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, 46.4Ϯ9.4% at 10 Ϫ5 mol/L, PϽ0.01). 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 (PϽ0.05 and PϽ0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60Ϯ6% versus 39Ϯ6%, PϽ0.05). In addition, BEC restored depressed L-arginine (10 Ϫ4 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.

In a mouse chronic hypoxia model of pulmonary hypertension, we discovered a novel hypoxia-inducible gene in lung, FIZZ1/RELM␣, first through a cDNA array analysis and then confirmed by RT-PCR. Western blot and immunohistochemistry revealed that its expression was induced by hypoxia only in lung. The hypoxia-upregulated gene expression was located in the pulmonary vasculature, bronchial epithelial cells, and type II pneumocytes. 3H-thymidine incorporation demonstrated that the recombinant protein stimulated rat pulmonary microvascular smooth muscle cell (RPSM) proliferation dose-dependently ranging from 3.3؋10؊9 to 3.3؋10 ؊8 mol/L. Therefore, we renamed this gene as hypoxia-induced mitogenic factor (HIMF). HIMF strongly activated Akt phosphorylation. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (10 mol/L) inhibited HIMF-activated Akt phosphorylation. It also inhibited HIMF-stimulated RPSM proliferation. Thus, the PI3K/Akt pathway, at least in part, mediates the proliferative effect of HIMF. Further studies showed that HIMF had angiogenic and vasoconstrictive properties. HIMF increased pulmonary arterial pressure and vascular resistance more potently than either endothelin-1 or angiotensin II. P ulmonary vascular remodeling, characterized by pulmonary microvascular smooth muscle cell proliferation, is implicated in the development of hypoxic pulmonary arterial hypertension (PAH). To search for the genes that may participate in the pulmonary remodeling, a cDNA microarray analysis (Incyte Genomics; 9415 genes) was performed using lung samples from mice exposed to 10% O 2 or room air for 4 days. EST AA712003 was found to be induced by hypoxia.A literature search revealed that EST AA712003 had just been reported as FIZZ1 (found in inflammatory zone 1), a protein induced in murine lung in an ovalbumin-induced asthma model. 1 Besides its induction in the bronchial mucosal epithelial cells, FIZZ1 was also induced in type II pneumocytes and it inhibited NGF-induced survival of DRG neurons and NGF-mediated increase in neuronal CGRP content. 1 Holcomb et al also reported that FIZZ1 is a secreted protein sharing the consensus sequence of 10 cysteine residues in the C-terminus ( C 10 C) with two other murine genes expressed respectively in intestinal crypt epithelial (FIZZ2) and white adipose tissue (FIZZ3) and two related human genes (human FIZZ1 and human FIZZ3). 1 Later, FIZZ3 was shown to be implicated in type II diabetes mellitus and was renamed as resistin. 2 FIZZ1 and FIZZ2 were renamed as resistin-like molecule ␣ (RELM␣) and ␤ (RELM␤), respectively. 3 Human FIZZ1, however, was renamed as human RELM␤. 3 Recently, FIZZ1 was found in macrophages 4 and in the stromal vascular fraction of adipose tissue, 5 and it inhibited adipocyte differentiation. 6 However, the function of FIZZ1 remains unclear.We hypothesized that FIZZ1 participates in the process of hypoxia-induced pulmonary remodeling. FIZZ1 could be induced at or near the pulmonary vasculature by hypoxia, and the secreted FIZZ1 might have a prolif...

Although interactions between superoxide (O 2•؊ ) and nitric oxide underlie many physiologic and pathophysiologic processes, regulation of this crosstalk at the enzymatic level is poorly understood. Here, we demonstrate that xanthine oxidoreductase (XOR), a prototypic superoxide O 2•؊ -producing enzyme, and neuronal nitric oxide synthase (NOS1) coimmunoprecipitate and colocalize in the sarcoplasmic reticulum of cardiac myocytes. Deficiency of NOS1 (but not endothelial NOS, NOS3) leads to profound increases in XOR-mediated O 2•؊ production, which in turn depresses myocardial excitation-contraction coupling in a manner reversible by XOR inhibition with allopurinol. These data demonstrate a unique interaction between a nitric oxide and an O 2•؊ -generating enzyme that accounts for crosstalk between these signaling pathways; these findings demonstrate a direct antioxidant mechanism for NOS1 and have pathophysiologic implications for the growing number of disease states in which increased XOR activity plays a role.

Challenge of the airways of sensitized guinea pigs with aerosolized ovalbumin resulted in an early phase of microvascular protein leakage and a delayed phase of eosinophil accumulation in the airway lumen, as measured using bronchoalveolar lavage (BAL). Immunoreactive eotaxin levels rose in airway tissue and BAL fluid to a peak at 6 h falling to low levels by 12 h. Eosinophil numbers in the tissue correlated with eotaxin levels until 6 h but eosinophils persisted until the last measurement time point at 24 h. In contrast, few eosinophils appeared in BAL over the first 12 h, major trafficking through the airway epithelium occurring at 12–24 h when eotaxin levels were low. Constitutive eotaxin was present in BAL fluid. Both constitutive and allergen-induced eosinophil chemoattractant activity in BAL fluid was neutralized by an antibody to eotaxin. Allergen-induced eotaxin appeared to be mainly in airway epithelium and macrophages, as detected by immunostaining. Allergen challenge of the lung resulted in a rapid release of bone marrow eosinophils into the blood. An antibody to IL-5 suppressed bone marrow eosinophil release and lung eosinophilia, without affecting lung eotaxin levels. Thus, IL-5 and eotaxin appear to cooperate in mediating a rapid transfer of eosinophils from the bone marrow to the lung in response to allergen challenge.

Abstract-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 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.

. Attenuation of chronic hypoxic pulmonary hypertension by simvastatin. Am J Physiol Heart Circ Physiol 285: H938-H945, 2003. First published May 15, 2003 10.1152/ ajpheart.01097.2002The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to improve multiple normal endothelial cell functions and inhibit vascular wall cell proliferation. We hypothesized that one such agent, simvastatin, would attenuate chronic hypoxic pulmonary hypertension. Male adult Sprague-Dawley rats were exposed (14 days) to normoxia (N), normoxia plus once-a-day administered simvastatin (20 mg/kg ip) (NS), hypoxia (10% inspired O 2 fraction) (H), or hypoxia plus simvastatin (HS). Mean pulmonary artery pressure, measured in anesthetized, ventilated rats with an open-chest method, was reduced from 25 Ϯ 2 mmHg in H to 18 Ϯ 1 in HS (P Ͻ 0.001) but did not reach normoxic values (12 Ϯ 1 mmHg). Similarly, right ventricular/left ventricular plus interventricular septal weight was reduced from 0.53 Ϯ 0.02 in the H group to 0.36 Ϯ 0.02 in the HS group (P Ͻ 0.001). The increased hematocrit in H (0.65 Ϯ 0.02) was prevented by simvastatin treatment (0.51 Ϯ 0.01, P Ͻ 0.001). Hematocrit was similar in N versus NS. Alveolar vessel muscularization and medial thickening of vessels 50-200 M in diameter induced by hypoxia were also significantly attenuated in the HS animals. Lung endothelial nitric oxide synthase (eNOS) protein expression in the HS group was less than H (P Ͻ 0.01) but was similar in N versus NS. We conclude that simvastatin treatment potently attenuates chronic hypoxic pulmonary hypertension and polycythemia in rats and inhibits vascular remodeling. Enhancement of lung eNOS expression does not appear to be involved in mediating this effect. pulmonary vascular remodeling; 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition; nitric oxide; polycythemia; small G proteins CHRONIC PULMONARY HYPERTENSION is characterized by a component of abnormal pulmonary vasoconstriction and by structural remodeling of the small pulmonary arteries. Both processes lead to a progressive increase in pulmonary vascular resistance, which, when extreme, culminates in right ventricular (RV) failure and death. The syndrome occurs in diverse clinical settings, including lung disease associated with alveolar hypoxia. Endothelial cell injury-dysfunction is considered to be a key factor in the pathogenesis of pulmonary hypertension (3), leading to increased vascular smooth muscle tone, cell proliferation in the vascular wall, and activation of thrombotic mechanisms, all of which participate in the process of remodeling. Currently available therapies have a beneficial clinical effect yet cannot reverse the disease process. Therefore, the response is variable with considerable morbidity and mortality despite therapy (33). Clearly, there is an urgent need for new therapies.The 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitors (statins) have been shown to exert numerous effects on vascular wall function, independent of t...

Obesity is a major health care problem and is associated with significant cardiovascular morbidity. Leptin, a neuroendocrine hormone released by adipose tissue, is important in modulating obesity by signaling satiety and increasing metabolism. Moreover, leptin receptors are expressed on vascular endothelial cells (ECs) and mediate angiogenesis. We hypothesized that leptin may also play an important role in vasoregulation. We investigated vasoregulatory mechanisms in the leptin-deficient obese (ob/ob) mouse model and determined the influence of leptin replacement on endothelial-dependent vasorelaxant responses. The direct effect of leptin on EC nitric oxide (NO) production was also tested by using 4, 5-diaminofluorescein-2 diacetate staining and measurement of nitrate and nitrite concentrations. Vasoconstrictor responses to phenylephrine, norepinephrine, and U-46619 were markedly enhanced in aortic rings from ob/ob mice and were modulated by NO synthase inhibition. Vasorelaxant responses to ACh were markedly attenuated in mesenteric microvessels from ob/ob mice. Leptin replacement resulted in significant weight loss and reversal of the impaired endothelial-dependent vasorelaxant responses observed in ob/ob mice. Preincubation of ECs with leptin enhanced the release of NO production. Thus leptin-deficient ob/ob mice demonstrate marked abnormalities in vasoregulation, including impaired endothelial-dependent vasodilation, which is reversed by leptin replacement. These findings may be partially explained by the direct effect of leptin on endothelial NO production. These vascular abnormalities are similar to those observed in obese, diabetic, leptin-resistant humans. The ob/ob mouse may, therefore, be an excellent new model for the study of the cardiovascular effects of obesity.

Abstract-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. Key Words: xanthine oxidoreductase Ⅲ remodeling Ⅲ gene expression Ⅲ heart failure E merging data implicates oxidative stress (OS) in heart failure (HF) pathophysiology, contributing to cardiac remodeling, 1,2 mechanoenergetic uncoupling, 3,4 and depressed myofilament calcium sensitivity. 5,6 The major enzymatic sources of reactive oxygen species (ROS) in HF are xanthine oxidoreductase (XOR) 7 and nicotinamide adenine dinucleotide 2Ј-phosphate (NADPH) oxidase. 8 Several studies demonstrate XOR upregulation in animal models 4 -7,9,10 and in human dilated cardiomyopathy. 3,11 Functionally, XOR inhibition (XOI) acutely enhances myocardial mechanical efficiency in both animals and humans with HF. 3,4 However, whereas NADPH oxidase is implicated in ␣ 1 -adrenoreceptor stimulated hypertrophic signaling 12 and contributes to OS in reperfused hearts, playing a major role in post-myocardial infarction (MI) microvascular obstruction ("no-reflow" phenomenon) 13 and, like XOR, is increased in human HF, 8 the relative contribution of XOR and NADPH oxidase to HF pathophysiology requires further clarification.A recent series of studies has begun to examine the role of XOR in the cardiac remodeling process. 2,6,14 These data contribute to the growing argument implicating XOR as a key source of ROS in evolving HF. Whether inhibition of XOR can elicit reverse remodeling in established dilated cardiomyopathy remains unknown.Here we tested the hypothesis that cardiac XOR adversely affects cardiac remodeling in established cardiomyopathy in spontaneously hypertensive/HF (SHHF) rats. We show that chronic XOI reverses maladaptive cardiac remodeling through effects on cardiac structure, function, and fetal gene activation in SHHF rats, and that this process occurs independently of NADPH oxidase.