Sildenafil Improves Alveolar Growth and Pulmonary Hypertension in Hyperoxia-induced Lung Injury
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美国呼吸和危急护理医学 2005年第9期
Vascular Biology Research Group, Department of Pediatrics, Division of Neonatology
Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada
ABSTRACT
Rationale: Bronchopulmonary dysplasia (BPD), the chronic lung disease of preterm infants, and pulmonary emphysema, both significant global health problems, are characterized by an arrest in alveolar growth/loss of alveoli structures. Mechanisms that inhibit distal lung growth are poorly understood, but recent studies suggest that impaired vascular endothelial growth factor signaling and reduced nitric oxide (NO) production decreases alveolar and vessel growth in the developing lung, features observed in experimental oxygen-induced BPD. NO exerts its biological activity by stimulating guanosine 3',5'-cyclic monophosphate (cGMP) production. Objectives: Because cGMP is inactivated by phosphodiesterase (PDE) enzymes, we hypothesized that the cGMP-specific PDE5 inhibitor sildenafil would promote angiogenesis and attenuate oxygen-induced lung injury in newborn rats. Methods, Measurements, and Main Results: In vitro, sildenafil (10eC4 M) increased endothelial capillary network formation of human pulmonary endothelial cells exposed to hyperoxia. In vivo, rat pups were randomly exposed from birth to normoxia, hyperoxia (95% O2, BPD model), and hyperoxia+sildenafil (100 mg/kg/day subcutaneously). Rat pups exposed to hyperoxia showed fewer and enlarged air spaces as well as decreased capillary density, mimicking pathologic features seen in human BPD. These structural anomalies were associated with echographic (decreased pulmonary acceleration time) and structural (right ventricular hypertrophy and increased medial wall thickness) signs of pulmonary hypertension. Sildenafil preserved alveolar growth and lung angiogenesis, and decreased pulmonary vascular resistance, right ventricular hypertrophy and medial wall thickness. Conclusions: Our findings suggest a role for the NO/cGMP pathway during alveolar development. Sildenafil may have therapeutic potential in diseases associated with impaired alveolar structures.
Key Words: alveoli;angiogenesis;cGMP; nitric oxide;oxygen
In spite of advancements in perinatal care, bronchopulmonary dysplasia (BPD) remains a major cause of morbidity in premature babies with birth weights of less than 1,000 g (1eC3). BPD is the chronic lung disease that results from mechanical ventilation and oxygen therapy to treat acute respiratory failure in preterm infants (4). Perinatal lung injury during the late canalicular stage disrupts the normal sequence of lung development, resulting in the histologic pattern of alveolar simplification (larger but fewer alveoli with decreased septation) and impaired vascular growth (5, 6), and can be complicated by pulmonary hypertension (7). However, the mechanisms that regulate normal alveolar development and the mechanisms that disrupt lung growth in premature infants are unknown. Currently, with no treatment for BPD, neonatologists are faced with a major therapeutic challenge (8).
Recent evidence suggests a link between angiogenesis, the formation of blood vessels, and alveolarization (9). Various animal models of impaired alveolar development also display abnormal lung vascular development. Rat pups exposed to high oxygen levels (60eC95%) during the alveolar period have decreased alveolarization associated with decreased number of capillaries/mm2, increased muscularization of peripheral arteries, and medial hypertrophy of muscular arteries (10eC14). Similar findings exist in dexamethasone-induced inhibition of alveolarization (15) and in Fawn-hooded rat pups, a rat strain that develops spontaneous pulmonary hypertension as it ages (16). Lung histology of premature baboons (17, 18) and lambs (19) treated similarly to human preterm infants have arrested alveolar development and decreased pulmonary capillary vasculature, features observed, at autopsy, in infants who died with BPD (20). These findings suggest that vascular-specific growth factors are likely to play key roles during normal and impaired alveolar development.
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor crucial for proper vascular development (21). Recent data suggest that VEGF-driven angiogenesis contributes to lung development. Decreased lung VEGF levels have been observed in experimental (22) and human BPD (23). Pharmacologic and genetic inhibition of VEGF signaling results in reduced alveolarization in the neonatal rat (24eC26). Adenovirus-mediated VEGF gene transfer preserves normal lung growth in the oxygen-induced BPD model in newborn rats (26).
VEGF-induced angiogenesis is in part mediated by nitric oxide (NO) (27). NO plays an important role in regulating pulmonary vascular tone during transition at birth (28), but recent evidence suggests that NO also promotes structural changes in the developing lung, including branching morphogenesis (29) and alveolarization (30). Moreover, inhaled NO preserves normal lung growth in several neonatal animal models with structural and functional features of BPD (31, 32). Recent clinical trials suggest that early treatment with inhaled NO may decrease the risk of BPD in premature infants with acute respiratory failure (33, 34). Thus, the VEGF pathway may promote angiogenesis via an increase in NO production in the developing lung. NO stimulates the production of guanosine 3',5'-cyclic monophosphate (cGMP). cGMP is catabolized by phosphodiesterase enzymes (PDE). We hypothesized that sildenafil, a cGMP-specific PDE5 inhibitor, would promote angiogenesis and reduce hyperoxia-induced lung injury in newborn rats. Our data show that sildenafil increased pulmonary capillary density, preserved alveolar growth, and alleviated echographic and structural signs of pulmonary hypertension, suggesting that the NOeCcGMP pathway may be an effective target to protect the lung from alveolar damage. Some of the results of these studies have been previously reported in the form of an abstract (35).
METHODS
Endothelial Network Formation Assay In Vitro
Endothelial cord-like structure formation was assessed using Matrigel-coated wells. Human pulmonary artery endothelial cells (HPAEC; 80,000 cells/well) were seeded into 24-well plates coated with Matrigel (BD Biosciences, Mississauga, ON, Canada) and incubated at 37°C for 8 to 12 hours under the three following conditions: 21% O2, hyperoxia (95%), and hyperoxia+sildenafil (10-4M). Sildenafil was a generous gift from Pfizer Pharmaceuticals (Sandwich, UK). Endothelial network formation was observed using an inverted phase contrast microscope (Olympus, Melville, NY) and quantified by measuring the number of intersects in random fields from each well using OpenLab (Quorum Technologies Inc., Guelph, ON, Canada).
Animal Model
All procedures and protocols were approved by the Animal Health Care Committee of the University of Alberta. From birth to postnatal age 14 (P14), Sprague-Dawley rat pups were exposed to normoxia (21%, control group) or hyperoxia (95% O2, BPD group) in sealed Plexiglas chambers (BioSpherix, Redfield, NY) with continuous oxygen monitoring. Dams were switched between the hyperoxic and normoxic chambers every 48 hours to prevent damage to their lungs (36). Litter size was adjusted so that all cages had an equal number of pups to control for effects of litter size on nutrition and growth. Rat pups were killed at P14 using an intraperitoneal injection of pentobarbital, and lungs and heart were processed according to the performed experiments.
Experimental Protocol
Newborn rat pups were randomized to three groups: (1) normoxia (21%, control group), (2) hyperoxia (95% O2, BPD group), and (3) hyperoxia+sildenafil. Sildenafil (100 mg/kg) was administered to hyperoxic rat pups daily from birth to P14 via subcutaneous injection. Preliminary doseeCresponse studies using 2 and 20 mg/kg/day had no protective effect on alveolar structures. Consequently, a larger dose of 100 mg/kg/day was used for this study based on the effective sildenafil dosage reported in other rodent studies. Although considerably higher than the dose appropriate for humans, the dose we used had been shown in chronic in vivo studies in rats to yield mean free plasma concentrations comparable to levels obtained in humans at doses of 1 mg/kg/day (37). This reflects the near 100-fold higher rate of metabolism of sildenafil in rats (38).
Lung Morphometry
Lungs were fixed via the trachea with a 4% formaldehyde solution under a constant pressure of 20 cm H2O. After ligation of the trachea, the lungs were immersed in fixative overnight at 4°C. Lung volume was measured by water displacement. Lungs were processed and embedded in paraffin. Four-micrometer-thick serial sections were taken along the longitudinal axis of the cranial lobe of the right lung. The fixed distance between the sections was calculated so as to allow systematic sampling of approximately 10 sections across the whole lobe. Lungs were stained with hematoxylin and eosin. Alveolar structures were quantified by the radial alveolar count (RAC) (39).
Barium-Gelatin Angiograms and Arterial Density Counts
A barium-gelatin mixture (60°C) was injected at 70 mm Hg pressure in the main pulmonary artery until surface filling of vessels with barium was seen uniformly over the surface of the lung (40). The main pulmonary artery was tied off under pressure, the lungs were inflation-fixed with formaldehyde, the right cranial lobe was embedded in paraffin, and sections were cut and stained with hematoxylin and eosin. Barium-filled pulmonary arteries were counted per high-powered field (x100 magnification). Lungs of four animals/group, five sections/lung, and 10 high-power fields/section were counted.
Echo-Doppler
Pulmonary hypertension was assessed using echo-doppler (SONOS 5500; Philips Medical Systems, Markham, ON, Canada) to measure the pulmonary arterial acceleration time (PAAT) (41, 42).
Right Ventricular Hypertrophy and Pulmonary Artery Remodeling
Right ventricle and left ventricle plus septum were weighed separately to determine the right ventricle to left ventricle+septum ratio (RV/LV+S) as an index of right ventricular hypertrophy (RVH) (42). To assess pulmonary artery remodeling, the percent medial wall thickness (MWT) was calculated as (2 x wall thickness/external diameter) x 100% (42).
cGMP Assay
Plasma cGMP levels were measured using a cGMP EIA Kit (Biomedical Technologies, Stoughton, MA) (43).
Statistics
Values are expressed as the mean ± SEM. Intergroup differences were assessed by Student's paired t test or a factorial ANOVA, as appropriate. Post hoc analysis used a Fisher's probable least significant difference test (Statview 5.1; Abacus Concepts, Berkeley, CA). Statistical significance was defined as p < 0.05.
RESULTS
Sildenafil Promotes Vessel Formation In Vitro
In an in vitro model of angiogenesis, HPAEC form endothelial cord-like structures on Matrigel (Figure 1). Hyperoxia (95%) significantly reduced the endothelial network formation by HPAEC as compared with control, room aireCexposed HPAEC. Inhibition of cGMP inactivation using the cGMP-specific PDE5 inhibitor sildenafil (10eC4 M) restored a dense capillary network in hyperoxia.
Sildenafil Improves Lung Angiogenesis in Hyperoxia-induced BPD in Newborn Rats
Hyperoxia-exposed rat pups showed a significant decrease in pulmonary capillary density compared with normoxic rat pups, as observed in barium angiograms (Figure 2). Sildenafil-treated rat pups had higher capillary density than untreated hyperoxic rat pups.
Sildenafil Improves Alveolarization in Hyperoxia-induced BPD in Newborn Rats
Rat pups exposed to hyperoxia from birth to P14 displayed the characteristic features of alveolar simplification, with larger and fewer alveolar airspaces and decreased septation as compared with normoxic animals (Figure 3). Sildenafil preserved alveolar growth in hyperoxic rats as assessed by the RAC.
Sildenafil Reduced Pulmonary Hypertension Associated with Oxygen-induced Lung Injury
Chronic exposure to hyperoxia was associated with a significant decrease in the PAAT (Figure 4A) and an increase in RVH and MWT of small pulmonary arteries (Figures 4B and 4C). Sildenafil attenuated these functional and structural features of pulmonary hypertension as indicated by the increase in mean PAAT, reduction in RV/LV+S, and decrease in MWT.
Sildenafil-induced Alveolar and Vascular Growth Is Associated with Increased cGMP Levels
Hyperoxic rat pups had increased levels of cGMP compared with normoxic rats (Figure 5). Consistent with sildenafil's mechanism of action as a cGMP-specific PDE5 inhibitor, the concentration of cGMP was further increased in hyperoxic rats treated with sildenafil.
DISCUSSION
This study shows that sildenafil prevents lung injury and pulmonary hypertension in experimental oxygen-induced BPD in newborn rats. In vitro, sildenafil preserves a rich endothelial network in hyperoxic-exposed HPAEC. In vivo, sildenafil increases pulmonary capillary density, preserves alveolar growth, and alleviates echographic and structural signs of pulmonary hypertension. Our findings suggest a role for the NOeCcGMP pathway in angiogenesis and alveolarization in the developing lung. This pathway may be an effective target to protect the lung from impaired alveolar development.
Early observations by Liebow showed that the alveolar septa in emphysema were remarkably thin and almost avascular (44). He postulated that a reduction in the blood supply of the small precapillary blood vessels might induce the disappearance of alveolar septa. Despite this early observation, pulmonary vessels were thought for many years to be passive bystanders in lung development, following the branching pattern of the airways. Recent data, however, suggest that VEGF/NO-driven lung angiogenesis actively participates in alveolar growth (25, 30) and homeostasis throughout adulthood (45). In preterm infants, inhaled NO promising results as a prophylactic therapy for BPD (33, 34). Thus, we hypothesized that manipulating a downstream target of NO, specifically cGMP, would have similar beneficial effects on lung growth. Blockage of cGMP degradation using the PDE5 inhibitor sildenafil did preserve vascular and alveolar growth in the hyperoxic rat BPD model. This beneficial effect might have been through promotion of lung angiogenesis, because in vitro, sildenafil preserved endothelial network formation in hyperoxia. Further investigation is needed to reveal the precise mechanism by which sildenafil is lung-protective, and whether this is exclusively through promoting lung angiogenesis (and therefore alveolar development) or whether sildenafil or the NOeCcGMP pathway also has a direct effect on alveolar epithelial cell growth.
We found that hyperoxic rat pups had cGMP plasma levels greater than those in control rats. This finding is consistent with other studies in which increases in lung NO synthase and cGMP levels were seen in hyperoxic rats compared with normoxic rats (46). In the latter study, NO synthase blockade reduced lung cGMP levels in the hyperoxic rat pups but did not reverse the pathologic consequences of hyperoxic exposure (46). In our study, sildenafil further increased cGMP levels in hyperoxic rats and attenuated hyperoxic-induced lung injury. Thus, one possible interpretation is that upregulation of cGMP in hyperoxia may be a compensatory mechanism to counteract the deleterious effects of oxygen on lung development. By analogy, cGMP levels are increased in monocrotalline-induced pulmonary hypertension, and treatment with E4010, another PDE5 inhibitor, further enhances cGMP levels (47). In our study sildenafil further increased cGMP levels, and this may have been enough to promote lung angiogenesis/alveolar growth in hyperoxic conditions. This further assumes that the NOeCcGMP pathway is crucial for alveolar development.
cGMP can also be produced by atrial natriuretic peptide (ANP). Interestingly, type 2 alveolar epithelial cells express ANP receptors and ANP activation of guanylate cyclase A receptors produces cGMP (48, 49). The response to sildenafil can be influenced by the ANP pathway. Mice lacking NPR-A (NPR-AeC/eC), a guanylyl cyclaseeClinked natriuretic peptide receptor A, have higher basal RV systolic pressures (50). Furthermore, the beneficial effect of sildenafil on hypoxia-induced pulmonary vascular muscularization and cyclic GMP levels is blunted in NPR-AeC/eC mice, suggesting that a functional ANP pathway is crucial to the effectiveness of sildenafil (50). These recent findings warrant further investigations of the ANP pathway during lung development.
Pulmonary hypertension can contribute to morbidity and mortality in human BPD (7). The hyperoxic BPD model in rats also exhibits pulmonary hypertension characterized by increased pulmonary vasoconstriction, RVH, and MWT of pulmonary arteries. Decreased NO and excessive reactive oxygen species and endothelin pathway activation may account for pulmonary hypertension in this model (51eC53). Sildenafil alleviated pulmonary hypertension in our animal model. This is consistent with studies showing beneficial hemodynamic and structural effects of sildenafil in various adult animal models (54, 55) and in humans (56eC59) with pulmonary hypertension. Sildenafil is currently being investigated in newborns in clinical trials for persistent pulmonary hypertension, just like inhaled NO 10 years ago.
In conclusion, this is the first report on the potential therapeutic benefit of sildenafil in protecting from oxygen-induced alveolar damage. The relative pulmonary vascular specificity of sildenafil, its low cost, and its postmarketing safety makes it an attractive therapeutic option and warrants further study. Clinical trials are indicated to determine whether sildenafil can decrease the incidence/severity of BPD in premature infants and potentially other lung diseases characterized by alveolar damage.
B.T. is supported by the Canada Foundation for Innovation (CFI), the Alberta Heart and Stroke Foundation (H&SF), the Alberta Heritage Foundation for Medical Research (AHFMR), the Canadian Institutes for Health Research (CIHR), and by the Stollery Children's Hospital Foundation.
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Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada
ABSTRACT
Rationale: Bronchopulmonary dysplasia (BPD), the chronic lung disease of preterm infants, and pulmonary emphysema, both significant global health problems, are characterized by an arrest in alveolar growth/loss of alveoli structures. Mechanisms that inhibit distal lung growth are poorly understood, but recent studies suggest that impaired vascular endothelial growth factor signaling and reduced nitric oxide (NO) production decreases alveolar and vessel growth in the developing lung, features observed in experimental oxygen-induced BPD. NO exerts its biological activity by stimulating guanosine 3',5'-cyclic monophosphate (cGMP) production. Objectives: Because cGMP is inactivated by phosphodiesterase (PDE) enzymes, we hypothesized that the cGMP-specific PDE5 inhibitor sildenafil would promote angiogenesis and attenuate oxygen-induced lung injury in newborn rats. Methods, Measurements, and Main Results: In vitro, sildenafil (10eC4 M) increased endothelial capillary network formation of human pulmonary endothelial cells exposed to hyperoxia. In vivo, rat pups were randomly exposed from birth to normoxia, hyperoxia (95% O2, BPD model), and hyperoxia+sildenafil (100 mg/kg/day subcutaneously). Rat pups exposed to hyperoxia showed fewer and enlarged air spaces as well as decreased capillary density, mimicking pathologic features seen in human BPD. These structural anomalies were associated with echographic (decreased pulmonary acceleration time) and structural (right ventricular hypertrophy and increased medial wall thickness) signs of pulmonary hypertension. Sildenafil preserved alveolar growth and lung angiogenesis, and decreased pulmonary vascular resistance, right ventricular hypertrophy and medial wall thickness. Conclusions: Our findings suggest a role for the NO/cGMP pathway during alveolar development. Sildenafil may have therapeutic potential in diseases associated with impaired alveolar structures.
Key Words: alveoli;angiogenesis;cGMP; nitric oxide;oxygen
In spite of advancements in perinatal care, bronchopulmonary dysplasia (BPD) remains a major cause of morbidity in premature babies with birth weights of less than 1,000 g (1eC3). BPD is the chronic lung disease that results from mechanical ventilation and oxygen therapy to treat acute respiratory failure in preterm infants (4). Perinatal lung injury during the late canalicular stage disrupts the normal sequence of lung development, resulting in the histologic pattern of alveolar simplification (larger but fewer alveoli with decreased septation) and impaired vascular growth (5, 6), and can be complicated by pulmonary hypertension (7). However, the mechanisms that regulate normal alveolar development and the mechanisms that disrupt lung growth in premature infants are unknown. Currently, with no treatment for BPD, neonatologists are faced with a major therapeutic challenge (8).
Recent evidence suggests a link between angiogenesis, the formation of blood vessels, and alveolarization (9). Various animal models of impaired alveolar development also display abnormal lung vascular development. Rat pups exposed to high oxygen levels (60eC95%) during the alveolar period have decreased alveolarization associated with decreased number of capillaries/mm2, increased muscularization of peripheral arteries, and medial hypertrophy of muscular arteries (10eC14). Similar findings exist in dexamethasone-induced inhibition of alveolarization (15) and in Fawn-hooded rat pups, a rat strain that develops spontaneous pulmonary hypertension as it ages (16). Lung histology of premature baboons (17, 18) and lambs (19) treated similarly to human preterm infants have arrested alveolar development and decreased pulmonary capillary vasculature, features observed, at autopsy, in infants who died with BPD (20). These findings suggest that vascular-specific growth factors are likely to play key roles during normal and impaired alveolar development.
Vascular endothelial growth factor (VEGF) is an angiogenic growth factor crucial for proper vascular development (21). Recent data suggest that VEGF-driven angiogenesis contributes to lung development. Decreased lung VEGF levels have been observed in experimental (22) and human BPD (23). Pharmacologic and genetic inhibition of VEGF signaling results in reduced alveolarization in the neonatal rat (24eC26). Adenovirus-mediated VEGF gene transfer preserves normal lung growth in the oxygen-induced BPD model in newborn rats (26).
VEGF-induced angiogenesis is in part mediated by nitric oxide (NO) (27). NO plays an important role in regulating pulmonary vascular tone during transition at birth (28), but recent evidence suggests that NO also promotes structural changes in the developing lung, including branching morphogenesis (29) and alveolarization (30). Moreover, inhaled NO preserves normal lung growth in several neonatal animal models with structural and functional features of BPD (31, 32). Recent clinical trials suggest that early treatment with inhaled NO may decrease the risk of BPD in premature infants with acute respiratory failure (33, 34). Thus, the VEGF pathway may promote angiogenesis via an increase in NO production in the developing lung. NO stimulates the production of guanosine 3',5'-cyclic monophosphate (cGMP). cGMP is catabolized by phosphodiesterase enzymes (PDE). We hypothesized that sildenafil, a cGMP-specific PDE5 inhibitor, would promote angiogenesis and reduce hyperoxia-induced lung injury in newborn rats. Our data show that sildenafil increased pulmonary capillary density, preserved alveolar growth, and alleviated echographic and structural signs of pulmonary hypertension, suggesting that the NOeCcGMP pathway may be an effective target to protect the lung from alveolar damage. Some of the results of these studies have been previously reported in the form of an abstract (35).
METHODS
Endothelial Network Formation Assay In Vitro
Endothelial cord-like structure formation was assessed using Matrigel-coated wells. Human pulmonary artery endothelial cells (HPAEC; 80,000 cells/well) were seeded into 24-well plates coated with Matrigel (BD Biosciences, Mississauga, ON, Canada) and incubated at 37°C for 8 to 12 hours under the three following conditions: 21% O2, hyperoxia (95%), and hyperoxia+sildenafil (10-4M). Sildenafil was a generous gift from Pfizer Pharmaceuticals (Sandwich, UK). Endothelial network formation was observed using an inverted phase contrast microscope (Olympus, Melville, NY) and quantified by measuring the number of intersects in random fields from each well using OpenLab (Quorum Technologies Inc., Guelph, ON, Canada).
Animal Model
All procedures and protocols were approved by the Animal Health Care Committee of the University of Alberta. From birth to postnatal age 14 (P14), Sprague-Dawley rat pups were exposed to normoxia (21%, control group) or hyperoxia (95% O2, BPD group) in sealed Plexiglas chambers (BioSpherix, Redfield, NY) with continuous oxygen monitoring. Dams were switched between the hyperoxic and normoxic chambers every 48 hours to prevent damage to their lungs (36). Litter size was adjusted so that all cages had an equal number of pups to control for effects of litter size on nutrition and growth. Rat pups were killed at P14 using an intraperitoneal injection of pentobarbital, and lungs and heart were processed according to the performed experiments.
Experimental Protocol
Newborn rat pups were randomized to three groups: (1) normoxia (21%, control group), (2) hyperoxia (95% O2, BPD group), and (3) hyperoxia+sildenafil. Sildenafil (100 mg/kg) was administered to hyperoxic rat pups daily from birth to P14 via subcutaneous injection. Preliminary doseeCresponse studies using 2 and 20 mg/kg/day had no protective effect on alveolar structures. Consequently, a larger dose of 100 mg/kg/day was used for this study based on the effective sildenafil dosage reported in other rodent studies. Although considerably higher than the dose appropriate for humans, the dose we used had been shown in chronic in vivo studies in rats to yield mean free plasma concentrations comparable to levels obtained in humans at doses of 1 mg/kg/day (37). This reflects the near 100-fold higher rate of metabolism of sildenafil in rats (38).
Lung Morphometry
Lungs were fixed via the trachea with a 4% formaldehyde solution under a constant pressure of 20 cm H2O. After ligation of the trachea, the lungs were immersed in fixative overnight at 4°C. Lung volume was measured by water displacement. Lungs were processed and embedded in paraffin. Four-micrometer-thick serial sections were taken along the longitudinal axis of the cranial lobe of the right lung. The fixed distance between the sections was calculated so as to allow systematic sampling of approximately 10 sections across the whole lobe. Lungs were stained with hematoxylin and eosin. Alveolar structures were quantified by the radial alveolar count (RAC) (39).
Barium-Gelatin Angiograms and Arterial Density Counts
A barium-gelatin mixture (60°C) was injected at 70 mm Hg pressure in the main pulmonary artery until surface filling of vessels with barium was seen uniformly over the surface of the lung (40). The main pulmonary artery was tied off under pressure, the lungs were inflation-fixed with formaldehyde, the right cranial lobe was embedded in paraffin, and sections were cut and stained with hematoxylin and eosin. Barium-filled pulmonary arteries were counted per high-powered field (x100 magnification). Lungs of four animals/group, five sections/lung, and 10 high-power fields/section were counted.
Echo-Doppler
Pulmonary hypertension was assessed using echo-doppler (SONOS 5500; Philips Medical Systems, Markham, ON, Canada) to measure the pulmonary arterial acceleration time (PAAT) (41, 42).
Right Ventricular Hypertrophy and Pulmonary Artery Remodeling
Right ventricle and left ventricle plus septum were weighed separately to determine the right ventricle to left ventricle+septum ratio (RV/LV+S) as an index of right ventricular hypertrophy (RVH) (42). To assess pulmonary artery remodeling, the percent medial wall thickness (MWT) was calculated as (2 x wall thickness/external diameter) x 100% (42).
cGMP Assay
Plasma cGMP levels were measured using a cGMP EIA Kit (Biomedical Technologies, Stoughton, MA) (43).
Statistics
Values are expressed as the mean ± SEM. Intergroup differences were assessed by Student's paired t test or a factorial ANOVA, as appropriate. Post hoc analysis used a Fisher's probable least significant difference test (Statview 5.1; Abacus Concepts, Berkeley, CA). Statistical significance was defined as p < 0.05.
RESULTS
Sildenafil Promotes Vessel Formation In Vitro
In an in vitro model of angiogenesis, HPAEC form endothelial cord-like structures on Matrigel (Figure 1). Hyperoxia (95%) significantly reduced the endothelial network formation by HPAEC as compared with control, room aireCexposed HPAEC. Inhibition of cGMP inactivation using the cGMP-specific PDE5 inhibitor sildenafil (10eC4 M) restored a dense capillary network in hyperoxia.
Sildenafil Improves Lung Angiogenesis in Hyperoxia-induced BPD in Newborn Rats
Hyperoxia-exposed rat pups showed a significant decrease in pulmonary capillary density compared with normoxic rat pups, as observed in barium angiograms (Figure 2). Sildenafil-treated rat pups had higher capillary density than untreated hyperoxic rat pups.
Sildenafil Improves Alveolarization in Hyperoxia-induced BPD in Newborn Rats
Rat pups exposed to hyperoxia from birth to P14 displayed the characteristic features of alveolar simplification, with larger and fewer alveolar airspaces and decreased septation as compared with normoxic animals (Figure 3). Sildenafil preserved alveolar growth in hyperoxic rats as assessed by the RAC.
Sildenafil Reduced Pulmonary Hypertension Associated with Oxygen-induced Lung Injury
Chronic exposure to hyperoxia was associated with a significant decrease in the PAAT (Figure 4A) and an increase in RVH and MWT of small pulmonary arteries (Figures 4B and 4C). Sildenafil attenuated these functional and structural features of pulmonary hypertension as indicated by the increase in mean PAAT, reduction in RV/LV+S, and decrease in MWT.
Sildenafil-induced Alveolar and Vascular Growth Is Associated with Increased cGMP Levels
Hyperoxic rat pups had increased levels of cGMP compared with normoxic rats (Figure 5). Consistent with sildenafil's mechanism of action as a cGMP-specific PDE5 inhibitor, the concentration of cGMP was further increased in hyperoxic rats treated with sildenafil.
DISCUSSION
This study shows that sildenafil prevents lung injury and pulmonary hypertension in experimental oxygen-induced BPD in newborn rats. In vitro, sildenafil preserves a rich endothelial network in hyperoxic-exposed HPAEC. In vivo, sildenafil increases pulmonary capillary density, preserves alveolar growth, and alleviates echographic and structural signs of pulmonary hypertension. Our findings suggest a role for the NOeCcGMP pathway in angiogenesis and alveolarization in the developing lung. This pathway may be an effective target to protect the lung from impaired alveolar development.
Early observations by Liebow showed that the alveolar septa in emphysema were remarkably thin and almost avascular (44). He postulated that a reduction in the blood supply of the small precapillary blood vessels might induce the disappearance of alveolar septa. Despite this early observation, pulmonary vessels were thought for many years to be passive bystanders in lung development, following the branching pattern of the airways. Recent data, however, suggest that VEGF/NO-driven lung angiogenesis actively participates in alveolar growth (25, 30) and homeostasis throughout adulthood (45). In preterm infants, inhaled NO promising results as a prophylactic therapy for BPD (33, 34). Thus, we hypothesized that manipulating a downstream target of NO, specifically cGMP, would have similar beneficial effects on lung growth. Blockage of cGMP degradation using the PDE5 inhibitor sildenafil did preserve vascular and alveolar growth in the hyperoxic rat BPD model. This beneficial effect might have been through promotion of lung angiogenesis, because in vitro, sildenafil preserved endothelial network formation in hyperoxia. Further investigation is needed to reveal the precise mechanism by which sildenafil is lung-protective, and whether this is exclusively through promoting lung angiogenesis (and therefore alveolar development) or whether sildenafil or the NOeCcGMP pathway also has a direct effect on alveolar epithelial cell growth.
We found that hyperoxic rat pups had cGMP plasma levels greater than those in control rats. This finding is consistent with other studies in which increases in lung NO synthase and cGMP levels were seen in hyperoxic rats compared with normoxic rats (46). In the latter study, NO synthase blockade reduced lung cGMP levels in the hyperoxic rat pups but did not reverse the pathologic consequences of hyperoxic exposure (46). In our study, sildenafil further increased cGMP levels in hyperoxic rats and attenuated hyperoxic-induced lung injury. Thus, one possible interpretation is that upregulation of cGMP in hyperoxia may be a compensatory mechanism to counteract the deleterious effects of oxygen on lung development. By analogy, cGMP levels are increased in monocrotalline-induced pulmonary hypertension, and treatment with E4010, another PDE5 inhibitor, further enhances cGMP levels (47). In our study sildenafil further increased cGMP levels, and this may have been enough to promote lung angiogenesis/alveolar growth in hyperoxic conditions. This further assumes that the NOeCcGMP pathway is crucial for alveolar development.
cGMP can also be produced by atrial natriuretic peptide (ANP). Interestingly, type 2 alveolar epithelial cells express ANP receptors and ANP activation of guanylate cyclase A receptors produces cGMP (48, 49). The response to sildenafil can be influenced by the ANP pathway. Mice lacking NPR-A (NPR-AeC/eC), a guanylyl cyclaseeClinked natriuretic peptide receptor A, have higher basal RV systolic pressures (50). Furthermore, the beneficial effect of sildenafil on hypoxia-induced pulmonary vascular muscularization and cyclic GMP levels is blunted in NPR-AeC/eC mice, suggesting that a functional ANP pathway is crucial to the effectiveness of sildenafil (50). These recent findings warrant further investigations of the ANP pathway during lung development.
Pulmonary hypertension can contribute to morbidity and mortality in human BPD (7). The hyperoxic BPD model in rats also exhibits pulmonary hypertension characterized by increased pulmonary vasoconstriction, RVH, and MWT of pulmonary arteries. Decreased NO and excessive reactive oxygen species and endothelin pathway activation may account for pulmonary hypertension in this model (51eC53). Sildenafil alleviated pulmonary hypertension in our animal model. This is consistent with studies showing beneficial hemodynamic and structural effects of sildenafil in various adult animal models (54, 55) and in humans (56eC59) with pulmonary hypertension. Sildenafil is currently being investigated in newborns in clinical trials for persistent pulmonary hypertension, just like inhaled NO 10 years ago.
In conclusion, this is the first report on the potential therapeutic benefit of sildenafil in protecting from oxygen-induced alveolar damage. The relative pulmonary vascular specificity of sildenafil, its low cost, and its postmarketing safety makes it an attractive therapeutic option and warrants further study. Clinical trials are indicated to determine whether sildenafil can decrease the incidence/severity of BPD in premature infants and potentially other lung diseases characterized by alveolar damage.
B.T. is supported by the Canada Foundation for Innovation (CFI), the Alberta Heart and Stroke Foundation (H&SF), the Alberta Heritage Foundation for Medical Research (AHFMR), the Canadian Institutes for Health Research (CIHR), and by the Stollery Children's Hospital Foundation.
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