Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs

Abstract

-Arginase is an enzyme that limits substrate L-arginine bioavailability for the production of nitric oxide by the nitric oxide synthases and produces L-ornithine, which is a precursor for collagen formation and tissue remodeling. We studied the pulmonary vascular effects of arginase inhibition in an established model of repeated systemic bleomycin sulfate administration in neonatal rats that results in pulmonary hypertension and lung injury mimicking the characteristics typical of bronchopulmonary dysplasia. We report that arginase expression is increased in the lungs of bleomycin-exposed neonatal rats and that treatment with the arginase inhibitor amino-2-borono-6-hexanoic acid prevented the bleomycininduced development of pulmonary hypertension and deposition of collagen. Arginase inhibition resulted in increased L-arginine and L-arginine bioavailability and increased pulmonary nitric oxide production. Arginase inhibition also normalized the expression of inducible nitric oxide synthase, and reduced bleomycin-induced nitrative stress while having no effect on bleomycin-induced inflammation. Our data suggest that arginase is a promising target for therapeutic interventions in neonates aimed at preventing lung vascular remodeling and pulmonary hypertension. nitric oxide; oxidative stress; chronic lung disease ARGINASES METABOLIZE L-ARGININE to form L-ornithine and urea. L-Ornithine is the precursor for the production of polyamines and collagen, which have been implicated in tissue repair and remodeling, respectively. Arginases also regulate nitric oxide (NO) production by controlling substrate L-arginine availability for NO synthases (NOSs) (46). There is accumulating evidence that arginase activity contributes to the development of pulmonary fibrosis and pulmonary hypertension (PHT) (9,14,25,29,32,63).PHT is a common complication of moderate-severe bronchopulmonary dysplasia (BPD), a chronic lung disease affecting extremely premature infants (6). The underlying pathogenesis of BPD is complex and multifactorial (39). Previous studies from our group have established a model of repeated systemic bleomycin injection in neonatal rats that results in lung injury mimicking characteristics typical of BPD. This model is a useful tool to help understand mechanisms contributing to lung development and remodeling (31) but also to study the effects of therapeutic interventions aimed at preventing lung parenchymal and vascular injury (31,50,56).The chemotherapeutic agent bleomycin sulfate causes a pulmonary inflammatory and fibrotic response in rodents when instilled as a single intratracheal dose (27) or by repeated intraperitoneal injection (3). Bleomycin-induced lung injury is characterized by induction of proinflammatory cytokines (18), influx of macrophages and other inflammatory cells (23), "emphysematous" lung morphology, and severe PHT (49, 60). Herein, we report that arginase expression is greatly increased in the lungs of bleomycin-exposed neonatal rats and that concomitant treatment with an arginase inhibitor, ami...

Leukotriene B4mediates macrophage influx and pulmonary hypertension in bleomycin-induced chronic neonatal lung injury

“…This divergence is consistent with our laboratory's own previous observations employing alternative interventions [e.g., therapeutic hypercapnia, arginase inhibition (15,44)] that were also efficacious in preventing inflammation and vascular remodeling, but not in preventing vascular hypoplasia or emphysematous lung structure. It appears, therefore, at least in bleomycinexposed neonatal rats, that inflammation and vascular remodeling are regulated by pathways distinct from those that regulate angiogenesis and alveologenesis.…”

“…The degree of increase in LTB 4 secondary to bleomycin exposure was small, which we speculate may have reflected a dilutional effect inherent to measurement in whole tissue. LTB 4 has been previously described to be predominantly produced by inflammatory cells, particularly activated macrophages and neutrophils (32,50), which are both present in abundance in the bleomycin-exposed lung (15,26,30,44). In the present study, we were unable to determine the cellular sources of LTB 4 , due to a lack of commercially available antibodies suitable for in situ immunostaining.…”

“…In neonatal rats, our laboratory and others have reported that repeated systemic administration of bleomycin leads to a selective decrease in lung growth, along with arrested alveolarization, vascular hypoplasia, and chronic PHT (15,30,44,52), similar to the lung pathology observed in human infants with severe BPD. We have employed this model to demonstrate a critical role for macrophage influx and macrophagederived tumor necrosis factor (TNF)-␣ in the pathogenesis of chronic PHT (26,44).…”

HIF2α–arginase axis is essential for the development of pulmonary hypertension

“…This occurs through HIF1α regulation of the NOS2/iNOS gene, and HIF-2α regulation of the Arg-1 and Arg-2 genes (21-25). The enzyme Arg-2 in particular has been implicated in reducing airway NO and promoting remodeling and collagen deposition in pulmonary arterial hypertension (PAH) patients (26,27). We found that hypoxic up-regulation of arg-1 and -2 was reduced in hypoxia-conditioned isolated murine primary pulmonary endothelial cells (Fig.…”

“…The causal link between pulmonary hypertension and NO homeostasis has been extensively documented (26,36), and this is reflected clinically in the finding that intrapulmonary nitrates, biochemical reaction products of NO in bronchoalveolar fluid, and exhaled NO are all diminished in human pulmonary hypertension (37,38). Interestingly, primary pulmonary endothelial cells isolated from PAH patients have substantially increased expression of arg-2 (27), which would be predicted to decrease available L-arginine and reduce NOS-derived NO formation.…”