Abstract
Introduction: Pulmonary arterial hypertension (PAH) is characterized by complex remodeling of pulmonary arteries. In addition to impaired bone morphogenic protein (BMP) signaling, deficiency of the cell cycle regulator p53 has been implicated in PAH pathogenesis. Endothelial cells (ECs) from PAH patients have reduced levels of toll-like receptor 3 (TLR3), impairing EC function. This study investigated whether the p53, TLR3 and BMP pathways regulate endothelial phenotype and function in PAH. Methods: Human pulmonary artery ECs (PAECs) and rat CD117 + ECs were used. Knockdown experiments were carried out by targeting p53 and TLR3. Cells were further treated with the MDM2 antagonist Nutlin 3a, reducing p53 degradation, and with the TLR3 agonist Poly(I:C). Rats received CD117 + EC clones or SU5416 and were subjected to chronic hypoxia and then treated with Nutlin-3a, Poly(I:C) or vehicle. Results: We detected reduced p53 levels in PAECs from PAH patients. Clonal expansion of rat CD117 + ECs resulted in hyperproliferative cells with reduced p53 and TLR3 levels and evidence for histone methylation. Then, knockdown of p53 or TLR3 increased tube formation in surviving human and rat ECs. In addition, knockdown or activation of p53 indicated regulation of TLR3 mRNA by p53. TLR3 knockdown reduced expression of BMP2, whereas the TLR3 agonist Poly(I:C) induced BMP receptor 2 expression. Upstream, reduced p53 degradation by Nutlin 3a not only increased TLR3, but also BMP2 expression and increased BMP signaling in rat ECs. Interestingly, clonal selection resulted in EC clones with high BMP2 expression. TLR3 knockdown caused partial endothelial mesenchymal transition with increased expression of Snail, which was ameliorated by Poly(I:C). In vivo, Nutlin-3a treatment reduced PH while increasing TLR3 expression in rats. Poly(I:C) reduced PH and Smad2 phosphorylation in rats, and the protective effects were abolished in TRIF KO mice. Conclusion: The RNA receptor and p53 transcriptional target TLR3 regulates the BMP pathway in ECs and this interaction may explain some of the protective effects of TLR3 stimulation in PH. However as clonally selected ECs retain high BMP2 expression, differences between EC subpopulations can be suggested and are under investigation.
Published Version
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