Role of increased circulating microparticles in adverse outcomes of COVID-19 patients with diabetes

循环微粒增加对患有糖尿病的 COVID-19 患者不良后果的影响

• 批准号: 10547868
• 负责人: PINGNIAN HE
• 金额: $ 32.47万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10547868
• 关键词: 2019-nCoV; ACE2; ANXA5 gene; Accounting; Adhesions; Adhesives; Angiotensin II; Angiotensin Receptor; Angiotensin-Converting Enzyme Inhibitors; Animal Model; Animals; Area; Binding; Binding Proteins; Binding Sites; Biological Assay; Blood Cells; Blood Circulation; Blood Plasma Volume; Blood Vessels; Blood specimen; Body Weight; Breathing; Bronchoalveolar Lavage; COVID-19; COVID-19 morbidity; COVID-19 pathogenesis; COVID-19 patient; Caco-2 Cells; Cardiovascular Diseases; Cell membrane; Cells; Characteristics; Child; Clinical; Coagulants; Communicable Diseases; Conflict (Psychology); Confocal Microscopy; Coronavirus; Data; Dental crowns; Development; Diabetes Mellitus; Disease; Dose; Down-Regulation; Drug Targeting; Economics; Electron Microscopy; Endocytosis; Endothelial Cells; Endothelium; Enzymes; Experimental Designs; Flow Cytometry; Fluorescence; Functional disorder; Future; Hamsters; Harvest; Histology; Hour; Human; In Vitro; Incubated; Individual; Infection; Infection Control; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Influenza; Influenza A Virus, H1N1 Subtype; Injections; K-18 conjugate; Laboratories; Leukocytes; Lipid Binding; Lung; Measures; Mediating; Mesocricetus auratus; Meta-Analysis; Methods; Microfluidic Microchips; Microfluidics; Microvascular Dysfunction; Modeling; Monitor; Mus; Oral; Organ; Organelles; Outcome; Pathogenesis; Pathology; Patients; Perfusion; Peripheral; Persons; Phenotype; Phosphatidylserines; Plaque Assay; Plasma; Pneumonia; Posture; Proteins; Publishing; RNA; Rattus; Renin-Angiotensin-Aldosterone System; Reporting; Resolution; Resources; Reverse Transcription; Rivers; Role; SARS-CoV-2 infection; SARS-CoV-2 pathogenesis; SARS-CoV-2 spike protein; Severities; Streptozocin; Study models; Surface; Swab; Symptoms; Techniques; Temperature; Testing; Therapeutic; Time; Tissues; Transfusion; Up-Regulation; Upper Respiratory Infections; Virulent; Virus; Virus Diseases; Virus Replication; Virus-like particle; Western Blotting; adverse outcome; airway inflammation; base; cell type; co-infection; comorbidity; confocal imaging; cytokine; diabetic; diabetic patient; drug development; exosome; experimental study; high risk; human coronavirus; improved; in vivo; influenza infection; mortality; mortality risk; new therapeutic target; non-diabetic; novel; organ injury; pandemic disease; prevent; receptor; rectal; respiratory; response; severe COVID-19; social; socioeconomics; targeted treatment; therapeutic development; tissue culture; vascular inflammation; vasoconstriction; vector; vesicular release; viral RNA

A. Provide supporting data showing that diabetic MPs contribute to the increases in the propagation of the virus compared to non-diabetic MP. A1. Demonstrate higher number of cellular internalizations of S-protein-bound DB MPs than that of S-protein-bound normal MPs. Experiments will be conducted in microvessels developed in microfluidics. Our preliminary data showed no significant differences in ACE2 expression and ACE2-mediated MP interaction with S-protein between normal and DB MPs. The main factors that contribute to DB MP-mediated increases in virus entry are their much higher quantity and more adhesive surface due to their largely externalized phosphatidylserine (PS) than that of normal MPs. GFPtagged S-protein will be incubated with MPs isolated from normal and DB plasma and flow cytometry will be used to confirm the S-protein-bound MPs. To demonstrate the quality differences between normal and DB MPs, equal amount of S-protein-bound DB and normal MPs will be perfused into the in vitro microvessels. Confocal images will be collected to compare the internalized S-protein-bound DB MPs with that of normal MPs by quantification of GFP fluorescence. To demonstrate the role of externalized PS on DB MPs in the MP adhesion and internalization, the S-protein-bound DB MPs will be pre-coated with a lipid binding protein, Annexin V, before perfusion into microvessels. We predict that precoating of DB MPs with Annexin V will prevent the DB MP adhesion to ECs and reduce their cellular internalizations. A2. Demonstrate DB MP-mediated increases in HCoV-NL63 infection and propagation when compared to normal MPs. HCoV-NL63 (BEI Resources) will be propagated in Caco-2 cells to produce a working virus stock. Cultured ECs will be incubated with HCoV-NL63 with serial dilutions (1:10-1000) in the presence of DB and normal plasma or equal number of isolated DB and normal MPs, respectively. The incubation of HCoV-NL63 alone serves as viral infection control. To demonstrate the specific role of DB MPs in facilitating viral infection from that of other components in DB plasma such as inflammatory mediators and cytokines, cultured ECs will be incubated with HCoV-NL63 in the presence of MP-free DB plasma and the results will be compared with DB plasma containing increased MPs. For rigor, efficiency of HCoVNL63 infection will be determined using three distinct methods. Total virus will be harvested from cells and supernatant at 96 hpi and quantified by both plaque assay and 50% tissue culture infectious dose (TCID 50) assay as previously published (PMID 19014487). Infection will also be quantified using a one-step, real-time, quantitative reverse transcription PCR assay on RNA harvested from cells at 96 hpi using a commercially available kit (Liferiver). Supernatant will be collected daily to examine the release of MPs and cytokines, serving as indicators of viral infection-induced EC activation. Results will be compared among groups (3-4 replicates per group for all proposed preliminary studies). B. Provide preliminary data to support that HCoV-NL63 virus could produce the illness that is similar to SARS-CoV-2. HCoV-NL63 has been reported to cause upper respiratory tract infections in children, and also to cause pneumonia, as well as airway inflammation in K18-ACE2 mice. In this preliminary study, we will develop a hamster model of HCoVNL63 infection. Golden Syrian hamsters (8-10 weeks old, Charles River) will be used for this study. Normal and STZinduced diabetic hamsters will be intranasally inoculated with serial dilutions of HCoV-NL63 (5 x 104, 5 x 105, or 5 x 106 TCID50) in 200 μL PBS to establish the ID50 in vivo. Oral and rectal swabs will be collected every 24 hours starting 1-day post-inoculation (dpi) for up to five days. Body weight and clinical signs of illness as indicated by labored breathing, hunched posture and immobility, and temperature will be monitored twice a day. Hamsters will be euthanized on 3 and 5 dpi to harvest lungs and peripheral tissues. Remaining animals are sacrificed at 7 dpi (n = 3 per group). Hamster lungs will be assessed for histology and viral replication (measure viral RNA levels by qPCR). Cytokines will be measured in bronchoalveolar lavage and lung homogenate. Plasma will be collected for MP and cytokine analysis (details see application experimental designs C4B2). Results will be compared among groups. We predict that HCoV-NL63-infected DB hamsters have more severe respiratory inflammatory responses than HCoV-NL63-infected normal hamsters. Our previous study showed that cross-transfusion of DB plasma to normal rats caused immediate vascular inflammation in the recipient rat, indicating a role of DB MPs in mediating propagation of inflammation in the vasculature. To evaluate the specific role of DB MPs in facilitation and propagation of HCoV-NL63 infection, 40% plasma volume of a HCoV-NL63- infected normal hamster will be replaced by MP-rich and MP-free DB plasma (normal plasma as sham control) or vice versa and the viral infection-induced illness and vascular pathology will be compared between groups. In case HCoVNL63 infection alone causes very mild clinical symptoms in hamsters, the alternative is to coinfect hamster with human influenza H1N1 and HCoV-NL63. Hamster is an established animal model for the study of human influenza infection and coinfection of SRAS-CoV-2 and influenza has been shown to enhance the severity of pneumonia in hamsters. C. Provide evidence for a novel therapeutic target to alleviate the adverse outcomes of COVID-19 patients with diabetes. The focus of this application is to reveal a novel mechanism by which diabetes exacerbates the COVID-19 outcomes. The proposed mechanistic studies will lead to the identification of a novel target for therapeutic development. Our previous study showed that carotid injection of Annexin V (1 ml, 100 μg/Kg) to a DB rat reduced plasma MPs from 465 x 104/μL to 22 x 104/μL 1h after the injection. We will apply the same approach to HCoV-NL63 infected DB hamsters, and the plasma MPs and viral infection outcomes will be evaluated accordingly. The drug development is out of scope of this application. However, these preliminary studies will provide mechanistic evidence supporting the development of a Annexin V-based therapeutic product in the future studies.

A. 提供支持数据，表明糖尿病议员有助于病毒传播的增加 与非糖尿病议员相比。 A1。与 S 蛋白结合的 DB MP 相比，S 蛋白结合的 DB MP 的细胞内化数量更高 普通议员。实验将在微流体开发的微血管中进行。我们的初步数据显示 正常人和正常人之间 ACE2 表达以及 ACE2 介导的 MP 与 S 蛋白的相互作用没有显着差异 DB 议员。导致 DB MP 介导的病毒进入增加的主要因素是它们的数量要高得多 由于其大部分外化磷脂酰丝氨酸（PS），因此比普通 MP 具有更多的粘合表面。 GFP标记 S-蛋白将与从正常和 DB 血浆分离的 MP 一起孵育，并使用流式细胞术 确认 S 蛋白结合 MP。为了证明正常和 DB MP 之间的质量差异，等量 S蛋白结合的DB和正常MP将被灌注到体外微血管中。将收集共焦图像 通过 GFP 荧光定量将内化的 S 蛋白结合 DB MP 与正常 MP 进行比较。到 证明外化 PS 对 DB MP 在 MP 粘附和内化中的作用，S 蛋白结合的 DB MP 在灌注到微血管之前，将预先涂上脂质结合蛋白膜联蛋白 V。我们预测预涂 DB MP 与Annexin V 的结合将防止DB MP 粘附到EC 并减少其细胞内化。 A2。与正常 MP 相比，证明 DB MP 介导的 HCoV-NL63 感染和传播增加。 HCoV-NL63（BEI 资源）将在 Caco-2 细胞中繁殖，以产生工作病毒库。培养的 EC 将 在 DB 和正常血浆或等量存在的情况下，与连续稀释 (1:10-1000) 的 HCoV-NL63 一起孵育 分别为孤立的 DB 和正常 MP。单独孵育 HCoV-NL63 可作为病毒感染控制。到 证明 DB MP 在促进 DB 血浆中其他成分（例如 作为炎症介质和细胞因子，培养的 EC 将在无 MP 的情况下与 HCoV-NL63 一起孵育 DB 血浆和结果将与含有增加的 MP 的 DB 血浆进行比较。为了严谨、高效，HCoVNL63 将使用三种不同的方法来确定感染。将从细胞和上清液中收获总病毒 在 96 hpi 时，通过噬菌斑测定和 50% 组织培养感染剂量 (TCID 50) 测定（如前所述）进行定量 已发布（PMID 19014487）。感染也将使用一步、实时、定量反向进行量化 使用市售试剂盒（Liferiver）对 96 hpi 时从细胞中收获的 RNA 进行转录 PCR 测定。上清液 每天收集以检查 MP 和细胞因子的释放，作为病毒感染诱导 EC 的指标 激活。将在组间比较结果（所有拟议的初步研究每组重复 3-4 次）。 B. 提供初步数据支持 HCoV-NL63 病毒可以产生与 SARS-CoV-2 类似的疾病。 据报道，HCoV-NL63 可引起儿童上呼吸道感染，也可引起肺炎，例如 以及 K18-ACE2 小鼠的气道炎症。在这项初步研究中，我们将开发 HCoVNL63 的仓鼠模型 感染。本研究将使用金色叙利亚仓鼠（8-10 周龄，Charles River）。正常和 STZ 诱导的 糖尿病仓鼠将鼻内接种连续稀释的 HCoV-NL63（5 x 104、5 x 105 或 5 x 106 TCID50）溶于 200 μL PBS 以确定体内 ID50。从 1 天开始，每 24 小时收集一次口腔和直肠拭子 接种后 (dpi) 最多五天。体重和呼吸困难所表明的临床疾病体征， 弯腰驼背、不动，每天监测两次体温。仓鼠将于3日被安乐死 和 5 dpi 收获肺部和周围组织。其余动物在 7 dpi 处死（每组 n = 3 只）。仓鼠 将评估肺部的组织学和病毒复制（通过 qPCR 测量病毒 RNA 水平）。将测量细胞因子 支气管肺泡灌洗液和肺匀浆中。将收集血浆用于 MP 和细胞因子分析（详情参见 应用实验设计C4B2)。结果将在各组之间进行比较。我们预测 HCoV-NL63 感染者 DB仓鼠比感染HCoV-NL63的正常仓鼠有更严重的呼吸道炎症反应。我们的 先前的研究表明，将 DB 血浆交叉输注给正常大鼠会立即引起血管炎症 受体大鼠，表明 DB MP 在介导脉管系统炎症传播中的作用。评估 DB MP 在 HCoV-NL63 感染的促进和传播中的具体作用，HCoV-NL63 的 40% 血浆体积 感染的正常仓鼠将被富含 MP 和不含 MP 的 DB 血浆替代（正常血浆作为假对照）或反之 反之亦然，并且将在组之间比较病毒感染引起的疾病和血管病理学。如果是 HCoVNL63 单独感染会导致仓鼠出现非常轻微的临床症状，另一种方法是将仓鼠与人类共同感染 H1N1 流感和 HCoV-NL63。仓鼠是一种已建立的动物模型，用于研究人类流感感染和 SRAS-CoV-2 和流感的共同感染已被证明会加重仓鼠肺炎的严重程度。 C. 为新的治疗靶点提供证据，以减轻患有糖尿病的 COVID-19 患者的不良后果。 该应用的重点是揭示糖尿病加剧 COVID-19 结果的新机制。 拟议的机制研究将确定治疗开发的新靶点。我们的 先前的研究表明，向 DB 大鼠颈动脉注射膜联蛋白 V（1 ml，100 μg/Kg）可将血浆 MP 降低 465 x 注射后 1 小时从 104/μL 变为 22 x 104/μL。我们将对 HCoV-NL63 感染的 DB 仓鼠应用相同的方法，并且 血浆 MP 和病毒感染结果将进行相应评估。药品研发不属于药品研发范围 这个应用程序。然而，这些初步研究将提供支持发展的机制证据 未来研究中基于膜联蛋白 V 的治疗产品。