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.

摘要：动脉粥样硬化是心血管疾病的根本原因，其特征是动脉壁增厚。 血管壁因慢性炎症而形成脂肪充盈的泡沫细胞。单核细胞-内皮细胞间 相互作用在这一过程中是关键的，并由表达在 单核细胞表面表达内皮细胞及其同源受体。细胞间黏附 细胞间黏附分子-1(ICAM-1)是重要的内皮细胞表面黏附分子，其表达上调 使用促炎刺激。ICAM-1在翻译后通过N-糖基化修饰。后者可以是 被归类为高甘露糖、杂化或复杂的N-葡聚糖，典型的观点是 复杂的N-糖基化是蛋白质运输和表面表达所必需的。然而，我们已经表明， 无论是在人和小鼠体内的动脉粥样硬化，还是在用炎症刺激处理的内皮细胞中， ICAM-1以至少2种N-糖类、高甘露糖(HM)和复合体的形式表达。我们还证明了HM-ICAM- 1选择性地介导致炎(CD16+)单核细胞的黏附，而不是CD16-单核细胞的黏附。CD16+ 单核细胞与疾病呈正相关提示HM-ICAM-1在介导疾病中的重要作用 动脉粥样硬化的形成。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。我们提供的初步数据表明：(I)HM-ICAM-1存在于 人和小鼠动脉粥样硬化血管；(Ii)I类-甘露糖苷酶，催化转化的ER酶 在活化的内皮细胞中，HM和杂化N-聚糖到复合N-聚糖的形成受到抑制；(Iii)形成 内质网中的H_2O_2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-甘露糖苷酶在炎症期间被ER-H_2O_2抑制，导致HM- ICAM-1选择性地介导导致动脉粥样硬化的促炎性单核细胞黏附。我 提出两个目标。在目标1中，我们将测试HM-ICAM-1在动脉粥样硬化病变发展中的作用。 活着。在目标2中，我们将鉴定负责HM-形成的I类α-甘露糖苷酶亚型。 ICAM-1，并确定ER-H_2O_2抑制该异构体的机制。方法将利用部分 颈动脉结扎小鼠模型评价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，还对表面N-糖链进行了邻近连接分析和质谱分析， 并将确定单核细胞与α-甘露糖苷酶KO内皮细胞之间的相互作用。 我们预计，这些研究的完成将为氧化还原信号范式以及N-甲基-N-天冬氨酸的作用机制提供新的见解。 内皮细胞表面黏附分子的糖型介导炎症。