Vascular remodeling and smooth muscle cell proliferation are hallmark pathogenic features of pulmonary artery hypertension (PAH). 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% O2 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
- 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-ornithine and urea. Arginase is the first step in polyamine and proline synthesis. Polyamines and proline are critical for cell proliferation, differentiation, tissue repair, and growth (14, 19).
Both arginase isoforms are expressed in endothelial cells and have been implicated in mediating endothelial dysfunction in a variety of pathological vascular conditions such as aging blood vessels, ischemia-reperfusion injury, systemic hypertension, and PAH (8). It has previously been shown that there is an increase in arginase II expression in endothelial cells of patients with PAH (40). The overexpression of arginase I or arginase II in endothelial cells increases polyamine and proline production as well as cellular proliferation (19). Arginase has proven to be an important enzyme in the dysfunction of vascular endothelial cells; however, its expression in smooth muscle cells may also play a critical role in the pathogenesis of PAH via downstream proliferative effects. Given the central role of vascular smooth muscle cells in the remodeling and pulmonary vasoconstriction seen in PAH, we hypothesized that hypoxia would result in increased cellular proliferation via arginase induction in human PASMC (hPASMC).
MATERIALS AND METHODS
Human PASMC (Lonza) were grown in 21% O2, 5% CO2, balance N2 at 37°C in smooth muscle growth media (SmGM; Lonza), which includes smooth muscle basal medium (Lonza), 5% FBS, 0.5 ng/ml human recombinant epidermal growth factor, 2 ng/ml human recombinant fibroblast growth factor, 5 μg/ml insulin, and 50 μg/ml gentamicin. The hPASMC were used in experiments between the 5th and 8th passages, throughout which no changes in cell morphology were noted. The cells were grown to approximately 80–90% confluence and washed, fresh media was placed on the cells, and they were incubated in 21% O2, 5% CO2, balance N2 (normoxia) or 1% O2, 5% CO2, balance N2 (hypoxia) for 24, 48, or 120 h.
RNA was isolated from hPASMC as previously described (29). Briefly, 0.5 ml of TRIzol reagent (Invitrogen, Carlsbad, CA) was added to each well of a six-well plate containing the hPASMC and incubated for 5 min at room temperature. Cells were scraped, and the mixture was collected into 1.5-ml centrifuge tubes. Chloroform (0.1 ml) was added, and the tubes were shaken for 15 s and incubated at 30°C for 3 min. The mixture was centrifuged at 12,000 g for 15 min at 4°C before the supernatant was transferred to a fresh 1.5-ml centrifuge tube. Isopropyl alcohol (0.25 ml) was added, and the mixture was incubated at 30°C for 10 min and then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was then discarded, and the pellet was washed with 75% ethanol and centrifuged at 7,500 g for 5 min at 4°C. The supernatant was again discarded, and the pellet was partially dried, dissolved in RNase-free water, and stored at −80°C.
Protein was isolated from hPASMC as previously described (29). Briefly, hPASMC were washed with PBS, and 50–100 μl of lysis buffer (0.2 M NaOH, 0.2% SDS) was added to each plate or each well of a six-well plate. Thirty minutes before use, the following protease inhibitors were added to each milliliter of lysis buffer: 1 μl of aprotinin [10 mg/ml double distilled (dd) H2O], 1 μl of leupeptin (10 mg/ml ddH2O), and 1 μl of phenylmethylsulfonyl fluoride (34.8 mg/ml methanol). The lysis buffer solution was added to each plate or each well of a six-well plate. The hPASMC were scraped, pipetted into sterile centrifuge tubes, and placed on ice for 30 min. The cell lysates were centrifuged at 12,000 g for 10 min. The supernatant was stored in 1.5-ml tubes at −80°C. Total protein concentration was determined by the Bradford method (4) using a commercially available assay (Bio-Rad, Hercules, CA).
Reverse transcription and quantitative real-time PCR.
Reverse transcription was performed as previously described (5). Briefly, 2 μg of total RNA was reverse-transcribed in 2.5 μM dT16 (Applied Biosystems, Foster City, CA), 20 units of avian myeloblastosis virus (AMV) RT, 1 mM dNTP, 1× AMV RT buffer (Promega, Madison, WI), and RNase-free water for a total volume of 40 μl. The samples were incubated in a PCR iCycler (Bio-Rad) at 42°C for 60 min, 95°C for 5 min, and stored at −20°C. Quantitative real-time PCR was performed using the SYBR Green Jumpstart Taq ReadyMix with the Chromo4 real-time PCR detection system (Bio-Rad) with 40 cycles of real-time data collection using 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s, followed by melting curve analysis to verify the presence of a single product. Arginase I was amplified using the forward primer (5′-TTGGCAATTGGAAG-CATCTCTGGC-3′) and the reverse primer (5′-TCCACTTGTGGTTGTCAGTGGAGT-3′). Arginase II was amplified using the forward primer (5′-TTAGCAGAGCTGTGT-CAGATGGCT-3′) and the reverse primer (5′-GGGCATCAACCCAGACAACACAAA-3′). 18S was amplified using the forward primer (5′-CCAGAGCGAAAGCATTTGCCA-AGA-3′) and the reverse primer (5′-TCGGCATCGTTTATGGTCGGAACT-3′). For each reaction, negative controls containing reaction mixture and primers without cDNA were performed to verify that primers and reaction mixtures were free of template contamination. Relative arginase I and arginase II amounts were normalized to 18S expression using the ΔΔCT method (20). All samples were analyzed in duplicate. Data are shown as fold change relative to normoxia-exposed hPASMC controls at each respective time point.
The lysed hPASMC were assayed for arginase I and arginase II protein levels by Western blot analysis as previously described (28, 29). Aliquots of cell lysate were diluted 1:1 with SDS sample buffer, heated to 80°C for 15 min, and then centrifuged at 10,000 g at room temperature for 2 min. Aliquots of the supernatant were used for SDS-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes and blocked for 2 h in PBS with 0.1% Tween (PBS-T) containing 5% nonfat dried milk. The membranes were then incubated with primary antibody, arginase I (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) or arginase II (1:500; Santa Cruz Biotechnology), overnight and then washed three times with PBS-T. The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:10,000; Bio-Rad) for 1 h and then washed three times with PBS-T. The protein bands were visualized using enhanced chemiluminescence (ECL Plus reagent; Amersham Pharmacia Biotech, Piscataway, NJ) and quantified using densitometry (SigmaGel; Jandel Scientific, San Rafael, CA). To control for protein loading, the blots were stripped using a stripping buffer containing 62.5 mM Tris·HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol, and the blots were reprobed for β-actin (1:10,000; Abcam, Cambridge, MA) as described above. Data are shown as average mean density of the protein of interest normalized to the density of β-actin.
For the determination of arginase activity, cell lysates were prepared as described above. The cell lysate samples (200 μg) were added to 10% (vol/vol) MnCl2 with 0.2 M Tris buffer (pH 9.0) for a total volume of 150 μl. The mixtures were vortexed and incubated at 55°C for 5 min. Increasing concentrations of l-arginine (0, 0.03, 0.12, 0.3, 1.2, 3, 10, and 30 mM) were added to the heat-activated lysates in each test tube, vortexed, and incubated at 37°C for 1 h. The samples were colorimetrically assayed in duplicate for urea as previously described (29). Each sample (300 μl) was added to 1.5-ml chromogenic reagent [5 mg of thiosemicarbazide, 250 mg of diacetyl monoxime, and 25 mg of FeCl3 in 150 ml of 16.7% (vol/vol) H2SO4 and 13.3% (vol/vol) H3PO4]. The mixtures were vortexed, boiled at 100°C for 5 min, and then cooled to room temperature. Each sample with the added chromogenic reagent (200 μl) was pipetted into a microplate in duplicate, and the difference in absorbance at 530 nm was compared with a urea standard curve. The arginase activity was calculated as amount of urea (μmol)/min, and velocity was determined as μmol/(mg protein/min). The reaction velocity was plotted against the substrate concentration, and the Vmax and Km values were determined using the Michaelis-Menten equation (SigmaPlot 11.0; Jandel Scientific, Carlsbad, CA).
The effects of arginase inhibition on arginase activity in hPASMC were assessed by adding either vehicle or the arginase inhibitor, S-(2-boronoethyl)-l-cysteine [BEC (1 mM); Cayman Chemical, Ann Arbor, MI], to l-arginine (1 or 3 mM), and the assay was performed as described above. l-arginine at 1 and 3 mM were used because hypoxia-induced arginase activity was enhanced at these concentrations.
To determine cell proliferation, hPASMC (Lonza) were seeded in 6-well plates (∼5,000 cells per well) in SmGM and incubated in hypoxia for 120 h. Adherent cells were trypsinized, and viable cells were counted using a hemocytometer and trypan blue exclusion. To determine the role of arginase in the proliferative response of hPASMC in hypoxia, increasing concentrations (0.1, 0.5, and 1 mM) of BEC were added to the media, and cell proliferation was determined. In normoxia, 0.5 mM BEC was added to the media, and cell proliferation was determined. Data are shown as a percentage of normoxia controls.
Transfection of arginase II-siRNA.
To determine the effects of arginase II gene silencing on hPASMC proliferation, transient transfection of arginase II-small interfering RNA (siRNA) was performed with DharmaFECT 1 transfection reagent (Thermo Fisher Scientific, Lafayette, CO) according to manufacturer's protocol. Briefly, in 1.5-ml centrifuge tubes, 100 μl of 2 μM siRNA, arginase II-siRNA (Thermo Fisher Scientific), or nontargeting scramble siRNA (Thermo Fisher Scientific) was mixed with 100 μl of smooth muscle basal medium (Lonza), vortexed, and incubated at room temperature for 5 min. The DharmaFECT 1 transfection reagent was diluted 1:100 (total volume of 200 μl) and incubated at room temperature for 5 min. The transfection reagent was then added to each sample of siRNA, mixed, and incubated at room temperature for 20 min. SmGM (600 μl) was then added to each tube for a total volume of 1 ml. For the vehicle-treated hPASMC, ddH2O was used in place of siRNA.
Human PASMC (Lonza), grown to ∼70% confluence in six-well plates, were washed with PBS. Fresh media (1 ml) + siRNA-DharmaFECT reagent mixture (1 ml) were placed in each well of a six-well plate, and the hPASMC were incubated in normoxia for 24 h. The media was then removed, cells were washed, SmGM was added, and the hPASMC were incubated in normoxia for another 24 h. The hPASMC were used in experiments and incubated in either normoxia or hypoxia for 120 h, and protein was harvested for Western blot analysis or cells were prepared and counted for proliferation assay as described above.
Values are given as means ± SE. Unpaired t-test or one-way ANOVA was used to compare the data between groups. Differences were identified using Newman-Keuls post hoc testing for parametric data (Prism; GraphPad Software, San Diego, CA). Differences were considered significant when P < 0.05.
To determine the effects of hypoxia on arginase mRNA expression, hPASMC were grown to approximately 80–90% confluence and incubated in either normoxia or hypoxia for 24 or 48 h. RNA was harvested, and arginase I and arginase II mRNA expression were assessed by quantitative real-time PCR. There was a significant increase in arginase II mRNA expression in hPASMC exposed to hypoxia relative to those exposed to normoxia at both 24 and 48 h (Fig. 1). There was no difference in arginase I mRNA expression between hPASMC exposed to normoxia or hypoxia at 24 or 48 h (data not shown).
To determine the effects of hypoxia on arginase protein expression, hPASMC were grown to approximately 80–90% confluence and incubated in either normoxia or hypoxia for 24 or 48 h. Protein was harvested, and arginase I and arginase II protein expression were measured using Western blot analysis and quantified by densitometry. There was a ∼3-fold (P < 0.05) greater arginase II protein expression in hPASMC exposed to hypoxia than in those exposed to normoxia at 24 h and a ∼4-fold (P < 0.05) greater arginase II protein expression in hPASMC exposed to hypoxia than in those exposed to normoxia at 48 h (Fig. 2). Although both arginase I and arginase II isoforms were found in hPASMC, there was no difference in the levels of arginase I protein between hPASMC exposed to normoxia or hypoxia for either 24 or 48 h (Fig. 3).
Given that proliferation studies were measured at 120 h, arginase protein expression was also determined at this time point. Human PASMC grown to approximately 80–90% confluence were incubated in either normoxia or hypoxia for 120 h. Protein was harvested, and arginase I and arginase II protein expression were measured using Western blot analysis and quantified by densitometry. There was a ∼5-fold (P < 0.0002) greater arginase II protein expression in hPASMC exposed to hypoxia than in those exposed to normoxia at 120 h (Fig. 4). There was no difference between normoxic and hypoxic cells in the levels of arginase I protein (Fig. 5).
We determined the activity of arginase in hPASMC. Cell lysates were incubated with increasing concentrations of l-arginine in normoxic and hypoxic conditions, and urea production was measured. Michaelis-Menten kinetics for arginase were determined. There were no differences in arginase activity at any of the l-arginine concentrations studied at 24 h. There was no significant difference in Vmax or Km of arginase in hPASMC at 24 h (Fig. 6A). At 48 h, the arginase activity was greater in hypoxia for l-arginine concentrations ≥0.3 mM (Fig. 6B). The Vmax was significantly higher for hypoxic cells than normoxic cells [Fig. 6B, 0.25 ± 0.05 vs. 0.14 ± 0.02 μmol/(mg protein/min); P = 0.047], whereas the Km was not different between the two groups at 48 h (4.9 ± 1.3 vs. 3.3 ± 0.4 mM; P = 0.14).
We also determined the effects of the arginase inhibitor, BEC, on the hypoxia-induced increase in arginase activity in hPASMC. Cell lysates were incubated with either 1 or 3 mM l-arginine in normoxic and hypoxic conditions with or without the addition of BEC (1 mM), and arginase activity was measured at 48 h. In normoxic conditions, BEC inhibited the arginase activity compared with the vehicle-treated hPASMC at each tested l-arginine concentration (Fig. 7; 1 mM, P < 0.05; 3 mM, P < 0.001). BEC inhibited the hypoxia-induced increase in arginase activity for both concentrations of l-arginine used (Fig. 7; P < 0.001 for both 1 and 3 mM). For each concentration of l-arginine studied, treatment of hPASMC with BEC in both normoxic and hypoxic conditions inhibited arginase activity to similar degrees (Fig. 7).
Proliferation of hPASMC after exposure to either normoxia or hypoxia was determined by counting viable cells using trypan blue exclusion. Cells were seeded at ∼5,000 cells per well on a 6-well plate and incubated in normoxia or hypoxia for 120 h. After 120 h in normoxia, hPASMC number had increased from ∼5,000 cells per well to 93,667 ± 6,123 viable cells per well (Fig. 8A). The hPASMC exposed to hypoxia for 120 h had ∼1.7-fold (P < 0.0001) greater viable cell numbers than did hPASMC exposed to normoxia for 120 h (Fig. 8A).
To determine the role of arginase in the proliferative response, the arginase inhibitor, BEC, was used. Human PASMC were seeded on 6-well plates at a density of ∼5,000 cells per well, and BEC was added to the medium (0, 0.1, 0.5, and 1 mM). The cells were then incubated in hypoxia for 120 h, and viable cell numbers were determined using trypan blue exclusion. In addition, BEC-treated (0.5 mM) hPASMC were incubated in normoxia for 120 h, and viable cell numbers were determined. Each of the tested concentrations of BEC prevented the hypoxia-induced hPASMC proliferation, such that viable cell numbers in the hypoxic, BEC-treated hPASMC were not different from viable cell numbers in the normoxic, untreated hPASMC (Fig. 8A). At a concentration of 0.5 mM BEC, the number of viable normoxic, BEC-treated hPASMC was reduced to ∼54% of the normoxic, untreated hPASMC (Fig. 8B).
To further delineate the role of isoform-specific arginase in the hypoxia-induced proliferative response, transfection of arginase II-siRNA into hPASMC was performed. The selective knockdown of arginase II expression at 120 h, after transfection of arginase II-siRNA, was confirmed by Western blot analysis and quantified by densitometry. Scramble siRNA transfection resulted in greater arginase II protein levels in hypoxia than normoxia (Fig. 9, A and B). In normoxia, the arginase II protein expression in the arginase II-siRNA-transfected cells was not different from the normoxic, scramble siRNA-transfected cells (Fig. 9, A and B). In hypoxia, the arginase II-siRNA prevented the hypoxia-induced increase in arginase II protein levels, such that arginase II protein expression in the arginase II-siRNA-transfected cells was equivalent to levels seen in normoxic, scramble siRNA-transfected cells (Fig. 9, A and B).
Proliferation of the transfected hPASMC after exposure to either normoxia or hypoxia was then determined by counting viable cells using trypan blue exclusion. Cells were seeded at ∼5,000 cells per well on a 6-well plate and incubated in normoxia or hypoxia for 120 h. The viable cell numbers of the normoxic, arginase II-siRNA-transfected hPASMC was not statistically different from the normoxic, scramble siRNA-transfected controls (Fig. 9C). However, the arginase II-siRNA transfection prevented the hypoxia-induced hPASMC proliferation, such that viable cell numbers were not different from the normoxic, arginase II-siRNA-transfected cells or normoxic, scramble siRNA-transfected controls (Fig. 9C).
The main objective of the present study was to determine the effects of hypoxia, an important contributor to the pathogenesis of PAH, on arginase production, arginase activity, and proliferation of hPASMC. The major findings of this study were that 1) hypoxia increased arginase II mRNA and protein levels in hPASMC, 2) hypoxia had no effect on arginase I mRNA or protein levels, 3) hypoxia increased arginase activity, which was completely inhibited by the arginase inhibitor, BEC, 4) hypoxia resulted in greater cell proliferation, which was completely inhibited by BEC, and 5) siRNA targeting arginase II in hPASMC prevented hypoxia-induced arginase II expression and completely inhibited hypoxia-induced hPASMC proliferation. Taken together, these findings support our hypothesis that hypoxia results in hPASMC proliferation via arginase II induction.
We found that hPASMC express both arginase isoforms. Previously, arginase II was reported to be nondetectable in rat aortic smooth muscle cells (9, 37). Similar findings were reported in rabbit carotid artery smooth muscle cells (26). Recently, Belik et al. (3) found arginase II staining in the muscular layer of pulmonary arteries using immunohistochemistry. Thus it may be that PASMC differ from aortic/carotid smooth muscle cells in terms of arginase II expression.
To our knowledge, the effects of hypoxia on arginase expression in vascular smooth muscle cells have not been previously shown. Interestingly, we found that hypoxia did not result in either downregulation or upregulation of arginase I gene or protein expression. Conversely, arginase II gene and protein expression were both upregulated in hypoxia-exposed hPASMC. These data demonstrate that in hPASMC, arginase II is the hypoxia-inducible isoform, whereas arginase I expression levels are unaffected by hypoxia. Hypoxia has been shown to increase arginase activity or expression in a variety of other cell types. An increase in arginase activity was shown in the brain of rats exposed to hypoxic-ischemic insult (6). In wound-derived rat macrophages, an increase in arginase activity and total l-arginine metabolism was found when exposed to an anoxic environment (1). Louis et al. (22) described similar findings in rat wound-derived and mouse peritoneal macrophages in both hypoxic and anoxic culture. Interestingly, in the same study, arginase I mRNA levels were increased in both cell types, whereas arginase II mRNA levels, present only in the LPS-stimulated murine peritoneal macrophages, were suppressed by oxygen deprivation (22). In contrast, arginase II gene expression and activity were found to be increased in the hypoxic kidney of a sickle cell transgenic mouse model (34). In the present study, we found an increase in arginase II expression along with an increase in arginase activity in hypoxia-exposed hPASMC. Furthermore, the hypoxia-induced increase in arginase activity was completely inhibited by the arginase inhibitor, BEC. Thus, taken together, these studies demonstrate that arginase expression and activity are regulated by hypoxia in both a cell type- and isoform-specific manner. To our knowledge, we are the first to describe arginase kinetics in this specific cell type.
Controversy exists on the effect of hypoxia on hPASMC in vitro. Hypoxia has been found in various studies to inhibit or induce cellular proliferation. Several mechanisms have been proposed for the variation in proliferative response in this cell type, including the source of pulmonary artery cells, phenotypic variations of the smooth muscle cell within the arterial media, severity of hypoxia, seeding density, and the concentration of serum in the cell culture plates (30). Chronic hypoxia, however, does lead to proliferation of PASMC in vivo, with findings of thickened muscular layers in the pulmonary arteries of animals exposed to hypoxia (30), thereby resulting in PAH. We found a greater viable cell number after exposing hPASMC to hypoxia for 120 h than in normoxia-exposed hPASMC. Hence, in our in vitro cell culture model, hPASMC exposed to hypoxia have a proproliferative response. Of note, the commercially available hPASMC used in the present study were isolated from main pulmonary artery segments. This finding is consistent with other in vitro studies in a variety of species including human, ovine, porcine, and rat PASMC (2, 7, 11, 33). However, there may be differences in responses of vascular smooth muscle cells to hypoxia based on their location along the arterial tree, i.e., vascular smooth muscle cells from the main pulmonary artery may have different hypoxia-induced proliferative responses than smooth muscle cells from the smallest muscular pulmonary arteries.
The arginase inhibitor, BEC, was used to determine the mechanism of hypoxia-induced proliferation in hPASMC. Given the hypoxia-induced increase in arginase II expression and arginase activity, we hypothesized that hypoxia-induced proliferation of hPASMC was due to the increase in arginase activity. Interestingly, we did find that hPASMC proliferation and arginase activity were inhibited by BEC in normoxic conditions. Furthermore, at each dose tested, BEC completely inhibited hypoxia-induced cellular proliferation relative to normoxic, untreated hPASMC. Although both arginase I and arginase II are present in the hPASMC we studied, hypoxia induces an increase specifically in arginase II protein levels in our experimental conditions. Therefore, it is likely that it is the increase in arginase II protein that leads to the hypoxia-induced increase in arginase activity and thereby to greater cellular proliferation in hypoxic hPASMC. We used a siRNA targeting arginase II in hPASMC to address the specific role of arginase II in this response. Transfection with the arginase II-siRNA completely prevented the hypoxia-induced increase in cellular proliferation, supporting our hypothesis that arginase II is the specific arginase isoform involved in hypoxia-induced hPASMC proliferation.
An increase in arginase activity would result in greater levels of l-ornithine production, and l-ornithine can be further metabolized to produce polyamines, which are critical for cellular proliferation. In rat aortic smooth muscle cells transfected with arginase I, an increase in cell proliferation was observed, and polyamine production correlated with these proproliferative effects (38). In addition, aortic smooth muscle cell proliferation has also been shown to be inhibited by NG-hydroxy-l-arginine (NOHA), a potent competitive inhibitor of arginase, whereas the addition of the polyamine, putrescine, prevented the cytostatic action of NOHA (12). Therefore, we speculate that the hypoxia-induced proliferation of hPASMC occurs through an increase in the production of polyamines via arginase II induction.
Besides the augmented downstream effects of l-ornithine from elevated arginase activity, we speculate that the increase in proliferation of hPASMC exposed to hypoxia may also be a result of a decrease in NO production due to the increased arginase activity that may limit the bioavailability of l-arginine to NOS (36). Although the affinity of l-arginine is much higher for NOS than for arginase, the maximum activity of arginase is >1,000 times that of NOS, suggesting similar rates of substrate utilization at physiological l-arginine concentrations (39). NO is a recognized inhibitor of smooth muscle cell growth (21), and its direct role in hPASMC may be a contributor to vascular tone, whereas a decrease in production would promote proliferation (36).
The hypoxia-induced proliferation of PASMC may involve a variety of signal transduction networks. Cell proliferation under hypoxic conditions was found in one study to be associated with enhanced p27Kip1 degradation allowing cell cycle progression (17). Recently, platelet-activating factor has been found to induce fetal ovine PASMC proliferation via a NF-κB and cyclin-dependent kinase pathway (11). Furthermore, hypoxia-induced proliferation of vascular smooth muscle cells may include a role for reactive oxygen species through the regulation of hypoxia-inducible factor-1α and/or TGF-β1 (13, 41). In hypoxic macrophages, arginase I is induced via the CCAAT/enhancer binding protein-β (C/EBPβ); however, in those studies, arginase II expression was not increased in hypoxic macrophages (22). In rat aortic smooth muscle cells and a murine macrophage cell line, cytokine-induced arginase I expression is dependent on PKA activity (35, 37). Thus the cellular mechanism(s) triggered by hypoxia to increase arginase II expression in hPASMC remain to be elucidated.
In conclusion, we found that arginase II gene and protein expression were increased in hPASMC exposed to hypoxia, resulting in an increase in arginase activity. This was associated with an increase in cellular proliferation. Both pharmacological inhibition of arginase and siRNA targeting the arginase II gene completely prevented hypoxia-induced arginase activity and this hypoxia-induced proliferative response in hPASMC. These data are consistent with the concept that arginase II plays an integral role in the hypoxia-induced proliferation of hPASMC. Furthermore, these studies suggest that arginase inhibition may be a target for preventing the remodeling and smooth muscle cell proliferation seen in pulmonary hypertensive diseases associated with hypoxia including chronic obstructive pulmonary disease and bronchopulmonary dysplasia.
The authors have no conflict of interest to declare for this study.
- Copyright © 2009 the American Physiological Society