The role of innate immunity in idiopathic pulmonary arterial hypertension

摘要 损伤相关分子模式分子（DAMP）是细胞内的分子，其在细胞内与细胞内的蛋白质结合。 由受损细胞分泌或被动释放。一旦释放，DAMPs促进炎症和组织 修复.高迁移率盒1（HMGB1）蛋白是一种原型和高度保守的DAMP， 通过激活模式识别受体（PRR），包括Toll样受体， 2（TLR2）、TLR4和晚期糖基化终产物受体（Receptor for Advanced Glycation End Products，RECEPTOR）。有证据表明，在 HMGB1作为先天免疫的关键起始分子，协调 炎症和组织重塑。初步数据表明HMGB1、TLR2和TLR4参与了 肺动脉高压（PAH）的动物和组织培养模型。这个问题的核心假设是 HMGB1通过激活PRR，是先天免疫的关键介质， PAH中的内皮激活和血管重塑。提出了两个具体目标来检验这一点 假说.具体目标1将集中于表征HMGB1和PRR在人乳腺癌中的表达和定位。 特发性PAH（IPAH）和非PAH患者样品。我们将使用免疫染色定位HMGB1，TLR2， TLR4和TLR3表达到特定的肺结构和细胞类型，集中在血管 重塑我们将定量HMGB1、TLR2、TLR4的肺特异性表达。我们将使用ELISA， Luminex测定用于定量与先天免疫系统激活相关的细胞因子和趋化因子， 以及IPAH和非PAH患者血浆和肺中的HMGB1。这些数据会把地点联系起来 特异性DAMP释放、PRR表达模式和水平以及促炎细胞因子产生， 是我们主要假设的核心具体目标2将侧重于阐明HMGB1和PRR在血管内皮细胞中的作用。 重塑和内皮激活。我们将：A）测量TLR 2、TLR 4和TLR 3的mRNA和蛋白质表达 肺动脉内皮细胞（PAEC）和肺动脉平滑肌细胞（PASMC）的愤怒， IPAH和非PAH患者。B）测定HMGB1对PAEC和PASMC增殖、迁移、 和凋亡。C）确定HMGB1对PAEC活化的影响。D）使用siRNA来确定特异性 单个PRR在介导HMGB1对PAEC和PASMC的作用中的作用。这些数据将提供关键 HMGB1和PRR在促进血管重塑和内皮细胞增殖中作用的机制证据 PAH中的激活。拟议的研究是基于强大的初步数据的基础上。它旨在 将我们在动物中的初步发现扩展到人类，并进一步研究DAMP和PRR的作用， PAH先天免疫反应的关键介质。在完成拟议的研究后，我们将有 为1）进一步研究先天免疫在PAH中的作用和2）识别新的 与先天免疫相关的治疗靶点，用于治疗PAH。

Abstract Damage-associated molecular pattern molecules (DAMPs) are intracellular molecules that are actively secreted or passively released by damaged cells. Once released, DAMPs promote inflammation and tissue repair. The high-mobility box group 1 (HMGB1) protein is a prototypic and highly conserved DAMP which acts as a pro-inflammatory cytokine by activating pattern recognition receptors (PRRs) including Toll-Like Receptor 2 (TLR2), TLR4, and the Receptor for Advanced Glycation End Products (RAGE). Evidence suggests that in response to persistent tissue injury, HMGB1 acts as a key initiating molecule of innate immunity, orchestrating inflammation and tissue remodeling. Preliminary data implicate HMGB1, TLR2 and TLR4 in the development of pulmonary arterial hypertension (PAH) in animal and tissue culture models. The central hypothesis of this proposal is that HMGB1, via activation of PRRs, is a key mediator of innate immunity and contributes to endothelial activation and vascular remodeling in PAH. Two specific aims are proposed to test this hypothesis. Specific aim 1 will focus on characterizing the expression and location of HMGB1 and PRRs in idiopathic PAH (IPAH) and non-PAH patient samples. We will use immunostaining to localize HMGB1, TLR2, TLR4, and RAGE expression to specific lung structures and cell types, focusing on areas of vascular remodeling. We will quantify the lung-specific expression of HMGB1, TLR2, TLR4. We will use ELISA and Luminex assays to quantify cytokines and chemokines related to activation of the innate immune system as well as HMGB1 in plasma and lung from IPAH and non-PAH patients. These data will correlate the location- specific DAMP release, PRR-expression pattern and level and pro-inflammatory cytokine production that are central to our main hypothesis. Specific aim 2 will focus on elucidating a role for HMGB1 and PRRs in vascular remodeling and endothelial activation. We will: A) Measure mRNA and protein expression of TLR2, TLR4, and Rage in pulmonary artery endothelial cells (PAEC) and pulmonary artery smooth muscle cells (PASMC) from IPAH and non-PAH patients. B) Determine the effect of HMGB1 on PAEC and PASMC proliferation, migration, and apoptosis. C) Determine the effect of HMGB1 on PAEC activation. D) Use siRNA to determine a specific role for individual PRRs in mediating the effects of HMGB1 on PAEC and PASMC. These data will provide key mechanistic evidence of a role for HMGB1 and PRRs in promoting vascular remodeling and endothelial activation in PAH. The proposed research is based on a foundation of strong preliminary data. It is designed to extend our preliminary findings in animals to humans and further investigate the role of DAMPs and PRRs as key mediators of the innate immune response in PAH. Upon completion of the proposed research we will have a stronger foundation upon which to 1) further investigate the role innate immunity in PAH and 2) identify novel therapeutic targets related to innate immunity for the treatment of PAH.