Abstract
Activation of the endothelium by pro-inflammatory stimuli plays a key role in the pathogenesis of a multitude of vascular diseases. Angiogenesis is a crucial component of the vascular response associated with inflammatory signaling. The CD40/CD40 ligand dyad in endothelial cells (EC) has a central role in promoting vascular inflammatory response; however, the molecular mechanism underlying this component of inflammation and angiogenesis is not fully understood. Here we report a novel microRNA mediated suppression of endothelial CD40 expression. We found that CD40 is closely regulated by miR-424 and miR-503, which directly target its 3′ untranslated region. Pro-inflammatory stimuli led to increased endothelial CD40 expression, at least in part due to decreased miR-424 and miR-503 expression. In addition, miR-424 and miR-503 reduced LPS induced EC sprouting, migration and tube formation. Moreover, we found that miR-424 and miR-503 expression is directly regulated by peroxisome proliferator-activated receptor gamma (PPARγ), whose endothelial expression and activity are decreased in response to inflammatory factors. Finally, we demonstrate that mice with endothelial-specific deletion of miR-322 (miR-424 ortholog) and miR-503 have augmented angiogenic response to LPS in a Matrigel plug assay. Overall, these studies identify a PPARγ-dependent miR-424/503-CD40 signaling axis that is critical for regulation of inflammation mediated angiogenesis.
Highlights
Endothelial cells (ECs) have unique functions in vascular biology
We found that PPARγ is a direct transcriptional activator of miR-424 and miR-503, and its activation by pioglitazone leads to suppression of CD40 expression and LPS-induced angiogenesis in a miR-424 and miR-503 dependent manner
We found that subjecting human umbilical vein endothelial cells (HUVECs) to knockdown of argonaute 2 (AGO2), a key protein of the RNA-induced silencing complex (RISC) complex[28,29,30], or DICER, a key protein of miRNA processing, resulted in significant increase in CD40 protein levels (Fig. 1c), suggesting that baseline CD40 expression is miRNA dependent
Summary
Endothelial cells (ECs) have unique functions in vascular biology They can modulate immune response and angiogenesis, and have a key role in the maintenance of vascular homeostasis through protection of the vascular wall from pathological stimuli[1, 2]. Peroxisome proliferator-activated receptor gamma (PPARγ) receptors, transcription factors belonging to the nuclear hormone receptor superfamily, have been demonstrated to have widespread expression in the vasculature, including in ECs, vascular smooth muscle cells, and monocytes/macrophages[13,14,15]. We demonstrate enhanced angiogenic response to inflammatory stimuli in mice with endothelial specific deletion of miR-322 (miR-424 ortholog) and miR-503 These findings demonstrate a novel mechanism by which PPARγ signaling can augment the expression of homeostatic miRNAs, and likely serves as a key rheostat modulating endothelial inflammatory response
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