Barghouth, Mohammad

Introduction: Co-stimulatory proteins are master regulators of the immune system. We identified the co-stimulatory CD40-CD40L dyad as a key driver of inflammation during atherosclerosis and have shown that CD40 and CD40L exert cell-type divergent functions. Hypothesis: CD40 is abundantly expressed on endothelial cells (EC) undergoing endothelial mesenchymal transition (EndMT). We hypothesize that EC CD40 drives atherosclerosis via EndMT. Methods: We analyzed scRNAseq data from human carotid plaques, performed multiplex IHC, and used human aortic endothelial cells (HAECs) for in vitro studies. A tamoxifen-inducible EC specific CD40 deficient atherosclerotic mouse model (cadherin5 (cdh5)ERT2-CD40 flfl -ApoE -/- ) was generated. Results: Besides its presence on immune cells, CD40 is expressed on endothelial cells in both human and mouse atherosclerotic plaques. Analysis of scRNAseq data of human carotid atherosclerotic plaques showed that CD40 is highly expressed in COL1A2/FN1 expressing endothelial cells (ECs), a subset undergoing EndMT. Multiplex staining revealed that CD40 colocalized with the EndMT markers αSMA, NOTCH3, TWIST and VCAM1 in human plaques. In vitro , stimulation of human arterial endothelial cells (HAECs) with CD40L resulted in an increase in the EndMT markers markers fibronectin, vimentin, sm22α, FSP1 and snail. Using phosphoproteomics, CD40L was found to decrease phosphorylation of the junctional integrity proteins CTNNA1 [S652; S655] and ARHGAP21 [S1383]. To investigate the endothelial-specific effects of CD40 in atherosclerosis, 8-week-old cdh5ERT2-CD40 flfl -ApoE -/- mice and their CD40 flfl -ApoE -/- littermate controls were treated with tamoxifen and fed a high cholesterol diet for 10 wks. Deletion of EC-CD40 resulted in a significant reduction in plaque size. This reduction was accompanied by a shift from fibrous cap atheromas (FCA) towards intimal xanthoma (IX) and pathological intimal thickening (PIT). Flow cytometry analysis of the spleen showed that CD40 deletion in ECs results in a significant decrease in CD8 and CD4 effector T cells. This decrease in CD8 and CD4 effector T cells was accompanied by a diminished activation status, shown by a decrease in CD69 + CD8 + CD62 - CD44 + T cells, CD40L + CD8 + CD62 - CD44 + T cells and CD40L + CD4 + CD62 - CD44 + T cells. These findings demonstrate that endothelial CD40 drives atherosclerosis by inducing EndMT-driven plaque inflammation.

Introduction: Atherosclerosis, the underlying cause of cardiovascular disease (CVD), is a lipid-driven chronic inflammatory disease. We have identified the co-stimulatory CD40-CD40L immune checkpoint dyad as a key driver of inflammation during atherosclerosis. CD40 and CD40L exert cell-type divergent functions via different signaling pathways. Hypothesis: We hypothesize that CD40 on endothelial cell (ECs) induces endothelial-to-mesenchymal transition (EndMT)-mediated vascular inflammation in atherosclerosis. Methods & Results: Using single cell RNAseq, we found that CD40 (TNFRSF5) is expressed on PDE3A + GJA5 + and KDR + COL15A1 + ECs of advanced human carotid atherosclerotic plaques. CD40 is highly expressed in the endothelial lining of human and mouse atherosclerotic plaques, and colocalizes with the EndMT markers fibronectin and collagen and adhesion molecule ICAM-1. CD40L activation of human aortic endothelial cells (HAECs) results in expression of the EndMT markers fibronectin, vimentin, sm22α, FSP1 and snail. Activation of CD40 in EC induces filamin A dependent translocation of CD40 into lipid rafts, increases TNF-receptor associated factor (TRAFs) -2, -3 and 6 levels, induces Akt signaling and increases expression of VCAM1 and CCL2. Conclusions: The endothelial cell has been more and more regarded as a cell type that plays a major role in driving inflammation. Our data suggest that the CD40-CD40L axis plays a significant role in EC-mediated inflammation in atherosclerosis by driving EndMT.