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; 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.

答：提供支持数据，表明糖尿病MP有助于病毒的传播增加 与非糖尿病MP相比。 A1。与S蛋白结合的S蛋白结合DB MP的细胞内在化数量更高 正常MPS。实验将在微流体中开发的微血管中进行。我们显示的初步数据 ACE2表达和ACE2介导的MP相互作用与正常和S-蛋白的相互作用无显着差异 DB MPS。导致DB MP介导的病毒进入增加的主要因素是它们的数量更高 并且由于其大量外部化磷脂酰丝氨酸（PS）而比正常MPS更大，因此粘合剂表面更大。 gfptagged S蛋白将与从正常和DB血浆中分离的MP一起孵育，流式细胞仪将用于 确认与S蛋白结合的MPS。为了证明正常MP和DB MP之间的质量差异 S蛋白结合的DB和正常MP的体外微血管将被灌注。将收集共聚焦图像 将内在的S蛋白结合的DB MP与正常MP的GFP荧光进行比较。到 演示外部化PS在DB MPS中的作用在MP粘附和内在化中，S蛋白结合的DB MPS 在灌注成微血管之前，将与脂质结合蛋白A膜联蛋白V预先涂覆。我们预测这是预涂的 用膜联蛋白V的DB MPS的数量将防止DB MP对EC的粘附并减少其细胞内在化。 A2。与正常MP相比，HCOV-NL63感染和传播的DB MP介导的增加。 HCOV-NL63（BEI资源）将在CACO-2细胞中繁殖以产生工作病毒。培养的EC将是 与HCOV-NL63一起在存在DB和正常等离子体或相等数量的情况下与系列稀释液（1：10-1000）一起孵育 分别孤立的DB和正常MP的。单独的HCOV-NL63孵育可作为病毒感染对照。到 证明DB MP在支持DB血浆中其他成分的病毒感染中的特定作用 作为炎症介质和细胞因子，在不含MP的情况下，培养的EC将与HCOV-NL63一起孵育 DB血浆和结果将与含有MPS增加的DB血浆进行比较。严格，hcovnl63的效率 感染将使用三种不同的方法确定。总病毒将从细胞和上清液中收获 在96 hpi处，通过牙菌斑测定和50％组织培养感染剂量（TCID 50）分析进行定量 出版（PMID 19014487）。还将使用一步，实时的定量反向来量化感染 使用市售试剂盒（Lifereriver）从96 hpi收获的RNA上的转录PCR分析。上清液 每天都会收集以检查MP和细胞因子的释放，以作为病毒感染诱导的EC的指标 激活。将比较组之间的结果（所有提出的初步研究的每组3-4个重复）。 B.提供初步数据，以支持HCOV-NL63病毒可以产生与SARS-COV-2相似的疾病。 据报道，HCOV-NL63会引起儿童上呼吸道感染，并引起肺炎，如 以及K18-ACE2小鼠中的气道注射。在这项初步研究中，我们将开发HCOVNL63的仓鼠模型 感染。金叙利亚仓鼠（8-10周大，查尔斯河）将用于这项研究。正常且诱导的 糖尿病仓鼠将通过HCOV-NL63的系列稀释液（5 x 104、5 x 105或5 x 106 TCID50）在200μlPBS中以在体内建立ID50。从1天开始每24小时收集口服和直肠拭子 接种后（DPI）长达五天。体重和疾病的临床迹象如劳动呼吸所表明的 弯曲的姿势和不动，每天将两次监测温度。仓鼠将在3上安乐死 和5 dpi收集肺部和周围组织。剩余的动物在7 dpi时处死（每组n = 3）。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 在支气管肺泡灌洗和肺匀浆中。血浆将用于MP和细胞因子分析（详细信息请参阅 应用实验设计C4B2）。将比较组之间的结果。我们预测HCOV-NL63感染了 与HCOV-NL63感染的正常仓鼠相比，DB仓鼠具有更严重的呼吸道炎症反应。我们的 先前的研究表明，DB血浆向正常大鼠的交叉输血引起了立即的血管炎症 受体大鼠，表明DB MP在降低脉管中的炎症传播中的作用。评估 DB MP在HCOV-NL63感染的设施和传播中的特定作用，HCOV-NL63-的40％等离子体体积 受感染的正常仓鼠将被富含MP和MP的DB血浆（正常等离子体作为假对照）代替 在组之间将比较Versa和病毒感染引起的疾病和血管病理。如果HCOVNL63 仅感染就会引起仓鼠非常轻微的临床症状，另一种选择是与人类共感染仓鼠 影响力H1N1和HCOV-NL63。仓鼠是研究人类影响力感染和研究的既定动物模型 SRAS-COV-2和影响力的共感染已显示可增强仓鼠肺炎的严重程度。 C.提供一个新的治疗靶标的证据，以减轻199例糖尿病患者的不良后果。 该应用的重点是揭示一种新的机制，糖尿病加剧了19个结果。 提出的机械研究将导致鉴定出一种新型治疗性发育靶标。我们的 先前的研究表明，对DB大鼠的颈动脉注射纳克斯蛋白V（1 ml，100μg/kg）从465 x降低了血浆MPS 注射后104/μL至22 x 104/μl。我们将在HCOV-NL63感染的DB仓鼠中采用相同的方法，以及 血浆MP和病毒感染结果将相应评估。药物开发不超出 此应用程序。但是，这些初步研究将提供支持发展的机械证据 未来研究中基于Annexin v的治疗产品。