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 糖尿病会增加患中风的风险，使脑损伤恶化，使中风恢复恶化， 易导致中风后认知障碍 (PSCI) 本次竞争性更新的目标是进行调查。 内皮-周细胞信号传导受损在周细胞促炎表型转化中的作用 中风后和糖尿病小鼠 PSCI 的发展 亲本 R01 研究了破坏的作用。 糖尿病后常见的急性微血管功能障碍（“无复流”）中的内皮周细胞串扰 中风，并加剧急性缺血性脑损伤，我们的研究结果表明内皮信号传导丧失。 周细胞，通过内皮衍生的环氧二十碳三烯酸酯 (EET) 介导，作用于 G 蛋白偶联 周细胞上的受体 GPR39 会导致周细胞收缩并减少毛细血管血流量。 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经历表型 与细胞激活一致的转化，然后迁移到缺血区域并在缺血区域内增殖。 我们将检验糖尿病条件下 EETs/GPR39 信号传导持续中断的假设 将周细胞转化为有助于 PSCI 的促炎表型，目标 1 将确定内皮细胞是否具有相关性。 EET 以性别和年龄依赖性方式预防糖尿病 PSCI。 具有诱导性内皮特异性 EET 代谢酶缺失的糖尿病和非糖尿病小鼠 可溶性环氧化物水解酶内皮 sEH (iEC-sEHKO) 将经历短暂的 MCA 闭塞 (MCAO)，随后 通过 MCAO 后 1 个月和 3 个月的认知测试，我们将确定 T2D 对中风后的影响。 我们将使用单细胞 RNAseq 和单细胞质谱流式分析。 (scCyTOF) 表征 MCAO 后第 1、7 和 30 天的周细胞异质性和表型。 将从雄性和雌性、糖尿病和非糖尿病 NG2-DsRed 小鼠中分离出来，这些小鼠将周细胞标记为红色 神经炎症的新基因和标记物将使用荧光定位于周细胞中。 免疫组织化学 (IHC) 和 RNAscope 目标 3 将确定 GPR39 在 PSCI 和周细胞中的作用。 糖尿病和非糖尿病条件下中风后的表型转换我们将使用 GPR39 敲除 (KO)。 小鼠测试以下假设：内皮 EET 激活 GPR39 可预防 非糖尿病大脑中的 PSCI，并且糖尿病会减少内皮 EET，从而促进促炎 周细胞表型，导致持续性神经炎症和 PSCI 我们将使用无偏脂质组学进行分析。 oxylipins（氧化脂肪酸，包括 EET）以及 IHC 和 RNAscope，用于评估表达、分布和 GPR39 和目标 2 中鉴定的基因在糖尿病和非糖尿病大脑中的细胞定位。 转化提案使用创新方法来研究新型受体（GPR39）在周细胞中的作用 表型转换及其在糖尿病 PSCI 中的作用如果得到证实，结果将为 GPR39 的使用铺平道路。 激动剂作为治疗剂通过调节周细胞对中风的反应来预防糖尿病 PSCI。