L-citrulline attenuates lipopolysaccharide-induced inflammatory lung injury in neonatal rats.

BACKGROUND: Prenatal or postnatal lung inflammation and oxidative stress disrupt alveolo-vascular development leading to bronchopulmonary dysplasia (BPD) with and without pulmonary hypertension. L-citrulline (L-CIT), a nonessential amino acid, alleviates inflammatory and hyperoxic lung injury in preclinical models of BPD. L-CIT modulates signaling pathways mediating inflammation, oxidative stress, and mitochondrial biogenesis-processes operative in the development of BPD. We hypothesize that L-CIT will attenuate lipopolysaccharide (LPS)-induced inflammation and oxidative stress in our rat model of neonatal lung injury. METHODS: Newborn rats during the saccular stage of lung development were used to investigate the effect of L-CIT on LPS-induced lung histopathology and pathways involved in inflammatory, antioxidative processes, and mitochondrial biogenesis in lungs in vivo, and in primary culture of pulmonary artery smooth muscle cells, in vitro. RESULTS: L-CIT protected the newborn rat lung from LPS-induced: lung histopathology, ROS production, NFκB nuclear translocation, and upregulation of gene and protein expression of inflammatory cytokines (IL-1ß, IL-8, MCP-1α, and TNF-α). L-CIT maintained mitochondrial morphology, increased protein levels of PGC-1α, NRF1, and TFAM (transcription factors involved in mitochondrial biogenesis), and induced SIRT1, SIRT3, and superoxide dismutases protein expression. CONCLUSION: L-CIT may be efficacious in decreasing early lung inflammation and oxidative stress mitigating progression to BPD. IMPACT: The nonessential amino acid L-citrulline (L-CIT) mitigated lipopolysaccharide (LPS)-induced lung injury in the early stage of lung development in the newborn rat. This is the first study describing the effect of L-CIT on the signaling pathways operative in bronchopulmonary dysplasia (BPD) in a preclinical inflammatory model of newborn lung injury. If our findings translate to premature infants, L-CIT could decrease inflammation, oxidative stress and preserve mitochondrial health in the lung of premature infants at risk for BPD.

Maturational effect of leptin on CO2 chemosensitivity in newborn rats.

BACKGROUND: Leptin augments central CO2 chemosensitivity and stabilizes breathing in adults. Premature infants have unstable breathing and low leptin levels. Leptin receptors are on CO2 sensitive neurons in the Nucleus Tractus Solitarius (NTS) and locus coeruleus (LC). We hypothesized that exogenous leptin improves hypercapnic respiratory response in newborn rats by improving central CO2 chemosensitivity. METHODS: In rats at postnatal day (p)4 and p21, hyperoxic and hypercapnic ventilatory responses, and pSTAT and SOCS3 protein expression in the hypothalamus, NTS and LC were measured before and after treatment with exogenous leptin (6 µg/g). RESULTS: Exogenous leptin increased the hypercapnic response in p21 but not in p4 rats (P ≤ 0.001). At p4, leptin increased pSTAT expression only in the LC, and SOCS3 expression in the NTS and LC; while at p21 pSTAT and SOCS3 levels were higher in the hypothalamus, NTS, and LC (P ≤ 0.05). CONCLUSIONS: We describe the developmental profile of the effect of exogenous leptin on CO2 chemosensitivity. Exogenous leptin does not augment central CO2 sensitivity during the first week of life in newborn rats. The translational implication of these findings is that low plasma leptin levels in premature infants may not be contributing to respiratory instability. IMPACT: Exogenous leptin does not augment CO2 sensitivity during the first week of life in newborn rats, similar to the developmental period when feeding behavior is resistant to leptin. Exogenous leptin increases CO2 chemosensitivity in newborn rats after the 3rd week of life and upregulates the expression of pSTAT and SOC3 in the hypothalamus, NTS and LC. Low plasma leptin levels in premature infants are unlikely contributors to respiratory instability via decreased CO2 sensitivity in premature infants. Thus, it is highly unlikely that exogenous leptin would alter this response.

Adiponectin and the regulation of gastric content volume in the newborn rat.

BACKGROUND: Oral intake is dependent on the gastric ability to accommodate the food bolus. Comparatively, neonates have a smaller gastric capacity than adults and this may limit the volume of their milk intake. Yet, we previously reported that the newborn rat gastric milk volume is greatest after birth and, when normalized to body weight, decreases with postnatal age. Such age-dependent changes are not the result of intake differences, but greater gastric accommodation and reduced emptying rate. AIM: Hypothesizing that breastmilk-derived adiponectin is the factor regulating gastric accommodation in neonates, we comparatively evaluated its effects on the rat fundic muscle tone at different postnatal ages. METHODS: In freshly dispersed smooth muscle cells (SMC), we measured the adiponectin effect on the carbachol-induced length changes. RESULTS: Adiponectin significantly reduced the carbachol-stimulated SMC shortening independently of age. In the presence of the inhibitor iberiotoxin, the adiponectin effect on SMC shortening was suppressed, suggesting that it is mediated via large-conductance Ca2+ sensitive K+ channel activation. Lastly, we comparatively measured the newborn rat gastric milk curd adiponectin content in one- and two-week-old rats and found a 50% lower value in the latter. CONCLUSION: Adiponectin, a major component of breastmilk, downregulates fundic smooth muscle contraction potential, thus facilitating gastric volume accommodation. This rodent's adaptive response maximizes breastmilk intake volume after birth.

Recombinant adiponectin protects the newborn rat lung from lipopolysaccharide-induced inflammatory injury.

Preterm infants are at high risk for developing bronchopulmonary dysplasia and pulmonary hypertension from inflammatory lung injury. In adult models, adiponectin (APN)-an adipocyte-derived hormone-protects the lung from inflammatory injury and pulmonary vascular remodeling. Cord blood APN levels in premature infants born < 26 weeks gestation are 5% of the level in infants born at term. We previously reported the expression profile of APN and its receptors in neonatal rat lung homogenates during the first 3 weeks of postnatal development. Here, we characterize the expression profile of APN and its receptors in specific lung cells and the effects of exogenous recombinant APN (rAPN) on lipopolysaccharide-(LPS)-induced cytokine and chemokine production in total lung homogenates and specific lung cells. In vitro, rAPN added to primary cultures of pulmonary artery smooth muscle cells attenuated the expression of LPS-induced pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines. In vivo, intraperitoneal rAPN (2 mg/kg), given 4 hr prior to intrapharyngeal administration of LPS (5 mg/kg) to newborn rats at postnatal day 4, significantly reduced gene and protein expression of the pro-inflammatory cytokine IL-1ß and reduced protein expression of the chemokines monocyte chemoattractant protein (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1α) in the lung. LPS-induced histopathological changes in the lung were also decreased. Moreover, rAPN given 20 hr after intrapharyngeal LPS had a similar effect on lung inflammation. These findings suggest a role for APN in protecting the lung from inflammation during early stages of lung development.

Chronic Intermittent Hypoxia in Premature Infants: The Link Between Low Fat Stores, Adiponectin Receptor Signaling and Lung Injury.

Premature infants have chronic intermittent hypoxia (CIH) that increases morbidity, and the youngest and the smallest premature infants are at the greatest risk. The combination of lung injury from inflammation/oxidative stress causing low functional residual capacity combined with frequent short apneas leads to CIH. Adiponectin (APN) is an adipose-derived adipokine that protects the lung from inflammation and oxidative stress. Premature and small for gestational age (SGA) infants have minimal body fat and low levels of circulating APN. To begin to understand the potential role of APN in lung protection during lung development, we characterized the developmental profile of APN and APN receptors (AdipoR1 and AdipoR2) protein and mRNA expression in the newborn rat lung at fetal day (FD) 19, and postnatal days (PD) 1, 4, 7, 10, 14, 21, and 28. Protein levels in lung homogenates were measured by western blot analyses; relative mRNA expression was detected by quantitative PCR (qPCR); and serum high molecular weight (HMW) APN was measured using enzyme-linked immunosorbent assay (ELISA). Results: APN protein and mRNA levels were lowest at FD19 and PD1, increased 2.2-fold at PD4, decreased at PD10, and then increased again at PD21. AdipoR1 protein and mRNA levels peaked at PD1, followed by a threefold drop by PD4, and remained low until PD21. AdipoR2 protein and mRNA levels also peaked at PD1, but remained high at PD4, followed by a 1.7-fold drop by PD10 that remained low by PD21. Serum APN levels detected by ELISA did not differ from PD4 to PD28. To date, this is the first report characterizing APN and APN receptor protein and mRNA expression in the rat lung during development. The developmental stage of the newborn rat lung models that of the premature human infant; both are in the saccular stage of lung development. In the newborn rat lung, alveolarization begins at PD4, peaks at PD10, and ends at PD21. Importantly, we found that AdipoR1 receptor protein and mRNA expression is lowest during lung alveolarization (PD4 to PD21). Thus, we speculate that low levels of AdipoR1 during lung alveolarization contributes to the increased susceptibility to developing acute lung edema and chronic lung injury such as bronchopulmonary dysplasia (BPD) in premature human infants.

Sodium nitrite augments lung S-nitrosylation and reverses chronic hypoxic pulmonary hypertension in juvenile rats.

Deficient nitric oxide (NO) signaling plays a critical role in the pathogenesis of chronic neonatal pulmonary hypertension (PHT). Physiological NO signaling is regulated by S-nitrosothiols (SNOs), which act both as a reservoir for NO and as a reversible modulator of protein function. We have previously reported that therapy with inhaled NO (iNO) increased peroxynitrite-mediated nitration in the juvenile rat lung, although having minimal reversing effects on vascular remodeling. We hypothesized that sodium nitrite (NaNO2) would be superior to iNO in enhancing lung SNOs, thereby contributing to reversal of chronic hypoxic PHT. Rat pups were exposed to air or hypoxia (13% O2) from postnatal days 1 to 21. Dose-response prevention studies were conducted from days 1-21 to determine the optimal dose of NaNO2. Animals then received rescue therapy with daily subcutaneous NaNO2 (20 mg/kg), vehicle, or were continuously exposed to iNO (20 ppm) from days 14-21. Chronic PHT secondary to hypoxia was both prevented and reversed by treatment with NaNO2. Rescue NaNO2 increased lung NO and SNO contents to a greater extent than iNO, without causing nitration. Seven lung SNO proteins upregulated by treatment with NaNO2 were identified by multiplex tandem mass tag spectrometry, one of which was leukotriene A4 hydrolase (LTA4H). Rescue therapy with a LTA4H inhibitor, SC57461A (10 mg·kg-1·day-1 sc), partially reversed chronic hypoxic PHT. We conclude that NaNO2 was superior to iNO in increasing tissue NO and SNO generation and reversing chronic PHT, in part via upregulated SNO-LTA4H.

mTOR-Notch3 signaling mediates pulmonary hypertension in hypoxia-exposed neonatal rats independent of changes in autophagy.

BACKGROUND/AIM: Mammalian target of rapamycin (mTOR) is a pivotal regulator of cell proliferation, survival, and autophagy. Autophagy is increased in adult experimental chronic pulmonary hypertension (PHT), but its contributory role to pulmonary vascular disease remains uncertain and has yet to be explored in the neonatal animal. Notch is a major pro-proliferative pathway activated by mTOR. A direct relationship between autophagy and Notch signaling has not been previously explored. Our aim was to examine changes in mTOR-, Notch-, and autophagy-related pathways and the therapeutic effects of autophagy modulators in experimental chronic neonatal PHT secondary to chronic hypoxia. METHODS: Rat pups were exposed to normoxia or hypoxia (13% O2 ) from postnatal days 1-21, while receiving treatment with temsirolimus (mTOR inhibitor), DAPT (Notch inhibitor), or chloroquine (inhibitor of autophagic flux). RESULTS: Exposure to hypoxia up-regulated autophagy and Notch3 signaling markers in lung, pulmonary artery (PA), and PA-derived smooth muscle cells (SMCs). Temsirolimus prevented chronic PHT and attenuated PA and SMC signaling secondary to hypoxia. These effects were replicated by DAPT. mTOR or Notch inhibition also down-regulated smooth muscle content of platelet-derived growth factor ß-receptor, a known contributor to vascular remodeling. In contrast, chloroquine had no modifying effects on markers of chronic PHT. Knockdown of Beclin-1 in SMCs had no effect on hypoxia-stimulated Notch3 signaling. CONCLUSIONS: mTOR-Notch3 signaling plays a critical role in experimental chronic neonatal PHT. Inhibition of autophagy did not suppress Notch signaling and had no effect on markers of chronic PHT.

Intermittent hypoxia during recovery from neonatal hyperoxic lung injury causes long-term impairment of alveolar development: A new rat model of BPD.

Bronchopulmonary dysplasia (BPD) is a chronic lung injury characterized by impaired alveologenesis that may persist into adulthood. Rat models of BPD using varying degrees of hyperoxia to produce injury either cause early mortality or spontaneously recover following removal of the inciting stimulus, thus limiting clinical relevance. We sought to refine an established rat model induced by exposure to 60% O2 from birth by following hyperoxia with intermittent hypoxia (IH). Rats exposed from birth to air or 60% O2 until day 14 were recovered in air with or without IH (FIO2 = 0.10 for 10 min every 6 h) until day 28 Animals exposed to 60% O2 and recovered in air had no evidence of abnormal lung morphology on day 28 or at 10-12 wk. In contrast, 60% O2-exposed animals recovered in IH had persistently increased mean chord length, more dysmorphic septal crests, and fewer peripheral arteries. Recovery in IH also increased pulmonary vascular resistance, Fulton index, and arterial wall thickness. IH-mediated abnormalities in lung structure (but not pulmonary hypertension) persisted when reexamined at 10-12 wk, accompanied by increased pulmonary vascular reactivity and decreased exercise tolerance. Increased mean chord length secondary to IH was prevented by treatment with a peroxynitrite decomposition catalyst [5,10,15,20-Tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin iron (III) chloride, 30 mg/kg/day, days 14-28], an effect accompanied by fewer inflammatory cells. We conclude that IH during recovery from hyperoxia-induced injury prevents recovery of alveologenesis and leads to changes in lung and pulmonary vascular function lasting into adulthood, thus more closely mimicking contemporary BPD.

Simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of RhoA signaling.

Chronic neonatal pulmonary hypertension (PHT) frequently results in early death. Systemically administered Rho-kinase (ROCK) inhibitors prevent and reverse chronic PHT in neonatal rats, but at the cost of severe adverse effects, including systemic hypotension and growth restriction. Simvastatin has pleiotropic inhibitory effects on isoprenoid intermediates that may limit activity of RhoA, which signals upstream of ROCK. We therefore hypothesized that statin treatment would safely limit pulmonary vascular RhoA activity and prevent and reverse experimental chronic neonatal PHT via downstream inhibitory effects on pathological ROCK activity. Sprague-Dawley rats in normoxia (room air) or moderate normobaric hypoxia (13% O2) received simvastatin (2 mg·kg-1·day-1 ip) or vehicle from postnatal days 1-14 (prevention protocol) or from days 14-21 (rescue protocol). Chronic hypoxia increased RhoA and ROCK activity in lung tissue. Simvastatin reduced lung content of the isoprenoid intermediate farnesyl pyrophosphate and decreased RhoA/ROCK signaling in the hypoxia-exposed lung. Preventive or rescue treatment of chronic hypoxia-exposed animals with simvastatin decreased pulmonary vascular resistance, right ventricular hypertrophy, and pulmonary arterial remodeling. Preventive simvastatin treatment improved weight gain, did not lower systemic blood pressure, and did not cause apparent toxic effects on skeletal muscle, liver or brain. Rescue therapy with simvastatin improved exercise capacity. We conclude that simvastatin limits RhoA/ROCK activity in the chronic hypoxia-exposed lung, thus preventing or ameliorating hemodynamic and structural markers of chronic PHT and improving long-term outcome, without causing adverse effects.

Leukotriene B4 mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury.

Systemically-administered bleomycin causes inflammation, arrested lung growth, and pulmonary hypertension (PHT) in the neonatal rat, similar to human infants with severe bronchopulmonary dysplasia (BPD). Leukotrienes (LTs) are inflammatory lipid mediators produced by multiple cell types in the lung. The major LTs, LTB4 and cysteinyl LTs, are suggested to contribute to BPD, but their specific roles remain largely unexplored in experimental models. We hypothesized that LTs are increased in bleomycin-induced BPD-like injury, and that inhibition of LT production would prevent inflammatory cell influx and thereby ameliorate lung injury. Rat pups were exposed to bleomycin (1 mg·kg(-1)·day(-1) ip) or vehicle (control) from postnatal days 1-14 and were treated with either zileuton (5-lipoxygenase inhibitor), montelukast (cysteinyl LT1 receptor antagonist), or SC57461A (LTA4 hydrolase inhibitor) 10 mg·kg(-1)·day(-1) ip. Bleomycin led to increased lung content of LTB4, but not cysteinyl LTs. Bleomycin-induced increases in tissue neutrophils and macrophages and lung contents of LTB4 and tumor necrosis factor-α were all prevented by treatment with zileuton. Treatment with zileuton or SC57461A also prevented the hemodynamic and structural markers of chronic PHT, including raised pulmonary vascular resistance, increased Fulton index, and arterial wall remodeling. However, neither treatment prevented impaired alveolarization or vascular hypoplasia secondary to bleomycin. Treatment with montelukast had no effect on macrophage influx, PHT, or on abnormal lung structure. We conclude that LTB4 plays a crucial role in lung inflammation and PHT in experimental BPD. Agents targeting LTB4 or LTB4-mediated signaling may have utility in infants at risk of developing BPD-associated PHT.

Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs.

Arginase is an enzyme that limits substrate L-arginine bioavailability for the production of nitric oxide by the nitric oxide synthases and produces L-ornithine, which is a precursor for collagen formation and tissue remodeling. We studied the pulmonary vascular effects of arginase inhibition in an established model of repeated systemic bleomycin sulfate administration in neonatal rats that results in pulmonary hypertension and lung injury mimicking the characteristics typical of bronchopulmonary dysplasia. We report that arginase expression is increased in the lungs of bleomycin-exposed neonatal rats and that treatment with the arginase inhibitor amino-2-borono-6-hexanoic acid prevented the bleomycin-induced development of pulmonary hypertension and deposition of collagen. Arginase inhibition resulted in increased L-arginine and L-arginine bioavailability and increased pulmonary nitric oxide production. Arginase inhibition also normalized the expression of inducible nitric oxide synthase, and reduced bleomycin-induced nitrative stress while having no effect on bleomycin-induced inflammation. Our data suggest that arginase is a promising target for therapeutic interventions in neonates aimed at preventing lung vascular remodeling and pulmonary hypertension.

Rho kinase mediates right ventricular systolic dysfunction in rats with chronic neonatal pulmonary hypertension.

Chronic neonatal pulmonary hypertension frequently culminates in right ventricular (RV) failure and death. In juvenile rats, RV systolic dysfunction secondary to chronic hypoxia is rescued by systemic treatment with a Rho kinase (ROCK) inhibitor. To explore the relationship between ROCK inhibitor-mediated decreases in pulmonary vascular resistance and pressure, RV hypertrophy, and systolic dysfunction, we compared the effects of systemically administered to inhaled (pulmonary-selective) ROCK inhibitor on RV systolic function. Rat pups were exposed to air or hypoxia (13% O2) from Postnatal Days 1 to 21 and received rescue treatment with aerosolized fasudil (200 mM) for 15 minutes three times daily or intraperitoneal Y27632 (15 mg/kg twice daily) from Days 14 to 21. Chronic hypoxia differentially increased RhoA and ROCK activity in the right, but not left, cardiac ventricle. Inhaled ROCK inhibitor normalized pulmonary vascular resistance and caused regression of RV hypertrophy and pulmonary arterial wall remodeling but did not improve RV systolic dysfunction (decreased stroke volume and tricuspid annular plane systolic excursion). Systemic, but not inhaled, ROCK inhibitor normalized up-regulated ROCK and phosphodiesterase 5 activities in the right ventricle. Treatment with sildenafil (100 mg/kg/d intraperitoneally from Days 14 to 21) improved RV systolic function. Collectively, these data indicate that pressure unloading and regressed arterial and cardiac remodeling did not lead to recovery of systolic function while right ventricular ROCK activity remained increased. Right ventricle-specific up-regulation of RhoA/ROCK activity is critical to hypoxia-mediated systolic dysfunction, in part by regulating the activity of phosphodiesterase 5.

Therapeutic hypercapnia prevents inhaled nitric oxide-induced right-ventricular systolic dysfunction in juvenile rats.

Chronic pulmonary hypertension in the neonate and infant frequently presents with right-ventricular (RV) failure. Current clinical management may include protracted treatment with inhaled nitric oxide (iNO), with the goal of reducing RV afterload. We have previously reported that prolonged exposure to iNO causes RV systolic dysfunction in the chronic hypoxia-exposed juvenile rat, which was prevented by a peroxynitrite decomposition catalyst. Given that inhalation of CO2 (therapeutic hypercapnia) may limit oxidative stress and upregulated cytokine expression in the lung and other organs, we hypothesized that therapeutic hypercapnia would attenuate cytokine-mediated nitric oxide synthase (NOS) upregulation, thus limiting peroxynitrite generation. Sprague-Dawley rat pups were exposed to chronic hypoxia (13% O2) from postnatal day 1 to 21, while receiving iNO (20 ppm) from day 14 to 21, with or without therapeutic hypercapnia (10% CO2). Therapeutic hypercapnia completely normalized RV systolic function, RV hypertrophy, and remodeling of pulmonary resistance arteries in animals exposed to iNO. Inhaled nitric oxide-mediated increases in RV peroxynitrite, apoptosis, and contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and NOS-2 were all attenuated by therapeutic hypercapnia. Inhibition of NOS-2 activity with 1400 W (1 mg/kg/day) prevented iNO-mediated upregulation of peroxynitrite and led to improved RV systolic function. Blockade of IL-1 receptor signaling with anakinra (500 mg/kg/day) decreased NOS-2 content and had similar effects compared to NOS-2 inhibition on iNO-mediated effects, whereas blockade of TNF-α signaling with etanercept (0.4 mg/kg on alternate days) had no effects on these parameters. We conclude that therapeutic hypercapnia prevents the adverse effects of sustained exposure to iNO on RV systolic function by limiting IL-1-mediated NOS-2 upregulation and consequent nitration. Therapeutic hypercapnia also acts synergistically with iNO in normalizing RV hypertrophy, vascular remodeling, and raised pulmonary vascular resistance secondary to chronic hypoxia.

Rho-kinase inhibitor prevents bleomycin-induced injury in neonatal rats independent of effects on lung inflammation.

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation dominated by neutrophils and macrophages, inhibited distal airway and vascular development, and pulmonary hypertension, similar to human infants with severe bronchopulmonary dysplasia. Rho-kinase (ROCK) is known to mediate lung injury in adult animals via stimulatory effects on inflammation. We therefore hypothesized that inhibition of ROCK may ameliorate bleomycin-induced lung injury in the neonatal rat. Pups received daily intraperitoneal bleomycin or saline from Postnatal Days 1 through 14 with or without Y-27632, a ROCK inhibitor. Treatment with Y-27632 prevented bleomycin-induced pulmonary hypertension, as evidenced by normalized pulmonary vascular resistance, decreased right-ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. Bleomycin-induced changes in distal lung architecture, including septal thinning, inhibited alveolarization, and decreased numbers of peripheral arteries and capillaries, were partially or completely normalized by Y-27632. Treatment with Y-27632 or a CXCR2 antagonist, SB265610, also abrogated tissue neutrophil influx, while having no effect on macrophages. However, treatment with SB265610 did not prevent bleomycin-induced lung injury. Lung content of angiostatic thrombospondin-1 (TSP1) was increased significantly in the lungs of bleomycin-exposed animals, and was completely attenuated by treatment with Y-27632. Thrombin-stimulated TSP1 production by primary cultured rat pulmonary artery endothelial cells was also attenuated by Y-27632. Taken together, our findings suggest a preventive effect of Y-27632 on bleomycin-mediated injury by a mechanism unrelated to inflammatory cells. Our data suggest that improvements in lung morphology may have been related to indirect stimulatory effects on angiogenesis via down-regulation of TSP1.

Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle.

Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% ("physiologic") or 20% ("supraphysiologic") at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H2O2 (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H2O2 release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H2O2 content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.

The IGF-I/IGF-R1 pathway regulates postnatal lung growth and is a nonspecific regulator of alveologenesis in the neonatal rat.

IGF-I, IGF-II, and the IGF-I receptor are widely distributed throughout the neonatal rat lung on days 4, 7, 10, and 14 of life, with a similar abundance at each of these time points. Injection of 20 µg/g of a truncated soluble IGF-I receptor on days 2 and 5 of life, to decoy ligand away from the endogenous IGF-I receptor, reduced lung weight and lung-to-body weight ratio, reduced lung tissue fraction, and impaired alveolar formation, as assessed by secondary crest formation and mean linear intercepts on day 7 of life. Lung procollagen I content and elastin fiber density were also reduced. Injection of 100 µg/day of neutralizing anti-IGF-I, to prevent IGF-I from binding to the IGF-I receptor, on days 3, 4, and 5 of life reduced tissue fraction and elastin fiber density and impaired alveolar formation on day 6 of life. Both interventions reduced total lung cell and secondary crest cell DNA synthesis and small vessel counts per unit area, but these effects were lost after normalization to the reduced tissue fraction. These findings are consistent with a role for IGF-I binding to the IGF-I receptor in postnatal lung growth and on alveologenesis through a nonspecific positive effect on DNA synthesis. Injection of 100 µg/day of neutralizing anti-IGF-II, to prevent IGF-II from binding to the IGF-I receptor, on days 3, 4, and 5 of life had no effect on total lung cell DNA synthesis per unit area on day 6 of life, and a role for IGF-II in postnatal alveologenesis was not further pursued.

Therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-α.

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation, arrested lung growth, and pulmonary hypertension (PHT), as observed in human infants with severe bronchopulmonary dysplasia. Inhalation of CO(2) (therapeutic hypercapnia) has been described to limit cytokine production and to have anti-inflammatory effects on the injured lung; we therefore hypothesized that therapeutic hypercapnia would prevent bleomycin-induced lung injury. Spontaneously breathing rat pups were treated with bleomycin (1 mg/kg/d ip) or saline vehicle from postnatal days 1-14 while being continuously exposed to 5% CO(2) (Pa(CO(2)) elevated by 15-20 mmHg), 7% CO(2) (Pa(CO(2)) elevated by 35 mmHg), or normocapnia. Bleomycin-treated animals exposed to 7%, but not 5%, CO(2), had significantly attenuated lung tissue macrophage influx and PHT, as evidenced by normalized pulmonary vascular resistance and right ventricular systolic function, decreased right ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. The level of CO(2) neither prevented increased tissue neutrophil influx nor led to improvements in decreased lung weight, septal thinning, impaired alveolarization, or decreased numbers of peripheral arteries. Bleomycin led to increased expression and content of lung tumor necrosis factor (TNF)-α, which was found to colocalize with tissue macrophages and to be attenuated by exposure to 7% CO(2). Inhibition of TNF-α signaling with the soluble TNF-2 receptor etanercept (0.4 mg/kg ip from days 1-14 on alternate days) prevented bleomycin-induced PHT without decreasing tissue macrophages and, similar to CO(2), had no effect on arrested alveolar development. Our findings are consistent with a preventive effect of therapeutic hypercapnia with 7% CO(2) on bleomycin-induced PHT via attenuation of macrophage-derived TNF-α. Neither tissue macrophages nor TNF-α appeared to contribute to arrested lung development induced by bleomycin. That 7% CO(2) normalized pulmonary vascular resistance and right ventricular function without improving inhibited airway and vascular development suggests that vascular hypoplasia does not contribute significantly to functional changes of PHT in this model.

Sustained therapeutic hypercapnia attenuates pulmonary arterial Rho-kinase activity and ameliorates chronic hypoxic pulmonary hypertension in juvenile rats.

Sustained therapeutic hypercapnia prevents pulmonary hypertension in experimental animals, but its rescue effects on established disease have not been studied. Therapies that inhibit Rho-kinase (ROCK) and/or augment nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling can reverse or prevent progression of chronic pulmonary hypertension. Our objective in the present study was to determine whether sustained rescue treatment with inhaled CO(2) (therapeutic hypercapnia) would improve structural and functional changes of chronic hypoxic pulmonary hypertension. Spontaneously breathing pups were exposed to normoxia (21% O(2)) or hypoxia (13% O(2)) from postnatal days 1-21 with or without 7% CO(2) (Pa(CO(2)) elevated by ∼25 mmHg) or 10% CO(2) (Pa(CO(2)) elevated by ∼40 mmHg) from days 14 to 21. Compared with hypoxia alone, animals exposed to hypoxia and 10% CO(2) had significantly (P < 0.05) decreased pulmonary vascular resistance, right-ventricular systolic pressure, right-ventricular hypertrophy, and medial wall thickness of pulmonary resistance arteries as well as decreased lung phosphodiesterase (PDE) V, RhoA, and ROCK activity. Rescue treatment with 10% CO(2), or treatment with a ROCK inhibitor (15 mg/kg ip Y-27632 twice daily from days 14 to 21), also increased pulmonary arterial endothelial nitric oxide synthase and lung NO content. In contrast, cGMP content and cGMP-dependent protein kinase (PKG) activity were increased by exposure to 10% CO(2), but not by ROCK inhibition with Y-27632. In vitro exposure of pulmonary artery smooth muscle cells to hypercapnia suppressed serum-induced ROCK activity, which was prevented by inhibition of PKG with Rp-8-Br-PET-cGMPS. We conclude that sustained hypercapnia dose-dependently inhibited ROCK activity, augmented NO-cGMP-PKG signaling, and led to partial improvements in the hemodynamic and structural abnormalities of chronic hypoxic PHT in juvenile rats. Increased PKG content and activity appears to play a major upstream role in CO(2)-induced suppression of ROCK activity in pulmonary arterial smooth muscle.

Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension.

Chronic pulmonary hypertension in infancy and childhood is characterized by a fixed and progressive increase in pulmonary arterial pressure and resistance, pulmonary arterial remodeling, and right ventricular hypertrophy and systolic dysfunction. These abnormalities are replicated in neonatal rats chronically exposed to hypoxia from birth in which increased activity of Rho-kinase (ROCK) is critical to injury, as evidenced by preventive effects of ROCK inhibitors. Our objective in the present study was to examine the reversing effects of a late or rescue approach to treatment with a ROCK inhibitor on the pulmonary and cardiac manifestations of established chronic hypoxic pulmonary hypertension. Rat pups were exposed to air or hypoxia (13% O(2)) from postnatal day 1 and were treated with Y-27632 (15 mg/kg) or saline vehicle by twice daily subcutaneous injection commencing on day 14, for up to 7 days. Treatment with Y-27632 significantly attenuated right ventricular hypertrophy, reversed arterial wall remodeling, and completely normalized right ventricular systolic function in hypoxia-exposed animals. Reversal of arterial wall remodeling was accompanied by increased apoptosis and attenuated content of endothelin (ET)-1 and ET(A) receptors. Treatment of primary cultured juvenile rat pulmonary artery smooth muscle cells with Y-27632 attenuated serum-stimulated ROCK activity and proliferation and increased apoptosis. Smooth muscle apoptosis was also induced by short interfering RNA-mediated knockdown of ROCK-II, but not of ROCK-I. We conclude that sustained rescue treatment with a ROCK inhibitor reversed both the hemodynamic and structural abnormalities of chronic hypoxic pulmonary hypertension in juvenile rats and normalized right ventricular systolic function. Attenuated expression and activity of ET-1 and its A-type receptor on pulmonary arterial smooth muscle was a likely contributor to the stimulatory effects of ROCK inhibition on apoptosis. In addition, our data suggest that ROCK-II may be dominant in enhancing survival of pulmonary arterial smooth muscle.

Pulmonary vascular and cardiac effects of peroxynitrite decomposition in newborn rats.

Evidence implicates oxidative stress as playing a prominent role in the pathogenesis of pulmonary hypertension, to which peroxynitrite anion (ONOO(-)) may make a major contribution. Hypothesizing that removal of ONOO(-) would attenuate chronic neonatal pulmonary hypertension, we examined the effects of a ONOO(-) decomposition catalyst (FeTPPS) on pulmonary arteries in vitro, on primary cultured pulmonary artery smooth muscle cell (PASMC) and cardiomyocyte survival and growth, and on central hemodynamics in rat pups exposed to hypoxia (13% O(2)) for 7 days from birth. Daily FeTPPS (30 mg/kg ip) reduced lung nitrotyrosine content, attenuated vascular remodeling, and normalized pulmonary vascular resistance in hypoxia-exposed animals. FeTPPS attenuated proliferation and increased apoptosis of neonatal PASMCs in vitro. Isolated neonatal pulmonary arteries treated with FeTPPS showed reduced agonist-induced force development and enhanced endothelium-dependent and -independent relaxation, possibly via increased nitrate. However, we observed endothelial dysfunction, enhanced lung tissue phosphodiesterase 5 activity, and biventricular cardiac hypertrophy in air-exposed animals receiving FeTPPS. Further, in contrast to PASMCs, FeTPPS enhanced survival of newborn cardiomyocytes. We conclude that decomposition of ONOO(-) with FeTPPS attenuates chronic hypoxia-induced pulmonary hypertension; however, it may negatively influence the modulation of normal pulmonary arterial relaxation function, cell survival, and growth.