Novel Involvement of NAMPT and TLR4 in PAH Vascular Remodeling

摘要 这项申请旨在解决尚未得到满足的肺动脉高压的治疗需求。 (PAH)，一种预后不佳的致命疾病。PAH病理生物学的一个重要特征是肺脏深部病变。 没有血管重塑，遗憾的是缺乏新的治疗策略。我们成功地聘用了 基因组密集型方法鉴定烟酰胺磷酸核糖转移酶(NAMPT)为新的多环芳烃 PAH患者外周血单核细胞强烈上调的靶点。我们还发现显著增加了 NAMPT在人PAH受试者和临床前PAH模型(小鼠和 NAMPT定位于肺内皮细胞(ECs)。我们进一步报告说，减少可获得的 分泌或细胞外NAMPT或eNAMPT(通过eNAMPT中和抗体、siRNAs、NAMPT+/-小鼠)dra- 在我们的临床前PAH模型中有效地减轻了PAH的严重程度。要检验NAMPT促进的假设 PH发生过程中的血管重塑，可作为一种新的PAH治疗靶点，特异性靶点1(SA #1)将进一步表征NAMPT表达对PAH刺激的响应(PDGF，VEGF， PHD2抑制剂，内皮素-1)专注于启动子活性和表观遗传调节(DNA甲基化，miR- NAS)和特定的转录因子(HIF-2a、STAT5、SOX17)来调控NAMPT反式调控。 脚本。SA#2将机械地检测细胞外NAMPT(ENAMPT)对血管的贡献 重塑通过影响内皮细胞的凋亡和SMC的激活(Ca~(2+)信号和延长 停工)。我们将特别关注eNAMPT与Toll样受体4(TLR4)的相互作用，这是我们最近 NAMPT-TLR4介导的NF-κB转录活性的研究 ENAMPT影响血管重塑的机制。SA#3将利用我们之前发布的研究- IES和初步数据表明NAMPT 5‘启动子多态性(SNPs)改变NAMPT启动子 活动也会显著增加急性呼吸窘迫的易感性和严重性 综合征(ARDS)，并将在PAH中进行评估。SA#3研究将确定NAMPT SNPs对 转录调控，对eNAMPT-TLR4结合和核因子κB在PAH中的激活。最后，SA#4将在 建立PAH，降低NAMPT表达(条件性EC基因敲除小鼠)的治疗效果，EN- AMPT消除(中和抗体)，抑制NAMPT酶活性(新型FK-866类似物)， 和TLR4拮抗剂(新型多肽抑制剂)。由杰出调查人员的密切参与提供支持 和大量高度翻译性的已发表/初步数据强调NAMPT是一种新的先天性免疫 调制器，这项应用将成功地定义NAMPT在PAH易感性、病理生物学和 严肃性。

ABSTRACT This application seeks to address the unmet need for curative therapies in pulmonary arterial hypertension (PAH), a fatal disease with a dismal prognosis. A key feature of the pathobiology of PAH is the profound pulmo- nary vascular remodeling for which new therapeutic strategies are woefully lacking. We successfully employed genomic–intensive approaches to identify nicotinamide phosphoribosyltransferase (NAMPT) as a novel PAH target that is robustly upregulated in PBMCs from PAH patients. We also demonstrated markedly increased NAMPT expression in remodeled vessels from human PAH subjects and in preclinical PAH models (mice and rats) with NAMPT localized to lung endothelial cells (ECs). We further reported that reducing the availability of secreted or extracellular NAMPT or eNAMPT (via eNAMPT neutralizing antibodies, siRNAs, Nampt+/- mice) dra- matically attenuated PAH severity in our preclinical PAH models. To test the hypothesis that NAMPT promotes vascular remodeling during PH development and serves as a novel PAH therapeutic target, Specific Aim #1 (SA #1) will further characterize the regulation of NAMPT expression in response to PAH stimuli (PDGF, VEGF, PHD2 inhibitor, endothelin-1) focusing on promoter activity and epigenetic regulation (DNA methylation, miR- NAs) and specific transcription factors (HIF-2a, STAT5, SOX17) that we have shown to regulate NAMPT tran- scription. SA #2 will mechanistically examine the contribution of extracellular NAMPT (eNAMPT) to vascular remodeling via influences on EC apoptosis and smooth muscle cell (SMC) activation (Ca2+ signaling and prolif- eration). We will specifically focus on the interaction of eNAMPT with Toll–like receptor 4 (TLR4), that we recently identified as the NAMPT receptor and explore eNAMPT-TLR4 mediated NFκB transcriptional activities as a novel mechanism by which eNAMPT may influence vascular remodeling. SA #3 will leverage our prior published stud- ies and preliminary data demonstrating that NAMPT 5’ promoter polymorphisms (SNPs) alter NAMPT promoter activity which also confer significantly increased risk for susceptibility and severity in acute respiratory distress syndrome (ARDS) and will be assessed in PAH. SA #3 studies will determine the influence of NAMPT SNPs on transcriptional regulation, on eNAMPT-TLR4 binding, and NFκB activation in PAH. Finally, SA #4 will define in established PAH, the therapeutic efficacy of reduced NAMPT expression (conditional EC knockout mice), eN- AMPT elimination (neutralizing antibodies), inhibition of NAMPT enzymatic activity (novel FK-866 analogues), and TLR4 antagonism (novel peptide inhibitors). Supported by intimate involvement of outstanding investigators and substantial highly translational published/preliminary data highlighting NAMPT as a novel innate immunity modulator, this application will successfully define NAMPT participation in PAH susceptibility, pathobiology, and severity.