Effect Of Canagliflozin On Retinal Angiogenesis And Its Mechanism

Objective:The formation of blood vessels is a complex process subject to multiple regulation.Endothelial cells are highly plastic cells arranged in the lumen of blood vessels,which can obtain oxygen in the blood and rapidly change from a static state to a highly migratory and proliferative state under the stimulation of anoxia or growth factors.Retinal angiogenesis is involved in a variety of pathophysiological processes,the most important of which is diabetic retinopathy.As a new type of hypoglycemic drug,Canagliflozin inhibits sodium-glucose cotransporters 1 and 2 at the same time,which on the one hand reduces the absorption of glucose through the intestine,on the other hand reduces the reabsorption of glucose by the kidney,and increases the excretion of urine sugar,thus achieving the dual hypoglycemic effect.The main purpose of this study was to investigate the effects of Canagliflozin on retinal angiogenesis and its possible mechanism of action,so as to provide new ideas for related clinical diagnosis and treatment.Method:1.To clarify the influence of CANA on angiogenesisTwo representative experimental methods to measure angiogenesis were selected,namely tube formation experiment of endothelial cells and aortic ring experiment.Human umbilical vein endothelial cells were selected to establish the cell model of angiogenesis.Vascular endothelial growth factor VEGF was used as the inducer to observe the tube forming ability and complete tube forming number of endothelial cells after CANA treatment.The aorta from the same healthy adult C57 mouse was used to observe the budding ability of the aorta ring after VEGF and CANA treatment.2.To explore the related mechanism of CANA affecting angiogenesisHuman umbilical vein endothelial cells were selected to establish the cell model of angiogenesis.Endothelial cells were treated with VEGF at different time points.Western blot was used to detect the expression of H3K27 ac,and the optimal time point for VEGF to induce H3K27 ac was determined.The endothelial cells were treated with different concentrations of VEGF.Western blot was used to detect the expression of H3K27 ac,and the optimal concentration of VEGF induced H3K27 ac was determined.The optimal concentration of VEGF and CANA were used to treat cells.The expression of H3K27 ac was detected by western blot to determine the influence of CANA on histone H3 acetylation and the optimal concentration.The optimal concentration of VEGF and CANA was used to detect the expression of H3K27 ac and Ang2 by western blot.3.To explore the molecular mechanism of CANA regulating angiogenesisEndothelial cells were treated with VEGF and CANA,cell RNA was extracted at appropriate time points,and a total RNA library was established after passing the detection,for sequencing,to observe the differential expression of VEGF and CANA-treated related genes,and to further carry out Gene Ontology functional enrichment analysis of the differential gene sets.The sequencing results were verified by q PCR.Result:1.To clarify the influence of CANA on angiogenesisVEGF enhanced the tube-forming ability of endothelial cells and the budding ability of aortic ring,while CANA inhibited the tube-forming ability of endothelial cells,resulting in discontinuity and decreased number of tubes,and inhibited the budding ability of aortic ring.2.To explore the related mechanism of CANA affecting angiogenesisVEGF significantly increased the expression of H3K27 ac in a time-dependent and concentration-dependent manner.CANA inhibited the expression of H3K27 ac induced by VEGF in a concentration-dependent manner.In addition,CANA significantly inhibited the expression of Ang2,a protein associated with angiogenesis.3.To explore the molecular mechanism of CANA regulating angiogenesisAfter VEGF induction,the expression of some genes was significantly up-regulated,while CANA significantly down-regulated the expression of some genes.GO functional enrichment analysis of the differential gene set showed that the expression of differential genes after VEGF induction and CANA treatment was most correlated with angiogenesis,and the differential genes were mostly related to cellular connectivity and other biological functions.q PCR verification showed that VEGF induced the expression of angiogeny-related genes ANGPT2,DLL4,and HEY1,while CANA inhibited the expression of angiogeny-related genes ANGPT2,DLL4,and HEY1.Conclusion:In this study,we found that CANA inhibited retinal angiogenesis and regulates retinal angiogenesis by inhibiting histone H3 acetylation and regulating retinal angiogenesis related transcription factors and genes.