Alterations in the metabolism of L-arginine via arginase and nitric oxide synthase play a critical role in the endothelial dysfunction seen in PAH. L-arginine metabolism by arginase produces L-ornithine and urea. L-ornithine is a precursor for polyamine and proline synthesis, ultimately leading to an increase in cellular proliferation. Given the integral role of the smooth muscle layer in the pathogenesis of hypoxia-induced PAH, we hypothesized that hypoxia would increase cellular proliferation via arginase induction in human pulmonary artery smooth muscle cells (hPASMC). We found that arginase II mRNA and protein expression were significantly increased in cultured hPASMC exposed to 1% O 2 for 24 and 48 h, which coincided with an increase in arginase activity at 48 h. There were no hypoxia-induced changes in levels of arginase I mRNA or protein in cultured hPASMC. Exposure to hypoxia resulted in more than one and a half times as many viable cells after 120 h than normoxic exposure. The addition of the arginase inhibitor, S-(2-boronoethyl)-L-cysteine, completely prevented both the hypoxia-induced increase in arginase activity and proliferation in hPASMC. Furthermore, transfection of small interfering RNA (siRNA) targeting arginase II in hPASMC resulted in knockdown of arginase II protein levels and complete prevention of the hypoxia-induced cellular proliferation. These data support our hypothesis that hypoxia increases proliferation of hPASMC through the induction of arginase II. pulmonary hypertension; L-arginine; vascular remodeling PULMONARY ARTERY HYPERTENSION (PAH) is a life-threatening complication of chronic hypoxic lung diseases characterized by vasoconstriction, thrombosis, and the pathogenic hallmark of vascular remodeling that involves all layers of the vessel wall (10, 15). The smooth muscle layer plays an integral role in the pathogenesis of PAH with the extension of smooth muscle into smaller, normally nonmuscular pulmonary arteries within the respiratory acinus, a feature common to all forms of PAH remodeling (10, 25). In addition, pulmonary artery smooth muscle cells (PASMC) markedly proliferate, resulting in decreased luminal diameters and ultimately the obstruction of resistance level pulmonary arteries (10, 32).The L-arginine metabolic pathway has been shown to be important in maintaining vascular tone. L-arginine is the substrate for nitric oxide synthase (NOS) that generates the signaling molecule nitric oxide (NO) with the coproduct L-citrulline (31). Endogenous NO maintains vascular integrity by maintaining vasodilator tone and modulating vascular smooth muscle cell proliferation (12,18,23). Additionally, L-arginine is the substrate for arginase, of which there are two described isoforms (16,24). Arginase I is a cytosolic enzyme that is highly expressed in the liver. Conversely, arginase II is a mitochondrial protein that is not expressed in the liver (8). Both arginase isoforms are expressed in the lung (27). The metabolism of L-arginine by arginase results in the production of L-ornithin...
We hypothesized that hypoxia would activate epidermal growth factor receptor (EGFR) tyrosine kinase, leading to increased arginase expression and resulting in proliferation of human pulmonary microvascular endothelial cell (hPMVEC). To test this hypothesis, hPMVEC were incubated in normoxia (20% O(2), 5% CO(2)) or hypoxia (1% O(2), 5% CO(2)). Immunoblotting for EGFR and proliferating cell nuclear antigen was done, and protein levels of both total EGFR and proliferating cell nuclear antigen were greater in hypoxic hPMVEC than in normoxic hPMVEC. Furthermore, hypoxic hPMVEC had greater levels of EGFR activity than did normoxic hPMVEC. Hypoxic hPMVEC had a twofold greater level of proliferation compared with normoxic controls, and this increase in proliferation was prevented by the addition of AG-1478 (a pharmacological inhibitor of EGFR). Immunoblotting for arginase I and arginase II demonstrated a threefold induction in arginase II protein levels in hypoxia, with little change in arginase I protein levels. The hypoxic induction of arginase II protein was prevented by treatment with AG-1478. Proliferation assays were performed in the presence of arginase inhibitors, and hypoxia-induced proliferation was also prevented by arginase inhibition. Finally, treatment with an EGFR small interfering RNA prevented hypoxia-induced proliferation and urea production. These findings demonstrate that hypoxia activates EGFR tyrosine kinase, leading to arginase expression and thereby promoting proliferation in hPMVEC.
Prostacyclin is a pulmonary vasodilator and is produced by prostacyclin synthase and stimulates adenylate cyclase (AC) via the prostacyclin receptor (IP) to produce cAMP. Forskolin is a direct stimulant of AC. Phosphodiesterase 3 hydrolyzes cAMP and is inhibited by milrinone. Objective-To characterize the prostacyclin-AC-cAMP pathway in the ovine ductal ligation model of persistent pulmonary hypertension of the newborn (PPHN).Setting-University-based laboratory animal facility. Subjects-Lambs delivered to time-dated pregnant ewes.Interventions-Fifth generation pulmonary arteries (PA) and lung parenchyma were isolated from control fetal lambs (n = 8) and fetal lambs with PPHN induced by antenatal ductal ligation (n = 9). We studied relaxation responses to various agonists (milrinone, forskolin, prostacyclin, and iloprost, a prostacyclin analog) that increase cAMP in PA after half-maximal constriction with norepinephrine and pretreatment with propranolol ± indo-methacin. Lung protein levels of prostacyclin synthase, IP, AC2, and phosphodiesterase 3A were analyzed by Western blot and cAMP by enzyme-linked immunoassay.Main Results-Milrinone relaxed control and PPHN PA and pretreatment with indomethacin significantly impaired this response. Relaxation to milrinone, prostacyclin, and iloprost were significantly impaired in PA from PPHN lambs. Pretreatment with milrinone markedly enhanced relaxation to prostacyclin and iloprost in PPHN PA, similar to relaxation in control PA. Relaxation to forskolin was similar in control and PPHN PAs indicating normal AC activity. Protein levels of prostacyclin synthase and IP were decreased in PPHN lungs compared with control, but AC2, cAMP, and phosphodiesterase 3A remained unchanged.Conclusions-Prostacyclin and iloprost are dilators of PAs from PPHN lambs and their effect is enhanced by milrinone. This combination therapy may be an effective strategy in the management of patients with PPHN.For information regarding this article, E-mail: slakshmi@buffalo.edu. The authors have not disclosed any potential conflicts of interest. NO (9,10). Iloprost is a recently approved analog of prostacyclin approved as a nebulized treatment of pulmonary arterial hypertension in adults (11) and has been used anecdotally in neonates with PPHN (12). NIH Public AccessEndogenous prostacyclin is produced by cyclo-oxygenase (COX) and prostacyclin synthase (PGIS) enzymes (Fig. 1). Indomethacin is an inhibitor of COX. Prostacyclin stimulates adenylate cyclase (AC) by a prostacyclin receptor (IP)-coupled mechanism. Approximately ten isoforms of AC are found in the lung, but isoform 2 (AC2) is found predominantly in the pulmonary arterial smooth muscle cells (13). Forskolin is a receptor independent, direct stimulant of AC (14). AC catalyzes the formation of cAMP, and cAMP is hydrolyzed to AMP by at least three phosphodiesterase (PDE) isozymes in the pulmonary vasculature (15,16), namely PDE1, PDE3, and PDE4. Of all the inhibitors of these enzymes, milrinone, a PDE3 inhibitor, is widely used in intens...
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