Altered vasoreactivity in neonatal rats with pulmonary hypertension associated with bronchopulmonary dysplasia: Implication of both eNOS phosphorylation and calcium signaling

Bronchopulmonary dysplasia (BPD) consists of an arrest of pulmonary vascular and alveolar growth, with persistent hypoplasia of the pulmonary microvasculature and alveolar simplification. In 25 to 40% of the cases, BPD is complicated by pulmonary hypertension (BPD-PH) that significantly increases the risk of morbidity. In vivo studies suggest that increased pulmonary vascular tone could contribute to late PH in BPD. Nevertheless, an alteration in vasoreactivity as well as the mechanisms involved remain to be confirmed. The purpose of this study was thus to assess changes in pulmonary vascular reactivity in a murine model of BPD-PH. Newborn Wistar rats were exposed to either room air (normoxia) or 90% O2 (hyperoxia) for 14 days. Exposure to hyperoxia induced the well-known features of BPD-PH such as elevated right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodeling and decreased pulmonary vascular density. Intrapulmonary arteries from hyperoxic pups showed decreased endothelium-dependent relaxation to acetylcholine without any alteration of relaxation to the NO-donor sodium nitroprusside. This functional alteration was associated with a decrease of lung eNOS phosphorylation at the Ser1177 activating site. In pups exposed to hyperoxia, serotonin and phenylephrine induced exacerbated contractile responses of intrapulmonary arteries as well as intracellular calcium response in pulmonary arterial smooth muscle cells (PASMC). Moreover, the amplitude of the store-operated Ca2+ entry (SOCE), induced by store depletion using a SERCA inhibitor, was significantly greater in PASMC from hyperoxic pups. Altogether, hyperoxia-induced BPD-PH alters the pulmonary arterial reactivity, with effects on both endothelial and smooth muscle functions. Reduced activating eNOS phosphorylation and enhanced Ca2+ signaling likely account for alterations of pulmonary arterial reactivity.

Introduction Preterm birth causes infants to be born while the lung is still immature and unable to maintain adequate gas exchange [1]. Consequently, preterm infants often require respiratory support including assisted ventilation and supplemental oxygen therapy. However, these life-saving interventions can induce complications, among which bronchopulmonary dysplasia (BPD) is one of the most serious. BPD consists more prominently of an arrest of pulmonary vascular and alveolar growth, with persistent hypoplasia of the pulmonary microvasculature and alveolar simplification [2]. A dysmorphic growth and an impaired function of the pulmonary vasculature can result in pulmonary hypertension (PH) and subsequently lead to right ventricular hypertrophy, in 25 to 40% of the cases of BPD [2,3]. PH severely increases morbidity of BPD and, since the underlying pathophysiology is poorly understood, there is no definitive treatment to prevent BPD associated PH (BPD-PH).
Pulmonary vascular histopathology anomalies are observed in BPD-PH. Disruption of vascular growth results in decreased vessel density throughout the pulmonary micro-capillary network [4][5][6]. Moreover, intimal hyperplasia and increased muscularization of small pulmonary arteries have also been described [6][7][8]. Although structural remodeling and dysmorphic vascular growth can contribute to PH in patients with BPD, PH in children with BPD remains commonly responsive to a combined treatment with oxygen and inhaled nitric oxide (NO) which then decreases pulmonary artery pressures to near-normal levels [9], thus suggesting that the increased pulmonary vascular tone also significantly contributes to late PH. Nevertheless, alterations of vasoreactivity remain to be confirmed and underlying mechanisms to be characterized.
Interestingly, animal studies demonstrated that an increase in pulmonary vascular pressure itself could induce impaired vascular growth and alveolarization in the developing lung, suggesting that hemodynamic stress may be an additional mechanism for abnormal lung structure in BPD. Indeed, in a fetal ovine model, chronically elevated pulmonary vascular pressure induced by surgical partial constriction of the ductus arteriosus led to morphological pulmonary alteration characteristic of BPD, such as increased pulmonary arteriolar wall thickness, reduced small pulmonary vascular density and reduced alveolarization [10]. Furthermore, reducing pulmonary vascular pressure with inhaled NO attenuated these lung injuries in a rat model of BPD [11]. Since alterations in vascular reactivity are involved in the increase in pulmonary vascular pressure and since the latter contributes to structural abnormalities of the lung observed in BPD, vasoreactivity thus appears as an important element to take into consideration in BPD. Pulmonary vascular tone is controlled by the variations in the cytosolic calcium concentration ([Ca 2+ ] cyt ) in pulmonary arterial smooth muscle cells (PASMC) [12,13] and by the release of vasorelaxant factors, such as NO by endothelial cells. Then, endothelial NO-synthase (eNOS) is a key determinant of perinatal pulmonary vascular tone, whose activity is regulated by phosphorylation, interaction with substrate and co-factors, shear stress and numerous agonists [14,15].
In this study, we used a previously described hyperoxic murine model to mimic BPD-PH [16]. Although lung signaling pathways involved in BPD-PH pathogenesis have been extensively studied in this model, there is little information on vasoreactivity. To assess pulmonary vascular reactivity, we evaluated ex vivo contractile and relaxant responses of intrapulmonary arteries isolated from control vs hyperoxic pups. We also studied cytosolic calcium responses to vasocontractile agents as well as both expression and phosphorylation of eNOS in the BPD-PH model. We found that hyperoxia induces endothelial dysfunction and increased vasoconstriction in pulmonary arteries. These functional alterations were associated with a decreased eNOS phosphorylation in the lung and with an increased [Ca 2+ ] cyt in PASMC.

Materials and methods Animals
All animal studies conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). Agreement (number A33-063-907) was obtained from the French authorities and all the protocols used were approved by the local ethics committee ("Comité d'éthique régional d'Aquitaine", protocol number 2015091112352424APAFIS#1739). Aged-matched Wistar rat litters (Elevage Janvier, Le Genest St Isle, France) were exposed within 24 hours of birth either to room air (normoxia) or to 90% O 2 (hyperoxia) in a plexiglass chamber with continuous O 2 monitoring. Dams were rotated daily between normoxic and hyperoxic chambers to prevent maternal O 2 toxicity. Pups were maintained in the chamber for 14 days and were analyzed at postnatal day 14. During this 14-day period, if the animals showed any signs of illness or distress, they were immediately euthanized with an intraperitoneal injection of pentobarbital (0.3 mg / g body weight). Exposure to hyperoxia resulted in a survival rate of 82.6%.

Right ventricular systolic pressure and hypertrophy
Hemodynamic measurements were performed in nonventilated pups under isoflurane anesthesia (1.5%-2.5%, 2 L air / min). After thoracotomy, a heparin-filled hypodermic needle connected to a polyethylene catheter was placed into the right ventricular cavity by direct puncture of the right ventricle, as previously described [17]. The right ventricular systolic pressure was measured by using a fluid-filled force transducer (Baxter Uniflow gauge pressure transducer, Guyancourt, France) connected to the PowerLab recording unit (AD Instruments, Colorado Springs, CO) and analyzed with the LabChart software (AD Instruments). The whole procedure lasted less than 5 min for each animal. After completion of the measurement, animals were euthanized and their hearts and lungs were removed en bloc. The weight ratio or Fulton's index: right ventricle / (left ventricle + septum) was calculated to assess right ventricular hypertrophy.

Morphometric analysis of pulmonary arterioles
Pulmonary vascular remodeling was assessed by measuring the percentage of wall thickness of the arterioles as described previously [17]. Briefly, formaldehyde-fixed, paraffin-embedded, 4 μm thick left lung sections were stained with hematoxylin and eosin. All the arteries with external diameters below 100 μm on each lung section from 6 normoxic animals and 4 hyperoxic animals were considered; this corresponds to 35 to 43 vessels per lung section for normoxic animals and 12 to 32 vessels per lung section for hyperoxic animals. Analysis was performed in a blinded fashion by using the Image J software. The percentage of wall thickness was calculated as [(external wall areas)-(internal wall areas)] x 100 / external wall areas.

Vessel density
Vessel density was evaluated by immunostaining of lung sections with anti-von Willebrand factor (vWF; Millipore, Temacula, CA) to visualize even the small non-muscularized vessels. Images were captured using NanoZoomer 2.0HT at 20X magnification. For vessel counting, 4 well inflated randomly acquired fields (3.5 mm 2 ) were imaged per animal. The number of vessels (<100 μm) per field were counted and averaged per animal in a blinded fashion.

Wire myograph studies
Intrapulmonary arteries (first order) were dissected and segments of 1.8-2.0 mm length were mounted in a Mulvany myograph (Danish Myo Technology, Aarhus, Denmark). Arteries were bathed in Krebs buffer (119 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl 2 , 1.17 mM MgSO 4 , 1.18 mM KH 2 PO 4 , 25 mM NaHCO 3 and 5.5 mM glucose) and maintained at 37˚C. Arteries from control and hyperoxic pups were studied under a resting tension corresponding to equivalent transmural pressures of 30 and 60 mmHg, respectively, which were previously demonstrated to be the optimal resting tension in a passive length-tension relationship. The arteries functionality was checked by adding 80 mM KCl and no difference was observed between normoxic and hyperoxic arteries. The intrapulmonary artery contraction was evaluated with serotonin and phenylephrine. The relaxant responses to acetylcholine, sodium nitroprusside (SNP) and isoprenaline were determined after precontraction with phenylephrine (3.10 −7 M, which corresponds approximately to 90% of the maximal contraction).

PASMC isolation and microspectrofluorimetric measurement of cytosolic calcium
PASMC were isolated from first order intrapulmonary artery as previously described [18]. Arteries from 3 different animals were simultaneously dissociated for each experiment and PASMC were plated on 25 mm coverslips and placed in a recording chamber on the stage of an inverted Nikon Eclipse/TE 200 microscope. The Ca 2+ -sensitive fluorescent probe indo-1 was used to record changes in [Ca 2+ ] cyt in cells [18]. PASMC were incubated at room temperature during 30 min in a physiological solution (130 mM NaCl, 5.6 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 8 mM HEPES and 11 mM glucose) containing 5 μM of indo-1 penta-acetoxymethyl ester (indo1/AM; I-1223, Molecular Probes, Eugene, OR). Cells were then washed for 15 min in an indo-1/AM free solution and maintained at room temperature in the same solution before conducting the fluorescence measurements. Indo-1 was excited at 355 nm and the emitted fluorescence was recorded at 405 nm and 480 nm. The fluorescence ratio (λ 405 /λ 480 ) was calculated and recorded online by using the pCLAMP software (pCLAMP 10 system, Molecular Devices, Foster City, CA). Δ ratio λ 405 /λ 480 is the difference between the basal ratio and the ratio at maximal peak amplitude. Cells were constantly superfused with physiological solution at a rate of 3 mL / min. Serotonin (10 μM) or phenylephrine (10 μM) was delivered during 20 seconds by a pressure ejection from a pipette located close to the recorded cell. In order to study the store-operated Ca 2+ entry (SOCE), PASMC were preincubated with thapsigargin (1 μM) for 15 min in a 0 mM Ca 2+ solution prior to stimulation with a 2 mM Ca 2+ solution.

Data analysis
Data were expressed as mean ± SEM of n cells for calcium measurements, n vessels for wire myograph studies or n rats for all other studies. Statistical evaluation was performed by the non-parametric Mann-Whitney test for the right ventricular systolic pressure and hypertrophy, the vessel morphometry and density, the protein expression and phosphorylation as well as calcium measurements. The concentration-response curves were compared using analysis of variance. Values were considered statistically significant when P < 0.05.

Characterization of the BPD-PH model
Right ventricular systolic pressure was significantly increased in pups exposed to hyperoxia for 14 days (Fig 1A). Hearts from those pups also shown a right ventricular hypertrophy, as evidenced by a significant increase in the weight ratio right ventricle / (left ventricle + septum) ( Fig 1B). Hyperoxia also induced vascular remodeling by expansion of medial wall thickness in small pulmonary arteries (Fig 1C and 1D). Immunostaining of vWF showed that neonatal hyperoxia significantly decreased pulmonary vascular density (Fig 1E and 1F). All these modifications have previously been reported as features of BPD [19].

Hyperoxia-induced alteration of vasomotor responses
Endothelium-dependent relaxation to acetylcholine was significantly decreased in intrapulmonary arteries from pups exposed to hyperoxia, compared with normoxic controls (Fig 2A). However, relaxation to the NO-donor SNP was not altered following chronic hyperoxia ( Fig  2B). This suggests that the decreased response to acetylcholine induced by hyperoxia is due to endothelial dysfunction. In this connection, relaxant response to the β 2 -adrenergic receptor agonist isoprenaline, which is NO-and endothelium-independent in rat intrapulmonary arteries [20] was not influenced by hyperoxia exposure (Fig 2C).
In addition, serotonin and phenylephrine-induced contraction were both significantly exacerbated in intrapulmonary arteries from pups exposed to hyperoxia, compared with normoxic controls (Fig 3).

Hyperoxia-induced alteration of eNOS expression and phosphorylation
Since NO-dependent relaxation of pulmonary arteries was decreased after exposure to hyperoxia, we investigated eNOS expression and phosphorylation. We found that chronic neonatal hyperoxia up-regulated eNOS expression in the lungs (Fig 4A and 4C), but reduced the proportion of eNOS phosphorylated at the Ser1177 activation site (Fig 4B and 4C).

Hyperoxia-induced increase in basal [Ca 2+ ] cyt and Ca 2+ response to serotonin and phenylephrine in PASMC
We investigated Ca 2+ signaling in the context of increased vascular contractile response by hyperoxia. Basal [Ca 2+ ] cyt was slightly but significantly enhanced in PASMC isolated from pups exposed to hyperoxia, compared with normoxic controls (Fig 5A, 5B and 5C). Serotonin and phenylephrine both induced a rapid [Ca 2+ ] cyt increase in PASMC (Fig 5A and 5B). This (F) Vessel density was significantly decreased by hyperoxia (n = 9) compared with normoxia (n = 9). ** P < 0.01; *** P < 0.001. Ca 2+ response was strongly amplified in PASMC isolated from pups exposed to hyperoxia, compared with normoxic controls (Fig 5A, 5B and 5D). Serotonin-mediated [Ca 2+ ] cyt increase in PASMC is due to a Ca 2+ release from reticulum store as it was reduced in the presence of thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) ( Fig  5E). Replenishment of reticulum by SOCE mechanism was greatly enhanced in PASMC from hyperoxic pups as compared with normoxic controls (Fig 5F).

Discussion
Although altered vascular tone contributes to the pathophysiology of BPD-PH, vasoreactivity has been poorly examined in this disease. The aim of this study was thus to characterize the modification in contractile and relaxant pulmonary vascular responses and to determine underlying mechanisms. We demonstrate that, in a murine model of BPD-PH, intrapulmonary  arterial reactivity is reduced in response to the endothelium-dependent relaxing-agent acetylcholine and exacerbated in response to contractile agents. Because acetylcholine induces vasorelaxation through stimulation of NO production by eNOS, the altered relaxant response to acetylcholine can be explained by the reduced eNOS phosphorylation at Ser1177, which decreases its activity. On the other hand, the enhanced contractility could be related to an Altered vasoreactivity in PH-BPD increase in [Ca 2+ ] cyt in PASMC, that may be attributed to heightened SOCE activity and/or calcium release from intracellular calcium stores.
In this study, we used a previously described murine model to mimic BPD-PH [16]. Rodents are well suited for the study of BPD because their lungs at birth are in the saccular stage [16], structurally similar to human neonates born at 24 to 28 weeks of gestation. This model was reliable because pups exposed to hyperoxia consistently developed lung lesions, such as decreased vascular density and increased pulmonary vascular remodeling. These changes in lung structure closely mimic the histological features of the disorganized lung architecture observed in human infants suffering from BPD-PH [3,16,21]. These morphologic changes are accompanied by elevated right ventricular pressure which leads to right ventricular hypertrophy, a cardinal feature of PH.
We ascribed the altered vascular relaxation to eNOS activity although conflicting data exist regarding the effect of chronic hyperoxia on eNOS lung expression in the newborn rat. Such discrepancies could result from differences in the experimental design regarding percentage of O 2 , age of onset and duration of hyperoxia. While some authors report, in accordance with our results, an increase in eNOS lung protein content after exposure of pups to 60% O 2 from day 0 to day 14 or 95% O 2 from day 0 to day 8 or from day 3 to day 10 [22][23][24], others report that eNOS lung expression was either reduced after exposure of pups to 95% O 2 from day 1 to day 7 [25] or unaltered after exposure of pups to 60% O 2 from day 1 to day 14 [26]. Moreover, several studies demonstrated an alteration of the NO-soluble guanylate-cyclase (sGC)-cGMP signaling pathway in the hyperoxic newborn rat model. Physiologically, endothelial cells can release NO that stimulates sGC in PASMC to produce cGMP, which then induces vasodilatation. The 14-day exposure of pups to 75% O 2 decreased sGC activity and lowered cGMP levels in small pulmonary arteries [6]. Exposure of pups to 95% O 2 from day 5 to day 12 also reduced cGMP levels in the lungs [27]. Consistent with those observations, the 14-day exposure of pups to 60% O 2 decreased NO release from pulmonary arteries [26]. These results are in accordance with our demonstration that, despite an increase in total eNOS expression, phosphorylation of eNOS at the Ser1177 activating site [15] is reduced by hyperoxia, suggesting that eNOS is less able to produce NO and to activate sGC. Furthermore, a decrease in eNOS activity is consistent with the altered relaxant response to acetylcholine we observed in pulmonary arteries from hyperoxic newborn rats. In the present study, endothelial dysfunction was demonstrated by altered relaxant response to acetylcholine without altered response to the NO-donor SNP in intrapulmonary arteries from hyperoxic pups. The unaltered relaxant capacity of smooth muscle was also confirmed in this model by an unchanged relaxant response to the β 2adrenergic receptor agonist isoprenaline, which is NO-and endothelium-independent in rat intrapulmonary arteries [20].
The additional role of NO in regulating normal lung maturation is supported by studies in which NO-donors increase branching morphogenesis of cultured fetal rat lungs [28]. Furthermore, other studies demonstrated that eNOS-deficient mouse pup lungs had decreased both microvascular development and expression of angiogenic factors, such as vascular endothelial growth factor-A, Flk-1, and TIE2 [29]. NO also seems to regulate the development of the injured newborn lung in various models. For example, eNOS-deficient mouse pups exposed to mild hypoxia show decreased pulmonary vessel density and alveolarization [30]. Furthermore, exposure to exogenous NO decreased abnormal PASMC proliferation in rat pups treated with monocrotaline, a toxic alkaloid that induces pulmonary hypertension [31], and improved pulmonary vessel density and alveolarization in rat pups exposed to hyperoxia [25]. These studies suggest a protective effect of NO on the injured newborn lung. Consequently, the alterations we observed in eNOS phosphorylation and endothelial NO-dependent relaxation may not only be implicated in vascular tone and PH but also in altered pulmonary development, although this hypothesis requires further investigation. Finally, since the NO-sGC-cGMP axis modulates vascular tone by regulating PASMC [Ca 2+ ] cyt , the decrease in eNOS phosphorylation at Ser1177 could also contribute to the increase in basal [Ca 2+ ] cyt in PASMC isolated from pups exposed to hyperoxia as later discussed.
This study provides the first evidence that Ca 2+ signaling is modified in PASMC from newborn rat with BPD-PH. Interestingly, Ca 2+ signaling in PASMC plays a central role both in vasoconstriction, through its pivotal effect on PASMC contraction, and in vascular remodeling, through its stimulatory effect on PASMC proliferation and migration [32,33]. We demonstrated that the resting [Ca 2+ ] cyt is higher in the PASMC from hyperoxic newborn rats than in those from controls. Additionally, the amplitude of SOCE, induced by store depletion using thapsigargin, a SERCA inhibitor, was significantly greater in PASMC from pups with BPD-PH. Moreover, in PASMC from pups with BPD-PH, the Ca 2+ response to serotonin is increased compared with control cells. SOCE, first described as capacitative Ca 2+ entry, corresponds to the opening of store-operated Ca 2+ channels (SOC) on the plasma membrane, due to depletion of Ca 2+ from the endoplasmic reticulum. This process allows Ca 2+ to fiow into the cytosol and Ca 2+ is then sequestered into the endoplasmic reticulum by the SERCA, thus replenishing Ca 2+ stores. As previously demonstrated in PASMC from adult rats [34], Ca 2+ response to serotonin involves the release of Ca 2+ from intracellular stores, since it is inhibited by thapsigargin. The enhanced SOCE activity induced by hyperoxia allows better replenishment of the reticulum, which may explain the increase in serotonin-mediated Ca 2+ response, and consequently the increase in contractile response.
In accordance with our results, studies in fetal airway smooth muscle cells have also demonstrated that hyperoxia enhances fetal airway contractility via an increase in the amplitude of Ca 2+ responses to histamine [35,36]. Furthermore, as in the current model, the resting [Ca 2 + ] cyt is higher in PASMC from adult patients with idiopathic pulmonary arterial hypertension or adult rats with hypoxic PH [37,38]. Similarly, the contraction to serotonin is also increased in adult rats with hypoxic PH [38]. Additionally, the amplitude of SOCE is increased in PASMC from adult patients with idiopathic pulmonary arterial hypertension compared with control cells [32,39]. When [Ca 2+ ] cyt increases, Ca 2+ binds to calmodulin, which then activates myosin light chain kinase and consequently induces phosphorylation of contractile proteins and vessel contraction. Sustained pulmonary vasoconstriction is partly responsible for the elevated pulmonary vascular resistance and for the pulmonary arterial pressure observed in adult patients with idiopathic pulmonary arterial hypertension [32]. Likewise, we can hypothesize that, in our model of BPD-PH, the elevated [Ca 2+ ] cyt we observed contributes to elevated pulmonary arterial pressure, reflected by the increase in right ventricular systolic pressure.
Besides its promotion of contraction, the elevation of intracellular Ca 2+ , and especially SOCE, can activate signal transduction pathways important for the stimulation of transcription factors required for cell-cycle progression and proliferation, a key component of vascular remodeling [32]. In normal PASMC, the resting [Ca 2+ ] cyt as well as the increase in [Ca 2+ ] cyt due to SOCE are both greater in proliferating PASMC than in growth-arrested cells [40,41]. In animal models of adult PH as well as in human PASMC exposed to hypoxia, it has been demonstrated that increased PASMC proliferation is dependent on enhanced SOCE [42][43][44]. In the same line of evidence, treatments that reduce SOCE in these models also reduce PASMC proliferation. These results suggest that elevated [Ca 2+ ] cyt and intracellularly stored Ca 2+ play an important role in PASMC growth, especially in the context of PH. In our model of BPD-PH, we report remodeling of pulmonary arteries with increased medial wall thickness, reflecting an increase in PASMC proliferation. Since we observed enhanced SOCE in PASMC, SOCE and proliferation in PASMC in BPD-PH may be closely related, although this assumption requires further investigation.
In conclusion, in the present study, we have shown that pulmonary vascular reactivity is altered in a model of murine BPD-PH with both endothelial and smooth muscle dysfunctions. The cellular mechanisms involve a decreased eNOS phosphorylation at its activating site and an increase in resting [Ca 2+ ] cyt and SOCE in PASMC. The present findings argue in favor of future studies to investigate the influence of decreased eNOS activity and Ca 2+ signaling modification on altered lung maturation, including PASMC proliferation and migration in order to closely link vascular contractility and remodeling in this disease.