MicroRNA-146-5p Promotes Pulmonary Artery Endothelial Cell Proliferation under Hypoxic Conditions through Regulating USP3

Objective. MicroRNAs play a pivotal role in the progression of pulmonary hypertension (PAH). Although microRNA-146-5p is specifically expressed in many diseases, but in PAH, its role remains elusive. Patients and Methods. 30 patients with PAH and 20 healthy volunteers in our hospital were enrolled, and their serum samples were extracted for the detection of microRNA-146-5p and ubiquitin specific protease 3 (USP3) expression. In addition, the interaction between microRNA-146-5p and USP3 was examined by luciferase reporting assay. Furthermore, the potential mechanism was explored by cell counting kit-8 (CCK-8), 5-ethynyl-2 ′ -deoxyuridine (EdU), and Western blotting experiments. Results. It was found that microRNA-146-5p was higher in PAH patients than in healthy volunteers. Meanwhile, in hypoxia-induced human pulmonary artery endothelial cell lines (HPAECs), microRNA-146-5p expression was dramatically downregulated while USP3 protein expression was conversely upregulated. Under hypoxic conditions, microRNA-146-5p mimics was able to prompt the growth of HPAECs. In addition, after overexpression of microRNA-146-5p, luciferase reporting assay revealed a reduced luciferase activity of the reporter gene containing the USP3 3 ′ -untranslated region, and a reduction of USP3 protein expression was also confirmed. However, USP3 overexpression partially attenuated the impact of upregulated microRNA-146-5p on the proliferation capacity of HPAECs. Conclusions. MicroRNA-146-5p was able to enhance the proliferation ability of HPAEC cells under hypoxic conditions through targeting USP3, suggesting the microRNA-146-5p/USP3 axis may act as a target for PAH treatment.

Effects of varying shear stress profiles on the regulation of pulmonary artery endothelial cell gene expression: a focus on selected mediators of vascular tone, inflammation and angiogenesis

Background Pulmonary arterial hypertension (PAH) is a complex pathology characterized by obliterative vascular remodeling that leads to right heart failure and death. Predisposition to PAH is associated with mutations in the BMPR2 gene in approximately 70–80% of familial cases and around 30% for that of sporadic PAH. The study of the pathogenetic basis of PAH is often performed in static endothelial cultures. Such two-dimensional, isolated cell microenvironments fail to consider the heterogeneity in mechanical stress acting on endothelial cells in various regions of the pulmonary vascular tree. In the remodeled pulmonary vasculature, low and oscillatory shear stress is observed in the proximal pulmonary artery with high shear stress in distal pre-capillary pulmonary arterioles. Therefore, the impact of varied shear profiles (including both laminar and oscillatory flow) on pulmonary artery endothelial cell (and that of BMPR2-deplete) gene expression of common vasoactive (EDN1, ENOS), proinflammatory (IL6, IL8) and angiogenic mediators (ANG2, VEGFA), are poorly described. Purpose To evaluate the effects of shear stress magnitude, including unidirectional and oscillatory flow on BMPR2-knockdown human pulmonary artery endothelial cell (HPAEC) gene expression of EDN1, ENOS, IL6, IL8, ANG2 and VEGFA. Methods HPAECs were transfected with siRNA directed against BMPR2 (siB2) or with a non-targeting control (siCon). Cells were exposed to 10 hours of laminar or oscillatory flow (1Hz; 1.5 dyn/cm2, 15 dyn/cm2 or 90 dyn/cm2) using a parallel-plate fluid flow chamber system. Measurement of mRNA expression was performed using qPCR. Results Shear stress intensity and flow type (unidirectional and oscillatory) mediated diverse effects on HPAEC gene expression across the markers studied. Changes in gene expression were calculated relative to that of static siCon-transfected HPAECs and in such a manner are summarized as fold changes in the table below. Asterisks are shown where significant fold differences are reported. *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001. aP≤0.05, bP≤0.05, cP≤0.05, denote comparisons between groups. Of note, no significant differences in gene expression were observed between static siCon and static siB2. Conclusions For the markers studied, different magnitudes of shear stress and flow profiles (together with BMPR2 loss) exhibit varied patterns of gene expression in the pulmonary vascular endothelium. As such, this illustrates the need for wider study of in vitro endothelial-shear stress interactions in understanding mechanisms of remodeling in PAH. FUNDunding Acknowledgement Type of funding sources: None. Table 1

Circulating Plasma miRNA and Clinical/Hemodynamic Characteristics Provide Additional Predictive Information About Acute Pulmonary Thromboembolism, Chronic Thromboembolic Pulmonary Hypertension and Idiopathic Pulmonary Hypertension

Idiopathic pulmonary artery hypertension (IPAH), chronic thromboembolic pulmonary hypertension (CTEPH), and acute pulmonary embolism (APTE) are life-threatening cardiopulmonary diseases without specific surgical or medical treatment. Although APTE, CTEPH and IPAH are different pulmonary vascular diseases in terms of clinical presentation, prevalence, pathophysiology and prognosis, the identification of their circulating microRNA (miRNAs) might help in recognizing differences in their outcome evolution and clinical forms. The aim of this study was to describe the APTE, CTEPH, and IPAH-associated miRNAs and to predict their target genes. The target genes of the key differentially expressed miRNAs were analyzed, and functional enrichment analyses were carried out. The miRNAs were detected using RT-PCR. Finally, we incorporated plasma circulating miRNAs in baseline and clinical characteristics of the patients to detect differences between APTE and CTEPH in time of evolution, and differences between CTEPH and IPAH in diseases form. We found five top circulating plasma miRNAs in common with APTE, CTEPH and IPAH assembled in one conglomerate. Among them, miR-let-7i-5p expression was upregulated in APTE and IPAH, while miRNA-320a was upregulated in CTEP and IPAH. The network construction for target genes showed 11 genes regulated by let-7i-5p and 20 genes regulated by miR-320a, all of them regulators of pulmonary arterial adventitial fibroblasts, pulmonary artery endothelial cell, and pulmonary artery smooth muscle cells. AR (androgen receptor), a target gene of hsa-let-7i-5p and has-miR-320a, was enriched in pathways in cancer, whereas PRKCA (Protein Kinase C Alpha), also a target gene of hsa-let-7i-5p and has-miR-320a, was enriched in KEGG pathways, such as pathways in cancer, glioma, and PI3K-Akt signaling pathway. We inferred that CTEPH might be the consequence of abnormal remodeling in APTE, while unbalance between the hyperproliferative and apoptosis-resistant phenotype of pulmonary arterial adventitial fibroblasts, pulmonary artery endothelial cell and pulmonary artery smooth muscle cells in pulmonary artery confer differences in IPAH and CTEPH diseases form. We concluded that the incorporation of plasma circulating let-7i-5p and miRNA-320a in baseline and clinical characteristics of the patients reinforces differences between APTE and CTEPH in outcome evolution, as well as differences between CTEPH and IPAH in diseases form.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.