Endothelial Toll-like Receptor 3 in the pathogenesis and therapy of Pulmonary Arterial Hypertension

相当多的知识表明，血管细胞变得过度增殖和癌症样 肺动脉高压(PAH)。这一概念导致了抗癌药物的临床试验 治疗多环芳烃。但要想成功，就需要了解血管对肺动脉的异常反应。 血管内皮细胞(PAEC)损伤及其在闭塞性动脉病发展中的作用。 这种动脉病变的特征是一系列损伤、内皮细胞凋亡和克隆性选择 异常的ECs。这些内皮细胞表现出抗凋亡和无节制的增殖。它也是至关重要的 将这些发现与促进PAH的EC功能受损联系起来。这种受损的EC功能是 表现为血管生成受损和肺动脉平滑肌细胞(PASMC)调节失调 功能。为了解决我们知识中的这些重要差距，该提案将研究Toll-like的作用 受体3(TLR3)介导的内皮RNA信号转导。RNA信号通路激活促进 生理上的EC功能。TLR3调节病毒和死亡细胞向RNA发出的信号。试点工作 显示PAH内皮细胞TLR3表达减少，从而导致内皮细胞功能缺陷。 RNA信号向维甲酸诱导基因-I和黑色素瘤分化抗原-5的转变 (MDA-5)。这些缺陷促进了PASMC功能的异常和肺动脉高压(PH)。在PAH， TLR3在内皮细胞中的抑制是由于细胞周期调节因子P53的丢失所致。P53和TLR3缺失， 通过克隆选择加压获得高增殖的内皮细胞。双链(DS)RNA是 TLR3、RIG-I和MDA-5的配体。大剂量dsRNA可改善EC功能，降低重度PH。 高剂量但不是低剂量dsRNA通过白介素10(IL-10)介导的TLR3上调 保护内皮细胞的途径。这一途径在PAH中受损。总体假设是，在PAH中， 选择压力会产生P53缺失的内皮细胞，从而导致TLR3抑制。TLR3 缺乏通过失调的RNA信号通路促进PAH。相比之下，高剂量dsRNA 治疗通过IL-10介导的内皮细胞上调改善EC功能并降低PAH TLR3.这一假说将通过以下具体目标进行检验：目标1.确定克隆 P53的选择和缺失抑制了内皮细胞TLR3的表达。目的2.确定内皮细胞的抑制 TLR3通过RIG-I和MDA-5促进重度PH。目的3.检测dsRNA是否通过 IL-10，通过恢复生理性EC功能逆转重度PH。这一假说解释了一个 PAH中内皮功能障碍的研究不足：肺内皮细胞的调节 通过TLR3介导的RNA信号发挥作用。一种基于TLR3的定向恢复策略 生理性EC功能和逆转重度PH将得到发展。这种方法使用脂质体。 具有广泛适用性的载体技术用于肺血管疾病的靶向给药。

A sizable body of knowledge shows that vascular cells become hyper-proliferative and cancer-like in pulmonary arterial hypertension (PAH). This concept has led to clinical trials of anticancer drugs to treat PAH. But success requires to understand the aberrant vascular response to pulmonary arterial endothelial cell (PAEC) injury and how it contributes to the development of the occlusive arteriopathy. This arteriopathy is characterized by a sequence of injury, EC apoptosis and clonal selection of aberrant ECs. These ECs show apoptosis-resistance and unchecked proliferation. It is also crucial to relate these findings to the crippled EC function that promotes PAH. This impaired EC function is shown by impaired angiogenesis and dysregulation of pulmonary artery smooth muscle cell (PASMC) function. To address these important gaps in our knowledge, the proposal will study the role of Toll-like receptor 3 (TLR3)-mediated endothelial RNA signaling. RNA signaling activates pathways promoting physiologic EC function. TLR3 regulates signaling to RNA from viruses and dying cells. Pilot work shows that PAH ECs have reduced expression of TLR3, which causes functional deficits in ECs via shift in RNA signaling to retinoid acid-inducible gene-I (RIG-I) and melanoma differentiation antigen-5 (MDA-5). The deficits promote aberrant PASMC function and pulmonary hypertension (PH). In PAH, repression of TLR3 in ECs results from loss of the cell cycle regulator p53. p53- and TLR3-deficient, hyperproliferative ECs were generated through clonal selection pressure. Double-stranded (ds) RNA is a ligand for TLR3, RIG-I and MDA-5. High dose dsRNA improves EC function and reduces severe PH. High dose, but not low dose dsRNA upregulates TLR3 through an interleukin-10 (IL-10)-mediated pathway that protects ECs. This pathway is impaired in PAH. The overall hypothesis is that in PAH, selection pressure yields p53-deficient endothelium, which causes TLR3 repression. TLR3 deficiency promotes PAH via dysregulated RNA signaling. In contrast, high dose dsRNA therapy improves EC function and reduces PAH via IL-10-mediated upregulation of endothelial TLR3. The hypothesis will be tested with the following specific aims: Aim 1. To identify whether clonal selection and loss of p53 repress TLR3 in ECs. Aim 2. To determine whether repression of endothelial TLR3 promotes severe PH via RIG-I and MDA-5. Aim 3. To test whether dsRNA upregulates TLR3 via IL-10 and reverses severe PH by restoring physiologic EC function. The hypothesis explains an underexplored angle of endothelial dysfunction in PAH: the regulation of pulmonary endothelial function through TLR3-mediated RNA signaling. A targeted strategy based on TLR3 to restore physiologic EC function and reverse severe PH will be developed. This approach uses liposomal carrier technology with broad applicability for targeted drug delivery in lung vascular diseases.