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

摘要 糖尿病增加了中风的风险，加重了脑损伤，阻碍了中风的恢复， 易患中风后认知障碍（PSCI）。这次竞争性续约的目的是调查 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R 01研究了干扰的作用。 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，内皮细胞信号的丢失 通过内皮细胞衍生的环氧二十碳三烯酸（Epoxyeicosatrienoates，ESTs）作用于G蛋白偶联物， 周细胞上的GPR 39受体导致周细胞收缩和毛细血管血流减少。沿着，我们 观察到在中风的亚急性/慢性阶段，周细胞从毛细血管收缩并经历表型 转化与细胞活化一致，随后迁移到缺血区域并在缺血区域内增殖。 我们将检验在糖尿病条件下持续破坏EkB/GPR 39信号传导的假设， 将周细胞转化为促炎表型，从而导致PSCI。目标1将确定内皮 依地平以性别和年龄依赖的方式保护糖尿病PSCI。无论男女老少， 具有EET代谢酶的可诱导内皮特异性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 在MCAO后1个月和3个月进行认知测试。在目标2中，我们将确定T2 D对卒中后 周细胞异质性和表型。我们将使用单细胞RNAseq和单细胞质谱细胞术 在MCAO后第1、7和30天，使用SCyTOF（scCyTOF）来表征周细胞异质性和表型。周细胞 将从雄性和雌性、糖尿病和非糖尿病NG 2-DsRed小鼠中分离，其将周细胞标记为红色 荧光。神经炎症的新基因和标记物将被定位在周细胞中， 免疫组化（IHC）和RNA显微镜。目的3探讨GPR 39在PSCI和周细胞中的作用 在糖尿病和非糖尿病条件下中风后的表型转换。我们将使用GPR 39敲除（KO） 小鼠来测试GPR 39被内皮细胞活化对内皮细胞增殖的发展具有保护作用的假设。 PSCI在非糖尿病大脑中，糖尿病降低内皮细胞Ehrs，这促进了促炎症反应， 周细胞表型，导致持续的神经炎症和PSCI。我们将使用无偏见的脂质组学来分析 氧化脂肪酸（氧化脂肪酸，包括雌二醇），以及IHC和RNAscope，以评估表达，分布和 GPR 39和Aim 2中鉴定的基因在糖尿病和非糖尿病脑中的细胞定位。高度 翻译建议使用创新的方法来研究周细胞中一种新受体（GPR 39）的作用 表型转换及其在糖尿病PSCI中的作用。如果得到证实，该结果将为GPR 39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。

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.