The role of innate immunity in idiopathic pulmonary arterial hypertension

摘要 损伤相关的分子模式分子(DAMP)是细胞内活跃的分子。 由受损的细胞分泌或被动释放。一旦释放，湿气就会促进炎症和组织 修理。高迁移率盒组1(HMGB1)蛋白是一种典型的高度保守的DAMP，其作用是 通过激活包括Toll样受体在内的模式识别受体(PRRs)作为促炎细胞因子 2(TLR2)、TLR4和晚期糖基化终产物受体(RAGE)。有证据表明， 对持续性组织损伤的反应，HMGB1作为天然免疫的关键启动分子，协调 炎症和组织重塑。初步数据表明HMGB1、TLR2和TLR4在发育中起作用 在动物和组织培养模型中研究肺动脉高压(PAH)。这一点的中心假设是 有观点认为，HMGB1通过激活PRRs，是先天免疫的关键介体，并有助于 对血管内皮细胞活化和血管重塑的影响。提出了两个具体的目标来测试这一点 假设。具体目标1将集中在表征HMGB1和PRRs的表达和定位 特发性PAH(IPAH)和非PAH患者样本。我们将使用免疫染色来定位HMGB1，TLR2， TLR4和RAGE在特定的肺结构和细胞类型中表达，主要集中在血管区域 改建。我们将对HMGB1、TLR2、TLR4的肺特异性表达进行量化。我们将使用ELISA卡和 Luminex检测与激活天然免疫系统相关的细胞因子和趋化因子 IPH患者和非PAH患者血浆和肺组织中HMGB1的含量也不同。这些数据将把位置关联起来- 特异性湿释放、PRR表达模式和水平以及促炎细胞因子的产生 这是我们主要假设的核心。具体目标2将集中于阐明HMGB1和PRRs在血管中的作用 血管重塑和内皮激活。我们将：a)检测TLR2、TLR4和TLR4的mRNA和蛋白表达 大鼠肺动脉内皮细胞(PAEC)和肺动脉平滑肌细胞(PASMC)中RAGE的表达 IPAH和非PAH患者。B)确定HMGB1对PAEC和PASMC增殖、迁移、 和细胞凋亡。C)确定HMGB1对PAEC激活的影响。D)使用siRNA确定特定的 单个PRRs在调节HMGB1对PAEC和PASMC影响中的作用。这些数据将提供关键 HMGB1和PRRs在促进血管重塑和血管内皮细胞中作用的机制证据 在多环芳烃中激活。拟议的研究是建立在强大的初步数据基础上的。它的设计目的是 将我们在动物身上的初步发现推广到人类，并进一步研究阻尼剂和PRR作为 PAH先天免疫反应的关键介质。在完成拟议的研究后，我们将拥有 1)进一步研究先天免疫在PAH中的作用和2)发现新的 治疗PAH的先天免疫相关靶点。

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.