Utilization of PHBI resources to study the role of Innate Immunity in Idiopathic

描述（申请人提供）：损伤相关分子模式分子（DAMP）是受损细胞主动分泌或被动释放的细胞内分子。一旦释放，DAMP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，其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））作为促炎细胞因子发挥作用。有证据表明，在对持续性组织损伤的反应中，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在动物和组织培养模型中参与肺动脉高压（PAH）的发生。该建议的中心假设是，HMGB1通过激活PRR，是先天免疫的关键介质，并有助于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假设。具体目标1将侧重于表征特发性PAH（IPAH）和非PAH患者样本中HMGB1和PRR的表达和位置。我们将使用免疫染色定位HMGB1，TLR2，TLR4和HMGB1表达到特定的肺结构和细胞类型，重点是血管重塑的领域。我们将定量HMGB1、TLR2、TLR4的肺特异性表达。我们将使用ELISA和Luminex测定来定量IPAH和非PAH患者血浆和肺中与先天免疫系统激活相关的细胞因子和趋化因子以及HMGB1。这些数据将与位置特异性DAMP释放、PRR表达模式和水平以及促炎细胞因子产生相关，这些是我们主要假设的核心。具体目标2将集中于阐明HMGB1和PRR在血管重塑和内皮激活中的作用。我们将：A）测量来自IPAH和非PAH患者的肺动脉内皮细胞（PAEC）和肺动脉平滑肌细胞（PASMC）中TLR 2、TLR 4和Rage的mRNA和蛋白质表达。B）确定HMGB1对PAEC和PASMC增殖、迁移和凋亡的影响。C）确定HMGB1对PAEC活化的影响。D）使用siRNA来确定单个PRR在介导HMGB1对PAEC和PASMC的作用中的特定作用。这些数据将为HMGB1和PRR在PAH中促进血管重塑和内皮激活的作用提供关键机制证据。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作为PAH先天免疫反应的关键介质的作用。在完成拟议研究后，我们将有一个更坚实的基础，在此基础上1）进一步研究先天免疫在PAH中的作用，2）确定与先天免疫相关的新治疗靶点，用于治疗PAH。 公共卫生相关性：尽管出现了新的治疗方法，肺动脉高压患者的预后仍然很差。本研究项目的目的是利用肺动脉高压突破计划中的人体组织来研究先天免疫系统在肺动脉高压中的作用。完成拟议的研究将增加我们对肺动脉高压分子发病机制的理解，并有可能为治疗这种毁灭性疾病定义新的治疗靶点。(End摘要）

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Despite the advent of new therapies the prognosis for pulmonary hypertension patients remains poor. The goal of this research project is utilize human tissue from the Pulmonary Hypertension Breakthrough Initiative to investigate the role of the innate immune system in pulmonary hypertension. Completion of the proposed research will increase our understanding of the molecular pathogenesis of pulmonary hypertension and has the potential to define novel therapeutic targets for the treatment of this devastating disease. (End of Abstract)