Jennifer M. Kleinhenz

The Nox4 Inhibitor GKT137831 Attenuates Hypoxia-Induced Pulmonary Vascular Cell Proliferation

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)β1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARγ and TGF-β1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-β1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-β1 and reductions in PPARγ expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxia-induced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.

Disruption of endothelial peroxisome proliferator-activated receptor-γ reduces vascular nitric oxide production

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-gamma (PPARgamma), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPARgamma ligands attenuate, whereas the loss of function mutations in PPARgamma stimulate, endothelial dysfunction, suggesting that PPARgamma may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPARgamma in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPARgamma gene to produce endothelial PPARgamma null mice (ePPARgamma(-/-)). When compared with littermate controls, ePPARgamma(-/-) animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPARgamma(-/-) mice. Similarly, intact aortic segments from ePPARgamma(-/-) mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPARgamma(-/-) aortas. Reduced nitric oxide production in ePPARgamma(-/-) aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-kappaB in aortic homogenates. These findings demonstrate that endothelial PPARgamma regulates vascular nitric oxide production and that the disruption of endothelial PPARgamma contributes to endothelial dysfunction in vivo.

The PPARγ ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo

Peroxisome proliferator-activated receptor (PPAR) γ activation attenuates hypoxia-induced pulmonary hypertension (PH) in mice. The current study examined the hypothesis that PPARγ attenuates hypoxia-induced endothelin-1 (ET-1) signaling to mediate these therapeutic effects. To test this hypothesis, human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia or hypoxia (1% O(2)) for 72 h and treated with or without the PPARγ ligand rosiglitazone (RSG, 10 μM) during the final 24 h of exposure. HPAEC proliferation was measured with MTT assays or cell counting, and mRNA and protein levels of ET-1 signaling components were determined. To explore the role of hypoxia-activated transcription factors, selected HPAECs were treated with inhibitors of hypoxia-inducible factor (HIF)-1α (chetomin) or nuclear factor (NF)-κB (caffeic acid phenethyl ester, CAPE). In parallel studies, male C57BL/6 mice were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for 3 wk with or without gavage with RSG (10 mg·kg(-1)·day(-1)) for the final 10 days of exposure. Hypoxia increased ET-1, endothelin-converting enzyme-1, and endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated hypoxia-induced activation of HIF-1α, NF-κB activation, and ET-1 signaling pathway components. Similarly, treatment with chetomin or CAPE prevented hypoxia-induced increases in HPAEC ET-1 mRNA and protein levels. These findings indicate that PPARγ activation attenuates a program of hypoxia-induced ET-1 signaling by inhibiting activation of hypoxia-responsive transcription factors. Targeting PPARγ represents a novel therapeutic strategy to inhibit enhanced ET-1 signaling in PH pathogenesis.

Endothelial Cell Peroxisome Proliferator–Activated Receptor γ Reduces Endotoxemic Pulmonary Inflammation and Injury

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator–activated receptor γ (PPARγ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARγ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARγ. Endothelial cell PPARγ (ePPARγ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARγ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARγ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARγ ligands. Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARγ, but not in its absence, and also inhibited neutrophil mobility in a PPARγ-dependent manner. Our results demonstrate a key protective role of ePPARγ against endotoxemic injury and a potential ePPARγ-mediated anti-inflammatory role for NFAs.

Hypoxia downregulates PPARγ via an ERK1/2–NF-κB–Nox4-dependent mechanism in human pulmonary artery smooth muscle cells

The ligand-activated transcription factor peroxisome proliferator-activated receptor γ (PPARγ) regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. This study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo. Hypoxia reduced PPARγ mRNA and protein levels, PPARγ activity, and the expression of PPARγ-regulated genes in human pulmonary artery smooth muscle cells (HPASMCs) exposed to 1% oxygen for 72 h. Similarly, exposure of mice to hypoxia (10% O₂) for 3 weeks reduced PPARγ mRNA and protein in mouse lung. Inhibiting ERK1/2 with PD98059 or treatment with siRNA directed against either NF-κB p65 or Nox4 attenuated hypoxic reductions in PPARγ expression and activity. Furthermore, degradation of H₂O₂ using PEG-catalase prevented hypoxia-induced ERK1/2 phosphorylation and Nox4 expression, suggesting sustained ERK1/2-mediated signaling and Nox4 expression in this response. Mammalian two-hybrid assays demonstrated that PPARγ and p65 bind directly to each other in a mutually repressive fashion. We conclude from these results that hypoxic regimens that promote PH pathogenesis and HPASMC proliferation reduce PPARγ expression and activity through ERK1/2-, p65-, and Nox4-dependent pathways. These findings provide novel insights into mechanisms by which pathophysiological stimuli such as hypoxia cause loss of PPARγ activity and pulmonary vascular cell proliferation, pulmonary vascular remodeling, and PH. These results also indicate that restoration of PPARγ activity with pharmacological ligands may provide a novel therapeutic approach in selected forms of PH.

Peroxisome proliferator-activated receptor gamma depletion stimulates Nox4 expression and human pulmonary artery smooth muscle cell proliferation

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia-induced cell proliferation include sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase 4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels associated with PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Depletion of PPARγ for 24-72 h activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ depletion, indicating that ERK 1/2 lies upstream of NF-κB activation. Depletion of PPARγ for 72 h increased NF-κB-dependent Nox4 expression and H2O2 production. Inhibition of NF-κB or Nox4 attenuated PPARγ depletion-induced HPASMC proliferation. Degradation of PPARγ depletion-induced H2O2 by PEG-catalase prevented HPASMC proliferation and also ERK 1/2 and NF-κB activation and Nox4 expression, indicating that H2O2 participates in feed-forward activation of the above signaling events. Contrary to the effects of PPARγ depletion, HPASMC PPARγ overexpression reduced ERK 1/2 and NF-κB activation, Nox4 expression, and cell proliferation. Taken together these findings provide novel evidence that PPARγ plays a central role in the regulation of the ERK1/2-NF-κB-Nox4-H2O2 signaling axis in HPASMC. These results indicate that reductions in PPARγ caused by pathophysiological stimuli such as prolonged hypoxia exposure are sufficient to promote the proliferation of pulmonary vascular smooth muscle cells observed in PH pathobiology.

Peroxisome Proliferator-Activated Receptor γ Regulates the V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1/microRNA-27a Axis to Reduce Endothelin-1 and Endothelial Dysfunction in the Sickle Cell Mouse Lung.

&NA; Pulmonary hypertension (PH), a serious complication of sickle cell disease (SCD), causes significant morbidity and mortality. Although a recent study determined that hemin release during hemolysis triggers endothelial dysfunction in SCD, the pathogenesis of SCD‐PH remains incompletely defined. This study examines peroxisome proliferator‐activated receptor &ggr; (PPAR&ggr;) regulation in SCD‐PH and endothelial dysfunction. PH and right ventricular hypertrophy were studied in Townes humanized sickle cell (SS) and littermate control (AA) mice. In parallel studies, SS or AA mice were gavaged with the PPAR&ggr; agonist, rosiglitazone (RSG), 10 mg/kg/day, or vehicle for 10 days. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with vehicle or hemin for 72 hours, and selected HPAECs were treated with RSG. SS mice developed PH and right ventricular hypertrophy associated with reduced lung levels of PPAR&ggr; and increased levels of microRNA‐27a (miR‐27a), v‐ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1), endothelin‐1 (ET‐1), and markers of endothelial dysfunction (platelet/endothelial cell adhesion molecule 1 and E selectin). HPAECs treated with hemin had increased ETS1, miR‐27a, ET‐1, and endothelial dysfunction and decreased PPAR&ggr; levels. These derangements were attenuated by ETS1 knockdown, inhibition of miR‐27a, or PPAR&ggr; overexpression. In SS mouse lung or in hemin‐treated HPAECs, activation of PPAR&ggr; with RSG attenuated reductions in PPAR&ggr; and increases in miR‐27a, ET‐1, and markers of endothelial dysfunction. In SCD‐PH pathogenesis, ETS1 stimulates increases in miR‐27a levels that reduce PPAR&ggr; and increase ET‐1 and endothelial dysfunction. PPAR&ggr; activation attenuated SCD‐associated signaling derangements, suggesting a novel therapeutic approach to attenuate SCD‐PH pathogenesis.

PPARγ Regulates Mitochondrial Structure and Function and Human Pulmonary Artery Smooth Muscle Cell Proliferation

&NA; Pulmonary hypertension (PH) is a progressive disorder that causes significant morbidity and mortality despite existing therapies. PH pathogenesis is characterized by metabolic derangements that increase pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling. PH‐associated decreases in peroxisome proliferator‐activated receptor &ggr; (PPAR&ggr;) stimulate PASMC proliferation, and PPAR&ggr; in coordination with PPAR&ggr; coactivator 1&agr; (PGC1&agr;) regulates mitochondrial gene expression and biogenesis. To further examine the impact of decreases in PPAR&ggr; expression on human PASMC (HPASMC) mitochondrial function, we hypothesized that depletion of either PPAR&ggr; or PGC1&agr; perturbs mitochondrial structure and function to stimulate PASMC proliferation. To test this hypothesis, HPASMCs were exposed to hypoxia and treated pharmacologically with the PPAR&ggr; antagonist GW9662 or with siRNA against PPAR&ggr; or PGC1&agr; for 72 hours. HPASMC proliferation (cell counting), target mRNA levels (qRT‐PCR), target protein levels (Western blotting), mitochondria‐derived H2O2 (confocal immunofluorescence), mitochondrial mass and fragmentation, and mitochondrial bioenergetic profiling were determined. Hypoxia or knockdown of either PPAR&ggr; or PGC1&agr; increased HPASMC proliferation, enhanced mitochondria‐derived H2O2, decreased mitochondrial mass, stimulated mitochondrial fragmentation, and impaired mitochondrial bioenergetics. Taken together, these findings provide novel evidence that loss of PPAR&ggr; diminishes PGC1&agr; and stimulates derangements in mitochondrial structure and function that cause PASMC proliferation. Overexpression of PGC1&agr; reversed hypoxia‐induced HPASMC derangements. This study identifies additional mechanistic underpinnings of PH, and provides support for the notion of activating PPAR&ggr; as a novel therapeutic strategy in PH.

Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells.

Background: Pathogenesis of pulmonary hypertension is complex and involves activation of the transcription factor, hypoxia‐inducible factor‐1 (HIF‐1) that shifts cellular metabolism from aerobic respiration to glycolysis, in part, by increasing the expression of its downstream target pyruvate dehydrogenase kinase‐1 (PDK‐1), thereby promoting a proliferative, apoptosis‐resistant phenotype in pulmonary vascular cells. Activation of the nuclear hormone transcription factor, peroxisome proliferator‐activated receptor gamma (PPAR&ggr;), attenuates pulmonary hypertension and pulmonary artery smooth muscle cell (PASMC) proliferation. In the current study, we determined whether PPAR&ggr; inhibits HIF‐1&agr; and PDK‐1 expression in human PASMCs. Methods: HPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) for 2‐72 hours ± treatment with the PPAR&ggr;‐ligand, rosiglitazone (RSG, 10 &mgr;M). Results: Compared to normoxia, HIF‐1&agr; mRNA levels were elevated in HPASMC at 2 hours hypoxia and reduced to baseline levels by 24‐72 hours. HIF‐1&agr; protein levels increased following 4 and 8 hours of hypoxia and returned to baseline levels by 24 and 72 hours. PDK‐1 protein levels increased following 24 hours hypoxia and remained elevated by 72 hours. RSG treatment at the onset of hypoxia attenuated HIF‐1&agr; protein and PDK‐1 mRNA and protein levels at 4, 8 and 24 hours of hypoxia, respectively. However, RSG treatment during final 24 hours of 72‐hour hypoxia, an intervention that inhibits HPASMC proliferation, failed to prevent hypoxia‐induced PDK‐1 expression. Conclusion: Hypoxia causes transient activation of HPASMC HIF‐1&agr; that is attenuated by RSG treatment initiated at hypoxia onset. These findings provide novel evidence that PPAR&ggr; modulates fundamental and acute cellular responses to hypoxia through both HIF‐1‐dependent and HIF‐1‐independent mechanisms.

Smooth Muscle-Targeted Overexpression of Peroxisome Proliferator Activated Receptor-γ Disrupts Vascular Wall Structure and Function

Activation of the nuclear hormone receptor, PPARγ, with pharmacological agonists promotes a contractile vascular smooth muscle cell phenotype and reduces oxidative stress and cell proliferation, particularly under pathological conditions including vascular injury, restenosis, and atherosclerosis. However, pharmacological agonists activate both PPARγ-dependent and -independent mechanisms in multiple cell types confounding efforts to clarify the precise role of PPARγ in smooth muscle cell structure and function in vivo. We, therefore, designed and characterized a mouse model with smooth muscle cell-targeted PPARγ overexpression (smPPARγOE). Our results demonstrate that smPPARγOE attenuated contractile responses in aortic rings, increased aortic compliance, caused aortic dilatation, and reduced mean arterial pressure. Molecular characterization revealed that compared to littermate control mice, aortas from smPPARγOE mice expressed lower levels of contractile proteins and increased levels of adipocyte-specific transcripts. Morphological analysis demonstrated increased lipid deposition in the vascular media and in smooth muscle of extravascular tissues. In vitro adenoviral-mediated PPARγ overexpression in human aortic smooth muscle cells similarly increased adipocyte markers and lipid uptake. The findings demonstrate that smooth muscle PPARγ overexpression disrupts vascular wall structure and function, emphasizing that balanced PPARγ activity is essential for vascular smooth muscle homeostasis.