ENHO Gene Regulate Angiogenesis After Hindlimb Ischemia And Its Possible Mechanisms

Objective:More than 200 million patients suffer from peripheral arterial disease over the world.With the development of aging society,things are getting worse.However,the mechanism of peripheral arterial disease has not been fully elucidated and the treatment of the disease is relatively limited.Endothelial function and angiogenesis are crucial to the recovery of peripheral arterial disease.Endothelial cells play an important role in protecting vessels and regulating angiogenesis.Energy homeostasis（ENHO）associated genes can regulate energy metabolism and protect endothelial cells.This research intends to explore the regulation of ENHO gene in endothelial cells on angiogenesis and its molecular mechanism after hindlimb ischemia（HLI）.Methods:About 8 weeks ENHO gene endothelial-specific knockout mice(ENHO^ΔEC)and control mice(ENHO^WT)were used in this research.After injection of tamoxifen for 1 week,a hindlimb ischemia model was established.The result of loss of ENHO in endothelial cells on angiogenesis after ischemia was collected to analyze the function of ENHO.Laser Doppler was used to assess the recovery of blood supply in lower limb of mice 0 days、3 days、7 days、14 days and 28 days after the ischemia.The gastrocnemius muscle was harvested 3days、14days and 28days after the ischemia,and the capillary density was detected by immunofluorescence.Lentivirus was used to knock out the ENHO gene in human umbilical vein endothelial cells（HUVEC）which were cultured in a hypoxic environment with 1%O₂in order to establish an in vitro hypoxia model.Through tube formation experiments,Transwell migration experiments,wound healing experiments,and Ed U experiments,we observed the effects of ENHO gene on proliferation,migration,and tubular formation of HUVEC.si RNA and plasmid were used to knout down or overexpress GPR19 in ENHO gene knout-out cells.Transwell migration experiment,wound healing experiment and Ed U experiment were performed to make it clear that whether GPR19 is the straight downstream of ENHO.Results:The study showed that the speed of blood supply recovery in ENHO^ΔECmice was lower than that of control mice after the establishment of hindlimb ischemia model and the difference was statistically significant（P<0.001）.The CD31 immunofluorescence staining also verified that knock-out of ENHO in endothelial cells made the new vessel density in ENHO^ΔECsignificantly lower compared with control mice.These results all indicate that the ENHO gene in endothelial cells is essential for angiogenesis and blood supply restoration.In vitro experiments,the expression of CD31 in endothelial cells under hypoxia increased,and the loss of ENHO significantly inhibited the increase of CD31.The results of VEGF and P-AMPK are similar to CD31.And the results of HUVEC tube formation experiments,transwell migration experiments,wound healing experiments,and Ed U proliferation experiments showed that the loss of ENHO or GPR19 significantly inhibited proliferation,migration,and vascularization.What’s more,the inhibition of GPR19 can further aggravate the functional inhibition in HUVEC with ENHO gene knockout.Conversely,overexpression of GPR19 can reduce the impact of ENHO knockout.Conclusion:The deletion of ENHO gene in endothelial cells inhibits the recovery of blood supply after ischemia.The deletion of ENHO gene in HUVEC inhibits cell proliferation,migration,and angiogenesis.And GPR19 seem to be the straight downstream of ENHO.These results indicate that the ENHO gene plays an important role in the process of angiogenesis through activation of GPR19.This research provides a new theoretical basis for in-depth understanding of the process and mechanism of angiogenesis,and provides new directions and theoretical support for the treatment of peripheral vascular diseases.