Role of plasma membrane ATPase4 in the pathophysiology of pulmonary arterial hypertension

Background Pulmonary Arterial Hypertension (PAH) is a progressive vascular disease characterised by pulmonary vascular remodelling, inflammation, and excessive apoptosis of pulmonary arterial endothelial cells (PAECs). Elevated levels of pro- inflammatory cytokines such as TNF-α are a hallmark of PAH and are known to induce endothelial injury. Calcium dysregulation contributes to apoptotic signalling, suggesting that calcium transporters like Plasma Membrane Calcium ATPase 4 (PMCA4) may play an important regulatory role. PMCA4, encoded by ATP2B4, maintains calcium homeostasis and modulates signalling pathways through its scaffolding function at the plasma membrane. This study investigates the contribution of PMCA4 to inflammation-induced endothelial apoptosis and its regulation at the RNA level. Methods and Results To determine the relevance of PMCA4 in PAH, the expression of PAH-associated receptors (BMPR2, ALK1, TGFβR1, Endoglin) was evaluated in lung RNA from PMCA4 wild-type and knockout mice by qPCR, showing no significant differences. In vitro, stimulation of human PAECs and HUVECs with TNF-α and cycloheximide (CHX) significantly reduced PMCA4 mRNA and protein expression. Silencing PMCA4 using siRNA markedly enhanced TNF-α + CHX–induced apoptosis, confirmed by increased TUNEL staining and caspase-3 cleavage. In contrast, BMP9-induced Smad1/5 and TGF-β–induced Smad2 phosphorylation remained unaltered, indicating that PMCA4’s anti-apoptotic role is independent of canonical Smad signaling. A luciferase reporter construct harboring the PMCA4 3′UTR was engineered to assess post-transcriptional regulation. Luciferase assays demonstrated that TNF-α destabilizes PMCA4 transcripts through specific 3′UTR elements. Cloning and validation of these constructs confirmed the integrity of the engineered vector and its recombination efficiency in HEK293T cells, establishing a reliable system for RNA stability studies. Conclusion The downregulation of PMCA4 by TNF-α promotes endothelial apoptosis through caspase-dependent mechanisms without altering BMP9, TGF-β, or MAPK pathways. Post-transcriptional destabilization of PMCA4 mRNA via its 3′UTR represents a novel mechanism of inflammatory regulation in the pulmonary endothelium. Together, these findings highlight PMCA4 as a key protective factor against inflammation-induced endothelial injury and a potential therapeutic target in the pathophysiology of PAH.

Citation

David Onesimu, E,L.S. (2026) Role of plasma membrane ATPase4 in the pathophysiology of pulmonary arterial hypertension. University of Wolverhampton. https://wlv.openrepository.com/handle/2436/626400

Publisher

University of Wolverhampton

URI

https://wlv.openrepository.com/handle/2436/626400

Type

Thesis or dissertation

Language

en

Description

A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.

Collections

Theses and Dissertations