Pulmonary arterial hypertension (PAH) is a severe disease affecting the pulmonary arteries, causing increased blood pressure due to narrowing of the pulmonary artery lumen. Aberrant proliferation of endothelial cells (ECs) and smooth muscle cells (SMCs), along with a dysregulation of the immune response, contributes to arterial remodeling. 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. In lung tissue from control patients and patients with PAH, MDA5 immunoreactivity was widely distributed throughout the pulmonary artery wall. After exposing whole body MDA5(-/-) mice to the chronic hypoxia/SU5416 (Hx/Su) protocol, we detected reduced right ventricular systolic pressure and pulmonary artery media wall thickness compared with wild-type mice. Proinflammatory mediators, interferon-regulated genes, and perivascular accumulation of CD11b(+) myeloid cells were decreased in the lung tissue of Hx/Su-exposed MDA5(-/-) mice. In cultured human pulmonary artery ECs, MDA5 gene silencing disrupted DNA synthesis and angiogenic network formation. Bulk RNA sequencing analysis revealed the differential expression of 2,533 genes, affecting gene ontologies such as immune response, cell cycle, and cholesterol metabolism. Analysis of a publicly available single-cell RNA-sequence dataset indicates increased expression of MDA5 in monocytes/macrophages in pulmonary arteries from patients with PAH, which is associated with an interferon gene signature. Knockdown of MDA5 in macrophage-like cells reduced the type I interferon gene signature. Our data suggest a protective effect of whole body MDA5 knockout in mice, which may be due to reduced immune dysregulation. NEW & NOTEWORTHY This study identifies the role of melanoma differentiation-associated protein 5 (MDA5) in the development of pulmonary hypertension and pulmonary artery muscularization. The results indicate that MDA5 is important for pulmonary artery mural cell function, and that MDA5 knockout reduces immune dysregulation by decreasing a type I interferon gene signature in immune cells, particularly monocytes and macrophages.

Background: Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung endothelial dysfunction, but the exact cause remains unknown. We performed in-depth bulk transcriptomic analysis of human lung microvascular endothelial cells (HLMVECs) and pulmonary artery endothelial cells (PAECs) following in vitro SARS-CoV-2 infection as surrogates to compartment-specific transcriptome changes. Methods: HLMVECs and PAECs were infected with a multiplicity of infection (MOI) of 2 of SARS-CoV-2. After 6h, the cells were removed for RNA isolation, followed by bulk RNA sequencing (RNA-seq), read trimming and alignment to the human genome, and analysis of differentially expressed genes (DEGs). Ingenuity pathway analysis was conducted on DEGs. Infection was verified using quantitative real-time PCR (qRT-PCR) of SARS-CoV-2 Nucleocapsid (N), envelope (E), and spike (S) gene mRNAs prior to sequencing. Results: HLMVECs and PAECs showed amplification of SARS-CoV-2 N, E, and S protein mRNA following infection. HLMVECs had 1097 DEGs, with 576 downregulated DEGs and 521 upregulated DEGs. Main gene ontology (GO) cell functions that were affected in SARS-CoV-2 infected HLMVECs belonged to the categories “cell cycle, survival, and DNA repair”, “Cell movement and trafficking” and “Development”. Surprisingly, immune function was only represented in a fraction of the genes. In a detailed analysis of pathway activation, we discovered that many pathways affecting stem cell signaling, cell death, and immune signaling were predicted to be inhibited. Few pathways were predicted to be activated, including Coagulation System and PTEN Signaling. In contrast, PAECs exhibited 3813 DEGs, with 1756 downregulated and 2057 upregulated DEGs. Here, the main GO cell functions were similar to HLMVECs. Also, similar to HLMVECs, most identified pathways were predicted to be inhibited. However, the clustering of the pathways was different from HLMVECs and focused on mitochondrial function, cancer cell signaling, and endocrine signaling. Pathways with predicted activation included EIF2 Signaling, oxidative phosphorylation, and RHOA Signaling. Conclusion: Lung ECs exhibit fundamental transcriptomic changes in response to SARS-CoV-2 infection, demonstrating a predominant pattern of predicted inhibition. There are distinct differences between microvascular and pulmonary artery ECs that could explain functional differences. Further verification in vivo is needed to determine the translational value of our findings.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from patients with PAH. Endothelial haploinsufficiency of RAB7 caused spontaneous pulmonary hypertension (PH) in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA-Seq, and RAB7-silenced ECs showed impaired angiogenesis and expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the predegradation level. Furthermore, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in rats with chronic hypoxia/SU5416. In conclusion, we demonstrate for the first time to our knowledge the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation to be a potential therapeutic strategy in a preclinical model of PH.

Introduction: Pulmonary arterial hypertension (PAH) is a progressive and lethal disease. The current treatment options do not target pulmonary artery endothelial cell (PAEC) dysfunction. We have previously shown a role for the endosomal double stranded (ds) RNA receptor toll-like receptor 3 (TLR3) and its regulation via interleukin-10 (IL-10) in promoting PAEC dysfunction and experimental pulmonary hypertension (PH). Yet the role of the cytosolic dsRNA receptor melanoma differentiation factor 5 (MDA-5) in endothelial dysfunction is not known. Hypothesis: We hypothesized that a deficient MDA-5/IL-10 receptor A (IL-10RA) signaling axis expression exists in PAH PAECs as a potential cause of endothelial dysfunction. Goals: Our goal is to test the role of a potential new axis of MDA-5 and IL10RA in EC homeostasis and pulmonary hypertension. Methods: Western blot analysis of control and PAH PAECs and PA smooth muscle cells (SMCs) was conducted. Gene silencing was done using siRNA targeting MDA-5 and IL-10RA, followed by bulk RNA sequencing. Apoptosis was tested using Annexin V binding. IL-10 whole body knockout (IL-10 -/- ) and endothelial-specific IL-10RA haploinsufficient mice (IL-10RA fl/wt Cdh5-Cre + ) and appropriate controls were exposed to chronic hypoxia and SU5416 (Hx/Su), followed by hemodynamics. Results: Our data show a reduction of MDA-5 in PAECs, but not in PASMCs. PAH PAECs further had reduced IL-10RA expression and application of dsRNA elevated IL-10RA expression in PAH PAECs. Knockdown of MDA-5 and IL-10RA caused differential expression of 2533 and 2638 genes, respectively. Comparative analysis revealed an overlap of differentially expressed genes (DEGs) in canonical pathways including reduced nitric oxide, VEGF, Apelin and Integrin signaling. Comparison also yielded overlapping DEGs for reduced cell survival, impaired cytoskeleton organization and migration. Gene silencing of MDA-5 resulted in increased PAEC apoptosis. IL-10 -/- and IL-10RA fl/wt Cdh5-Cre + mice showed exaggerated PH. Conclusions: Our data indicate an impaired cytosolic dsRNA receptor MDA-5 and IL-10RA axis in PAH PAECs which contributes to endothelial dysfunction and PH. Experiments are underway to identify the detailed mechanisms underlying our findings.

Pulmonary arterial hypertension (PAH) features pathogenic and abnormal endothelial cells (ECs), and one potential origin is clonal selection. We studied the role of p53 and toll-like receptor 3 (TLR3) in clonal expansion and pulmonary hypertension (PH) via regulation of bone morphogenetic protein (BMPR2) signaling. ECs of PAH patients had reduced p53 expression. EC-specific p53 knockout exaggerated PH, and clonal expansion reduced p53 and TLR3 expression in rat lung CD117+ ECs. Reduced p53 degradation (Nutlin 3a) abolished clonal EC expansion, induced TLR3 and BMPR2, and ameliorated PH. Polyinosinic/polycytidylic acid [Poly(I:C)] increased BMPR2 signaling in ECs via enhanced binding of interferon regulatory factor-3 (IRF3) to the BMPR2 promoter and reduced PH in p53-/- mice but not in mice with impaired TLR3 downstream signaling. Our data show that a p53/TLR3/IRF3 axis regulates BMPR2 expression and signaling in ECs. This link can be exploited for therapy of PH.

Abstract Pulmonary arterial hypertension (PAH) is a progressive, devastating disease, and its main histological manifestation is an occlusive pulmonary arteriopathy. One important functional component of PAH is aberrant endothelial cell (EC) function including apoptosis‐resistance, unchecked proliferation, and impaired migration. The mechanisms leading to and maintaining physiologic and aberrant EC function are not fully understood. Here, we tested the hypothesis that in PAH, ECs have increased expression of the transmembrane protein integrin‐β5, which contributes to migration and survival under physiologic and pathological conditions, but also to endothelial‐to‐mesenchymal transition (EnMT). We found that elevated integrin‐β5 expression in pulmonary artery lesions and lung tissue from PAH patients and rats with PH induced by chronic hypoxia and injection of CD117+ rat lung EC clones. These EC clones exhibited elevated expression of integrin‐β5 and its heterodimerization partner integrin‐αν and showed accelerated barrier formation. Inhibition of integrin‐ανβ5 in vitro partially blocked transforming growth factor (TGF)‐β1‐induced EnMT gene expression in rat lung control ECs and less in rat lung EC clones and human lung microvascular ECs. Inhibition of integrin‐ανβ5 promoted endothelial dysfunction as shown by reduced migration in a scratch assay and increased apoptosis in synergism with TGF‐β1. In vivo, blocking of integrin‐ανβ5 exaggerated PH induced by chronic hypoxia and CD117+ EC clones in rats. In summary, we found a role for integrin‐ανβ5 in lung endothelial survival and migration, but also a partial contribution to TGF‐β1‐induced EnMT gene expression. Our results suggest that integrin‐ανβ5 is required for physiologic function of ECs and lung vascular homeostasis.

Cardiovascular sequelae of severe acute respiratory syndrome (SARS) coronavirus-2 (CoV-2) disease 2019 (COVID-19) contribute to the complications of the disease. One potential complication is lung vascular remodeling, but the exact cause is still unknown. We hypothesized that endothelial TLR3 insufficiency contributes to lung vascular remodeling induced by SARS-CoV-2. In the lungs of COVID-19 patients and SARS-CoV-2 infected Syrian hamsters, we discovered thickening of the pulmonary artery media and microvascular rarefaction, which were associated with decreased TLR3 expression in lung tissue and pulmonary artery endothelial cells (ECs). In vitro , SARS-CoV-2 infection reduced endothelial TLR3 expression. Following infection with mouse-adapted (MA) SARS-CoV-2, TLR3 knockout mice displayed heightened pulmonary artery remodeling and endothelial apoptosis. Treatment with the TLR3 agonist polyinosinic:polycytidylic acid reduced lung tissue damage, lung vascular remodeling, and endothelial apoptosis associated with MA SARS-CoV-2 infection. In conclusion, repression of endothelial TLR3 is a potential mechanism of SARS-CoV-2 infection associated lung vascular remodeling and enhancing TLR3 signaling is a potential strategy for treatment.