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Objective: Mutations in BMPR2 (bone morphogenetic protein receptor 2) are associated with familial and sporadic pulmonary arterial hypertension (PAH). The functional and molecular link between loss of BMPR2 in pulmonary artery smooth muscle cells (PASMC) and PAH pathogenesis warrants further investigation, as most investigations focus on BMPR2 in pulmonary artery endothelial cells. Our goal was to determine whether and how decreased BMPR2 is related to the abnormal phenotype of PASMC in PAH. Methods: SMC-specific Bmpr2 −/− mice ( BKO SMC ) were created and compared to controls in room air, after 3 weeks of hypoxia as a second hit, and following 4 weeks of normoxic recovery. Echocardiography, right ventricular systolic pressure, and right ventricular hypertrophy were assessed as indices of pulmonary hypertension. Proliferation, contractility, gene and protein expression of PASMC from BKO SMC mice, human PASMC with BMPR2 reduced by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation were compared to controls, to investigate the phenotype and underlying mechanism. Results: BKO SMC mice showed reduced hypoxia-induced vasoconstriction and persistent pulmonary hypertension following recovery from hypoxia, associated with sustained muscularization of distal pulmonary arteries. PASMC from mutant compared to control mice displayed reduced contractility at baseline and in response to angiotensin II, increased proliferation and apoptosis resistance. Human PASMC with reduced BMPR2 by small interference RNA, and PASMC from PAH patients with a BMPR2 mutation showed a similar phenotype related to upregulation of pERK1/2 (phosphorylated extracellular signal related kinase 1/2)-pP38-pSMAD2/3 mediating elevation in ARRB2 (β-arrestin2), pAKT (phosphorylated protein kinase B) inactivation of GSK3-beta, CTNNB1 (β-catenin) nuclear translocation and reduction in RHOA (Ras homolog family member A) and RAC1 (Ras-related C3 botulinum toxin substrate 1). Decreasing ARRB2 in PASMC with reduced BMPR2 restored normal signaling, reversed impaired contractility and attenuated heightened proliferation and in mice with inducible loss of BMPR2 in SMC, decreasing ARRB2 prevented persistent pulmonary hypertension. Conclusions: Agents that neutralize the elevated ARRB2 resulting from loss of BMPR2 in PASMC could prevent or reverse the aberrant hypocontractile and hyperproliferative phenotype of these cells in PAH.

Reduced expression of bone morphogenetic protein receptor 2 subverts a stress granule response, heightens GM-CSF mRNA translation, and increases inflammatory cell recruitment to exacerbate pulmonary arterial hypertension., Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34–PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)–positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

Rationale: Hypoxia-inducible factor-1α (HIF-1α), an oxygen (O 2 )-sensitive transcription factor, mediates transcriptional responses to low-O 2 tension states. Although acute hypoxia causes pulmonary vasoconstriction and chronic hypoxia can cause vascular remodeling and pulmonary hypertension, conflicting data exist on the role of HIF-1α in modulating pulmonary vascular tone. Objective: To investigate the role of smooth muscle cell (SMC)–specific HIF-1α in regulating pulmonary vascular tone. Methods and Results: Mice with an SMC-specific deletion of HIF-1α (SM22α-HIF-1α −/− ) were created to test the hypothesis that pulmonary artery SMC (PASMC) HIF-1α modulates pulmonary vascular tone and the response to hypoxia. SM22α-HIF-1α −/− mice exhibited significantly higher right ventricular systolic pressure compared with wild-type littermates under normoxia and with exposure to either acute or chronic hypoxia in the absence of histological evidence of accentuated vascular remodeling. Moreover, myosin light chain phosphorylation, a determinant of SMC tone, was higher in PASMCs isolated from SM22α-HIF-1α −/− mice compared with wild-type PASMCs, during both normoxia and after acute hypoxia. Further, overexpression of HIF-1α decreased myosin light chain phosphorylation in HIF-1α–null SMCs. Conclusions: In both normoxia and hypoxia, PASMC HIF-1α maintains low pulmonary vascular tone by decreasing myosin light chain phosphorylation. Compromised PASMC HIF-1α expression may contribute to the heightened vasoconstriction that characterizes pulmonary hypertension.

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.

Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

S100A4/Mts-overexpressing mice have thick elastic laminae and mild pulmonary arterial hypertension (PAH), and the occasional older mouse develops occlusive neointimal lesions and perivascular inflammation. We hypothesized that a vasculotropic virus could induce neointimal lesions in the S100A4/Mts1 mouse by facilitating breakdown of elastin and migration and proliferation of smooth muscle cells. To test this hypothesis, we infected S100A4/Mts1 mice with gammaherpesvirus 68 (γHV68). We observed, 6 mo after γHV68 [4 × 103 plaque-forming units (PFU)], perivascular inflammation in 10/15 S100A4/Mts1 mice and occlusive neointimal formation in 3/10 mice, accompanied by striking degradation of elastin. We then compared the early response after high-dose γHV68 (4 × 106 PFU) in C57Bl/6 and S100A4/Mts1 mice. In S100A4/Mts1 mice only, significant PAH, muscularization of distal vessels, and elastase activity were observed 6 wk after γHV68. These features resolved by 3 mo without neointimal formation. We therefore infected mice with the M1-γHV68 strain that reactivates from latency with higher efficiency and observed neointimal lesions at 3 mo in 2/5 C57Bl/6 (5–9% of vessels) and in 5/5 S100A4/Mts1 mice (13–40% of vessels) accompanied by mild PAH, heightened lung elastase activity, and intravascular viral expression. This suggested that enhanced generation of elastin peptides in S100A4/Mts1 mice may promote increased viral entry in the vessel wall. Using S100A4/Mts1 PA organ culture, we showed, in response to elastase activity, heightened production of elastin peptides associated with invasion of inflammatory cells and intravascular viral antigen. We therefore propose that early viral access to the vessel wall may be a critical determinant of the extent of vascular pathology following reactivation.

Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zinc. In this study, we examined the cellular mechanisms responsible for Zn2+-induced IL-8 expression. Zn2+ stimulation resulted in pronounced increases in both IL-8 mRNA and protein expression in the human airway epithelial cell line (BEAS-2B). IL-8 promoter activity was significantly increased by Zn2+ exposure in BEAS-2B cells, indicating that Zn2+-induced IL-8 expression is transcriptionally mediated. Mutation of the activating protein (AP)-1 response element in an IL-8 promoter-enhanced green fluorescent protein construct reduced Zn2+-induced IL-8 promoter activity. Moreover, Zn2+ exposure of BEAS-2B cells induced the phosphorylation of the AP-1 proteins c-Fos and c-Jun. We observed that Zn2+ exposure induced the phosphorylation of ERK, JNK, and p38 MAPKs, whereas inhibition of ERK or JNK activity blocked IL-8 mRNA and protein expression in BEAS-2B cells treated with Zn2+. In addition, we investigated the role of protein tyrosine phosphatases in the activation of signaling by Zn2+. Zn2+ treatment inhibited ERK- and JNK-directed phosphatase activities in BEAS-2B cells. These results suggested that Zn2+-induced inhibition of phosphatase activity is an initiating event in MAPK and AP-1 activation that leads to enhanced IL-8 expression by human airway epithelial cells.

Previously, we observed that hypoxia increases the expression of the β1-subunit ( KCNMB1) of the calcium-sensitive potassium channel (BKCa). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-α ( HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between −3,540 bp and −3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1α transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the β1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at −3,540 to −3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.

Background: Kawasaki disease (KD) is a leading cause of acquired heart disease in children. However, the molecular mechanisms leading to coronary aneurysms, and the host factors determining high-risk patients are unknown. We have previously shown that mice haploinsufficient for tropoelastin (EL ±), develop more severe destruction of the coronary elastin compared to wild type (WT) mice after injection with Lactobacillus casei wall extract (LCWE), an inducer of coronary arteritis. In contrast to the beneficial effect of TGFβ blockade on the aneurysms that form in Marfan syndrome (MS), we hypothesized that TGFβ protects against aneurysm formation in this inflammatory model by: enhancing tropoelastin; and suppressing proteolytic activity. Methods: WT and EL ± mice were given either TGFβ neutralizing antibody (TGFβ-Nab) or control IgG in addition to LCWE. At 14d, tropoelastin and plasminogen activator inhibitor-1 (PAI-1) protein was determined, and matrix metalloproteinase (MMP)-2 and -9 and serine elastase activity was measured. Elastin degradation and total medial elastin content was assessed by morphometric analysis at 42d. Results: TGFβ blockade worsened the LCWE-mediated fragmentation of the coronary elastin, with areas of complete dissolution of the external lamina in WT mice, and even more severe features in EL ± mice. Tropoelastin protein was 40% lower in the EL ± vs. WT mice after LCWE (P < 0.05), and TGFβ blockade further exaggerated this discrepancy. TGFβ-Nab markedly reduced PAI-1 in the WT mice after LCWE (P < 0.05), and produced a similar pattern in the EL ± mice, and induced a four-fold induction of MMP-9 activity in both genotypes. This effect was associated with increased elastin fragmentation (P < 0.05), and decreased total medial elastin content (P < 0.05), despite inhibition of the LCWE-mediated induction of serine elastase activity. Conclusions: We conclude that: diminished tropoelastin expression worsens elastin destruction in the setting of acute coronary arteritis; and TGFβ protects the vasculature in this model by maintaining PAI-1 levels and suppressing activation of MMP-9. Thus strategies to block TGFβ, currently in use to prevent aneurysms in MS, are unlikely to be beneficial in KD, and may be detrimental. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Human γ herpesvirus infection is linked to idiopathic pulmonary arterial hypertension (IPAH). S100A4, a metastasis gene, is highly expressed in the plexiform lesions of IPAH patients. Previously, we reported that murine γherpesvirus-68 (M1-MHV-68) infection in S100A4 overexpressing but not in C57 wild-type (WT) mice induced mild neointimal formation in pulmonary arteries (PA) associated with heightened serine elastase activity. We hypothesized that these neointimal lesions would progress during an extended reactivation phase, in the S100A4 mice in association with ongoing elastolysis and that the susceptibility in S100A4 mice was related either to viral load, inflammation or differential induction of host miRNAs in response to infection. We now report that the % of vessels with neointimal lesions progressively increased from 25 to 40%, between 3 and 6 mo after viral infection (P < 0.05) in S100A mice but were not observed in WT. The extent of neointimal lesions correlated with progressive fragmentation of the elastic laminae, (P < 0.001). Interestingly, these features were not sufficient to induce elevated RVSP or RVH, in the absence of muscularization or loss of distal PAs. Susceptibilty of S100A4 mice was not related to an increase in viral DNA in intra-pulmonary arteries assessed by laser capture microscopy, nor could we establish a relationship with the severity of perivascular inflammation or cytokine expression (IFN-γ and TNF-α). However, we confirmed higher lung serine elastase activity in S100A4 vs. C57 mice as early as 1 wk after viral infection. Since expression of miRNAs may be responsible for host susceptibility, we compared lungs of C57 and S100A4 mice at 6 mo using microArrays followed by qRT-PCR. Induction of miR-466 in WT mice infected with virus (P < 0.01), was not observed in S100A4 mice. miR-466 can target the 3′ UTR of RUNX1, a transcription factor for serine elastase, and reduce its expression. The inability to reduce RUNX1 in S100A4 mice in response to viral infection may be related to elevated serine elastase activity. Our studies show that viral infection can lead to progressive neointimal formation and that the susceptibility of S100A4 mice associated with heightened elastase may be linked to differential expression of miR-466. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).

Zinc is an essential micronutrient, but is proinflammatory when inhaled into the lung. While it is recognized that zinc exposure of airway epithelial cells activates the transcription factor NF-kappaB and increases the expression of inflammatory cytokines to mediate this response, the underlying mechanism of NF-kappaB activation remains to be characterized. In this study, we investigated these Zn2+-induced signaling mechanisms in the BEAS-2B human airway epithelial cell line. Fifty micromolars Zn2+ induced NF-kappaB-dependent transcriptional activity. However, this occurred independently of IkappaBalpha degradation, an essential event in activation of the canonical NF-kappaB pathway, which is induced by physiological stimuli such as TNFalpha and IL-1beta. We also observed that 50 microM Zn2+ exposure caused p65/RelA phosphorylation on Ser 276, Ser 529, and Ser 536 in both cytoplasmic and nuclear cell fractions. Mutational analysis pointed to Ser 536 of p65/RelA as the determinant of Zn2+-induced NF-kappaB transactivation in BEAS-2B cells. Pharmacological inhibition of IKKalpha/beta activity reduced both Zn2+-induced p65/RelA phosphorylation at Ser 536 and NF-kappaB-dependent transcriptional activity, suggesting that IKKalpha/beta is necessary for these Zn2+-induced effects. Taken together, these data show that exposure to supraphysiological concentrations of Zn2+ induces NF-kappaB-dependent transcription through an alternate mechanism, suggesting a novel pathway for cellular responses to environmental stress.

Epidemiological studies suggest that ultrafine particles contribute to particulate matter-induced adverse health effects. Interleukin (IL)-8 is an important proinflammatory cytokine in the human lung that is induced in respiratory cells exposed to a variety of environmental insults, including ambient air ultrafine particles. In this study, we examined the effect of a model ultrafine particle on IL-8 expression and the cellular mechanisms responsible for this event. Here, we report that carbonaceous ultrafine particles consisting of synthetic elemental carbon particles (UfCP) markedly increase the expression of IL-8 mRNA and protein in normal human bronchial epithelial (NHBE) cells. IL-8 promoter activity was increased by UfCP exposure in NHBE cells, indicating UfCP-induced IL-8 expression is transcriptionally regulated. IL-8 expression in NHBE is known to be regulated by nuclear factor (NF)-kappaB activation. However, UfCP did not induce inhibitory factor kappaBalpha degradation, NF-kappaB-DNA binding, or NF-kappaB-dependent promoter activity in NHBE cells, indicating that UfCP induces IL-8 expression through a mechanism that is independent of NF-kappaB activation. Additionally, we observed that UfCP exposure induces the phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in a biphasic manner and that the inhibition of p38 MAPK activity can block IL-8 mRNA expression induced by UfCP in NHBE cells. These results demonstrate that UfCP-induced expression of IL-8 involves a transcriptional mechanism and activation of p38 MAPK in NHBE cells.