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: 10794921
• 负责人: Alexandria Hernandez-Nichols
• 金额: $ 4.12万
• 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/10794921
• 关键词: Address; Adhesions; Affect; Alpha-mannosidase; Anabolism; Aorta; Arterial Fatty Streak; Atherosclerosis; Binding; Biological Assay; C57BL/6 Mouse; CRISPR/Cas technology; Cardiovascular Diseases; Carotid Arteries; Categories; 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; Macrophage; 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; Testing; Topical application; Weight; Western Blotting; atherogenesis; drinking water; endothelial dysfunction; glycoproteomics; glycosylation; hypercholesterolemia; in vivo; inhibitor; insight; migration; 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。 血管壁由于慢性炎症而形成充满脂肪的泡沫细胞。单核细胞-内皮细胞间质 相互作用是这个过程的关键，并且是由表达于表面的粘附分子之间的结合介导的。 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 分子1（ICAM-1）是关键的表面内皮粘附分子之一，其表达上调 具有促炎刺激作用。 ICAM-1 通过 N-糖基化进行翻译后修饰。后者可以是 被归类为高甘露糖、杂合或复杂的 N-聚糖，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 在人和小鼠的体内动脉粥样硬化中，以及在用炎症刺激处理的内皮细胞中， ICAM-1 以至少 2 种 N-糖型、高甘露糖 (HM) 和复合物的形式表达。我们还表明 HM-ICAM- 1 选择性介导促炎 (CD16+) 单核细胞的粘附，但不介导 CD16- 单核细胞的粘附。 CD16+ 单核细胞与疾病呈正相关，表明 HM-ICAM-1 在介导中发挥重要作用 动脉粥样硬化。本提案的重点是确定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 α-甘露糖苷酶，催化转化的 ER 酶 HM 和杂合 N-聚糖形成复合 N-聚糖，在活化的内皮细胞中受到抑制； (三) 形成 ER 中的 H2O2 介导 α-甘露糖苷酶的抑制和 HM-ICAM-1 的形成。这些数据导致 假设 I 类 α-甘露糖苷酶在炎症过程中被 ER H2O2 抑制，导致 HM- ICAM-1 选择性介导促炎性单核细胞粘附，导致动脉粥样硬化。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 体内。在目标 2 中，我们将鉴定 I 类 α-甘露糖苷酶亚型，它负责形成 HM- ICAM-1 并确定 ER H2O2 抑制该亚型的机制。方法将利用部分 颈动脉结扎小鼠模型评估 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，通过邻近连接测定和质谱分析表面 N-聚糖， 并将确定单核细胞和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范式以及 N-如何 内皮表面粘附分子的糖型介导炎症。