AtRA Promotes Huvecs Tube Formation By Up-regulating The Expression Of VEGF In 3T3-L1 Preadipocytes And Its Molecular Mechanism

Backgrounds and aims: During the progression of obesity and its related metabolic diseases,adipocytes hypertrophy and oxygen diffusion disorder are the leading cause of adipose tissue hypoxia and adipocytes necrosis.Studies found that promoting angiogenesis in adipose tissue could reduce hypoxia and improve obesity and its related glucose and lipid metabolism disorder.The present study was designed to explore the effects of atRA on VEGF expression in adipocytes as well as the effects of conditioned media from atRA treated adipocytes on HUVECs tube formation,and to reveal its underlying mechanism.Methods:(1)3T3-L1 preadipocytes were employed to study the effect of atRA on the differentiation of 3T3-L1 preadipocytes by oil red O staining and intracellular fatty acid levels;(2)Matrigel tube formation assay and Transwell migration assay were used to study the migration and tube formation of Human Umbilical Vein Endothelial Cells(HUVECs)induced by the conditioned medium of Adipose cells;(3)Western blotting and Real-time polymerase chain reaction were used to detect the VEGF expression of 3T3-L1 preadipocytes,and to confirm for the best atRA concentration;(4)WB and qRT-PCR were used to detect the activation of PI3K/AKT/mTOR pathway in 3T3-L1 preadipocytes treated with 1?M atRA;(5)The 3T3-L1 preadipocytes were treated with both atRA and PI3 K inhibitor LY294002,and the expression of VEGF and PI3K/AKT/mTOR were detected with WB and qRT-PCR;the HUVECs were treated with conditioned medium of adipose cells,and the migration and tube formation of HUVECs were detected by Transwell migration assay and matrigel tube formation assay.Results:(1)The 3T3-L1 preadipocytes were cultured with atRA in different concentration(0nM?10nM?100nM?1?M?10?M)and induced for differentiation.On day 8th of differentiation,the lipid accumulation in adipocytes was decreased with high doses of atRA(1 and 10 ?M),but had no change with low doses of atRA(10 and 100 nM).And the lowest lipid level was 10?M,which both had a lowest degree of maturity;(2)The HUVECs treated with conditioned medium of adipose cells(AC-CM)showed that 1?M-CM group had the most migration and tube formation ability;(3)The result of WB and qRT-PCR showed that the expression of VEGF in adipocytes was increased with the increment of atRA concentration,and the highest expression of VEGF in adipocytes were 1?M group;(4)The PI3K/AKT/mTOR pathway were detected with WB and qRT-PCR in adipocytes treated with 1?M atRA,on day 8th of differentiation,PI3K/AKT/mTOR pathway was significantly activated;the protein expression of VEGF,PI3 K,p-AKT,p-mTOR were significantly increased in adipocytes with the induction of 1?M atRA,but highly decreased when treated with the PI3 K inhibitor LY294002,and the ability of migration and tube formation in HUVECs were significantly diminished.Conclusion: atRA induces vascular endothelial growth factor expression in 3T3-L1 preadipocytes through PI3K/AKT/mTOR pathway,which promote the tube formation of HUVECs.