Redox Signaling in the Endoplasmic Reticulum Regulates Endothelial Surface N-glycoforms: implications for vascular inflammation

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Abstract: Atherosclerosis is the underlying cause of cardiovascular disease characterized by thickening of the vessel wall due to chronic inflammation and formation of fat filled foam cells. Monocyte-endothelial intercellular interactions are key in this process and are mediated by binding between adhesion molecules expressed on the surface of the endothelial cells and their cognate receptors expressed on the monocytes. Intercellular adhesion molecule 1 (ICAM-1) is one of the key surface endothelial adhesion molecules whose expression is up-regulated with pro-inflammatory stimuli. ICAM-1 is post-translationally modified by N-glycosylation. The latter can be categorized as being high mannose, hybrid or complex N-glycans with the canonical perspective being that complex N-glycosylation is required for protein trafficking and surface expression. However, we have shown that both in human and mouse atherosclerosis in vivo, and in endothelial cells treated with inflammatory stimuli, ICAM-1 is expressed in at least 2 N-glycoforms, high mannose (HM) and complex. We also show that HM-ICAM- 1 selectively mediates adhesion of pro-inflammatory (CD16+) monocytes, but not CD16- monocytes. CD16+ monocytes positively associate with disease suggesting an important role for HM-ICAM-1 in mediating atherogenesis. The focus of this proposal is to determine the role of HM-ICAM-1 in vivo and the mechanism regulating formation of HM-ICAM-1. We present preliminary data showing that: (i) HM-ICAM-1 is present on human and mouse atherosclerotic vessels; (ii) class I -mannosidases, ER enzymes that catalyze conversion of HM and hybrid N-glycans to complex N-glycans, are inhibited in activated endothelial cells; (iii) formation of H2O2 in the ER mediates inhibition of α-mannosidases and formation of HM-ICAM-1. These data have led to the hypothesis that class I α-mannosidases are inhibited during inflammation by ER H2O2 resulting in a HM- ICAM-1 that selectively mediates pro-inflammatory monocyte adhesion leading to atherosclerosis. I propose two aims. In Aim 1, we will test the role of HM-ICAM-1 in the development of atherosclerotic lesions in vivo. In Aim 2, we will identify the Class I α-mannosidase isoform that is responsible for the formation of a HM- ICAM-1 and determine the mechanism by which ER H2O2 inhibits this isoform. Approaches will utilize a partial carotid ligation mouse model to assess the role of HM-ICAM-1 and α-mannosidase activity in atherosclerosis development in vivo. Also, analyses of surface N-glycans by proximity ligation assay and mass spectrometry, and assessment of interactions between monocytes and α-mannosidase KO endothelial cells will be determined. We anticipate completion of these studies will provide new insights into redox signaling paradigms and how N- glycoforms of endothelial surface adhesion molecules mediate inflammation.