Statins protect mice from high-decibel noise-induced hearing loss.

No medical interventions for noise induced hearing loss (NIHL) are approved by the Food and Drug Administration (USA). Here, we evaluate statins in CBA/CaJ mice as potential drugs for hearing loss. Direct delivery of fluvastatin to the cochlea and oral delivery of lovastatin were evaluated. Baseline hearing was assessed using Auditory Brain Stem Responses (ABRs). For fluvastatin, a cochleostomy was surgically created in the basal turn of the cochlea by a novel, laser-based procedure, through which a catheter attached to a mini-osmotic pump was inserted. The pump was filled with a solution of 50 µM fluvastatin+carrier or with the carrier alone for continuous delivery to the cochlea. Mice were exposed to one octave band noise (8-16 kHz x 2 h x 110 dB SPL). In our past work with guinea pigs, fluvastatin protected in the contralateral cochlea. In this study in CBA/CaJ mice, hearing was also assessed in the contralateral cochlea 1-4 weeks after noise exposure. At two weeks post exposure, ABR thresholds at 4, 8, 12, 16, and 32 kHz were elevated, as expected, in the noise+carrier alone treated mice by approximately 9-, 17-, 41-, 29-, and 34-dB, respectively. Threshold elevations were smaller in mice treated with noise+fluvastatin to about 2-, 6-, 20-,12- and 12-dB respectively. Survival of inner hair cell synapses were not protected by fluvastatin over these frequencies. Lovastatin delivered by gavage showed lower threshold shifts than with carrier alone. These data show that direct and oral statin delivery protects mice against NIHL.

Sex Genotyping of Archival Fixed and Immunolabeled Guinea Pig Cochleas.

For decades, outbred guinea pigs (GP) have been used as research models. Various past research studies using guinea pigs used measures that, unknown at the time, may be sex-dependent, but from which today, archival tissues may be all that remain. We aimed to provide a protocol for sex-typing archival guinea pig tissue, whereby past experiments could be re-evaluated for sex effects. No PCR sex-genotyping protocols existed for GP. We found that published sequence of the GP Sry gene differed from that in two separate GP stocks. We used sequences from other species to deduce PCR primers for Sry. After developing a genomic DNA extraction for archival, fixed, decalcified, immunolabeled, guinea pig cochlear half-turns, we used a multiplex assay (Y-specific Sry; X-specific Dystrophin) to assign sex to tissue as old as 3 years. This procedure should allow reevaluation of prior guinea pig studies in various research areas for the effects of sex on experimental outcomes.

Regulation of hypoxia-induced pulmonary hypertension by vascular smooth muscle hypoxia-inducible factor-1α.

RATIONALE: Chronic hypoxia induces pulmonary vascular remodeling, pulmonary hypertension, and right ventricular hypertrophy. At present, little is known about mechanisms driving these responses. Hypoxia-inducible factor-1α (HIF-1α) is a master regulator of transcription in hypoxic cells, up-regulating genes involved in energy metabolism, proliferation, and extracellular matrix reorganization. Systemic loss of a single HIF-1α allele has been shown to attenuate hypoxic pulmonary hypertension, but the cells contributing to this response have not been identified. OBJECTIVES: We sought to determine the contribution of HIF-1α in smooth muscle on pulmonary vascular and right heart responses to chronic hypoxia. METHODS: We used mice with homozygous conditional deletion of HIF-1α combined with tamoxifen-inducible smooth muscle-specific Cre recombinase expression. Mice received either tamoxifen or vehicle followed by exposure to either normoxia or chronic hypoxia (10% O2) for 30 days before measurement of cardiopulmonary responses. MEASUREMENTS AND MAIN RESULTS: Tamoxifen-induced smooth muscle-specific deletion of HIF-1α attenuated pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, right ventricular hypertrophy was unchanged despite attenuated pulmonary pressures. CONCLUSIONS: These results indicate that HIF-1α in smooth muscle contributes to pulmonary vascular remodeling and pulmonary hypertension in chronic hypoxia. However, loss of HIF-1 function in smooth muscle does not affect hypoxic cardiac remodeling, suggesting that the cardiac hypertrophy response is not directly coupled to the increase in pulmonary artery pressure.

Isolation of pulmonary artery smooth muscle cells from neonatal mice.

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al. that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 µM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

Developmental differences in hyperoxia-induced oxidative stress and cellular responses in the murine lung.

Exposure of newborn mice to high inspired oxygen elicits a distinct phenotype of compromised alveolar and vascular development, although lethality during long-term exposure is lower in newborns compared to adults. As the effects of hyperoxia are mediated by excessive reactive oxygen species (ROS) generation, we hypothesized that newborn mice may exhibit enhanced expression of antioxidant defenses or attenuated ROS generation compared with adults. We measured subcellular oxidant responses to acute hyperoxia in lung slices and alveolar epithelial cells at varying time points during postnatal murine lung development. Oxidant stress was assessed using RoGFP, a ratiometric protein thiol redox sensor, targeted to the cytosol or the mitochondrial matrix. In contrast to newborn resistance to oxygen-induced mortality, cells of lung slices from younger mice demonstrated exaggerated mitochondrial matrix oxidant stress compared to adults, whereas oxidant stress responses in the cytosol were absent. Cell death in lung slices from newborn mice exposed to 48h of hyperoxia was also greater than for adults. Consistent with these findings, expression of antioxidant enzymes in newborn lungs was lower than in adults, and induction of antioxidant levels and activity during 24h of in vivo exposure was absent. However, expression of the reactive oxygen species-generating enzyme NADPH oxidase 1 was increased with hyperoxic exposure in the young but not the adult lung. Collectively, these results suggest that the greater lethality in adult animals may be more likely attributed to processes such as inflammation than to differences in antioxidant defenses. Therapies for neonatal and adult oxidative lung injury should therefore consider and address developmental differences in oxidative stress responses.

Cyclic stretch induces inducible nitric oxide synthase and soluble guanylate cyclase in pulmonary artery smooth muscle cells.

In the pulmonary vasculature, mechanical forces such as cyclic stretch induce changes in vascular signaling, tone and remodeling. Nitric oxide is a potent regulator of soluble guanylate cyclase (sGC), which drives cGMP production, causing vasorelaxation. Pulmonary artery smooth muscle cells (PASMCs) express inducible nitric oxide synthase (iNOS), and while iNOS expression increases during late gestation, little is known about how cyclic stretch impacts this pathway. In this study, PASMC were subjected to cyclic stretch of 20% amplitude and frequency of 1 Hz for 24 h and compared to control cells maintained under static conditions. Cyclic stretch significantly increased cytosolic oxidative stress as compared to static cells (62.9 ± 5.9% vs. 33.3 ± 5.7% maximal oxidation), as measured by the intracellular redox sensor roGFP. Cyclic stretch also increased sGCß protein expression (2.5 ± 0.9-fold), sGC activity (1.5 ± 0.2-fold) and cGMP levels (1.8 ± 0.2-fold), as well as iNOS mRNA and protein expression (3.0 ± 0.9 and 2.6 ± 0.7-fold, respectively) relative to control cells. An antioxidant, recombinant human superoxide dismutase (rhSOD), significantly decreased stretch-induced cytosolic oxidative stress, but did not block stretch-induced sGC activity. Inhibition of iNOS with 1400 W or an iNOS-specific siRNA inhibited stretch-induced sGC activity by 30% and 68% respectively vs. static controls. In conclusion, cyclic stretch increases sGC expression and activity in an iNOS-dependent manner in PASMC from fetal lambs. The mechanism that produces iNOS and sGC upregulation is not yet known, but we speculate these effects represent an early compensatory mechanism to counteract the effects of stretch-induced oxidative stress. A better understanding of the interplay between these two distinct pathways could provide key insights into future avenues to treat infants with pulmonary hypertension.

Increased p22(phox)/Nox4 expression is involved in remodeling through hydrogen peroxide signaling in experimental persistent pulmonary hypertension of the newborn.

AIM: To determine if the NADPH oxidase isoform Nox4 contributes to increased H(2)O(2) generation in persistent pulmonary hypertension of the newborn (PPHN) pulmonary arteries (PA), and to identify downstream signaling targets of Nox4 that contribute to vascular remodeling and vasoconstriction. RESULTS: PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs, PA, and PA smooth muscle cells (PASMC) were isolated from control and PPHN lambs. Increased expression of p22(phox) and Nox4 in PPHN lungs, PA, and PASMC was associated with increased reactive oxygen species in PPHN PA, increased protein thiol oxidation in PPHN PASMC, and a decreased activity of extracellular superoxide dismutase (ecSOD) in the lungs and PASMC. Nox4 small interfering RNA (siRNA) decreased Nox4 expression and thiol oxidation and increased the ecSOD activity in PPHN PASMC. An increased activity of nuclear factor-kappa B (NFκB) and expression of its target gene cyclin D1 were detected in PPHN lungs, PA, and PASMC. Nox4 siRNA and catalase attenuated these increases in PASMC, and catalase decreased cyclin D1 expression in PPHN lungs. INNOVATION: This study demonstrates for the first time that Nox4 expression is elevated in a lamb model of neonatal pulmonary hypertension. It identifies increased NFκB and cyclin D1 expression and a decreased ecSOD activity as targets of increased Nox4 signaling. CONCLUSION: PPHN increases p22(phox) and Nox4 expression and activity resulting in elevated H(2)O(2) levels in PPHN PA. Increased H(2)O(2) induces vasoconstriction via mechanisms involving ecSOD inactivation, and stimulates vascular remodeling via NFκB activation and increased cyclin D1 expression. Approaches that inhibit the pulmonary arterial Nox4 activity may attenuate vasoconstriction and vascular remodeling in PPHN.

Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn.

NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O(2) for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-l-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-l-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22(phox) and p47(phox) and decreased the expression of Nox2 and p22(phox) in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity.

Hydrocortisone normalizes oxygenation and cGMP regulation in lambs with persistent pulmonary hypertension of the newborn.

In the pulmonary vasculature, cGMP levels are regulated by soluble guanylate cyclase (sGC) and phosphodiesterase 5 (PDE5). We previously reported that lambs with persistent pulmonary hypertension of the newborn (PPHN) demonstrate increased reactive oxygen species (ROS) and altered sGC and PDE5 activity, with resultant decreased cGMP. The objective of this study was to evaluate the effects of hydrocortisone on pulmonary vascular function, ROS, and cGMP in the ovine ductal ligation model of PPHN. PPHN lambs were ventilated with 100% O(2) for 24 h. Six lambs received 5 mg/kg hydrocortisone every 8 h times three doses (PPHN-hiHC), five lambs received 3 mg/kg hydrocortisone followed by 1 mg·kg(-1)·dose(-1) times two doses (PPHN-loHC), and six lambs were ventilated with O(2) alone (PPHN). All groups were compared with healthy 1-day spontaneously breathing lambs (1DSB). O(2) ventilation of PPHN lambs decreased sGC activity, increased PDE5 activity, and increased ROS vs. 1DSB lambs. Both hydrocortisone doses significantly improved arterial-to-alveolar ratios relative to PPHN lambs, decreased PDE5 activity, and increased cGMP relative to PPHN lambs. High-dose hydrocortisone also increased sGC activity, decreased PDE5 expression, decreased ROS, and increased total vascular SOD activity vs. PPHN lambs. These data suggest that hydrocortisone treatment in clinically relevant doses improves oxygenation and decreases hyperoxia-induced changes in sGC and PDE5 activity, increasing cGMP levels. Hydrocortisone reduces ROS levels in part by increasing SOD activity in PPHN lambs ventilated with 100% O(2.) We speculate that hydrocortisone increases cGMP by direct effects on sGC and PDE5 expression and by attenuating abnormalities induced by oxidant stress.

Mitochondrial oxidant stress increases PDE5 activity in persistent pulmonary hypertension of the newborn.

In the pulmonary vasculature, phosphodiesterase-5 (PDE5) degrades cGMP and inhibits nitric oxide-mediated, cGMP-dependent vasorelaxation. We previously reported that ventilation with 100% O2 increased PDE5 activity in pulmonary arteries (PAs) of pulmonary hypertension lambs (PPHN) more than in control lambs. In the present study, PA smooth muscle cells (PASMCs) from PPHN lambs had increased basal PDE5 activity, decreased cGMP-responsiveness to NO, and increased mitochondrial matrix oxidant stress compared to control PASMC. Hyperoxia (24 h) increased PDE5 activity and mitochondrial matrix oxidant stress above baseline to a similar degree in PPHN and control PASMC. Mitochondrially targeted catalase decreased PDE5 activity at baseline and after hyperoxia in PPHN PASMC. Similarly, catalase treatment of PPHN lambs ventilated with 100% O2 decreased PDE5 activity and increased cGMP in PA. We conclude that baseline PDE5 activity and oxidative stress is increased in PPHN PASMC, and scavenging H2O2 is sufficient to block oxidant-mediated increases in PDE5 activity in PPHN.

SOD and inhaled nitric oxide normalize phosphodiesterase 5 expression and activity in neonatal lambs with persistent pulmonary hypertension.

Phosphodiesterase 5 (PDE5) and soluble guanylate cyclase (sGC) are key regulators of cGMP and pulmonary vascular tone. We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled nitric oxide (iNO) or recombinant human Cu/Zn SOD (rhSOD) on sGC, PDE5, and cGMP in the ovine ductal ligation model of persistent pulmonary hypertension of the newborn (PPHN). PPHN lambs were ventilated with 100% O(2) for 24 h alone or combined with either inhalation of 20 parts per million (ppm) iNO continuously or a single intratracheal dose of rhSOD (5 mg/kg). Ventilated PPHN lambs were compared with PPHN fetuses, control fetuses, and 1-day-old spontaneously breathing lambs (1DSB). In the small pulmonary arteries of 1DSB lambs, sGC expression increased, PDE5 expression decreased, and cGMP concentrations increased relative to fetal levels. In PPHN lambs ventilated with 100% O(2), sGC activity increased to levels comparable with 1DSB levels. However, PDE5 expression and activity increased, and cGMP levels remained at fetal levels. Addition of either iNO or rhSOD decreased PDE5 expression and activity in PPHN lambs and increased cGMP levels to levels comparable with 1DSB lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) impairs cGMP-mediated vasodilation in part due to increased PDE5 expression and activity. The addition of either iNO or rhSOD normalized PDE5 and cGMP levels. Thus therapies designed to decrease PDE5 and increase cGMP, such as iNO and rhSOD, may prove useful in the treatment of PPHN in newborn infants.

Regulation of phosphodiesterase 3 in the pulmonary arteries during the perinatal period in sheep.

The role of cAMP in the pulmonary vasculature during the transition from intrauterine to extrauterine life is poorly understood. We hypothesized that cAMP levels are regulated by alterations in phosphodiesterase 3 (PDE3), which hydrolyzes cAMP. PDE3 protein expression and hydrolytic activity were increased in the resistance pulmonary arteries (PA) from spontaneously breathing 1-d-old (1dSB) lambs relative to equivalent-gestation fetuses. This was accompanied by a decrease in steady-state cAMP. Ventilation with 21% O2 and 100% O2 for 24 h disrupted the normal transition, whereas ventilation with 100% O2 + inhaled NO (iNO) for 24 h restored both PDE3 activity and cAMP to 1dSB levels. Consistent with these findings, relaxation to milrinone, a PDE3 inhibitor, was greater in the PA isolated from 1dSB and 100% O2 + iNO lambs, relative to fetal, 21% O2, and 100% O2 lambs. In conclusion, PDE3 expression and activity in the PA dramatically increase after birth, with a concomitant decrease in steady-state cAMP. Ventilation with either 21% O2 or 100% O2 blunts this PDE3 increase, whereas iNO restores PDE3 activity to levels equivalent to 1dSB lambs. The vasodilatory effects of milrinone were most pronounced in vessels from lambs with the highest PDE3 activity, i.e., 1dSB and 100% O2 + iNO lambs. Thus, milrinone may be most beneficial when used in conjunction with iNO.

Superoxide dismutase restores eNOS expression and function in resistance pulmonary arteries from neonatal lambs with persistent pulmonary hypertension.

Endothelial nitric oxide (NO) synthase (eNOS) expression and activity are decreased in fetal lambs with persistent pulmonary hypertension (PPHN). We sought to determine the impact of mechanical ventilation with O(2) with or without inhaled NO (iNO) or recombinant human SOD (rhSOD) on eNOS in the ductal ligation model of PPHN. PPHN lambs and age-matched controls were ventilated with 100% O(2) for 24 h alone or combined with 20 ppm iNO continuously or a single dose of rhSOD (5 mg/kg) given intratracheally at delivery. In 1-day spontaneously breathing lambs, eNOS expression in resistance pulmonary arteries increased relative to fetal levels. eNOS expression increased in control lambs ventilated with 100% O(2), but not in PPHN lambs. Addition of iNO or rhSOD increased eNOS expression and decreased generation of reactive oxygen species (ROS) in PPHN lambs relative to those ventilated with 100% O(2) alone. However, only rhSOD restored eNOS function, increased tetrahydrobiopterin (BH(4)), a critical cofactor for eNOS function, and restored GTP cyclohydrolase I expression in isolated vessels and lungs from PPHN lambs. These data suggest that ventilation of PPHN lambs with 100% O(2) increases ROS production, blunts postnatal increases in eNOS expression, and decreases available BH(4) in PPHN lambs. Although the addition of iNO or rhSOD diminished ROS production and increased eNOS expression, only rhSOD improved eNOS function and levels of available BH(4). Thus therapies designed to decrease oxidative stress and restore eNOS coupling, such as rhSOD, may prove useful in the treatment of PPHN in newborn infants.

Hyperoxia increases phosphodiesterase 5 expression and activity in ovine fetal pulmonary artery smooth muscle cells.

In the pulmonary vasculature, cGMP concentrations are regulated in part by a cGMP-dependent phosphodiesterase (PDE), PDE5. Infants with persistent pulmonary hypertension of the newborn (PPHN) are often mechanically ventilated with high oxygen concentrations. The effects of hyperoxia on the developing pulmonary vasculature and PDE5 are largely unknown. Here, we demonstrate that exposure of fetal pulmonary artery smooth muscle cells (FPASMCs) to high levels of oxygen for 24 hours leads to decreased responsiveness to exogenous NO, as determined by a decreased intracellular cGMP response, increased PDE5 mRNA and protein expression, as well as increased PDE5 cGMP hydrolytic activity. We demonstrate that inhibition of PDE5 activity with sildenafil partially rescues cGMP responsiveness to exogenous NO. In FPASMCs, hyperoxia leads to increased oxidative stress without increasing cell death. Treatment of normoxic FPASMCs with H2O2 is sufficient to induce PDE5 expression and activity, suggesting that reactive oxygen species mediate the effects of hyperoxia in FPASMCs. In support of this mechanism, a chemical antioxidant, N-acetyl-cysteine, is sufficient to block the hyperoxia-mediated increase in PDE5 expression and activity and rescue cGMP responsiveness to exogenous NO. Finally, ventilation of healthy neonatal sheep with 100% O2 for 24 hours leads to increased PDE5 protein expression in the resistance pulmonary arteries and increased PDE5 activity in whole lung extracts. These data suggest that PDE5 expression and activity play a critical role in modulating neonatal pulmonary vascular tone in response to common clinical treatments for PPHN, such as oxygen and inhaled NO.