The Effect Of Intermedin On Angiogenesis Of Glomerular Endothelial Cells Injured By Hypoxia

Objective:To explore the effect and mechanism of IMD on angiogenesis of rRGECs after hypoxia injury.Methods:(1)Rat renal glomerular endothelial cells(rRGECs)was donated by The Third Affiliated Hospital of Sun Yat-Sen University and grown in RPMI-1640 medium(HyClone,Logan,UT,USA)supplemented with 10% FBS(HyClone)at 37°C with 5%CO2.(2)The optimal concentration of IMD was selected by a quantitative colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT)assay.(3)The tube-like structure formation was detected by tube formation assay.(4)The cultured rRGECs were randomly divided into two groups: normal group(N group),hypoxia group.The normal group were incubated in normoxia and the hypoxia group was incubated in the hypoxia environment of 5% O2 for 6 hours which was randomly divided into five groups then: hypoxia group(H group);M0 group:IMD(0.1?mol/L)were added to the hypoxia group;M1 group:ERK inhibitor(PD98059,5uM)was added toM0group;M2 group:PI3K inhibitor(LY-294002,100uM)was added toM0 group;M3group:NOS inhibitor(L-NAME,10uM)was added toM0 group.(5)Western blot analysis was used to detect PI3 K ?AKT?NOS total protein and their phosphory.(6)Western blot analysis was used to detect VEGF?VE-cadherin?p-VEGFR2 protein expression.(3)Western Blotting results show that: compared with control group,hypoxia group and IMD group the expression of VEGF were increased,the difference was statistically significant(p < 0.05);IMD group: M0,MI group,M2,M3,no difference with eachother;Compared with normal control group,hypoxia group and IMD group the VE-cadherin were lower,the difference was statistically significant(p < 0.05),but the IMD group VE-cadherin expression increased than the hypoxia group,between IMD group VE-cadherin show no difference;Compared with control group,hypoxia group and IMD group p-VEGFR2 were increased,the difference was statistically significant(P < 0.05),compared with the hypoxia group,M2 group P-VEGFR2 express indifference,M0,MI,M3 group P-VEGFR2 expression reduced(P < 0.05),compared with group M0 MI,M3 group P-VEGFR2 express indifference,M2 group expression increased,and the difference was statistically significant(P < 0.05),compared with group of M1,M2 group P-VEGFR2 expression increased(P < 0.05),the M3 group P-VEGFR2 show no difference;Compared with group M2,M3 group p-VEGFR2 expression is reduced,the difference was statistically significant(p < 0.05);Results:(1)The rRGECs survival rate : compared with normal control group,hypoxia group and the IMD group absorbance value decline;But each absorbance value in the IMD group were increased than hypoxia group;Compared with M0 group,MI group,the absorbance value of M2 group were lower,M3 group no significant change;Compared with M1 group,M2 group absorbance value no difference,M3 group absorbance value were increased;Compared with the M2 group,M3 group absorbance value increases.(2)Detecyion of tubular structure : control group,hypoxia group,the IMD group tube cavity length of sample structure,cyclization,number,branch number in accordance with the time gradient for 12 hours,24 hours and 48 hours gradually decline,the difference was statistically significant(p < 0.05).With extended time,the connection between the endothelial cells gradually relaxed,lumen sample structure gradually degraded,48 hours lumen sample structure basic collapse,three sets of each are no difference.24 hours,compared with control group,hypoxia group and IMD group they were all decreased,the difference was statistically significant(p < 0.05);Compared with the hypoxia group,IMD group sample tube cavity structure branch number,number ofcavity length,cyclization,increasing,the difference was statistically significant(p <0.05),the change of the three indicators IMD group is lighter than the group of hypoxia,the difference was statistically significant(p < 0.05);48 hours has no difference between the three groups of experiment results;Compared with M0 group,MI group number of cavity ring down,M2,and M3 group sample tube cavity structure length,branch number decreases,the difference was statistically significant(p < 0.05);Compared with M1 group,M2,and M3 group increased number of lumen cyclization,lumen sample structure length and branch number decreases,the difference was statistically significant(p <0.05);Compared with the M2 group,M3 group sample tube cavity structure length,branch number were increased,the difference was statistically significant(p < 0.05).(3)IMD induced angiogenesis signal pathway changes : compared with control group,hypoxia group and IMD groupthe expression of VEGF were increased,the difference was statistically significant(p < 0.05);IMD group: M0,MI group,M2,M3,no difference with each other;Compared with control group,hypoxia group and IMD group the VE-cadherin were lower,the difference was statistically significant(p < 0.05),but the IMD group VE-cadherin expression increased than the hypoxia group,between IMD group VE-cadherin show no difference;Compared with control group,hypoxia group and IMD group p-VEGFR2 were increased,(P < 0.05),compared with the hypoxia group,M2 group p-VEGFR2 express indifference,M0,MI and M3 group p-VEGFR2 expression reduced(P < 0.05),compared with group M0,MI and M3 group p-VEGFR2 express indifference,M2 group expression increased,the difference was statistically significant(P < 0.05),compared with the group of M1,M2 group p-VEGFR2 expression increased(P < 0.05),the M3 group p-VEGFR2 show no difference;Compared with group M2,M3 group p-VEGFR2 expression is reduced,the difference was statistically significant(P < 0.05);Compared with normal control group,hypoxia group and the IMD group p-ERK1/2 expression is reduced,the difference was statistically significant(P <0.05),Compared with the hypoxia group,M0,M2,M3,p-ERK1/2 expression increased,and no difference between each other,and reduced M1 group expression,and Compared with M0 group,M1 group p-ERK1/2 expression is reduced,the difference wasstatistically significant(P < 0.05),p-ERK1 /2 in M2,M3,group express indifference,compared with M1 group,M2 AND M3 group p-ERK1/2 expression increased,the difference was statistically significant(P<0.05),compared with M2 group,,M3 group p-ERK1/2 show no difference;Compared with control group,Compared with normal control group,hypoxia group,the IMD group p-PI3 K expression decreased,compared with the hypoxia group,M0,M1,M3 group p-PI3 K increased expression,M2 group expression is reduced,and M0,M1,M3 compared group p-PI3 K express indifference,p-PI3 K expression decrease the M2 group,p < 0.05;Compared with the M1 group,the expression of p-PI3 K in the M2 group was decreased,p<0.05.There was no difference in the expression of M3 group.Compared with M2 group,the expression of p-PI3 K in M3 group increased,p<0.05.Compared with normal control group,hypoxia group,p-eNOS IMD group were decreased,compared with the hypoxia group,p < 0.05,p-M0,M1,M2 group increased expression of eNOS,M3 group expression decreased,p < 0.05,and M0,M1 compared group p-eNOS express indifference,M2,and M3 group p-eNOS expression decreased,p < 0.05,compared with M1 group,the group M2,M3,p-eNOS expression decreased,compared with group M2M3 group p-eNOS expression decreased,p < 0.05).Conclusion:Hypoxia can reduce rRGECs survival rate,and IMD under anoxic conditions can be applied to ERK,PI3K/AKT pathway to increase rRGECs survival rate,alleviate hypoxia injury,while ERK1/2,eNOS pathways are not involved in this process;Hypoxia can stimulate rRGECs to ncrease the expression of VEGF,the phosphorylation of VEGFR2 expression increased,IMD had no effect on the expression of VEGF.IMD can increase oxygen rRGECs VE-cadherin expression,at the same time reduce VEGFR2 phosphorylation expression,while blocking PI3 K pathway VEGFR2 phosphorylation expression increased,mechanism may be IMD by acting on VE-cadherin/VEGFR2 /PI3 K compounds,abate the expression of the phosphorylation of VEGFR2,blunting rRGECs to VEGF stimulation response,thus inhibiting excessive budding;IMD ininduced hypoxia injury rRGECs cavity expansion need to activate ERK1/2,but not limit vascular germination.IMD promote anoxic injury rRGECs length and branching vessels may depend on the action of PI3K/AKT/eNOS signal cascade,but eNOS is not the only pathways downstream of PI3K/AKT pathway to promote the growth of blood vessels germination;IMD is an endogenous angiogenic regulator that is critical for vascular normalization through the regulation of VE-cadherin and the downstream PI3K/AKT/eNOS and ERK1/2 pathways,eNOS is the downstream signaling pathway of PI3K/AKT,but it is not the only one.