Abstract 4365999: Knockout of the cytosolic RNA receptor melanoma differentiation-associated protein 5 reduces pulmonary hypertension

Background: Pulmonary arterial hypertension (PAH) is a severe disease affecting the pulmonary arteries, causing increased blood pressure due to lumen narrowing. Aberrant proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs), along with pro-inflammatory mediators and perivascular immune cell accumulation, contributes to arterial remodeling. Hypothesis: We hypothesized that the cytosolic RNA receptor melanoma differentiation-associated protein 5 (MDA5) contributes to PAH by dysregulating pulmonary vascular cell function and immune cell response. Methods: Lung tissue sections from control and PAH patients underwent immunofluorescence staining and confocal microscopy. siRNA targeting IFIH1 (gene for MDA5) was used for gene silencing in pulmonary artery ECs. BrdU incorporation and DNA staining analyzed the cell cycle; a Matrigel assay examined network formation, and a gap closure assay measured migration . Ifih1 -/- mice were exposed to the hypoxia/SU5416 (HX/Su) protocol for 21 days. Media wall thickness was quantified in histological sections. Immunohistochemistry assessed perivascular immune cell accumulation. PCR from lung tissue showed pro-inflammatory mRNA expression. Results: In lung tissue from control patients, MDA5 immunoreactivity was widely distributed throughout the pulmonary artery wall, with the strongest expression observed in immune cells. In the remodeled pulmonary arteries of PAH patients, MDA5 immunostaining was reduced in ECs. In cultured human pulmonary artery ECs, gene silencing of IFIH1 disrupted cell cycle progression, network formation, and gap closure. Bulk RNA sequencing analysis revealed the differential expression of 2,533 genes, affecting gene ontologies such as cell death and survival, as well as cell cycle. After exposing whole-body Ifih1 -/- mice to the Hx/Su protocol, we detected reduced right ventricular systolic pressure and pulmonary artery media wall thickness compared to wild-type mice. Pro-inflammatory mediators, interferon-regulated genes, and perivascular accumulation of CD11b-positive myeloid cells were decreased in the lung tissue of Hx/Su-exposed Ifih1 -/- mice. Conclusions: Our data suggest a protective effect of whole-body MDA5 knockout in mice, which may be due to a reduction in pro-inflammatory mediators and perivascular immune cell accumulation. Whether a decrease of MDA5 in ECs promotes or protects against PH will need further investigation.