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Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation.

ABSTRACT: We have previously demonstrated increased fibroblast growth factor-2 (FGF-2) expression in a lamb model of increased pulmonary blood flow secondary to congenital heart disease, which may contribute to the associated increases in pulmonary arterial muscularization. However, the mechanisms underlying these increases in FGF-2 expression remain to be identified. Initially, we found that exogenous FGF-2 increased endogenous FGF-2 promoter activity and protein levels in ovine pulmonary arterial smooth muscle cells (PASMC). Furthermore, we found that these increases in FGF-2 expression were mediated by increases in superoxide levels via NADPH oxidase activation. In addition, FGF-2-mediated increases in FGF-2 expression and PASMC proliferation were attenuated by inhibition of phosphatidylinositol 3-kinase, Akt, and NADPH oxidase. Increases in FGF-2 expression could be stimulated by other factors known to increase reactive oxygen species (ROS) signaling in PASMC (endothelin-1 and transforming growth factor-beta1), whereas antioxidants attenuated these increases. Deletion constructs localized the growth factor- and ROS-sensitive region within the proximal 103 bp of the FGF-2 promoter, and sequence analysis identified a putative hypoxia response element (HRE), a DNA binding site for the ROS-sensitive transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha). Stabilization of HIF-1alpha increased FGF-2 promoter activity, whereas mutation of the putative HRE attenuated FGF-2-induced FGF-2 promoter activity. Furthermore, FGF-2 increased HIF-1alpha protein levels and consensus HRE promoter activity in PASMC via antioxidant-sensitive mechanisms. Thus we conclude that FGF-2 can stimulate its own expression in PASMC via NADPH oxidase-mediated activation of ROS-sensitive transcription factors, including HIF-1alpha. This positive feedback mechanism may contribute to pulmonary vascular remodeling associated with increased pulmonary blood flow.

SUBMITTER: Black SM

PROVIDER: S-EPMC3970933 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation.

Black Stephen M SM DeVol Jennifer M JM Wedgwood Stephen S

American journal of physiology. Cell physiology 20071017 1

We have previously demonstrated increased fibroblast growth factor-2 (FGF-2) expression in a lamb model of increased pulmonary blood flow secondary to congenital heart disease, which may contribute to the associated increases in pulmonary arterial muscularization. However, the mechanisms underlying these increases in FGF-2 expression remain to be identified. Initially, we found that exogenous FGF-2 increased endogenous FGF-2 promoter activity and protein levels in ovine pulmonary arterial smooth ...[more]

PMID: 17942638

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Project description:Upon sensing a reduction in local oxygen partial pressure, pulmonary vessels constrict, a phenomenon known as hypoxic pulmonary vasoconstriction. Excessive hypoxic pulmonary vasoconstriction can occur with ascent to high altitude and is a contributing factor to the development of high-altitude pulmonary edema. The carbonic anhydrase inhibitor, acetazolamide, attenuates hypoxic pulmonary vasoconstriction through stimulation of alveolar ventilation via modulation of acid-base homeostasis and by direct effects on pulmonary vascular smooth muscle. In pulmonary arterial smooth muscle cells (PASMCs), acetazolamide prevents hypoxia-induced increases in intracellular calcium concentration ([Ca2+]i), although the exact mechanism by which this occurs is unknown. In this study, we explored the effect of acetazolamide on various calcium-handling pathways in PASMCs. Using fluorescent microscopy, we tested whether acetazolamide directly inhibited store-operated calcium entry or calcium release from the sarcoplasmic reticulum, two well-documented sources of hypoxia-induced increases in [Ca2+]i in PASMCs. Acetazolamide had no effect on calcium entry stimulated by store-depletion, nor on calcium release from the sarcoplasmic reticulum induced by either phenylephrine to activate inositol triphosphate receptors or caffeine to activate ryanodine receptors. In contrast, acetazolamide completely prevented Ca2+-release from the sarcoplasmic reticulum induced by hypoxia (4% O2). Since these results suggest the acetazolamide interferes with a mechanism upstream of the inositol triphosphate and ryanodine receptors, we also determined whether acetazolamide might prevent hypoxia-induced changes in reactive oxygen species production. Using roGFP, a ratiometric reactive oxygen species-sensitive fluorescent probe, we found that hypoxia caused a significant increase in reactive oxygen species in PASMCs that was prevented by 100 μM acetazolamide. Together, these results suggest that acetazolamide prevents hypoxia-induced changes in [Ca2+]i by attenuating reactive oxygen species production and subsequent activation of Ca2+-release from sarcoplasmic reticulum stores.

Project description:Rationale: Glycolytic shift is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). It remains unknown how glycolysis is increased and how increased glycolysis contributes to pulmonary vascular remodeling in PAH.Objectives: To determine whether increased glycolysis is caused by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) and how PFKFB3-driven glycolysis induces vascular remodeling in PAH.Methods: PFKFB3 levels were measured in pulmonary arteries of patients and animals with PAH. Lactate levels were assessed in lungs of animals with PAH and in pulmonary artery smooth muscle cells (PASMCs). Genetic and pharmacologic approaches were used to investigate the role of PFKFB3 in PAH.Measurements and Main Results: Lactate production was elevated in lungs of PAH rodents and in platelet-derived growth factor-treated PASMCs. PFKFB3 protein was higher in pulmonary arteries of patients and rodents with PAH, in PASMCs of patients with PAH, and in platelet-derived growth factor-treated PASMCs. PFKFB3 inhibition by genetic disruption and chemical inhibitor attenuated phosphorylation/activation of extracellular signal-regulated kinase (ERK1/2) and calpain-2, and vascular remodeling in PAH rodent models, and reduced platelet-derived growth factor-induced phosphorylation/activation of ERK1/2 and calpain-2, collagen synthesis and proliferation of PASMCs. ERK1/2 inhibition attenuated phosphorylation/activation of calpain-2, and vascular remodeling in Sugen/hypoxia PAH rats, and reduced lactate-induced phosphorylation/activation of calpain-2, collagen synthesis, and proliferation of PASMCs. Calpain-2 inhibition reduced lactate-induced collagen synthesis and proliferation of PASMCs.Conclusions: Upregulated PFKFB3 mediates collagen synthesis and proliferation of PASMCs, contributing to vascular remodeling in PAH. The mechanism is through the elevation of glycolysis and lactate that results in the activation of calpain by ERK1/2-dependent phosphorylation of calpain-2.

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Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation.

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Regulation of fibroblast growth factor-2 expression in pulmonary arterial smooth muscle cells involves increased reactive oxygen species generation.

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