Dechun Li

Arginase Reciprocally Regulates Nitric Oxide Synthase Activity and Contributes to Endothelial Dysfunction in Aging Blood Vessels

Background—Although abnormal l-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. l-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating l-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. Methods and Results—Inhibition of arginase with (S)-(2-boronoethyl)-l-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4±9.4% at 10−5 mol/L, P <0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-l-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P <0.05 and P <0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60±6% versus 39±6%, P <0.05). In addition, BEC restored depressed l-arginine (10−4 mol/L)–dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. Conclusions—These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular l-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Xanthine Oxidoreductase Inhibition Causes Reverse Remodeling in Rats With Dilated Cardiomyopathy

Increased reactive oxygen species (ROS) generation is implicated in cardiac remodeling in heart failure (HF). As xanthine oxidoreductase (XOR) is 1 of the major sources of ROS, we tested whether XOR inhibition could improve cardiac performance and induce reverse remodeling in a model of established HF, the spontaneously hypertensive/HF (SHHF) rat. We randomized Wistar Kyoto (WKY, controls, 18 to 21 months) and SHHF (19 to 21 months) rats to oxypurinol (1 mmol/L; n=4 and n=15, respectively) or placebo (n=3 and n=10, respectively) orally for 4 weeks. At baseline, SHHF rats had decreased fractional shortening (FS) (31±3% versus 67±3% in WKY, P<0.0001) and increased left-ventricular (LV) end-diastolic dimension (9.7±0.2 mm versus 7.0±0.4 mm in WKY, P<0.0001). Whereas placebo and oxypurinol did not change cardiac architecture in WKY, oxypurinol attenuated decreased FS and elevated LV end-diastolic dimension, LV end-systolic dimension, and LV mass in SHHF. Increased myocyte width in SHHF was reduced by oxypurinol. Additionally, fetal gene activation, altered calcium cycling proteins, and upregulated phospho–extracellular signal–regulated kinase were restored toward normal by oxypurinol (P<0.05 versus placebo-SHHF). Importantly, SHHF rats exhibited increased XOR mRNA expression and activity, and oxypurinol treatment reduced XOR activity and superoxide production toward normal, but not expression. On the other hand, NADPH oxidase activity remained unchanged, despite elevated subunit protein abundance in treated and untreated SHHF rats. Together these data demonstrate that chronic XOR inhibition restores cardiac structure and function and offsets alterations in fetal gene expression/Ca2+ handling pathways, supporting the idea that inhibiting XOR-derived oxidative stress substantially improves the HF phenotype.

Knockdown of arginase I restores NO signaling in the vasculature of old rats

Arginase, expressed in endothelial cells and upregulated in aging blood vessels, competes with NO synthase (NOS) for l-arginine, thus modulating vasoreactivity and attenuating NO signaling. Moreover, arginase inhibition restores endothelial NOS signaling and l-arginine responsiveness in old rat aorta. The arginase isoform responsible for modulating NOS, however, remains unknown. Because isoform-specific arginase inhibitors are unavailable, we used an antisense (AS) oligonucleotide approach to knockdown arginase I (Arg I). Western blot and quantitative PCR confirmed that Arg I is the predominant isoform expressed in endothelialized aortic rings and is upregulated in old rats compared with young. Aortic rings from 22-month-old rats were incubated for 24 hours with sense (S), AS oligonucleotides, or medium alone (C). Immunohistochemistry, immunoblotting, and enzyme assay confirmed a significant knockdown of Arg I protein and arginase activity in AS but not S or C rings. Conversely, calcium-dependent NOS activity and vascular metabolites of NO was increased in AS versus S or C rings. Acetylcholine (endothelial-dependent) vasorelaxant responses were enhanced in AS versus S or C treated rings. In addition, 1H-oxadiazolo quinoxalin-1-one (10 &mgr;mol/L), a soluble guanylyl cyclase inhibitor, increased the phenylephrine response in AS compared with S and C rings suggesting increased NO bioavailability. Finally, l-arginine (0.1 mmol/L)-induced relaxation was increased in AS versus C rings. These data support our hypothesis that Arg I plays a critical role in the pathobiology of age-related endothelial dysfunction. AS oligonucleotides may, therefore, represent a novel therapeutic strategy against age-related vascular endothelial dysfunction.

Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium.

Background— Neuronal nitric oxide synthase (NOS1) plays key cardiac physiological roles, regulating excitation-contraction coupling and exerting an antioxidant effect that maintains tissue NO-redox equilibrium. After myocardial infarction (MI), NOS1 translocates from the sarcoplasmic reticulum to the cell membrane, where it inhibits &bgr;-adrenergic contractility, an effect previously predicted to have adverse consequences. Counter to this idea, we tested the hypothesis that NOS1 has a protective effect after MI. Methods and Results— We studied mortality, cardiac remodeling, and upregulation of oxidative stress pathways after MI in NOS1-deficient (NOS1−/−) and wild-type C57BL6 (WT) mice. Compared with WT, NOS1−/− mice had greater mortality (hazard ratio, 2.06; P=0.036), worse left ventricular (LV) fractional shortening (19.7±1.5% versus 27.2±1.5%, P<0.05), higher LV diastolic diameter (5.5±0.2 versus 4.9±0.1 mm, P<0.05), greater residual cellular width (14.9±0.5 versus 12.8±0.5 &mgr;m, P<0.01), and equivalent &bgr;-adrenergic hyporesponsiveness despite similar MI size. Superoxide production increased after MI in both NOS1−/− and WT animals, although NO increased only in WT. NADPH oxidase (P<0.05) activity increased transiently in both groups after MI, but NOS1−/− mice had persistent basal and post-MI elevations in xanthine oxidoreductase activity. Conclusions— Together these findings support a protective role for intact NOS1 activity in the heart after MI, despite a potential contribution to LV dysfunction through &bgr;-adrenergic hyporesponsiveness. NOS1 deficiency contributes to an imbalance between oxidative stress and tissue NO signaling, providing a plausible mechanism for adverse consequences of NOS1 deficiency in states of myocardial injury.

Evidence for dysregulation of dimethylarginine dimethylaminohydrolase I in chronic hypoxia-induced pulmonary hypertension

Background—Chronic hypoxia–induced pulmonary hypertension is associated with increased pulmonary expression of nitric oxide synthase (NOS) enzymes. Nevertheless, some reports have indicated decreased pulmonary production of NO in the disease. To address this paradox, we determined pulmonary concentrations of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) in the hypoxia-induced pulmonary hypertension rat model. In addition, we determined whether dysregulation of the ADMA-metabolizing enzyme dimethylarginine dimethylaminohydrolase I (DDAH I) plays a role in this disease. Methods and Results—Adult male rats were exposed for 1 week to either normoxia or hypoxia (10% oxygen). Lung tissues were used for Western blot analysis of endothelial NOS and DDAH I expression, measurement of lung NO and ADMA content, and in vitro assay of DDAH enzyme activity. Western blot analysis revealed a 1.9-fold increase in endothelial NOS protein and a 37% decrease in DDAH I protein in the lungs of hypoxia-exposed rats. Both pulmonary DDAH enzyme activity and NO content were significantly decreased in the hypoxic group (by 37% and 22%, respectively), but pulmonary ADMA concentrations were increased by 2.3-fold compared with the normoxic group. Conclusions—These data demonstrate that the rat chronic hypoxia–induced pulmonary hypertension model is associated with increased pulmonary concentrations of the NOS inhibitor ADMA. Moreover, pulmonary hypertensive rats exhibit reduced pulmonary expression and activity of the ADMA-metabolizing enzyme DDAH I. The decreased DDAH I and increased ADMA concentrations may therefore contribute to pulmonary hypertension via the competitive inhibition of pulmonary NOS enzymes.

Expression of Endothelial Nitric Oxide Synthase in Ciliated Epithelia of Rats

Endothelial nitric oxide synthase (eNOS), originally found in the endothelium of vascular tissue, also exists in other cell types, including ciliated epithelia of airways. The eNOS is ultrastructurally localized to the basal body of the microtubules of the cilia, and nitric oxide (NO) stimulates ciliary beat frequency (CBF). We examined whether the expression of eNOS is present in ciliated cells of other organs. Western blotting analysis revealed that eNOS was expressed in the rat cerebrum, lung, trachea, testis, and oviduct. Immunohistochemical staining showed that eNOS was localized in the ciliated epithelia of airways, oviduct, testis, and ependymal cells of brain in addition to the endothelium and smooth muscle of the vasculature. To confirm the activation of eNOS in the ciliated epithelia, we examined the effect of l-arginine (L-Arg), the substrate of NOS, on the production of nitrite and nitrate (NOx) in the cultured explants of rat trachea. l-Arg (100 μM) increased NOx levels significantly (p<0.05). In explants exposed to inhibitors of NOS, the effect of l-Arg on the production of NOx was blocked. These findings suggest that epithelial NO plays an important role in signal transduction associated with ciliary functions.

Exogenous Nitric Oxide Upregulates p21waf1/cip1 in Pulmonary Microvascular Smooth Muscle Cells

The histopathology of chronic pulmonary hypertension includes microvascular proliferation and neointimal formation. Nitric oxide (NO) has been implicated in the regulation of these mechanisms, but how NO controls microvascular proliferation and its effect on pulmonary microvascular cells is still unclear. In this study, we characterized the in vitro effects of NO on rat pulmonary microvascular smooth muscle cell (PMVSMC) proliferation and investigated the contribution of the p42/44 mitogen-activated protein kinase (MAPK) pathway and p21waf1/cip1 induction to this response. NO donors inhibited PMVSMC proliferation in a dose-dependent manner. In the presence of hypoxia, the degree of inhibition was significantly enhanced. This inhibition was reversible and independent of apoptosis. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) had no impact on proliferation rates, suggesting a cyclic guanosine monophosphate-independent process. Administration of MEK1/2 inhibitors failed to abrogate the antimitotic effect of NO. There was a two- fold induction of the cyclin-dependent kinase inhibitor p21 in PMVSMC treated with NO donors. Under hypoxic conditions, NO caused a three-fold increase in p21 levels. These results demonstrate that NO inhibits PMVSMC proliferation and that this inhibition is not the result of p42/44 MAPK activation. The ability of NO to induce p21 upregulation may be a mechanism by which it exerts antiproliferative effects in PMVSMC.

Propofol stimulates ciliary motility via the nitric oxide-cyclic GMP pathway in cultured rat tracheal epithelial cells

Background Airway ciliary motility is impaired by inhaled anesthetics. Recent reports show that nitric oxide (NO) induces upregulation in ciliary beat frequency (CBF), and others report that propofol, an intravenous anesthetic, stimulates NO release; this raises the possibility that propofol increases CBF by stimulating the NO–cyclic guanosine monophosphate (cGMP) signal pathway. In this study, the authors investigated the effects of propofol on CBF and its relation with the NO–cGMP pathway using the pharmacologic blockers NG-monomethyl-l-arginine (l-NMMA), an NO synthase inhibitor; 1 H-[1,2,4]oxidazole[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor; and KT5823, a cGMP-dependent protein kinase inhibitor, in cultured rat tracheal epithelial cells. Methods Rat tracheal tissues were explanted and cultured for 3–5 days. Images of ciliated cells were videotaped using a phase-contrast microscope. Baseline CBF and CBF 25 min after exposure to propofol or blocker were measured using video analysis. Results Vehicle (0.1% dimethyl sulfoxide; n = 11) increased CBF by 0.2 ± 1.7% (mean ± SD) from baseline. Propofol stimulated CBF significantly (P < 0.01) and dose dependently (1 &mgr;m, 2.0 ± 1.9%, n = 6; 10 &mgr;m, 8.2 ± 6.7%, n = 9; 100 &mgr;m, 14.0 ± 4.7%, n = 10). Intralipid (0.05%), the clinical vehicle of propofol, did not affect CBF (−0.2 ± 2.2%; n = 5). The enhancement of CBF with use of 100 &mgr;m propofol was abolished (P < 0.01) by coadministration of 10 m&mgr;m l-NMMA (2.4 ± 3.6%; n = 5), 100 &mgr;m ODQ (−0.3 ± 2.2%; n = 6) or 30 &mgr;m KT5823 (−0.1 ± 4.1%; n = 8). l-NMMA, ODQ, or KT5823 alone did not change CBF. Conclusions These results show that propofol stimulates CBF via the NO–cGMP pathway in rat tracheal epithelial cells, suggesting a possible advantage of propofol in decreasing respiratory risk.

Attenuation of hypoxic pulmonary hypertension in rats by the HMG-CoA reductase inhibitor, simvastatin

Pulmonary arterial hypertension (PAH) is characterized by excessive vasoconstriction and vascular wall thickening which is frequently progressive leading to right heart failure and death. Current therapeutic options are limited. The HMG-CoA reductase inhibitors, statins, have been shown to have several beneficial effects in the systemic circulation beyond their cholesterollowering action, including improvements in endothelial cell function and inhibition of vascular smooth muscle cell proliferation. We hypothesized that these actions would attenuate hypoxic pulmonary hypertension in rats. Adult, male Sprague-Dawley rats were exposed to 4 days of 10% FiO2 (H) vs normoxia (N) and treated with pre-activated simvastatin 20 mg/kg/d sq (HS, NS) vs vehicle (N 4 in each group). H had signficantly greater right ventricular/LV septum and lung to body weight ratios and decreased proportion of non-muscularinized vessels 80 microns on smooth muscle alpha-actin stained sections, compared with N (0.36 /-0.005 vs 0.24 /0.006, 7.6 /0.16 mg/gm vs 5.5 /0.12, and 30 /4%vs 62 /2%, respectively; P 0.001 for all). Treatment with simvastatin (HS) was associated with 10% (0.32 /0.003) and 14% (6.5 /0.2 mg/gm) reduction in the first 2 indices and a 20% (49 /1.7%) increase in the latter (P 0.05 for all 3) compared with H. Conclusion: This preliminary data suggests that simvastatin is capable of attenuating hypoxic pulmonary hypertension and may play a role in the treatment of PAH.

In vivo magnetic resonance imaging of catheter-based vascular gene transfer

The purpose of this study was to develop an in vivo imaging tool to monitor vascular gene transfer. We produced gadolinium/blue-dye and gadolinium/gene-vector media by mixing Magnevist with a trypan-blue or a lentiviral vector carrying a green fluorescent protein (GFP) gene. The gadolinium was used as an imaging marker for MRI to visualize vessel wall enhancement, while the blue-dye/GFP was used as a tissue stain marker for histology/immunohistochemistry to confirm the success of the transfer. Using Remedy gene delivery catheters, we transferred the gadolinium/blue-dye (n=8) or gadolinium/GFP-lentivirus (n=4) into the arteries of 12 pigs, monitored. under high-resolution MR imaging. This technical development enabled dynamic visualization of: (i) where the gadolinium/genes distributed; (ii) how satisfactorily the target portion was marked; and (iii) whether the gene transfer procedure caused complications. Our study represents the first direct evidence that catheter-based vascular gene delivery/distribution can be monitored by MR imaging in vivo.