Angiogenic Activity And Mechanism Research Of Novel Isosteviol Anolog KFH-08

ObjectiveOur group showed that KP-4 protects myocardium.My previous study also found that KP-4 protects myocardial ischemia and promotes angiogenesis.In order to improve the myocardial protective activity,our group synthesized a series of anologs with KP-4 as the lead compound.KFH-08 with higher safety and stronger efficiency was screened.It was found that KFH-08 protects myocardial ischemia via VEGF signaling pathway.Regarding the relationship between VEGF signaling pathway and angiogenesis,we synthesized that KFH-08 also improves angiogenesis.Therefore,in this study,we will explore the angiogenesis acticity and molecular mechanism of KFH-08,including: 1)To explore the protective effect of KP-4,the leading compound of KFH-08,on myocardial ischemia and angiogenesis.2)To explore the effect of KFH-08 on angiogenesis.3)To explore the molecular mechanism of KFH-08 promoting angiogenesis.4)Vegfb knockout zebrafish line was constructed to investigate the molecular mechanism of KFH-08.Research contents 1.Kp-4 protects myocardial ischemia and promotes angiogenesisThe chronic myocardial ischemia model in rats was established.After treatment with KP-4 for four weeks,the echocardiography of the heart and the hemodynamics were performed to evaluate the myocardial protective activity of KP-4.Meanwhile,the effect of KP-4 on capillary was evaluated by immunohistochemistry and μCT imaging.According to these data,KP-4 probably has angiogenesis activity.Thus,in order to confirm the angiogenesis activity of KP-4,cell proliferation and tube formation in HUVEC in vitro and VRI inhibition angiogenesis in Tg [flk: EGFP] zebrafish in vivo were performed.2.KFH-08 promotes angiogenesisIn order to evaluate the angiogenesis activity of KFH-08,primary cell culture in vitro and VRI inhibition angiogenesis in Tg [flk: EGFP] zebrafish in vivo were performed.Finally,Tg [flk: EGFP]/Tg [gatal: d Red] double transgenic zebrafish model was used to evaluate the function of new vascular.3.KFH-08 activates angiogenesis,MAPK,NOTCH related gene and mitochondrial geneThe VRI inhibition angiogenesis in Tg [flk: EGFP] zebrafish model was constructed.The blank groups,VRI groups and KFH-08 groups were set.To explore the potential molecular mechanism of KFH-08,measuring the expression of genes by RNA seq.Then VEGF related genes were measured by q PCR.4.Vegfb knockout zebrafish line was constructed by CRISPR/cas9.The CRISPR/cas9 was used to knockout vegfb in Tg [flk: EGFP] zebrafish.The knockout homozygous zebrafish vegfb-/- was screened by sequencing.The effect of knockout vegfb on cardiovascular development was evaluated.Results1.KP-4,the leading compound of KFH-08,significantly increases EF、FS、CI、Max dp/dt and LVSP,which demonstrates that KP-4 promotes systolic function in chronic myocardial ischemia rats.Interestingly,KP-4 increases the coronary capillary density and the entire coronary arterial in myocardial ischemia rats,which indicateds that KP-4 probably has angiogenesis activity.Furthermore,KP-4 improves the proliferation and tube formation of HUVECs in vitro and the sprouting of intersegmental blood vessels in zebrafish embryos in vivo,which demonstrates that KP-4 enhances angiogenesis.2.KFH-08 significantly promoted the differentiation and proliferation of vascular endothelial cells in vitro and significantly restored the intersegmental blood vessels and head vessels of zebrafish after the inhibition of VRI.The blood circulation of new formly vessels was performed normally.Thus,KFH-08 promotes angiogenesis.3.To explore the possible molecular mechanism of KFH-08,the expression of genes was measured by RNA seq.We found that KFH-08 activated angiogenesis,MAPK,NOTCH related gene and mitochondrial gene.We also found that KFH-08 significantly increased VEGFA,VEGFB and VEGFR1 by q PCR.4.Vegfb knockout zebrafish line was constructed successfully by CRISPR/cas9.We found that knockout vegfb enduced vascular damage and pericardial edema in zebrafish embryos,which demonstrated that cardiovascular development was influenced by vegfb.Conclusions1、KP-4,the leading compound of KFH-08,protects myocardial ischemia and promotes angiogenesis.2、KFH-08 enhances angiogenesis.3、The molecular mechanism of KFH-08 promoting angiogenesis maybe related to VEGFA,VEGFB and VEGFR1.4、Vegfb knockout zebrafish line was constructed successfully by CRISPR/cas9.And the knockout of vegfb induced vascular damage and pericardial edema in zebrafish embryos.