Novel Involvement of NAMPT and TLR4 in PAH Vascular Remodeling

摘要 本申请旨在解决肺动脉高压治疗性治疗的未满足需求 (PAH)这是一种预后很差的致命疾病。PAH病理生物学的一个关键特征是深刻的肺- 几乎没有血管重塑，这是可悲的缺乏新的治疗策略。我们成功地雇用了 一种基因组密集的方法来鉴定烟酰胺磷酸核糖基转移酶（NAMPT）作为一种新的PAH 在PAH患者PBMC中显著上调的靶点。我们还发现， 来自人PAH受试者的重塑血管和临床前PAH模型（小鼠和小鼠）中的NAMPT表达 NAMPT定位于肺内皮细胞（EC）。我们进一步报告说，减少可用性， 分泌或细胞外NAMPT或eNAMPT（通过eNAMPT中和抗体，siRNA，Nampt+/-小鼠） 在我们的临床前PAH模型中显著减轻PAH严重程度。为了验证NAMPT促进 PH发展过程中的血管重塑，并作为一种新的PAH治疗靶点，特异性目标#1（SA #1）将进一步表征响应于PAH刺激（PDGF，VEGF， PHD 2抑制剂，内皮素-1），重点是启动子活性和表观遗传调控（DNA甲基化，miR- NAs）和特异性转录因子（HIF-2a，STAT 5，SOX 17），我们已经证明，调节NAMPT trans-factors。 笔迹。SA #2将从机制上检查细胞外NAMPT（eNAMPT）对血管生成的贡献。 通过影响EC凋亡和平滑肌细胞（SMC）活化（Ca 2+信号传导和增殖）， eration）。我们将特别关注eNAMPT与Toll样受体4（TLR 4）的相互作用，我们最近发现， 作为NAMPT受体，探索eNAMPT-TLR 4介导的NFκB转录活性作为一种新的 eNAMPT可能影响血管重塑的机制。SA #3将利用我们之前发布的研究报告- 事实和初步数据表明，NAMPT 5'启动子多态性（SNP）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风险 综合征（ARDS），并将在PAH中进行评估。SA #3研究将确定NAMPT SNP对 转录调控、eNAMPT-TLR 4结合和PAH中NFκB活化。最后，SA #4将在 建立的PAH，NAMPT表达降低的治疗效果（条件性EC敲除小鼠），eN- AMPT消除（中和抗体），NAMPT酶活性抑制（新型FK-866类似物）， 和TLR 4拮抗作用（新型肽抑制剂）。在杰出调查人员的密切参与下， 大量高度翻译的已发表/初步数据突出了NAMPT作为一种新型先天免疫 调节剂，该应用将成功定义NAMPT参与PAH易感性，病理生物学， 严重性。

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.