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
基本信息
- 批准号:10547868
- 负责人:
- 金额:$ 32.47万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:2019-nCoVACE2ANXA5 geneAccountingAdhesionsAdhesivesAngiotensin IIAngiotensin ReceptorAngiotensin-Converting Enzyme InhibitorsAnimal ModelAnimalsAreaBindingBinding ProteinsBinding SitesBiological AssayBlood CellsBlood CirculationBlood Plasma VolumeBlood VesselsBlood specimenBody WeightBreathingBronchoalveolar LavageCOVID-19COVID-19 morbidityCOVID-19 pathogenesisCOVID-19 patientCaco-2 CellsCardiovascular DiseasesCell membraneCellsCharacteristicsChildClinicalCoagulantsCommunicable DiseasesConflict (Psychology)Confocal MicroscopyCoronavirusDataDental crownsDevelopmentDiabetes MellitusDiseaseDoseDown-RegulationDrug TargetingEconomicsElectron MicroscopyEndocytosisEndothelial CellsEndotheliumEnzymesExperimental DesignsFlow CytometryFluorescenceFunctional disorderFutureHamstersHarvestHistologyHourHumanIn VitroIncubatedIndividualInfectionInfection ControlInflammationInflammation MediatorsInflammatoryInflammatory ResponseInfluenzaInfluenza A Virus, H1N1 SubtypeInjectionsK-18 conjugateLaboratoriesLeukocytesLipid BindingLungMeasuresMediatingMesocricetus auratusMeta-AnalysisMethodsMicrofluidic MicrochipsMicrofluidicsMicrovascular DysfunctionModelingMonitorMusOralOrganOrganellesOutcomePathogenesisPathologyPatientsPerfusionPeripheralPersonsPhenotypePhosphatidylserinesPlaque AssayPlasmaPneumoniaPostureProteinsPublishingRNARattusRenin-Angiotensin-Aldosterone SystemReportingResolutionResourcesReverse TranscriptionRiversRoleSARS-CoV-2 infectionSARS-CoV-2 pathogenesisSARS-CoV-2 spike proteinSeveritiesStreptozocinStudy modelsSurfaceSwabSymptomsTechniquesTemperatureTestingTherapeuticTimeTissuesTransfusionUp-RegulationUpper Respiratory InfectionsVirulentVirusVirus DiseasesVirus ReplicationVirus-like particleWestern Blottingadverse outcomeairway inflammationbasecell typeco-infectioncomorbidityconfocal imagingcytokinediabeticdiabetic patientdrug developmentexosomeexperimental studyhigh riskhuman coronavirusimprovedin vivoinfluenza infectionmortalitymortality risknew therapeutic targetnon-diabeticnovelorgan injurypandemic diseasepreventreceptorrectalrespiratoryresponsesevere COVID-19socialsocioeconomicstargeted treatmenttherapeutic developmenttissue culturevascular inflammationvasoconstrictionvectorvesicular releaseviral RNA
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
PINGNIAN HE其他文献
PINGNIAN HE的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('PINGNIAN HE', 18)}}的其他基金
Red blood cell released ATP in disturbed blood flow-initiated site specific vascular inflammation and atherosclerosis
红细胞在血流紊乱引发的特定部位血管炎症和动脉粥样硬化中释放 ATP
- 批准号:
10457975 - 财政年份:2019
- 资助金额:
$ 32.47万 - 项目类别:
Red blood cell released ATP in disturbed blood flow-initiated site specific vascular inflammation and atherosclerosis
红细胞在血流紊乱引发的特定部位血管炎症和动脉粥样硬化中释放 ATP
- 批准号:
10180296 - 财政年份:2019
- 资助金额:
$ 32.47万 - 项目类别:
Red blood cell released ATP in disturbed blood flow-initiated site specific vascular inflammation and atherosclerosis
红细胞在血流紊乱引发的特定部位血管炎症和动脉粥样硬化中释放 ATP
- 批准号:
10221039 - 财政年份:2019
- 资助金额:
$ 32.47万 - 项目类别:
Nitric oxide and microvessel permeability in vivo
一氧化氮和体内微血管通透性
- 批准号:
9258790 - 财政年份:2016
- 资助金额:
$ 32.47万 - 项目类别:
Microparticles and microvascular dysfunction in diabetes
糖尿病中的微粒和微血管功能障碍
- 批准号:
8680231 - 财政年份:2013
- 资助金额:
$ 32.47万 - 项目类别:
Microparticles and microvascular dysfunction in diabetes
糖尿病中的微粒和微血管功能障碍
- 批准号:
9120245 - 财政年份:2013
- 资助金额:
$ 32.47万 - 项目类别:
Microparticles and microvascular dysfunction in diabetes
糖尿病中的微粒和微血管功能障碍
- 批准号:
8578849 - 财政年份:2013
- 资助金额:
$ 32.47万 - 项目类别:
Microparticles and microvascular dysfunction in diabetes
糖尿病中的微粒和微血管功能障碍
- 批准号:
8996443 - 财政年份:2013
- 资助金额:
$ 32.47万 - 项目类别:
Nitric Oxide and Microvessel Permeability In Vivo
体内一氧化氮和微血管通透性
- 批准号:
7747939 - 财政年份:2007
- 资助金额:
$ 32.47万 - 项目类别:
Nitric Oxide and Microvessel Permeability In Vivo
体内一氧化氮和微血管通透性
- 批准号:
7213844 - 财政年份:2007
- 资助金额:
$ 32.47万 - 项目类别:
相似国自然基金
新型蝙蝠MERS簇冠状病毒HKU5的ACE2细胞受体识别及其分子机制研究
- 批准号:
- 批准年份:2024
- 资助金额:0.0 万元
- 项目类别:省市级项目
铁皮石斛通过肠道 ACE2 修复 Trp/GPR142 介
导“肠-胰岛 ”轴血糖调控功能的降糖机制研
究
- 批准号:Y24H280055
- 批准年份:2024
- 资助金额:0.0 万元
- 项目类别:省市级项目
人类ACE2变构抑制剂的成药性及其抗广谱冠状病毒感染的机制研究
- 批准号:82330111
- 批准年份:2023
- 资助金额:220 万元
- 项目类别:重点项目
CAFs来源的外泌体负性调控ACE2促进肾透明细胞癌癌栓新辅助靶向耐药的机制研究
- 批准号:82373169
- 批准年份:2023
- 资助金额:49 万元
- 项目类别:面上项目
新型蝙蝠MERS簇冠状病毒HKU5的ACE2受体识别及细胞入侵机制研究
- 批准号:32300137
- 批准年份:2023
- 资助金额:30 万元
- 项目类别:青年科学基金项目
基于AT2/ACE2/Ang(1-7)/MAS轴调控心脏-血管-血液系统性重构演变规律研究心衰气虚血瘀证及其益气通脉活血化瘀治法生物学基础
- 批准号:82305216
- 批准年份:2023
- 资助金额:30 万元
- 项目类别:青年科学基金项目
基于外泌体miRNAs介导细胞通讯的大豆ACE2激活肽调控血管稳态机制研究
- 批准号:32302080
- 批准年份:2023
- 资助金额:30 万元
- 项目类别:青年科学基金项目
感毒清经ACE2/Ang(1-7)/MasR信号通路抑制PM2.5诱导慢性气道炎症的机制:聚焦肺泡巨噬细胞极化与“胞葬”的表型串扰
- 批准号:82305171
- 批准年份:2023
- 资助金额:30 万元
- 项目类别:青年科学基金项目
刺参自溶引发机制中ACE2调控靶点的调控网络研究
- 批准号:32372399
- 批准年份:2023
- 资助金额:50 万元
- 项目类别:面上项目
Spike变异对新冠病毒抗原性及ACE2种属嗜性的影响研究
- 批准号:82272305
- 批准年份:2022
- 资助金额:52 万元
- 项目类别:面上项目
相似海外基金
新型コロナウイルス感染阻害能を有する抗ACE2抗体の阻害機構に関する構造基盤解明
阐明具有抑制新型冠状病毒感染能力的抗ACE2抗体抑制机制的结构基础
- 批准号:
24K09338 - 财政年份:2024
- 资助金额:
$ 32.47万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
ACE2のユビキチン化を介したコロナウイルス感染機構の解明と創薬への挑戦
通过ACE2泛素化阐明冠状病毒感染机制和药物发现的挑战
- 批准号:
22KJ2499 - 财政年份:2023
- 资助金额:
$ 32.47万 - 项目类别:
Grant-in-Aid for JSPS Fellows
ACE2阻害薬およびERK経路阻害薬による慢性腎炎進展抑制効果の検証
ACE2抑制剂和ERK通路抑制剂抑制慢性肾炎进展的效果验证
- 批准号:
23K14982 - 财政年份:2023
- 资助金额:
$ 32.47万 - 项目类别:
Grant-in-Aid for Early-Career Scientists
Large-scale compatibility assessments between ACE2 proteins and diverse sarbecovirus spikes
ACE2 蛋白和多种 sarbecovirus 尖峰之间的大规模兼容性评估
- 批准号:
10722852 - 财政年份:2023
- 资助金额:
$ 32.47万 - 项目类别:
一次線毛とコロナウイルス感染におけるACE2の役割の解明
阐明 ACE2 在原发菌毛和冠状病毒感染中的作用
- 批准号:
22KF0004 - 财政年份:2023
- 资助金额:
$ 32.47万 - 项目类别:
Grant-in-Aid for JSPS Fellows
The regulatory roles of ACE2 and its interaction with Nrf2 in arsenic-induced endothelial dysfunction in experimental and epidemiological studies
实验和流行病学研究中 ACE2 的调节作用及其与 Nrf2 的相互作用在砷诱导的内皮功能障碍中的作用
- 批准号:
23K16310 - 财政年份:2023
- 资助金额:
$ 32.47万 - 项目类别:
Grant-in-Aid for Early-Career Scientists
Role of ACE2 in the mechanism of intestinal regeneration
ACE2在肠道再生机制中的作用
- 批准号:
23K15078 - 财政年份:2023
- 资助金额:
$ 32.47万 - 项目类别:
Grant-in-Aid for Early-Career Scientists
Research and development of a novel pediatric anti-obesity medicine via ACE2 activation in DIZE
通过 DIZE 中 ACE2 激活研发新型儿科抗肥胖药物
- 批准号:
23K15417 - 财政年份:2023
- 资助金额:
$ 32.47万 - 项目类别:
Grant-in-Aid for Early-Career Scientists
Lung delivery of novel ACE2 variants for COVID-19
针对 COVID-19 的新型 ACE2 变体的肺部输送
- 批准号:
10483042 - 财政年份:2022
- 资助金额:
$ 32.47万 - 项目类别:
ACE2 on gut barrier dysfunction and BRB disruption
ACE2 对肠道屏障功能障碍和 BRB 破坏的影响
- 批准号:
10535485 - 财政年份:2022
- 资助金额:
$ 32.47万 - 项目类别: