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.

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.

Ca2+ Mediates HIF-dependent Upregulation of Aquaporin 1 in Pulmonary Arterial Smooth Muscle Cells

ABSTRACTProlonged exposure to hypoxia causes structural remodeling and sustained contraction of the pulmonary vasculature, resulting in the development of pulmonary hypertension. Both pulmonary arterial smooth muscle cell (PASMC) proliferation and migration contribute to the vascular remodeling. We previously showed that the protein expression of aquaporin 1 (AQP1), a membrane water channel protein, is elevated in PASMCs during following in vivo or in vitro exposure to hypoxia. Studies in other cell types suggest that AQP1 is a direct transcriptional target of hypoxia inducible factor (HIF)-1. Moreover, we and others have shown that an increase in intracellular calcium concentration ([Ca2+]i) is a hallmark of hypoxic exposure in PASMCs. Thus, we wanted to determine whether HIF regulates AQP1 in PASMCs and, if so, whether the process occurred via transcriptional regulation or was Ca2+-dependent. PASMCs were exposed to hypoxia, incubated with DMOG, which inhibits HIFα protein degradation or infected with constitutively active forms of HIF-1α or HIF-2α. Hypoxia, DMOG and HIF1/2α produced a time-dependent increase in AQP1 protein, but not mRNA. Interestingly, incubation with increasing HIF1/2α levels and DMOG increased [Ca2+]i in PASMCs, and this elevation was prevented by the voltage-gated Ca2+ channel inhibitor, verapamil (VER) and nonselective cation channel inhibitor SKF96365 (SKF). VER and SKF also blocked upregulation of AQP1 protein by DMOG or HIF1/2α, but had no effect on expression of GLUT1, a canonical HIF transcriptional target. Silencing of AQP1 abrogated increases in PASMC migration and proliferation induced by HIF1/2α, suggesting induction of AQP1 protein by HIF1/2α has a functional outcome in these cells. Thus, our results show that contrary to reports in other cell types, in PASMCs, AQP1 does not appear to be a direct target for HIF transcriptional regulation. Instead, AQP1 protein may be upregulated by a mechanism involving HIF-dependent increases in [Ca2+]i.

Mechanical programming of arterial smooth muscle cells in health and ageing

Arterial smooth muscle cells (ASMCs), the predominant cell type within the arterial wall, detect and respond to external mechanical forces. These forces can be derived from blood flow (i.e. pressure and stretch) or from the supporting extracellular matrix (i.e. stiffness and topography). The healthy arterial wall is elastic, allowing the artery to change shape in response to changes in blood pressure, a property known as arterial compliance. As we age, the mechanical forces applied to ASMCs change; blood pressure and arterial wall rigidity increase and result in a reduction in arterial compliance. These changes in mechanical environment enhance ASMC contractility and promote disease-associated changes in ASMC phenotype. For mechanical stimuli to programme ASMCs, forces must influence the cell’s load-bearing apparatus, the cytoskeleton. Comprised of an interconnected network of actin filaments, microtubules and intermediate filaments, each cytoskeletal component has distinct mechanical properties that enable ASMCs to respond to changes within the mechanical environment whilst maintaining cell integrity. In this review, we discuss how mechanically driven cytoskeletal reorganisation programmes ASMC function and phenotypic switching.