Bernadette Chen

Hypoxia promotes human pulmonary artery smooth muscle cell proliferation through induction of arginase.

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% 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.

Hypoxia-induced proliferation of human pulmonary microvascular endothelial cells depends on epidermal growth factor receptor tyrosine kinase activation.

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.

Mice deficient in Mkp-1 develop more severe pulmonary hypertension and greater lung protein levels of arginase in response to chronic hypoxia

The mitogen-activated protein (MAP) kinases are involved in cellular responses to many stimuli, including hypoxia. MAP kinase signaling is regulated by a family of phosphatases that include MAP kinase phosphatase-1 (MKP-1). We hypothesized that mice lacking the Mkp-1 gene would have exaggerated chronic hypoxia-induced pulmonary hypertension. Wild-type (WT) and Mkp-1(-/-) mice were exposed to either 4 wk of normoxia or hypobaric hypoxia. Following chronic hypoxia, both genotypes demonstrated elevated right ventricular pressures, right ventricular hypertrophy as demonstrated by the ratio of the right ventricle to the left ventricle plus septum weights [RV(LV + S)], and greater vascular remodeling. However, the right ventricular systolic pressures, the RV/(LV + S), and the medial wall thickness of 100- to 300-microm vessels was significantly greater in the Mkp-1(-/-) mice than in the WT mice following 4 wk of hypobaric hypoxia. Chronic hypoxic exposure caused no detectable change in eNOS protein levels in the lungs in either genotype; however, Mkp-1(-/-) mice had lower levels of eNOS protein and lower lung NO production than did WT mice. No iNOS protein was detected in the lungs by Western blotting in any condition in either genotype. Both arginase I and arginase II protein levels were greater in the lungs of hypoxic Mkp-1(-/-) mice than those in hypoxic WT mice. Lung levels of proliferating cell nuclear antigen were greater in hypoxic Mkp-1(-/-) than in hypoxic WT mice. These data are consistent with the concept that MKP-1 acts to restrain hypoxia-induced arginase expression and thereby reduces vascular remodeling and the severity of pulmonary hypertension.

Glutathione Reductase Facilitates Host Defense by Sustaining Phagocytic Oxidative Burst and Promoting the Development of Neutrophil Extracellular Traps

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione, which plays an important role in the bactericidal function of phagocytes. Because Gsr has been implicated in the oxidative burst in human neutrophils and is abundantly expressed in the lymphoid system, we hypothesized that Gsr-deficient mice would exhibit marked defects during the immune response against bacterial challenge. We report in this study that Gsr-null mice exhibited enhanced susceptibility to Escherichia coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Additionally, Gsr-null mice exhibited elevated sensitivity to Staphylococcus aureus. Examination of the bactericidal functions of the neutrophils from Gsr-deficient mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-deficient neutrophils paradoxically produced far less reactive oxygen species upon activation both ex vivo and in vivo. Unlike wild-type neutrophils that exhibited a sustained oxidative burst upon stimulation with phorbol ester and fMLP, Gsr-deficient neutrophils displayed a very transient oxidative burst that abruptly ceased shortly after stimulation. Likewise, Gsr-deficient neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Biochemical analysis revealed that the hexose monophosphate shunt was compromised in Gsr-deficient neutrophils. Moreover, Gsr-deficient neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism that operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.

Pharmacologic agents elevating cAMP prevent arginase II expression and proliferation of pulmonary artery smooth muscle cells.

Arginase II has been shown to be involved in the hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (hPASMCs). The signal transduction pathways responsible for the induction of arginase II are poorly understood. Cyclic AMP is involved in many intracellular processes, and cAMP levels are regulated by a balance between production via adenylate cyclases and degradation via phosphodiesterases. The purpose of this study was to determine the effects of cAMP on hypoxia-induced arginase expression, activity, and proliferation in hPASMCs. We found that the cAMP analog 8-Bromo-cAMP (8-Br-cAMP), the adenylate cyclase activator forskolin, and the phosphodiesterase 3 inhibitor cilostamide prevented the hypoxic induction of arginase II mRNA and protein expression in hPASMCs. The inhibition of arginase II protein was found to be mediated by exchange protein directly activated by cAMP. Arginase activity was decreased by 8-Br-cAMP, as evidenced by significantly lower V(max) for arginase in normoxia and hypoxia. The hypoxia-induced hPASMC proliferation was completely prevented by the addition of 8-Br-cAMP, forskolin, or cilostamide. These data are the first to describe the inhibitory effect of cAMP on arginase activity, expression, and resultant proliferation of hypoxic hPASMCs.

Overexpression of cationic amino acid transporter-1 increases nitric oxide production in hypoxic human pulmonary microvascular endothelial cells

1. The endogenous production of and/or the bioavailability of nitric oxide (NO) is decreased in pulmonary hypertensive diseases. l‐arginine (l‐arg) is the substrate for NO synthase (NOS). l‐arg is transported into cells via the cationic amino acid transporters (CAT), of which there are two isoforms in endothelial cells, CAT‐1 and CAT‐2.

Hypoxia induces arginase II expression and increases viable human pulmonary artery smooth muscle cell numbers via AMPKα1 signaling

Pulmonary artery smooth muscle cell (PASMC) proliferation is one of the hallmark features of hypoxia-induced pulmonary hypertension. With only supportive treatment options available for this life-threatening disease, treating and preventing the proliferation of PASMCs is a viable therapeutic option. A key promoter of hypoxia-induced increases in the number of viable human PASMCs is arginase II, with attenuation of viable cell numbers following pharmacologic inhibition or siRNA knockdown of the enzyme. Additionally, increased levels of arginase have been demonstrated in the pulmonary vasculature of patients with pulmonary hypertension. The signaling pathways responsible for the hypoxic induction of arginase II in PASMCs, however, remain unknown. Hypoxia is a recognized activator of AMPK, which is known to be expressed in human PASMCs (hPASMCs). Activation of AMPK by hypoxia has been shown to promote cell survival in PASMCs. In addition, pharmacologic agents targeting AMPK have been shown to attenuate chronic hypoxia-induced pulmonary hypertension in animal models. The present studies tested the hypothesis that hypoxia-induced arginase II expression in hPASMCs is mediated through AMPK signaling. We found that pharmacologic inhibitors of AMPK, as well as siRNA knockdown of AMPKα1, prevented hypoxia-induced arginase II. The hypoxia-induced increase in viable hPASMC numbers was also prevented following both pharmacologic inhibition and siRNA knockdown of AMPK. Furthermore, we demonstrate that overexpression of AMPK induced arginase II protein expression and viable cells numbers in hPASMCs.

The Src family tyrosine kinases src and yes have differential effects on inflammation-induced apoptosis in human pulmonary microvascular endothelial cells

Endothelial cells are essential for normal lung function: they sense and respond to circulating factors and hemodynamic alterations. In inflammatory lung diseases such as acute respiratory distress syndrome, endothelial cell apoptosis is an inciting event in pathogenesis and a prominent pathological feature. Endothelial cell apoptosis is mediated by circulating inflammatory factors, which bind to receptors on the cell surface, activating signal transduction pathways, leading to caspase-3-mediated apoptosis. We hypothesized that yes and src have differential effects on caspase-3 activation in human pulmonary microvascular endothelial cells (hPMVEC) due to differential downstream signaling effects. To test this hypothesis, hPMVEC were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls. After recovery, the hPMVEC were treated with cytomix (LPS, IL-1β, TNF-α, and IFN-γ). Treatment with cytomix induced activation of the extracellular signal-regulated kinase (ERK) pathway and caspase-3-mediated apoptosis. Treatment with siRNAsrc blunted cytomix-induced ERK activation and enhanced cleaved caspase-3 levels, while treatment with siRNAyes enhanced cytomix-induced ERK activation and attenuated levels of cleaved caspase-3. Inhibition of the ERK pathway using U0126 enhanced cytomix-induced caspase-3 activity. Treatment of hPMVEC with cytomix induced Akt activation, which was inhibited by siRNAsrc. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway using LY294002 prevented cytomix-induced ERK activation and augmented cytomix-induced caspase-3 cleavage. Together, our data demonstrate that, in hPMVEC, yes activation blunts the ERK cascade in response to cytomix, resulting in greater apoptosis, while cytomix-induced src activation induces the phosphatidylinositol 3-kinase pathway, which leads to activation of Akt and ERK and attenuation of apoptosis.

Hypoxic proliferation requires EGFR-mediated ERK activation in human pulmonary microvascular endothelial cells

We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation.