Effect And Mechanism Of Kctd10 On Angiogenesis In Mice

Kctd10 gene knockout mice model was established in our laboratory in 2008.The results published in 2014 found that Kctd10 gene knockout mice(Kctd10^-/-)showed severe cardiac AVC valve defects and angiogenesis inhibition,and embryo died on the 10.5 day(E10.5 d).Vascular endothelial cells are effector cells that play a major role in angiogenesis,and Kctd10 knockout homozygous mutant mice die early in embryo,which is not beneficial to study the role and molecular mechanism of Kctd10in mouse angiogenesis.Therefore,in this study,we first crossed Kctd10^Loxp/Loxpmouse knockout line with Tek-Cre transgenic mice specifically expressed in endothelial cells,and screened Kctd10endothelial cells specific knockout heterozygous mice(Kctd10^flox/-;Tek-Cre)by genotype identification.Embryos of E7.5,E8.5 and E11.5 were dissociated,and we found that Kctd10vascular endothelial cells specific knockout mice(Kctd10^flox/flox;Tek-Cre)died in the embryos of 8.5 days(E8.5);Effect of Kctd10 on retinal blood vessels of mice on the P5 was detected by tissue immunofluorescence,and the results showed that Kctd10 inhibited the branches and sprouts of blood vessels.Secondly,to further confirm the effect of Kctd10 on angiogenesis,we consider another possibility that Kctd10 can affect angiogenesis not only through vascular tissues but also other nonvascular tissues.Therefore,we then crossed Kctd10^Loxp/Loxpmouse knockout lines with Six3-Cre transgenic mice specifically expressed in retinal neurons and glial cells,and screenedKctd10retinaspecialknockoutmice(Kctd10^flox/flox;Six3-Cre)by genotype identification,then extracted the retina of Kctd10 retina specific knockout mice and expression of KCTD10 gene was analyzed by q RT-PCR and Western blot to confirm the preparation of Kctd10 retina specific knockout mice;The influence of Kctd10 on the retinal vessels of mice on the fifth day after birth was detected by tissue immunofluorescence,and it was discovered that Kctd10 can suppress the length,branches,sprouts and fliopodia of blood vessels.Finally,we extracted RNA and protein from P5 day retina of knockout mice and detected the expression of 18 genes related to angiogenesis by q RT-PCR.We found that VEGFR1,FGFR3,Bmp2 and Robo4 were significantly up regulated compared with control group.We selected Robo4 for subsequent research,and further confirmed that the expression of Robo4 was up regulated and the expression of p-Akt was down regulated compared with control group by Western blot.At the same time,KCTD10 cell line was constructed in human umbilical vein endothelial cells to detect the expression of Robo4,p-AKT.The results showed that overexpression of KCTD10 can inhibit the expression of Robo4and promote the expression of p-AKT.In conclusion,our findings revealed that vascular endothelial cells and retina specific Kctd10 knockout mice can inhibit angiogenesis and vascular endothelial cells specific Kctd10 knockout mice can lead to the death of the early embryo.The knockout of KCTD10 can promote the expression of Robo4,thus suppress the expression of p-AKT,and inhibit angiogenesis.The study provides a novel insight for promising therapeutic approaches of vascular disease.