PHD2 deletion in endothelial or arterial smooth muscle cells reveals vascular cell type-specific responses in pulmonary hypertension and fibrosis

Hypoxia plays an important regulatory role in the vasculature to adjust blood flow to meet metabolic requirements. At the level of gene transcription, the responses are mediated by hypoxia-inducible factor (HIF) the stability of which is controlled by the HIF prolyl 4-hydroxylase-2 (PHD2). In the lungs hypoxia results in vasoconstriction, however, the pathophysiological relevance of PHD2 in the major arterial cell types; endothelial cells (ECs) and arterial smooth muscle cells (aSMCs) in the adult vasculature is incompletely characterized. Here, we investigated PHD2-dependent vascular homeostasis utilizing inducible deletions of PHD2 either in ECs (Phd2∆ECi) or in aSMCs (Phd2∆aSMC). Cardiovascular function and lung pathologies were studied using echocardiography, Doppler ultrasonography, intraventricular pressure measurement, histological, ultrastructural, and transcriptional methods. Cell intrinsic responses were investigated in hypoxia and in conditions mimicking hypertension-induced hemodynamic stress. Phd2∆ECi resulted in progressive pulmonary disease characterized by a thickened respiratory basement membrane (BM), alveolar fibrosis, increased pulmonary artery pressure, and adaptive hypertrophy of the right ventricle (RV). A low oxygen environment resulted in alterations in cultured ECs similar to those in Phd2∆ECi mice, involving BM components and vascular tone regulators favoring the contraction of SMCs. In contrast, Phd2∆aSMC resulted in elevated RV pressure without alterations in vascular tone regulators. Mechanistically, PHD2 inhibition in aSMCs involved actin polymerization -related tension development via activated cofilin. The results also indicated that hemodynamic stress, rather than PHD2-dependent hypoxia response alone, potentiates structural remodeling of the extracellular matrix in the pulmonary microvasculature and respiratory failure.

Avaliação dos efeitos do inibidor multiquinase sorafenibe sobre as células musculares lisas arteriais de Rattus norvegicus: Estudo in vitro / Evaluation of the effects of the multikinase inhibitor sorafenib on the arterial smooth muscle cells of Rattus norvegicus: An in vitro study

A reestenose arterial é um processo inflamatório que pode ocorrer após colocação de stent por cateterismo. Os stents farmacológicos surgiram para reduzir esse problema e o inibidor multiquinase sorafenibe demonstrou ser um composto com ação efetiva. Este estudo in vitro avaliou os efeitos do sorafenibe sobre a citotoxicidade, migração celular e distribuição das células nas fases do ciclo celular. A linhagem celular de músculo liso de rato A7r5 foi tratada com sorafenibe em concentrações que variaram de 0 a 5 μM. Os efeitos citotóxicos foram avaliados por dois ensaios colorimétricos, MTT e SRB após 24 horas de tratamento. A distribuição das células nas fases do ciclo celular foi avaliada por citometria de fluxo e a capacidade de cicatrização/migração celular pelo ensaio scratch wound assay. Comparado com o controle positivo paclitaxel, o sorafenibe demonstrou um efeito 1,6 vezes maior na redução da proliferação celular. Na avaliação do ciclo celular, o sorafenibe mostrou um bloqueio na fase G0/G1. Além disso, o sorafenibe aumentou o número de A7r5 células na fase sub-G1, sugerindo morte celular. No entanto, no estudo de cicatrização/migração celular, não foi observado efeito quando comparado ao controle negativo. Assim, esses resultados in vitro sugerem que o sorafenibe é eficaz para uso em stents farmacológicos, sugerindo uma continuidade na investigação desse fármaco.

NF-κB/p65 Competes With Peroxisome Proliferator-Activated Receptor Gamma for Transient Receptor Potential Channel 6 in Hypoxia-Induced Human Pulmonary Arterial Smooth Muscle Cells

Objective: Peroxisome proliferator-activated receptor gamma (PPARγ) has an anti-proliferation effect on pulmonary arterial smooth muscle cells (PASMCs) via the transient receptor potential channel (TRPC) and protects against pulmonary artery hypertension (PAH), whereas nuclear factor-kappa B (NF-κB) has pro-proliferation and pro-inflammation effects, which contributes to PAH. However, the association between them in PAH pathology remains unclear. Therefore, this study aimed to investigate this association and the mechanisms underlying TRPC1/6 signaling-mediated PAH.Methods: Human pulmonary arterial smooth muscle cells (hPASMCs) were transfected with p65 overexpressing (pcDNA-p65) and interfering plasmids (shp65) and incubated in normal and hypoxic conditions (4% O2 and 72 h). The effects of hypoxia and p65 expression on cell proliferation, invasion, apoptosis, [Ca2+]i, PPARγ, and TRPC1/6 expression were determined using Cell Counting Kit-8 (CCK-8), Transwell, Annexin V/PI, Fura-2/AM, and western blotting, respectively. In addition, the binding of p65 or PPARγ proteins to the TRPC6 promoter was validated using a dual-luciferase report assay, chromatin-immunoprecipitation-polymerase chain reaction (ChIP-PCR), and electrophoretic mobility shift assay (EMSA).Results: Hypoxia inhibited hPASMC apoptosis and promoted cell proliferation and invasion. Furthermore, it increased [Ca2+]i and the expression of TRPC1/6, p65, and Bcl-2 proteins. Moreover, pcDNA-p65 had similar effects on hypoxia treatment by increasing TRPC1/6 expression, [Ca2+]i, hPASMC proliferation, and invasion. The dual-luciferase report and ChIP-PCR assays revealed three p65 binding sites and two PPARγ binding sites on the promoter region of TRPC6. In addition, hypoxia treatment and shPPARγ promoted the binding of p65 to the TRPC6 promoter, whereas shp65 promoted the binding of PPARγ to the TRPC6 promoter.Conclusion: Competitive binding of NF-κB p65 and PPARγ to TRPC6 produced an anti-PAH effect.

Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.

EXPRESS: Ubiquitin‑specific protease 7 mediates platelet derived growth factor-induced pulmonary arterial smooth muscle cells proliferation

Pulmonary arterial hypertension (PAH) is a devastating pulmonary vascular disease, in which the pathogenesis is complicated and unclear. Pulmonary arterial smooth muscle cells (PASMCs) proliferation is a key pathological feature of PAH. It has been shown that ubiquitin-specific protease 7 (USP7) is involved in cancer cell proliferation via deubiquitinating and stabilizing E3 ubiquitin ligase mouse double minute 2 (MDM2). However, the effect of USP7 and MDM2 on platelet derived growth factor (PDGF) -induced PASMCs proliferation is uncertain. This study aims to explore this issue. Our results indicated that PDGF up-regulated USP7 protein expression and stimulated PASMCs proliferation; this was accompanied with the increase of MDM2, forkhead box O4 (FoxO4) reduction and elevation of CyclinD1. While prior transfection of USP7 siRNA blocked PDGF-induced MDM2 up-regulation, FoxO4 down-regulation, increase of CyclinD1 and cell proliferation. Pre-depletion of MDM2 by siRNA transfection reversed PDGF-induced reduction of FoxO4, up-regulation of CyclinD1 and PASMCs proliferation. Furthermore, pre-treatment of cells with proteasome inhibitor MG-132 also abolished PDGF-induced FoxO4 reduction, CyclinD1 elevation and cell proliferation. Our study suggests that USP7 up-regulates MDM2, which facilitates FoxO4 ubiquitinated degradation, and subsequently increases the expression of CyclinD1 to mediate PDGF-induced PASMCs proliferation.