Introduction: Cold stress increases adrenergic stimulation of white adipose tissue (WAT) by upregulating uncoupling protein 1 (UCP1) to transform WAT into a brown adipose tissue -like phenotype, or “beige", “brite” adipocyte to increase heat production. WAT is highly vascularized. VEGFs and PDGFs mediated angiogenesis has been implicated in WAT beiging. However, the regulatory role of vascular endothelium in energy homeostasis is still less understood.
Hypothesis: We propose to examine whether Smad4 mediated angiocrine function is involved in WAT beiging.
Methods: To specifically knockdown Smad4 in endothelial cells, we used Smad4floxP/floxP as wild type (Smad4EC-WT) and Smad4floxP/floxP;Tie2CreERT2/+ as inducible endothelial-specific Smad4 knockout mice (Smad4EC-KO). Tamoxifen was injected to induce Cre expression in adult mice. Mice at 8-10 weeks old were housed at 8°C for 4 days or injected with β3 adrenoceptor agonist CL316,243 (CL) at 1 mg/kg subcutaneously for 10 day to induce beiging. RNA sequencing was performed in human endothelial cells (HUVECs) infected with lentiviral vector carrying scramble or SMAD4-shRNA.
Results: RNA-sequencing in HUVECs showed that SMAD4 knockdown downregulated genes involved in angiogenesis: VEGFA, CXCR4, etc; adipogenesis: PDGFA and PDGFB; and macrophage recruitment and infiltration: CCL2 and CX3CL1, etc; indicating the regulatory role of Smad4 in angiocrine function of endothelial cells. Smad4EC-KO mice had attenuated UCP1 upregulation induced by cold or CL injection. CD31+CD144+ endothelial cells reduced about 73% in the stromal vascular fraction of subcutaneous WAT after CL injection. Immunostaining also showed less CD31+ cells in sWAT. Adipogenic precursor cells (CD45-CD31-Sca-1+PDGFRa+) reduced about 58% in sWAT from Smad4EC-KO mice. We also detected reduced Ki67 expression in endothelial cells and adipogenic precursors indicating impaired proliferation.
Conclusions: Further experiments using VEGFA, PDGFA/B 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.