Karen C. Young

Inhibition of the SDF-1/CXCR4 Axis Attenuates Neonatal Hypoxia-Induced Pulmonary Hypertension

Exposure of the neonatal lung to chronic hypoxia produces significant pulmonary vascular remodeling, right ventricular hypertrophy, and decreased lung alveolarization. Given recent data suggesting that stem cells could contribute to pulmonary vascular remodeling and right ventricular hypertrophy, we tested the hypothesis that blockade of SDF-1 (stromal cell–derived factor 1), a key stem cell mobilizer or its receptor, CXCR4 (CXC chemokine receptor 4), would attenuate and reverse hypoxia-induced cardiopulmonary remodeling in newborn mice. Neonatal mice exposed to normoxia or hypoxia were randomly assigned to receive daily intraperitoneal injections of normal saline, AMD3100, or anti–SDF-1 antibody from postnatal day 1 to 7 (preventive strategy) or postnatal day 7 to 14 (therapeutic strategy). As compared to normal saline, inhibition of the SDF-1/CXCR4 axis significantly improved lung alveolarization and decreased pulmonary hypertension, right ventricular hypertrophy, vascular remodeling, vascular cell proliferation, and lung or right ventricular stem cell expressions to near baseline values. We therefore conclude that the SDF-1/CXCR4 axis both prevents and reverses hypoxia-induced cardiopulmonary remodeling in neonatal mice, by decreasing progenitor cell recruitment to the pulmonary vasculature, as well as by decreasing pulmonary vascular cell proliferation. These data offer novel insights into the role of the SDF-1/CXCR4 axis in the pathogenesis of neonatal hypoxia-induced cardiopulmonary remodeling and have important therapeutic implications.

Long-term reparative effects of mesenchymal stem cell therapy following neonatal hyperoxia-induced lung injury.

Background:Mesenchymal stem cell (MSC) therapy may prevent neonatal hyperoxia-induced lung injury (HILI). There are, however, no clear data on the therapeutic efficacy of MSC therapy in established HILI, the duration of the reparative effects, and the exact mechanisms of repair. The main objective of this study was to evaluate whether the long-term reparative effects of a single intratracheal (IT) dose of MSCs or MSC-conditioned medium (CM) are comparable in established HILI.Methods:Newborn rats exposed to normoxia or hyperoxia from postnatal day (P)2)–P16 were randomized to receive IT MSCs, IT CM, or IT placebo (PL) on P9. Alveolarization and angiogenesis were evaluated at P16, P30, and P100.Results:At all time periods, there were marked improvements in alveolar and vascular development in hyperoxic pups treated with MSCs or CM as compared with PL. This was associated with decreased expression of inflammatory mediators and an upregulation of angiogenic factors. Of note, at P100, the improvements were more substantial with MSCs as compared with CM.Conclusion:These data suggest that acute effects of MSC therapy in HILI are mainly paracrine mediated; however, optimum long-term improvement following HILI requires treatment with the MSCs themselves or potentially repetitive administration of CM.

The Association between Early Tracheal Colonization and Bronchopulmonary Dysplasia

OBJECTIVE:To evaluate the relationship between early tracheal colonization and bronchopulmonary dysplasia (BPD).STUDY DESIGN:This is a retrospective cohort study which included 308 inborn neonates admitted to the newborn intensive care unit at the University of Miami Jackson Memorial Medical Center between January 1997 and December 2000 with birthweight 500 to 1000 g, who required mechanical ventilation on the first day of life. Chorioamnionitis was diagnosed by maternal symptoms and histopathopathology. Tracheal cultures were obtained immediately after tracheal intubation. BPD was diagnosed in neonates who had supplemental oxygen requirement for more than 28 days. Pearsons χ2 and Logistic Regression Analysis were used to evaluate the relationship between chorioamnionitis, positive initial tracheal cultures and BPD, after adjusting for confounding variables.RESULTS:In patients with chorioamnionitis, the incidence of early positive tracheal cultures was 41% compared to 16% in those without chorioamnionitis, (p<0.00001). In patients with birthweight 700 to 1000 g, a positive early tracheal culture increased the risk of BPD (OR=2.42, CI 1.05 to 5.62, p<0.05).CONCLUSION:Preterm infants exposed to chorioamnionitis have an increased incidence of early tracheal colonization. This early tracheal colonization may predispose them to develop BPD.

Toll-like receptor 4–deficient mice are resistant to chronic hypoxia-induced pulmonary hypertension

ABSTRACT Current data suggest that Toll-like receptor 4 (TLR4), a key molecule in the innate immune response, may also be activated following tissue injury. Activation of this receptor is known to induce the production of several proinflammatory cytokines. Given that pulmonary inflammation has been shown to be a key contributor to chronic hypoxia-induced pulmonary vascular remodeling, the authors hypothesized that TLR4-deficient mice would be less susceptible to pulmonary hypertension (PH) as compared to mice with intact TLR4. TLR4-deficient and TLR4-intact strains of inbred mice were exposed to 4, 8, and 16 weeks of hypoxia (0.10 FiO2) or normoxia (0.21 FiO2) in a normobaric chamber. After chronic hypoxic exposure, TLR4-intact mice developed significant PH evidenced by increased right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary artery medial thickening. In contrast, TLR4-deficient mice had no significant change in any of these parameters and this was associated with decreased pulmonary vascular inflammatory response as compared to the TLR4-intact mice. These results suggest that TLR4 deficiency may decrease the susceptibility to developing PH by attenuating the pulmonary vascular inflammatory response to chronic hypoxia.

The Effect of Gender on Mesenchymal Stem Cell (MSC) Efficacy in Neonatal Hyperoxia-Induced Lung Injury.

Background Mesenchymal stem cells (MSC) improve alveolar and vascular structures in experimental models of bronchopulmonary dysplasia (BPD). Female MSC secrete more anti-inflammatory and pro-angiogenic factors as compared to male MSC. Whether the therapeutic efficacy of MSC in attenuating lung injury in an experimental model of BPD is influenced by the sex of the donor MSC or recipient is unknown. Here we tested the hypothesis that female MSC would have greater lung regenerative properties than male MSC in experimental BPD and this benefit would be more evident in males. Objective To determine whether intra-tracheal (IT) administration of female MSC to neonatal rats with experimental BPD has more beneficial reparative effects as compared to IT male MSC. Methods Newborn Sprague-Dawley rats exposed to normoxia (RA) or hyperoxia (85% O2) from postnatal day (P) 2- P21 were randomly assigned to receive male or female IT bone marrow (BM)-derived green fluorescent protein (GFP+) MSC (1 x 106 cells/50 μl), or Placebo on P7. Pulmonary hypertension (PH), vascular remodeling, alveolarization, and angiogenesis were assessed at P21. PH was determined by measuring right ventricular systolic pressure (RVSP) and pulmonary vascular remodeling was evaluated by quantifying the percentage of muscularized peripheral pulmonary vessels. Alveolarization was evaluated by measuring mean linear intercept (MLI) and radial alveolar count (RAC). Angiogenesis was determined by measuring vascular density. Data are expressed as mean ± SD, and analyzed by ANOVA. Results There were no significant differences in the RA groups. Exposure to hyperoxia resulted in a decrease in vascular density and RAC, with a significant increase in MLI, RVSP, and the percentage of partially and fully muscularized pulmonary arterioles. Administration of both male and female MSC significantly improved vascular density, alveolarization, RVSP, percent of muscularized vessels and alveolarization. Interestingly, the improvement in PH and vascular remodeling was more robust in the hyperoxic rodents who received MSC from female donors. In keeping with our hypothesis, male animals receiving female MSC, had a greater improvement in vascular remodeling. This was accompanied by a more significant decrease in lung pro-inflammatory markers and a larger increase in anti-inflammatory and pro-angiogenic markers in male rodents that received female MSC. There were no significant differences in MSC engraftment among groups. Conclusions Female BM-derived MSC have greater therapeutic efficacy than male MSC in reducing neonatal hyperoxia-induced lung inflammation and vascular remodeling. Furthermore, the beneficial effects of female MSC were more pronounced in male animals. Together, these findings suggest that female MSC maybe the most potent BM-derived MSC population for lung repair in severe BPD complicated by PH.

CXCR4 Blockade Attenuates Hyperoxia-Induced Lung Injury in Neonatal Rats

Background: Lung inflammation is a key factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Stromal-derived factor-1 (SDF-1) and its receptor chemokine receptor 4 (CXCR4) modulate the inflammatory response. It is not known if antagonism of CXCR4 alleviates lung inflammation in neonatal hyperoxia-induced lung injury. Objective: We aimed to determine whether CXCR4 antagonism would attenuate lung injury in rodents with experimental BPD by decreasing pulmonary inflammation. Methods: Newborn rats exposed to normoxia (room air, RA) or hyperoxia (FiO2 = 0.9) from postnatal day 2 (P2) to P16 were randomized to receive the CXCR4 antagonist, AMD3100 or placebo (PL) from P5 to P15. Lung alveolarization, angiogenesis and inflammation were evaluated at P16. Results: Compared to the RA pups, hyperoxic PL pups had a decrease in alveolarization, reduced lung vascular density and increased lung inflammation. In contrast, AMD3100-treated hyperoxic pups had improved alveolarization and increased angiogenesis. This improvement in lung structure was accompanied by a decrease in the macrophage and neutrophil counts in the bronchoalveolar lavage fluid and reduced lung myeloperoxidase activity. Conclusion: CXCR4 antagonism decreases lung inflammation and improves alveolar and vascular structure in neonatal rats with experimental BPD. These findings suggest a novel therapeutic strategy to alleviate lung injury in preterm infants with BPD.

The effect of a nebulized NO donor, DPTA/NO, on acute hypoxic pulmonary hypertension in newborn piglets.

NONOates, novel NO donors, are complexes of NO with nucleophiles which spontaneously and nonenzymatically release NO in aqueous solution. This study sought to determine the cardiopulmonary effects of the nebulized NONOate dipropylenetriamine (DPTA)/NO in newborn piglets with acute hypoxia-induced pulmonary hypertension. Twenty sedated and mechanically ventilated piglets (4–10 days old) exposed to hypoxia (FiO2 = 0.14) were randomly assigned to receive nebulized saline as placebo (PL) or DPTA/NO (75 mg) after 30 min of hypoxia. Pulmonary artery (Ppa) and wedge pressures, systemic (Psa) and right atrial pressures, cardiac output (CO) and arterial blood gas were measured at baseline and every 15 min for 2 h. Methemoglobin levels were measured at baseline and 1 h after drug nebulization. Data (means ± SD) were analyzed by repeated-measures analysis of variance. Acute hypoxia resulted in an increase in Ppa and pulmonary vascular resistance (PVR), which was significantly attenuated by DPTA/NO nebulization as compared to the PL group (p < 0.0001). Changes in Psa, CO, systemic vascular resistance (SVR), arterial blood gas and methemoglobin levels were not different between groups. In contrast to the increase in PVR/SVR observed during hypoxia in the PL group, there was a significant decrease in this ratio after NONOate administration (p < 0.0001). These data show that acute hypoxic pulmonary hypertension in newborn piglets is markedly attenuated by NONOate nebulization. This response is predominantly in the pulmonary vasculature as the PVR/SVR was significantly lower in the treated group. We speculate that NONOates may have clinical application in the treatment of persistent pulmonary hypertension of the newborn.

Stem cell factor improves lung recovery in rats following neonatal hyperoxia-induced lung injury

Background:Stem cell factor (SCF) and its receptor, c-kit, are modulators of angiogenesis. Neonatal hyperoxia-induced lung injury (HILI) is characterized by disordered angiogenesis. The objective of this study was to determine whether exogenous SCF improves recovery from neonatal HILI by improving angiogenesis.Methods:Newborn rats assigned to normoxia (RA: 20.9% O2) or hyperoxia (90% O2) from postnatal day (P) 2 to 15, received daily injections of SCF 100 μg/kg or placebo (PL) from P15 to P21. Lung morphometry was performed at P28. Capillary tube formation in SCF-treated hyperoxia-exposed pulmonary microvascular endothelial cells (HPMECs) was determined by Matrigel assay.Results:As compared with RA, hyperoxic-PL pups had decrease in alveolarization and in lung vascular density, and this was associated with increased right ventricular systolic pressure (RVSP), right ventricular hypertrophy, and vascular remodeling. In contrast, SCF-treated hyperoxic pups had increased angiogenesis, improved alveolarization, and attenuation of pulmonary hypertension as evidenced by decreased RVSP, right ventricular hypertrophy, and vascular remodeling. Moreover, in an in vitro model, SCF increased capillary tube formation in hyperoxia-exposed HPMECs.Conclusion:Exogenous SCF restores alveolar and vascular structure in neonatal rats with HILI by promoting neoangiogenesis. These findings suggest a new strategy to treat lung diseases characterized by dysangiogenesis.

Bone marrow-derived c-kit+ cells attenuate neonatal hyperoxia-induced lung injury.

Recent studies suggest that bone marrow (BM)-derived stem cells have therapeutic efficacy in neonatal hyperoxia-induced lung injury (HILI). c-kit, a tyrosine kinase receptor that regulates angiogenesis, is expressed on several populations of BM-derived cells. Preterm infants exposed to hyperoxia have decreased lung angiogenesis. Here we tested the hypothesis that administration of BM-derived c-kit+ cells would improve angiogenesis in neonatal rats with HILI. To determine whether intratracheal (IT) administration of BM-derived c-kit+ cells attenuates neonatal HILI, rat pups exposed to either normobaric normoxia (21% O2) or hyperoxia (90% O2) from postnatal day (P) 2 to P15 were randomly assigned to receive either IT BM-derived green fluorescent protein (GFP)+ c-kit–cells (PL) or BM-derived GFP+ c-kit+ cells on P8. The effect of cell therapy on lung angiogenesis, alveolarization, pulmonary hypertension, vascular remodeling, cell proliferation, and apoptosis was determined at P15. Cell engraftment was determined by GFP immunostaining. Compared to PL, the IT administration of BM-derived c-kit+ cells to neonatal rodents with HILI improved alveolarization as evidenced by increased lung septation and decreased mean linear intercept. This was accompanied by an increase in lung vascular density, a decrease in lung apoptosis, and an increase in the secretion of proangiogenic factors. There was no difference in pulmonary vascular remodeling or the degree of pulmonary hypertension. Confocal microscopy demonstrated that 1% of total lung cells were GFP+ cells. IT administration of BM-derived c-kit+ cells improves lung alveolarization and angiogenesis in neonatal HILI, and this may be secondary to an improvement in the lung angiogenic milieu.

Inhibition of Rac1 Signaling Downregulates Inflammasome Activation and Attenuates Lung Injury in Neonatal Rats Exposed to Hyperoxia.

Background: Inflammatory injury, particularly the production of active interleukin (IL)-1β plays a major role in the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. The release of active IL-1β is controlled by posttranscriptional modifications of its proform (pro-IL-1β) through the inflammasome. Rac1 is a member of the Rho family of GTPases that regulate the inflammatory process. Objective: This study tested the hypothesis that Rac1 signaling increases inflammasome activation that results in damaging inflammation, and that the inhibition of Rac1 signaling prevents lung injury, by inhibiting inflammasome activation in a newborn rat model of BPD induced by hyperoxia. Methods: Newborn rat pups were exposed to room air or hyperoxia (85% O2) and received daily intraperitoneal injections of placebo (normal saline) or NSC23766, a specific Rac1 inhibitor, for 10 days. The effects on lung inflammation, alveolarization, vascular development, vascular remodeling, right ventricular systolic pressure, and right ventricular hypertrophy (RVH) were then assessed. Results: Hyperoxia exposure upregulated Rac1 and increased the production of active IL-1β, which was accompanied by increasing expression of the inflammasome. In addition, hyperoxia induced the pathological hallmarks of BPD. However, treatment with NSC23766 significantly decreased inflammasome activation and macrophage infiltration, improved alveolar and vascular development, and reduced pulmonary vascular remodeling and RVH. Conclusion: These results indicate that Rac1 signaling regulates the expression of the inflammasome and plays a pivotal role in the pathogenesis of hyperoxia-induced neonatal lung injury. Therefore, targeting Rac1 signaling may provide a novel strategy to prevent and treat BPD in preterm infants.