity of blood vessels and reducing the occurrence of vascular exudation.Therefore,pericytes are the key to regulate the permeability of the microvascular wound and accelerate the wound healing.Vascular damage causes vascular leakage,because it can not transport sufficient oxygen in time,remove metabolic waste and inflammation,often make the wound in hypoxia.HIF-1α is the most important regulatory factor in the hypoxic environment,which is rapidly degraded under normal oxygen,and expressed steadily under hypoxia.It can form dimers with HIF-1 beta subunits,which regulate the biological processes such as inflammation and apoptosis,and control hundreds of genes including downstream vascular endothelial growth factor.Cobalt chloride is a commonly used hypoxia inducer at home and abroad.The Fe2+is replaced by Co2+of cobalt chloride,which affects oxygen utilization,causes hypoxia,and also inhibits the degradation of HIF-1 alpha.Vascular injury causes blood vessels leakage,and make the pericytes are in hypoxia.In hypoxia,the expression of HIF-1 alpha increased and the downstream gene VEGF is up-regulated.Therefore,in this study,we detected the effects of hypoxia on the expression of HIF-1,VEGF and Ang-1,and explored the effects of HIF-1 and VEGF on Ang-1 expression in hypoxia,so as to provide new ideas for clinical wound healing.ObjectiveTo investigate the effect of cobalt chloride-induced hypoxia on expression of Ang-1 in pericytes.MethodsInvestigating the effects of hypoxic environment on the expression of HIF-1-alpha,VEGF and Ang-1 in pericytes by establishing CoCl2-induced hypoxia model.Take the primary cell resuscitation and then expand and frozen.In the experiment,recover the frozen HBVP,then culture HBVP by pericyte medium.The 3-5 generation cells were used in the experiment.24h was intervened by different cobalt chloride when the cell was filled with 100mm culture dish 80-90%.The concentration are 0（control group）,50μmol/L,100μmol/L,200μmol/L,300μmol/L and 400μmol/L respectively.After samples were extracted,the expression of HIF-1 alpha protein was detected by Western-blot,the m RNA synthesis of HIF-1 alpha and Ang-1 were detected by RT-PCR,and the secretion of VEGF and Ang-1 was detected by ELISA.ResultsThe experimental results show that:（1）cobalt chloride-induced hypoxia promotes the expression of HIF-1 alpha and VEGF.The expression of HIF-1 alpha increased with cobalt chloride concentration showed a single peak curve,the peak appeared at 200 umol/L;at the same time hypoxia inhibits pericytes m RNA synthesis of HIF-1 alpha in a concentration dependent manner;（2）in hypoxia,the expression and m RNA synthesis of were affected.Expression and m RNA synthesis of Ang-1 showed a single peak curve with the increase of cobalt chloride concentration.The peak value appeared at 100 umol/L,and then decreased significantly compared with the control group.ConclusionCoCl₂-induced hypoxia affects the expression of Ang-1 in pericytes.