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

内质网中的氧化还原信号调节内皮表面 N-糖型：对血管炎症的影响

• 批准号: 10386275
• 负责人: Alexandria Hernandez-Nichols
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10386275
• 关键词: Address; Adhesions; Affect; Alpha-mannosidase; Anabolism; Arterial Fatty Streak; Atherosclerosis; Binding; Biological Assay; C57BL/6 Mouse; CRISPR/Cas technology; Cardiovascular Diseases; Carotid Arteries; Cell Adhesion Molecules; Cell Communication; Cell Culture Techniques; Cells; Chronic; Complex; Data; Development; Disease; Endoplasmic Reticulum; Endothelial Cells; Endothelium; Enzymes; Epitopes; Excision; FCGR3B gene; Fatty acid glycerol esters; Foam Cells; Gel; Golgi Apparatus; Human; Hybrids; Hydrogen Peroxide; Immune; In Vitro; Inflammation; Inflammatory; Intercellular adhesion molecule 1; Knock-out; Lead; Lectin; Left; Ligation; Link; Lipids; Mannose; Mannosidase; Mass Spectrum Analysis; Measures; Mediating; Modeling; Morbidity - disease rate; Mus; Oxidation-Reduction; Pluronics; Polysaccharides; Post-Translational Protein Processing; Process; Protein Isoforms; Proteomics; Publishing; Reporting; Role; Severities; Signal Transduction; Stimulus; Structure; Surface; TNF gene; Testing; Topical application; Weight; atherogenesis; drinking water; endothelial dysfunction; glycoproteomics; glycosylation; hypercholesterolemia; in vivo; inhibitor; insight; macrophage; monocyte; mortality; mouse model; new therapeutic target; novel; protein transport; receptor; sugar; vascular inflammation

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.

翻译后摘要：动脉粥样硬化是心血管疾病的根本原因，其特征在于增厚的 血管壁由于慢性炎症和脂肪填充的泡沫细胞的形成。单核细胞-内皮细胞间 相互作用是这一过程的关键，并通过在细胞表面表达的粘附分子之间的结合介导。 内皮细胞表面及其在单核细胞上表达的同源受体。细胞间粘附 细胞间粘附分子1（ICAM-1）是一种表达上调的关键表面内皮细胞粘附分子 促炎性刺激。ICAM-1通过N-糖基化后修饰。后者可以 被分类为高甘露糖、杂合或复合N-聚糖，其中典型的观点是， 蛋白质运输和表面表达需要复杂的N-糖基化。然而，我们已经证明， 在人和小鼠体内动脉粥样硬化中，以及在用炎症刺激物处理的内皮细胞中， ICAM-1以至少2种N-糖型、高甘露糖（HM）和复合物表达。我们还表明，HM-ICAM- 1选择性地介导促炎（CD 16+）单核细胞的粘附，但不介导CD 16-单核细胞的粘附。CD16+ 单核细胞与疾病正相关，提示HM-ICAM-1在介导 动脉粥样硬化本研究的重点是确定HM-ICAM-1在体内的作用及其机制 调节HM-ICAM-1的形成。我们目前的初步数据显示：（i）HM-ICAM-1存在于 人和小鼠动脉粥样硬化血管;（ii）I类β-甘露糖苷酶，催化转化的ER酶 HM和杂合N-聚糖到复合N-聚糖的形成在活化的内皮细胞中受到抑制; ER中的H2 O2介导α-甘露糖苷酶的抑制和HM-ICAM-1的形成。这些数据导致了 假设I类α-甘露糖苷酶在炎症过程中被ER H2 O2抑制，导致HM- 选择性介导促炎单核细胞粘附导致动脉粥样硬化的ICAM-1。我 提出两个目标。在目的1中，我们将检测HM-ICAM-1在动脉粥样硬化病变发展中的作用， vivo.在目的2中，我们将鉴定负责形成HM-1的I类α-甘露糖苷酶同种型。 ICAM-1，并确定ER H2 O2抑制这种亚型的机制。方法将利用部分 HM-ICAM-1和α-甘露糖苷酶活性在动脉粥样硬化中的作用 体内发育此外，通过邻位连接测定和质谱法分析表面N-聚糖， 并确定单核细胞和α-甘露糖苷酶KO内皮细胞之间相互作用的评估。 我们预计这些研究的完成将为氧化还原信号转导模式以及N- 内皮表面粘附分子的糖型介导炎症。