Fetal Pulmonary Artery Smooth Muscle Research Articles

In Vitro Consequences of Electronic-Cigarette Flavoring Exposure on the Immature Lung

Background: The developing lung is uniquely susceptible and may be at increased risk of injury with exposure to e-cigarette constituents. We hypothesize that cellular toxicity and airway and vascular responses with exposure to flavored refill solutions may be altered in the immature lung. Methods: Fetal, neonatal, and adult ovine pulmonary artery smooth muscle cells (PASMC) were exposed to popular flavored nicotine-free e-cigarette refill solutions (menthol, strawberry, tobacco, and vanilla) and unflavored solvents: propylene glycol (PG) or vegetable glycerin (VG). Viability was assessed by lactate dehydrogenase assay. Brochodilation and vasoreactivity were determined on isolated ovine bronchial rings (BR) and pulmonary arteries (PA). Results: Neither PG or VG impacted viability of immature or adult cells; however, exposure to menthol and strawberry flavored solutions increased cell death. Neonatal cells were uniquely susceptible to menthol flavoring-induced toxicity, and all four flavorings demonstrated lower lethal doses (LD50) in immature PASMC. Exposure to flavored solutions induced bronchodilation of neonatal BR, while only menthol induced airway relaxation in adults. In contrast, PG/VG and flavored solutions did not impact vasoreactivity with the exception of menthol-induced relaxation of adult PAs. Conclusion: The immature lung is uniquely susceptible to cellular toxicity and altered airway responses with exposure to common flavored e-cigarette solutions.

Cyclic Nucleotides Reduce Ryanodine Receptor Mediated Ca2+ Spark Activation Independent of Long Term Hypoxia in Ovine Fetal Pulmonary Arterial Myocytes

Ryanodine receptors (RyR) release Ca2+ from smooth muscle sarcoplasmic reticulum in rapid events known as Ca2+ sparks. These Ca2+ sparks activate voltage and Ca2+ gated potassium channels (BK channels) that cause cell repolarization. Ca2+ spark‐BK channel mediated cell repolarization dilates many types of vessels and is essential to pulmonary arterial (PA) vasodilation during the fetal transition to breathing air. Infants who do not successfully negotiate this transition are at risk of developing pulmonary hypertension, which has significant morbidity and mortality. Previous work shows that long term hypoxia (LTH) decreases Ca2+ spark events in fetal pulmonary arterial smooth muscle cells (PASMCs) even though the Ca2+ stores have functional RyRs. Additional studies illustrate that cyclic nucleotides can increase RyR activity, which led us to test the hypothesis that cyclic nucleotides could rescue RyR activity in myocytes of LTH fetuses. This was examined by treating PAs isolated from low (700m; LA) and high altitude (3801m; HA) near‐term fetal sheep with exogenous cyclic AMP (cAMP) and cyclic GMP (cGMP) or the methylxanthine IBMX, a non‐specific cAMP and cGMP phosphodiesterase inhibitor. RyR activity was measured by confocal imaging of Ca2+ sparks in fluo‐4 labeled PASMCs. The spatial and temporal aspects to the Ca2+ sparks were evaluated using SparkLab, a custom software package. Contrary to our hypothesis, the prevalence of Ca2+ sparks decreased in the presence of cAMP, cGMP, and IBMX for PASMC from both LA and HA fetal sheep. These data suggest that cyclic nucleotides reduce instead of increase vasodilatory capacity via RyR‐BK channel coupling mechanisms. Furthermore, increased cyclic nucleotides had no effect on the spatial‐temporal aspects to the Ca2+ sparks. Even though the signaling pathway remains unclear, the reduction of Ca2+ sparks by cyclic nucleotides is potentially a novel mechanism of RyR modulation in sheep fetal PASMC. This data suggests that increasing cyclic nucleotides may be contra‐indicated in newborn infants who are at risk of developing pulmonary hypertension.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. SH was a Loma Linda University Summer Undergraduate Research Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxia-induced cytosolic calcium influx is mediated primarily by the reverse mode of Na+/Ca2+ exchanger in smooth muscle cells of fetal small pulmonary arteries.

Constriction of small pulmonary arteries and high resistance of pulmonary circulation are important for maintaining fetal circulation before birth. In this study, we investigated how cytosolic free calcium concentration ([Ca(2+)]i) in fetal lamb small pulmonary artery smooth muscle cells (SPASMC) was affected by hypoxia and regulated by calcium pumps during this process. (Ca(2+))i in response to acute hypoxia was determined spectrofluorometrically with fluo-3AM in cultured fetal SPASMC. Chemicals or solutions, including ryanodine, 2-aminoethoxydiphenyl borate, Ca(2+)-free solution with 20 mmol ethyleneglycoltetraacetic (EGTA), nimodipine, Na(+)-free medium and KB-R7943, were administrated at the same time point when samples were exposed to acute hypoxia. (Ca(2+))i in fetal lamb SPASMC increased under acute hypoxia. 2-Aminoethoxydiphenyl borate, an inhibitor of inositol triphosphate calcium store, partially attenuated the (Ca(2+))i increase after 6-min treatment. Ryanodine, an inhibitor of ryanodine-sensitive calcium stores, had no effect on the (Ca(2+))i increase. Ca(2+)-free solution with EGTA completely abolished this increase. Both nimodipine, that blocks the voltage-gated calcium channel, and KB-R7943, that inhibits the reverse mode of Na(+)/Ca(2+) exchanger, greatly diminished the hypoxia-induced (Ca(2+))i increase. The inhibitory effect of KB-R7943 was stronger than nimodipine, evidenced by the fact that (Ca(2+))i dropped near to the baseline level in the presence of KB-R7943 at a later time point. Low extracellular Na(+) concentration enhanced the hypoxia-induced increase of (Ca(2+))i. These results suggest that hypoxia-induced Ca(2+) increase in fetal SPASMC results from cytosolic Ca(2+) influx mediated primarily by the reverse mode of Na(+)/Ca(2+) exchanger.

Cyclic nucleotides down regulate PAF receptor mediated effects in newborn ovine pulmonary arterial smooth muscle: significance in oxygen therapy of persistent pulmonary hypertension of the newborn (LB605)

Platelet activating factor (PAF) is implicated in the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN). Baseline PAFr activity is higher in fetal ovine pulmonary artery smooth muscle cells (PASMC) and is augmented by hypoxia. In fetal PASMC we have shown evidence of crosstalk between PAFr‐mediated cell signaling and activity of cyclic nucleotides (Cnucs) via respective receptors PKG and PKA. Since these mechanisms are not well described in the newborn counterpart, we studied effects of Cnucs on PAFr protein expression in newborn PASMC. Because long term effects of oxygen therapy on newborn lung are not well understood, we included various oxygen tension study conditions. We hypothesize that PPHN results from failure of newborn lamb pulmonary system to down regulate PAFr activity or to up regulate vasodilatory Cnuc activity.PASMC harvested from ovine newborns 6‐12d old were incubated for 24h with culture media or media containing 10μM cGMP or cAMP analogs or 0.1μM of PAF in normoxia (pO2~100torr), hypoxia (pO2 <40 torr) and hyperoxia (5% CO2, O2). Prostacyclin and TxA2 release were measured and compared to release by fetal PASMC. Proliferation studies were performed under the three conditions and PAFr, PKA and PKG expression were measured.Newborn PA baseline prostacyclin level was 9‐fold greater than in fetal PA, whereas baseline production of TxA2 by newborn PA was 7‐fold less. Overall, 24h hyperoxia attenuated cell proliferation across all treatments. Cnuc treatment attenuated PAF stimulation of proliferation and decreased PAFr expression.Elevated prostacyclin production over TxA2 suggests greater cyclooxygenase activity in newborn PA postnatally. Down regulation of PAFr effects by Cnucs indicates that normal newborn PA physiology favors vasodilatory pathways to minimize PAF‐induced hypertrophy or hyperplasia. Thus failure of newborn lung to anchor down regulation of vasoconstrictors with up regulation of vasodilators leads to pathogenesis of PPHN.

Hydrocortisone normalizes phosphodiesterase-5 activity in pulmonary artery smooth muscle cells from lambs with persistent pulmonary hypertension of the newborn.

Phosphodiesterase-5 (PDE5) is the primary phosphodiesterase in the pulmonary vasculature. It degrades cyclic guanosine monophosphate (cGMP) and inhibits cGMP-mediated vasorelaxation. We previously reported that hydrocortisone treatment decreased hyperoxia-induced PDE5 activity and markers of oxidative stress in lambs with persistent pulmonary hypertension of the newborn (PPHN) ventilated with 100% O2. The objective of our study was to determine the molecular mechanism by which hydrocortisone downregulates PDE5 and oxidative stress in fetal pulmonary artery smooth muscle cells (FPASMCs) from PPHN lambs. PPHN FPASMC were incubated for 24 hours in either 21% or 95% O2. Some cells were treated with 100 nM hydrocortisone and/or ±1 μM helenalin, an inhibitor of nuclear factor κ B (NFκB), a redox-sensitive transcription factor. Exposure to hyperoxia led to increased PDE5 activity, oxidative stress, and NFκB activity. Pretreatment of PPHN FPASMC with hydrocortisone normalized PDE5 activity, decreased cytosolic oxidative stress, increased expression of extracellular superoxide dismutase and NFκB inhibitory protein, and decreased NFκB activity. Similarly, treatment with NFκB inhibitor, helenalin, decreased PDE5 activity. These data suggest that hyperoxia activates NFκB, which in turn induces PDE5 activity in PPHN FPASMC, whereas treatment with hydrocortisone attenuates these changes by blocking reactive oxygen species-induced NFκB activity.

Brief Hyperoxia Increases Mitochondrial Oxidation and Increases Phosphodiesterase 5 Activity in Fetal Pulmonary Artery Smooth Muscle Cells

Oxygen is a pulmonary vasodilator, but data suggest high O(2) concentrations impede that response. We previously reported 24 h of 100% O(2) increased phosphodiesterase 5 (PDE5) activity in fetal pulmonary artery smooth muscle cells (FPASMC) and in ventilated neonatal lambs. PDE5 degrades cyclic GMP (cGMP) and inhibits nitric oxide (NO)-mediated cGMP-dependent vasorelaxation. We sought to determine the mechanism by which hyperoxia initiates reactive oxygen species (ROS) production and regulates PDE5. Thirty minutes of hyperoxia increased mitochondrial ROS versus normoxia (30.3±1.7% vs. 21.1±2.8%), but had no effect on cytosolic ROS, measured by roGFP, a ratiometric protein thiol redox sensor. Hyperoxia increased PDE5 activity (220±39%) and decreased cGMP responsiveness to NO (37±17%). Mitochondrial catalase overexpression attenuated hyperoxia-induced mitochondrial roGFP oxidation, compared to FPASMC infected with empty adenoviral vector (50±3% of control) or mitochondrial superoxide dismutase. MitoTEMPO, mitochondrial catalase, and DT-3, a cGMP-dependent protein kinase I alpha inhibitor, decreased PDE5 activity (32±13%, 26±21%, and 63±10% of control, respectively), and restored cGMP responsiveness to NO (147±16%,172±29%, and 189±43% of control, respectively). C57Bl6 mice exposed to 90%-100% O(2) for 45 min±mechanical ventilation had increased PA PDE5 activity (206±39% and 235±75%, respectively). This is the first description that hyperoxia induces ROS in the mitochondrial matrix prior to the cytosol. Our results indicate that short hyperoxia exposures can produce significant changes in critical cellular signaling pathways. These results indicate that mitochondrial matrix oxidant signals generated during hyperoxia, specifically H(2)O(2), activate PDE5 in a cGMP-dependent protein kinase-dependent manner in pulmonary vascular smooth muscle cells.

Hypoxic Regulation of Pulmonary Vascular Smooth Muscle Cyclic Guanosine Monophosphate–Dependent Kinase by the Ubiquitin Conjugating System

We previously reported that hypoxia attenuates nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated fetal pulmonary vessel relaxation by inhibiting cGMP-dependent protein kinase 1 (PKG1) activity, but not all the mechanisms by which acute hypoxia inhibits PKG1 activity have been delineated. Here we demonstrate for the first time, to the best of our knowledge, that acute hypoxia induces an accumulation of ubiquitinated PKG1 in ovine fetal and newborn pulmonary artery smooth muscle cells. Such a modification was not evident in ovine fetal systemic (cerebral) artery smooth muscle cells. The accumulation of polyubiquitinated PKG1 observed after 4 hours of hypoxia was affected neither by the activation of PKG1 kinase activity with the cell-permeable cGMP analogue 8-bromo-cGMP, nor by its inhibition with DT-3 in fetal pulmonary artery smooth muscle cells. Ubiquitinated PKG1α was unable to bind the cGMP analogue 8-(2-aminoethyl)thioguanosine-3',5' (AET)-cGMP, a ligand for the unmodified protein. Inhibition of the proteasomal complex with MG132 led to the accumulation of polyubiquitinated PKG1 in normoxia, indicating the involvement of the ubiquitin-26S proteasomal system in degradation and clearance of this protein under normoxic conditions. The ubiquitinated PKG1 under hypoxic conditions, however, was not predominantly targeted for proteasomal degradation. Importantly, reoxygenation reversed the acute hypoxia-induced accumulation of ubiquitinated PKG1. Our results suggest that the PKG1 ubiquitination induced by acute hypoxia plays a unique role in the regulation of the pulmonary vascular smooth muscle cell vasoreactivity and relaxation mediated by the NO-cGMP-PKG1 pathway.

Hypoxia Modulates the Expression of Leucine Zipper‐Positive MYPT1 and its Interaction with Protein Kinase G and Rho Kinases in Pulmonary Arterial Smooth Muscle Cells

We have shown previously that acute hypoxia downregulates protein kinase G (PKG) expression and activity in ovine fetal pulmonary vessels and pulmonary arterial smooth muscle cells (SMC). Here, we report that acute hypoxia also reduces the expression of leucinezipper-positive MYPT1 (LZ+MYPT1), a subunit of myosin light chain (MLC) phosphatase, in ovine fetal pulmonary arterial SMC. We found that in hypoxia, there is greater interaction between LZ+ MYPT1 and RhoA and Rho kinase 1 (ROCK1)/Rho kinase 2 (ROCK2) and decreased interaction between LZ+ MYPT1 and PKG, resulting in increased MLC20 phosphorylation, a higher pMLC20/MLC20 ratio and SMC contraction. In normoxic SMC PKG overexpression, LZ+ MYPT1 expression is upregulated while PKG knockdown had an opposite effect. LZ+ MYPT1 overexpression enhanced the interaction between PKG and LZ+ MYPT1. Overexpression of a mutant LZ- MYPT1 isoform in SMC mimicked the effects of acute hypoxia and decreased pMLC20/MLC20 ratio. Collectively, our data suggest that hypoxia downregulates LZ+ MYPT1 expression by suppressing PKG levels, reduces the interaction of LZ+ MYPT1 with PKG and promotes LZ+ MYPT1 interaction with RhoA or ROCK1/ROCK2, thereby promoting pulmonary arterial SMC contraction.

Cinaciguat, a soluble guanylate cyclase activator, causes potent and sustained pulmonary vasodilation in the ovine fetus

Impaired nitric oxide-cGMP signaling contributes to severe pulmonary hypertension after birth, which may in part be due to decreased soluble guanylate cyclase (sGC) activity. Cinaciguat (BAY 58-2667) is a novel sGC activator that causes vasodilation, even in the presence of oxidized heme or heme-free sGC, but its hemodynamic effects have not been studied in the perinatal lung. We performed surgery on eight fetal (126 +/- 2 days gestation) lambs (full term = 147 days) and placed catheters in the main pulmonary artery, aorta, and left atrium to measure pressures. An ultrasonic flow transducer was placed on the left pulmonary artery to measure blood flow, and a catheter was placed in the left pulmonary artery for drug infusion. Cinaciguat (0.1-100 microg over 10 min) caused dose-related increases in pulmonary blood flow greater than fourfold above baseline and reduced pulmonary vascular resistance by 80%. Treatment with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an sGC-oxidizing inhibitor, enhanced cinaciguat-induced pulmonary vasodilation by >120%. The pulmonary vasodilator effect of cinaciguat was prolonged, decreasing pulmonary vascular resistance for >1.5 h after brief infusion. In vitro stimulation of ovine fetal pulmonary artery smooth muscle cells with cinaciguat after ODQ treatment resulted in a 14-fold increase in cGMP compared with non-ODQ-treated cells. We conclude that cinaciguat causes potent and sustained fetal pulmonary vasodilation that is augmented in the presence of oxidized sGC and speculate that cinaciguat may have therapeutic potential for severe neonatal pulmonary hypertension.

Developmental regulation of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth muscle cells.

The transcriptional machinery involved in the transition of an infant from intrauterine to air-breathing life is developmentally regulated, as the fetus and adult manifest differential genetic expression. The low oxygen (O(2)) environment of the mammalian fetus and the increase in O(2) tension that occurs at birth may account for the developmentally regulated alterations in gene expression. We tested the hypothesis that hypoxia-inducible factor 1 (HIF-1) expression, an O(2)-sensitive transcription factor, is developmentally regulated. We found that in fetal pulmonary artery (PA) smooth muscle cells (SMC), fetal HIF-1 protein levels were O(2)-insensitive, whereas in adult PA SMC, hypoxia increased HIF-1 protein expression. Surprisingly, hypoxia increased HIF-1 mRNA expression in fetal, but not in adult, PA SMC. HIF-1 degradation and transcriptional activity is contingent on prolyl- and asparagyl-hydroxylases. To determine whether developmental differences in O(2) sensitivity or expression of these enzymes accounts for the divergence of HIF-1 sensitivity between fetus and adult, we studied the expression of the three most well characterized prolyl-hydroxylases, PHD1, PHD2, and PHD3, and the expression of regulators of HIF-1 transcriptional activity, asparagyl-hydroxylase, factor inhibiting HIF, and the oncogenic factor, CITED2 (CREB-binding protein/p300 interacting transactivator with ED-rich tail). We found that, as in the case of HIF-1, these genes are differentially regulated in the fetus, enabling the mammalian fetus to thrive in the low O(2) tension intrauterine environment even while rendering a newborn infant uniquely well adapted to respond to the acute increase in O(2) tension that occurs at birth.

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.

Cyclic Nucleotides Down‐Regulate Platelet Activating Factor Receptor (PAFr) Protein Expression in Ovine Fetal Pulmonary Arterial Smooth Muscle Cells (SMC‐PA)

Cyclic Nucleotides Down‐Regulate Platelet Activating Factor Receptor (PAFr) Protein Expression in Ovine Fetal Pulmonary Arterial Smooth Muscle Cells (SMC‐PA)

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.

Hypoxia Down-Regulates Cyclic Guanidine Monophosphate-Dependent Protein Kinase in Fetal Pulmonary Vascular Smooth Muscle Cell Through Generation of Reactive Oxygen Species and Promotes Development of Pulmonary Hypertension

We have previously reported that cyclic guanidine monophosphate (cGMP) mediates vasodilation primarily through protein kinase G (PKG) in fetal pulmonary vessels 1 Dhanakoti SN Gao Y Nguyen MQ et al. Involvement of cGMP-dependent protein kinase in the relaxation of ovine pulmonary arteries to cGMP and cAMP. J Appl Physiol. 2000; 88: 1637-1642 PubMed Google Scholar and that acute hypoxia down-regulates cGMP-PKG-mediated relaxation in these vessels. 2 Gao Y Dhanakoti SN Trevino EM et al. Effect of oxygen on cyclic GMP-dependent protein kinase-mediated relaxation in ovine fetal pulmonary arteries and veins. Am J Physiol Lung Cell Mol Physiol. 2003; 285: L611-L618 Crossref Scopus (34) Google Scholar The mechanism by which this occurs is not known. Using a microarray, we found that in human pulmonary vascular smooth muscle cells (PVSMCs) 4 h of hypoxia altered the expression of > 900 genes fourfold or more. This was reduced to 121 genes by the reactive oxygen species (ROS) scavenger ascorbate, suggesting a role for ROS in hypoxic gene expression. Hence, first we used dichlorodihydrofluorescein (DCF) to measure ROS production in the pulmonary artery and pulmonary vein SMC during hypoxia. We determined identity of ROS by using scavengers for superoxide, hydrogen peroxide, and peroxynitrite during DCF fluorescence measurement. Ovine fetal pulmonary artery smooth muscle cells and PVSMCs were grown in Dulbecco minimum essential medium, preloaded with DCF and incubated under conditions of hypoxia (0% O2-5% CO2) or normoxia (20% O2-5% CO2). After 30 min, hypoxia ROS production increased fourfold to sixfold compared to normoxia but returned to normoxic levels after 4 h. Pretreatment of PVSMCs with ROS scavengers (Tiron for superoxide radical and Trolox for peroxynitrite radical) or with mitochondria complex III inhibitor myxothiazol significantly decreased ROS generated by hypoxia. After 4 h, hypoxia reduced PKG protein and PKG1 messenger RNA expression in PVSMCs. The scavenging of superoxide and peroxynitrite generated during hypoxia prevented the hypoxic decrease in PKG protein and PKG1 messenger RNA expression. Our findings indicate that the hypoxic generation of ROS in PVSMCs leads to the down-regulation of cGMP-mediated vasodilation by the inhibition of PKG and predisposes the patient to the development of pulmonary hypertension.

Rat pulmonary arterial smooth muscle myosin light chain kinase and phosphatase activities decrease with age

We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced compared with the adult. Whether these developmental changes relate to age differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1cdelta) and regulatory (MYPT) subunits in late fetal, early newborn, and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blot analysis showed that the MLCK, MYPT, and PP1cdelta contents increased significantly with age and were highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y-27632 was greater than the adult tissue. In addition to the 130-kDa isoform of MLCK, we documented the presence of minor higher-molecular-weight embryonic isoforms in the fetus and newborn. During fetal life, the lung pulmonary arterial MLCK- and MLCP-specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.

Reactive Oxygen Species are Generated in Hypoxia in Ovine Fetal Pulmonary Artery and Vein Smooth Muscle and are Likely to be Superoxide and Peroxynitrite

Reactive Oxygen Species are Generated in Hypoxia in Ovine Fetal Pulmonary Artery and Vein Smooth Muscle and are Likely to be Superoxide and Peroxynitrite

Effect of normoxia and hypoxia on K+ current and resting membrane potential of fetal rabbit pulmonary artery smooth muscle

At birth, the increase in O(2) tension (pO(2)) is an important cause of the decrease in pulmonary vascular resistance. In adult animals there are impressive interspecies differences in the level of hypoxia required to elicit a pulmonary vasoconstrictor response and in the amplitude of the response. Hypoxic inhibition of some potassium (K(+)) channels in the membrane of pulmonary arterial smooth muscle cells (PASMCs) helps to initiate hypoxic pulmonary vasoconstriction. To determine the effect of the change in pO(2) on fetal rabbit PASMCs and to investigate possible species-dependent differences, we measured the current-voltage relationship and the resting membrane potential, in PASMCs from fetal resistance arteries using the amphotericin-perforated patch-clamp technique under hypoxic and normoxic conditions. Under hypoxic conditions, the K(+) current in PASMCs was small, and could be inhibited by 4-aminopyridine, iberiotoxin and glibenclamide, reflecting contributions by Kv, K(Ca) and K(ATP) channels. The average resting membrane potential was -44.3+/-1.3 mV (n=29) and could be depolarized by 4-AP (5 mM) and ITX (100 nM) but not by glibenclamide (10 microM). Changing from hypoxia, that mimicked fetal life, to normoxia dramatically increased the K(Ca) and consequently hyperpolarized (-9.3+/-1.7 mV; n=8) fetal rabbit PASMCs. Under normoxic conditions K(+) current was reduced by 4-AP with a significant change in resting membrane potential (11.1+/-1.7 mV; n=8). We conclude that resting membrane potential in fetal rabbit PASMCs under both hypoxic and normoxic conditions depends on both Kv and K(Ca) channels, in contrast to fetal lamb or porcine PASMCs. Potential species differences in the K(+) channels that control resting membrane potential must be taken into consideration in the interpretation of studies of neonatal pulmonary vascular reactivity to changes in O(2) tension.

Combined superoxide dismutase/catalase mimetics alter fetal pulmonary arterial smooth muscle cell growth.

Reactive oxygen species (ROS) are known to play an important role in the proliferation and viability of vascular smooth muscle cells. We have shown previously that treatment of fetal pulmonary arterial smooth muscle cells (FPASMC) with concentrations of 25 microM and higher of EUK-134, a superoxide dismutase/catalase mimetic, decreased cell viability via the induction of apoptosis. Here we demonstrate a dose-dependent decrease in serum-induced FPASMC growth at lower doses of EUK-134. This was due to the attenuation of FPASMC proliferation rather than the induction of apoptosis. Moreover, we found that the inhibition of FPASMC proliferation was observed using EUK-134 at concentrations as low as 5 microM. This inhibition of proliferation correlated with a 31% decrease in superoxide levels, as estimated using the oxidation of dihydroethidium. Flow cytometry revealed an increase in FPASMC in G2 after 24 h of exposure to 10 microM EUK-134. This was associated with a twofold increase in levels of the cell-cycle regulatory protein p21. This, together with our previous data, suggests that ROS levels determine the rate of FPASMC proliferation and, when below a threshold level, trigger apoptosis. Titration of ROS with antioxidants may help to prevent, or reverse, the vascular remodeling manifest in many cardiovascular disease states.

Molecular mechanisms of nitric oxide-induced growth arrest and apoptosis in fetal pulmonary arterial smooth muscle cells

Superoxide plays an important role in pulmonary arterial smooth muscle cell (SMC) proliferation and survival. The rapid reaction between superoxide and nitric oxide (NO) to form peroxynitrite suggests that endothelium-derived NO may influence adjacent SMC growth. To investigate this possibility, we determined the dose-dependent effects of NO on the proliferation and viability of pulmonary arterial SMC isolated from fetal lambs (FPASMC). Using fluorescence microscopy we found a dose-dependent decrease in superoxide levels in FPASMC treated with the NO donor spermine NONOate. This was associated with an increase in peroxynitrite-mediated protein nitration. At doses between 50 and 250 μM, spermine NONOate attenuated serum-induced FPASMC proliferation resulting in a G 0/G 1 cell cycle arrest. This process involved a decrease in levels of cyclin A and an increase in the nuclear localization of p21 and p27. Furthermore, 500 μM spermine NONOate decreased viable cell number by inducing programmed cell death: FPASMC treated with 500 μM spermine NONOate displayed a loss of mitochondrial membrane potential, followed by caspase activation and DNA fragmentation. These data suggest that NO inhibits superoxide-induced proliferation of FPASMC and at higher doses induces apoptosis. NO donors may therefore prove to be useful therapeutic tools to treat diseases resulting from excessive proliferation of vascular smooth muscle.

Fetal pulmonary hypertension prolongs the endothelin-1 induced increase in pulmonary artery smooth muscle cell cytosolic calcium concentration

Introduction: At birth, pulmonary artery (PA) blood flow increases 8–10 fold and PA pressure falls by 50% within 24 hours. The mechanisms responsible for postnatal adaptation of the pulmonary circulation remain incompletely understood. High serum endothelin-1 (ET-1) levels are found in infants with persistent pulmonary hypertension of the newborn. We hypothesized that chronic intrauterine pulmonary hypertension (PHT) potentiates agonist induced increase of cytosolic calcium concentration ([Ca2+]i) in fetal PA smooth muscle cells(SMC). Methods: Chronic intrauterine pulmonary hypertension was induced by intrauterine ligation of the ductus arteriosus (DA) in late-gestation fetal lambs. 7–10 days following DA ligation, animals were euthanized. Distal PAs (>4th generation) were removed from the lung and PA SMC were isolated and maintained in normoxic (95%O2) primary culture. Age-matched non-PHT controls were sacrificed at ∼132 days gestation and cells were cultured in the same manner. Fluorescence microscopy using fura-2 was used to determine the effect of ET-1 (10−6 -10−8 M) on PA SMC [Ca2+]i from PHT and control animals. To determine the effect of PHT on endothelin A and B (ETA and ETB) receptor mRNA expression, quantitative, internally controlled RT-PCR was performed on distal PAs and SMC in culture. Results: ET-1 (10−8 M) caused a monophasic increase in [Ca2+]i (206 +/- 47%, p < 0.01 vs baseline) above baseline in controls (n = 25), while PHT SMC [Ca2+]i did not increase significantly. When PHT SMC were treated with 10−6M ET-1, in those cells that responded, [Ca2+]i increased 174 +/- 81% above baseline(p < 0.01 vs baseline). Only 3/33 (9%) of PHT SMC responded to ET-1 compared to 90% of controls. Additionally, the duration of the increase in [Ca2+]i was longer in the PHT SMC: PHT SMC [Ca2+]i fell to baseline after 328 +/- 16 sec, where control [Ca2+]i fell to baseline after 117 sec. ETA and ETB receptor mRNA expression are both increased in PHT. Conclusions: PHT decreases PA SMC sensitivity to ET-1 by ∼100 fold, however, PHT increases the duration of the ET-1 induced increase in [Ca2+]i. We conclude that PHT prolongs the response of PA SMC to ET-1. This prolongation may lead to significant increases in PA SMC [Ca2+]i that results in attenuated postnatal adaptation of the pulmonary circulation.