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

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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.