Targeting Pathogenic Endothelial Dysfunction in Lupus Nephritis

系统性红斑狼疮(SLE)是一种免疫复合物介导的疾病，临床表现为 在先天免疫反应后表现出来。血管内皮细胞(EC)是一种条件效应细胞，由 产生细胞内活性氧(ROS)的炎症刺激和炎症流入的信号 细胞转化为组织。我们已经发表了内皮型一氧化氮合酶(ENOS)调节炎症反应的研究。 狼疮性肾炎(LN)与EC eNOS解偶联(减少抗炎一氧化氮的产生 (NO)和增加促炎ROS的产生)是SLE的潜在治疗靶点。我们假设 Sepiapterin，一种与eNOS偶联的化合物，将改善LN的结果和小鼠LN的血管功能 并通过调节PI3Kinase/Akt通路来减少炎症和纤维化基因的表达 小鼠和培养的肾小球内皮细胞。我们进一步假设，拟议的方法可能具有 脱靶效应，特别是在T细胞、单核/巨噬细胞和足细胞。为了解决这个突出的问题 假设，提出以下具体目标：具体目标1)确定七叶蝶呤在 小鼠LN对临床和组织学转归、大血管内皮功能的影响 治疗反应的药效学标志物。我们假设Sepiapterin将改善LN 小鼠狼疮性肾炎的预后和血管功能。目的：确定本病的疗效和最佳治疗指征。 ECD靶向治疗，我们将采用NZM2410模型自发性LN，并比较有无LN组 以确定对三个不同终点的疗效。1)通过以下方式预防/延长LN的发病 Sepiapterin单一疗法，2)霉酚酸酯治疗的小鼠，改善/加速缓解的诱导 3)诱导缓解、延缓/预防狼疮发作。 停用霉酚酸酯后再单用七叶蝶呤。临床和组织学参数与大血管 内皮功能将是主要的终点。系统性氧化应激的生物标记物将作为 探索性药效学指标。特定目标2.确定差异基因表达和 在人肾小球中诱导的以PI3K/Akt通路为重点的磷酸激酶信号转导事件 用层粘连蛋白血清诱导内皮细胞(HRGECs)，并经七叶蝶呤调节。基于我们发布的和 初步研究认为，炎症基因表达与PDGF受体/PI3K/Akt/FoxO1 Sepiapterin对LN Flare血清诱导的HRGEC信号转导有调节作用。对路径的了解 由LN Flare血清激活，既启动氧化还原调节通路，又可通过恢复 偶联eNOS对于合理开发针对LN ECD的治疗方法具有重要意义。我们将培养 活动性肾炎或细胞因子混合的SLE患者血清与HRGEC结合并通过干扰信号传导 PDGFR、PI3K和Akt，以确定对基因表达和NO和超氧化物歧化产物的影响。特定的 目的3.确定炎症和纤维化基因的差异表达和途径丰富 肾小球组织和T细胞中的内皮细胞、炎性细胞和足细胞 递增剂量的七叶蝶呤处理小鼠的脾细胞。基于我们发布的和 初步研究，我们假设候选的PI3K-Akt信号基因丰富(两者都被和增强 以前馈方式诱导氧化应激)将由西蝶呤调节。NZM2410小鼠将被 递增剂量七叶蝶呤治疗肾皮质和脾组织的单细胞RNA分析 测序。我们翻译小组的最终目标是以ECD为目标来改善心血管和肾脏 在LN的结果。鉴于LN心血管疾病的盛行率，我们将研究内皮依赖性 血管功能，为早期人类心血管预防研究提供信息。这里提出的目标将是 展示最有效的治疗适应症、反应的药效学标志物和替代或 减少退伍军人LN和其他内皮功能障碍疾病的炎症性ECD的辅助目标。

Systemic lupus erythematosus (SLE) is an immune complex-mediated disease in which clinical disease manifests after an innate immune response. Endothelial cells (EC) are conditional effector cells activated by inflammatory stimuli to produce intracellular reactive oxygen species (ROS) and signal for influx of inflammatory cells into tissue. We have published that endothelial nitric oxide synthase (eNOS) modulates inflammation in lupus nephritis (LN) and that uncoupling of EC eNOS (which reduces production of anti-inflammatory nitric oxide (NO) and increases production of pro-inflammatory ROS) is a potential therapeutic target in SLE. We hypothesize that sepiapterin, a compound that couples eNOS, will improve LN outcomes and vascular function in murine LN and work by modulating the PI3Kinase/Akt pathway to reduce expression of inflammation and fibrosis genes in mice and in cultured glomerular endothelial cells. We further hypothesize that the proposed approach may have off target effects, particularly in T cells, monocyte/macrophages, and podocytes. To address this overarching hypothesis, the following specific aims are proposed: Specific Aim 1) Determine the effect of sepiapterin in murine LN on clinical and histologic LN outcomes, large vessel endothelial function, and pharmacodynamic markers of treatment response. We hypothesize that sepiapterin will improve LN outcomes and vascular function in murine LN. To determine the effect of and optimal therapeutic indication for ECD targeted therapy, we will use the NZM2410 model of spontaneous LN and compare groups with and without sepiapterin treatment to determine effect on three different endpoints. 1) preventing/prolonging LN onset with sepiapterin monotherapy, 2) in mycophenolate-treated mice, improving/hastening induction of remission with sepiapterin as an adjunctive therapy, and 3) after inducing remission, prolonging/preventing LN flare with sepiapterin monotherapy after mycophenolate withdrawal. LN clinical and histologic parameters and large vessel endothelial function will be the primary endpoints. Biomarkers of systemic oxidative stress will serve as exploratory pharmacodynamic indicators. Specific Aim 2. Determine differential gene expression and phosphokinase signaling events, with a focus on the PI3K/Akt pathway, induced in human glomerular endothelial cells (HRGECs) by LN serum and modulated by sepiapterin. Based on our published and preliminary studies, we hypothesize that inflammatory gene expression and PDGF receptor/PI3K/Akt/FoxO1 signaling induced by LN flare serum can be modulated by sepiapterin in HRGECs. Knowledge of pathways activated by LN flare serum that both initiate redox-regulated pathways and that can be modulated by restoring coupling to eNOS will be important for rational development of therapies to target ECD in LN. We will culture HRGECs with serum from SLE patients with active nephritis or a cytokine mix and perturb signaling through the PDGFR, PI3K, and Akt to determine the effect on gene expression and NO and superoxide production. Specific Aim 3. Determine differential inflammatory and fibrosis gene expression and pathway enrichment in endothelial cells, infiltrating inflammatory cells, and podocytes from renal glomerular tissue and in T cells from the spleen in mice treated with increasing doses of sepiapterin. Based on our published and preliminary studies, we hypothesize that candidate PI3K-Akt signaling gene enrichment (both potentiated by and inducing of oxidative stress in a feed forward fashion) will be modulated by sepiapterin. NZM2410 mice will be treated with increasing doses of sepiapterin and renal cortical and spleen tissue analyzed by single cell RNA sequencing. The ultimate goal of our translational group is to target ECD to improve cardiovascular and renal outcomes in LN. Given the prevalence of cardiovascular disease in LN, we will study endothelium-dependent vascular function to inform early human studies of cardiovascular prevention. The aims proposed here will demonstrate the most effective treatment indications, pharmacodynamic markers of response, and alternate or adjunctive targets to reduce inflammatory ECD in LN and other diseases of endothelial dysfunction in Veterans.