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

摘要 糖尿病增加中风的风险，加剧脑损伤，恶化中风的康复和 易患中风后认知障碍(PSCI)。这次竞争更新的目标是调查 内皮-周细胞信号受损在周细胞致炎表型转化中的作用 卒中后糖尿病小鼠PSCI的发生发展。亲本R01调查了中断的作用 内皮周细胞在糖尿病后常见的急性微血管功能障碍(“无复流”)中的串扰 中风，这会加剧急性缺血性脑损伤。我们的发现表明内皮信号的丢失 通过内皮源性二十碳三烯酸酯(EETs)作用于G蛋白偶联的周细胞 周细胞上的受体GPR39导致周细胞收缩和毛细血管血流量减少。一路走来，我们 观察到在卒中的亚急性期/慢性期，周细胞从毛细血管回缩，并经历表型。 转化与细胞激活一致，随后迁移到缺血区并在缺血区内增殖。 我们将测试在糖尿病条件下EETs/GPR39信号持续中断的假设 将周细胞转化为促进PSCI的促炎表型。目标1将确定内皮细胞 EETs以性别和年龄相关的方式预防糖尿病PSCI。有男有女，有少有老， 内皮特异性EETs代谢酶可诱导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细胞(IEC-sEHKO)将经历短暂的MCA闭塞(MCAO)，随后 分别于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T2D对卒中后的影响 周细胞异质性和表型。我们将使用单细胞RNAseq和单细胞质量细胞术 (ScCyTOF)检测MCAO后第1、7、30天周细胞的异质性和表型。周细胞 将从雄性和雌性、糖尿病和非糖尿病NG2-DsRed小鼠身上分离出来，NG2-DsRed小鼠用红色标记周细胞 荧光。新的神经炎症基因和标志物将被定位在周细胞中 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卒中后的表型转换。我们将使用GPR39淘汰赛(KO) 小鼠验证内皮源性EETs激活GPR39可预防糖尿病发生的假说 非糖尿病患者大脑中的PSCI，糖尿病减少内皮EET，从而促进促炎 周细胞表型，导致持续性神经炎和PSCI。我们将使用无偏见的脂质组学来分析 氧脂(氧化脂肪酸，包括EETs)和IHC和RNAScope以评估表达、分布和 GPR39和AIM 2基因在糖尿病和非糖尿病脑中的细胞定位。高度的 翻译提案使用创新的方法研究一种新的受体(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 激动剂作为治疗药物，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.