Smad4 regulates angiogenesis and induction of beige adipocytes in adipose tissue

[Speaker] Chenguang Wang:1
[Co-author] Hongsong Zhang:1, Mingyu Huo:1, Ronald C Ma:2, Yu Huang:1, Xiaoyu Tian:1
1:Biomedical Sciences, Chinese University of Hong Kong, Hong Kong, 2:Medicine and Therapeutics, Chinese University of Hong Kong, Hong Kong

Brown adipose tissue (BAT) contains higher uncoupling protein 1(UCP1) expression and mitochondrial content which increases heat production. Induction of BAT from white adipose tissue(WAT) is a new strategy of treatment obesity and insulin resistance. cold stress increases adrenergic stimulation of WAT by upregulating UCP1. Previous studies showed that angiogenesis regulated by VEGFs and PDGFs contribute to BAT formatioin. However, the regulatory role of vascular endothelium in energy homeostasis is still poorly understood.
To specifically knockdown Smad4 in endothelial cells, we used Smad4 floxPfloxP as wild type (Smad4ECWT) and Smad4 floxPfloxP Tie2CreERT2 as inducible endothelial specific Smad4 knockout mice (Smad4ECKO). Cre recombinase expression was induced by Intraperitoneal injection of tamoxifen in adult mice.8 to 10 week old mice were housed at 8C for 4 days or injected with beta3 adrenoceptoragonist CL316,243 (CL) at dosage of 1 mg per kg subcutaneously for 10 day to induce beiging. RNA sequencing, qPCR and tube formation were performed on human endothelial cells (HUVECs) infected with lentiviral vector carrying scramble or SMAD4 shRNA.
RNA sequencing in HUVECs showed that SMAD4 knockdown downregulated genes involved in angiogenesis, macrophage recruitment and infiltration and adipogenesis, which is further validation by quantitative real time PCR. SMAD4 knockdown HUVECs showed reduced capacity of tube formation. Upon cold exposure at 8C, Smad4ECKO mice showed attenuated UCP1 upregulation in subcutaneous WAT measured by Western blotting and immunohistochemistry. Flow cytometric analysis of sWAT stromal vascular fraction showed that CD31CD144 plus endothelial cells reduced about 73 percent in Smad4 ECKO mice comparing to Smad4 ECWT mice.In addition, adipogenic precursor cells (CD45CD31 minus Sca-1PDGFRa plus) reduced about 58 percent in sWAT from Smad4 ECKO mice after cold exposure. We also detected reduced Ki67 expression in endothelial cells and adipogenic precursors indicating impaired proliferation.
Further experiments using VEGFA, PDGFaa peptides are being carried out to confirm the role of these growth factors in mediating the regulatory function of Smad4 on angiogenesis and adipogenesis contributing to WAT beiging. Our results suggest a possible role of vascular endothelium contributing to energy homeostasis.
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