Pericyte phenotypic switching in diabetic post-stroke cognitive impairment (PSCI)

糖尿病中风后认知障碍（PSCI）中的周细胞表型转换

• 批准号: 10855709
• 负责人: Nabil J Alkayed
• 金额: $ 69.62万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10855709
• 关键词: Acute; Age; Age Months; Agonist; Anti-Inflammatory Agents; Automobile Driving; Autopsy; Blood capillaries; Blood flow; Brain; Brain Injuries; Brain Ischemia; CSPG4 gene; Cells; Chronic Phase; Cognitive deficits; Cytometry; Data; Dementia; Development; Diabetes Mellitus; Diabetic mouse; DsRed; Endothelial Cells; Endothelium; Enzymes; Epoxide hydrolase; Fatty Acids; Female; Fluorescence; G-Protein-Coupled Receptors; GPR39 gene; Genes; Goals; Heterogeneity; Human; Immunohistochemistry; Impaired cognition; Impairment; Inflammatory; Ischemia; Ischemic Brain Injury; Knock-out; Knockout Mice; Label; Lipids; LoxP-flanked allele; Macrophage; Maps; Mediating; Microglia; Microvascular Dysfunction; Mus; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Parents; Pericytes; Phenotype; Play; Process; Proliferating; Recovery; Recovery of Function; Regulation; Reporting; Risk; Role; Signal Transduction; Signaling Molecule; Stroke; Testing; Therapeutic Agents; age related; aged; angiogenesis; cell type; cognitive testing; diabetic; functional outcomes; improved; innovation; lipidomics; male; migration; mouse model; neuroinflammation; non-diabetic; novel; post stroke; post stroke cognitive impairment; post stroke dementia; prevent; receptor; repaired; response; sex; single-cell RNA sequencing; stroke recovery; therapy development

ABSTRACT Diabetes increases the risk of developing stroke, exacerbates brain injury, worsens stroke recovery and predisposes to post-stroke cognitive impairment (PSCI). The goal of this competitive renewal is to investigate the role of impaired endothelial-pericyte signaling in the pro-inflammatory phenotypic transformation of pericytes after stroke and the development of PSCI in diabetic mice. The parent R01 investigated the role of disrupted endothelial pericyte crosstalk in the acute microvascular dysfunction (“no-reflow”) that is common after diabetic stroke, and that exacerbates acute ischemic brain damage. Our findings indicate that loss of endothelial signaling to pericytes, mediated via endothelial derived epoxyeicosatrienoates (EETs) acting on G protein coupled receptor GPR39 on pericytes, leads to pericyte contraction and reduced capillary blood flow. Along the way, we observed that in the subacute/chronic phases of stroke, pericytes retract from capillaries and undergo phenotypic transformation consistent with cell activation, followed by migration to and proliferation within the ischemic region. We will test the hypothesis that persistent disruption of EETs/GPR39 signaling under diabetic conditions transforms pericytes to a pro-inflammatory phenotype that contributes to PSCI. Aim 1 will determine if endothelial EETs protect against diabetic PSCI in a sex- and age-dependent manner. Male and female, young and old, diabetic and non-diabetic mice with an inducible endothelial specific deletion of EETs-metabolizing enzyme soluble epoxide hydrolase endothelial sEH (iEC-sEHKO) will undergo transient MCA occlusion (MCAO), followed by cognitive testing at 1 and 3 months after MCAO. In Aim 2, we will determine the effect of T2D on post-stroke pericyte heterogeneity and phenotype. We will use single-cell RNAseq and single-cell mass cytometry (scCyTOF) to characterize pericyte heterogeneity and phenotypes on days 1, 7 and 30 after MCAO. Pericytes will be isolated from male and female, diabetic and no-diabetic NG2-DsRed mice, which label pericytes in red fluorescence. Novel genes and markers of neuroinflammation will be localized in pericytes using immunohistochemistry (IHC) and RNAscope. Aim 3 will determine the role of GPR39 in PSCI and pericyte phenotypic switching after stroke under diabetic and non-diabetic conditions. We will use GPR39 knockout (KO) mice to test the hypothesis that GPR39 activation by endothelial EETs is protective against the development of PSCI in non-diabetic brain, and that diabetes decreases endothelial EETs, which promotes a pro-inflammatory pericyte phenotype, leading to persistent neuroinflammation and PSCI. We will use unbiased lipidomics to profile oxylipins (oxidized fatty acids, including EETs), and IHC and RNAscope to evaluate expression, distribution and cellular localization of GPR39 and genes identified in Aim 2 in diabetic and non-diabetic brain. The highly translational proposal uses innovative approaches to investigate the role of a novel receptor (GPR39) in pericyte phenotypic switching and its role in diabetic PSCI. If confirmed, the results will pave the way for the use of GPR39 agonists as therapeutic agents to prevent diabetic PSCI by modulating the pericyte response to stroke.

抽象的 糖尿病增加了中风的风险，加剧脑损伤，中风恢复和 触觉后认知障碍（PSCI）易感性。这种竞争更新的目标是调查 内皮 - 周期信号受损中的作用在周细胞的促炎表型转化中 中风和糖尿病小鼠中PSCI的发展。家长R01调查了中断的作用 急性微血管功能障碍（“无流量”）中的内皮周细胞串扰，糖尿病后常见 中风，加剧了急性缺血性脑损伤。我们的发现表明内皮信号的丧失 到周细胞，通过作用于G蛋白耦合的内皮衍生的环氧卫星酸酯（EET）介导 受体GPR39在周细胞上，导致周细胞收缩并减少毛细血管血流。一路上，我们 观察到，在中风的亚急性/慢性阶段，周细胞从毛细血管中缩回并进行表型 转化与细胞活化一致，然后在缺血区域内迁移和增殖。 我们将检验以下假设：糖尿病条件下EET/GPR39信号的持续破坏 将周细胞转化为有助于PSCI的促炎表型。 AIM 1将确定是否内皮 Eets以性别和年龄的方式预防糖尿病PSCI。男性和女性，年轻人， 糖尿病和非糖尿病小鼠具有可诱导的Eets-grotization酶的内皮特异性缺失 可溶性环氧化物水解酶内皮SEH（IEC-SEHKO）将经历短暂的MCA遮挡（MCAO），然后进行 通过MCAO后1和3个月的认知测试。在AIM 2中，我们将确定T2D对冲程后的影响 周细胞异质性和表型。我们将使用单细胞RNASEQ和单细胞质量细胞仪 （sccytof）在MCAO之后的第1、7和30天表征周细胞异质性和表型。周细胞 将从雄性和女性，糖尿病和无糖尿病NG2-DSRED小鼠中分离出来，将其标记为红色 荧光。新颖的基因和神经炎症的标志物将在周细胞中定位 免疫组织化学（IHC）和rnascope。 AIM 3将确定GPR39在PSCI和Pericyte中的作用 糖尿病和非糖尿病状况下中风后表型转换。我们将使用GPR39淘汰赛（KO） 小鼠检验以下假设：内皮EET的GPR39激活受到保护免受的发展 非糖尿病大脑中的PSCI，并且糖尿病会降低内皮EET，从而促进促炎性 周细胞表型，导致持续的神经炎症和PSCI。我们将使用公正的脂肪组学进行概况 oxylipins（包括EET在内的氧化脂肪酸）和IHC和RNASCOPE，以评估表达，分布和 糖尿病和非糖尿病大脑中AIM 2中鉴定出的GPR39的细胞定位和基因。高度 翻译建议使用创新方法来研究新型受体（GPR39）在周细胞中的作用 表型切换及其在糖尿病PSCI中的作用。如果得到确认，结果将为使用GPR39铺平道路 激动剂作为治疗剂，可通过调节对卒中的周围反应来预防糖尿病PSCI。