Impaired nitric oxide-mediated vasodilation in transgenic sickle mouse

Transgenic sickle mice expressing human beta(S)- and beta(S-Antilles)-globins show intravascular sickling, red blood cell adhesion, and attenuated arteriolar constriction in response to oxygen. We hypothesize that these abnormalities and the likely endothelial damage, also reported in sickle cell anemia, alter nitric oxide (NO)-mediated microvascular responses and hemodynamics in this mouse model. Transgenic mice showed a lower mean arterial pressure (MAP) compared with control groups (90 +/- 7 vs. 113 +/- 8 mmHg, P < 0.00001), accompanied by increased endothelial nitric oxide synthase (eNOS) expression. N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of NOS, caused an approximately 30% increase in MAP and approximately 40% decrease in the diameters of cremaster muscle arterioles (branching orders: A2 and A3) in both control and transgenic mice, confirming NOS activity; these changes were reversible after L-arginine administration. Aminoguanidine, an inhibitor of inducible NOS, had no effect. Transgenic mice showed a decreased (P < 0.02-0.01) arteriolar dilation in response to NO-mediated vasodilators, i.e., ACh and sodium nitroprusside (SNP). Indomethacin did not alter the responses to ACh and SNP. Forskolin, a cAMP-activating agent, caused a comparable dilation of A2 and A3 vessels ( approximately 44 and 70%) in both groups of mice. Thus in transgenic mice, an increased eNOS/NO activity results in lower blood pressure and diminished arteriolar responses to NO-mediated vasodilators. Although the increased NOS/NO activity may compensate for flow abnormalities, it may also cause pathophysiological alterations in vascular tone.     

Protective effect of arginine on oxidative stress in transgenic sickle mouse models

Sickle cell disease (SCD) is characterized by reperfusion injury and chronic oxidative stress. Oxidative stress and hemolysis in SCD result in inactivation of nitric oxide (NO) and depleted arginine levels. We hypothesized that augmenting NO production by arginine supplementation will reduce oxidative stress in SCD. To this end, we measured the effect of arginine (5% in mouse chow) on NO metabolites (NOx), lipid peroxidation (LPO), and selected antioxidants in transgenic sickle mouse models. Untreated transgenic sickle (NY1DD) mice (expressing  75% β^S-globin of all β-globins; mild pathology) and knockout sickle (BERK) mice (expressing exclusively hemoglobin S; severe pathology) showed reduced NOx levels and significant increases in the liver LPO compared with C57BL mice, with BERK mice showing maximal LPO increase in accordance with the disease severity. This was accompanied by reduced activity of antioxidants (glutathione, total superoxide dismutase, catalase, and glutathione peroxidase). However, GSH levels in BERK were higher than in NY1DD mice, indicating a protective response to greater oxidative stress. Importantly, dietary arginine significantly increased NOx levels, reduced LPO, and increased antioxidants in both sickle mouse models. In contrast, nitro-L-arginine methylester, a potent nonselective NOS inhibitor, worsened the oxidative stress in NY1DD mice. Thus, the attenuating effect of arginine on oxidative stress in SCD mice suggests its potential application in the management of this disease.     

Antihypertensive effect of quercetin in rats fed with a high-fat high-sucrose diet

The effects of different levels of quercetin on the blood pressure were studied in 6-week-old male Sprague-Dawley rats. The rats were fed with a control diet or a high-fat high-sucrose (HFS) diet containing 0, 0.02, 0.07, 0.2, or 0.5% quercetin for 4 weeks. The systolic blood pressure and the lipid peroxides in the plasma were both higher in the rats fed with the HFS diet without quercetin than in the rats fed with the control diet. The nitric oxide synthase (NOS) activity in the vascular tissues and nitric oxide (NO) metabolites in the plasma and urine were both lower in these rats. A distinct depression of the increase in blood pressure was found in the rats fed with the HFS diets containing quercetin. Each level of quercetin examined was effective, the 0.5% level being much more effective than other levels. Dietary quercetin decreased lipid peroxidation in the plasma of the rats fed with the HFS diets. Quercetin also suppressed the decrease in NO metabolites in the plasma and urine, and the NOS activity in the vascular tissues of these rats. These results suggest that the increased NO availability caused by the elevated NOS activity, and the antioxidative activity in these rats fed with quercetin may be sources of the antihypertensive effect of quercetin.     

Mechanism of acrolein-induced vascular toxicity.

Acrolein, an environmental pollutant and a lipid peroxidation product, is implicated in vascular pathogenesis. Although evidence indicates a link between vascular pathogenesis and acrolein, no direct studies relating to effects of acrolein on vascular function and responses are known. This study investigated the effects of acrolein on vascular function to understand the underlying mechanism of acrolein-induced vascular responses. Male Sprague-Dawley rats were treated with acrolein (2 or 4 mg/kg; i.p.) for 3 or 7 days. Urine and blood samples were collected. Changes in systolic blood pressure (SBP) and responses to acetylcholine and phenylephrine were determined. Acrolein (4 mg/kg, 7 days) significantly increased SBP by 25%, phenylephrine vasoconstriction by 2-fold, but decreased urinary excretion of nitrite by 25%. Acrolein inhibited generation of cyclic guanosine 3'5'-monophosphate (cGMP) by 98%, and did not alter expression of nitric oxide synthase (eNOS). Acrolein increased the generation of lipid hydroperoxide in plasma and aortic tissue by 21% and 124% respectively, increased glutathione-S-transferase (GST) and glutathione peroxidase (GSH-Px) activities. Acrolein up-regulated the expression of GST by 2 fold. These data suggest that induced SBP and altered vasoconstriction/vasodilatation in acrolein treated rats may be due to reduced availability of NO via increased free radical generation and reduced antioxidant defense.     

Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction

Superoxide, which can limit nitric oxide bioavailability, has been implicated in blood cell-vessel wall interactions observed in sickle cell transgenic (beta(S)) mice. Here we report that nonselective chemical inhibition of nitric oxide synthase isoforms dramatically reduces the enhanced leukocyte and platelet adhesion normally observed in cerebral venules of beta(S) mice. Although genetic deficiency of vascular wall inducible nitric oxide synthase does not alter adhesion responses in beta(S) mice, a significant attenuation is noted in beta(S) mice with vascular wall endothelial nitric oxide synthase (eNOS) deficiency, while the adhesion responses are exacerbated when eNOS is overexpressed in microvessels. The eNOS-mediated enhancement of blood cell adhesion is reversible by pretreatment with sepiapterin (which generates the eNOS cofactor tetrahydrobiopterin) or polyethyleneglycol-superoxide dismutase, implicating a role for eNOS-dependent superoxide production. These findings suggest that an imbalance between eNOS-derived nitric oxide and superoxide, both generated by the vessel wall, is critical to the proinflammatory and prothrombogenic phenotype that is assumed by the microvasculature in sickle cell disease.     

Exercise conditioning attenuates the hypertensive effects of nitric oxide synthase inhibitor in rat

Many individuals with cardiovascular diseases undergo periodic exercise conditioning with or with out medication. Therefore, this study investigated the interaction of exercise training and chronic nitric oxide synthase (NOS) inhibitor (Nitro-L-Arginine Methyl Ester, L-NAME) treatment on blood pressure and its correlation with aortic nitric oxide (NO), antioxidant defense system and oxidative stress parameters in rats. Fisher 344 rats were divided into four groups: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10 mg/kg, subcutaneous for 8 weeks) and (4) ET + L-NAME. Blood pressure (BP) was monitored weekly for 8 weeks with tail-cuff method. The animals were sacrificed 24 h after last treatments and thoracic aortic rings were isolated and analyzed. Exercise conditioning resulted in a significant increase in respiratory exchange ratio (RER), aortic NO production, NO synthase activity and inducible iNOS protein expression. Training significantly enhanced aortic GSH levels, GSH/GSSG ratio and up-regulation of aortic CuZn-SOD, Mn-SOD, catalase (CAT) glutathione peroxidase (GSH-Px) activity and protein expression and significantly decreased aortic lipid peroxidation. Chronic L-NAME administration resulted in a significant depletion of aortic NO, NOS activity, endothelial (eNOS) and iNOS protein expression, GSH level, GSH/GSSG ratio, down-regulation of aortic antioxidant enzyme activities and protein expressions. Aortic xanthine oxidase (XO) activity significantly increased with increased lipid peroxidation and protein oxidation after L-NAME administration. The biochemical changes were accompanied by increased in BP. Interaction of training and chronic NOS inhibitor treatment resulted in normalization of BP and aortic antioxidant enzyme activity and protein expression, up-regulation of aortic GSH/GSSG ratio, NO levels, Mn-SOD protein expression, depletion of GSSG, protein oxidation and lipid peroxidation. The data suggest that training attenuated the oxidative injury caused by chronic NOS inhibitor treatment by up-regulating the NO and antioxidant systems and lowering the BP in rats.     

Anti-inflammatory therapy ameliorates leukocyte adhesion and microvascular flow abnormalities in transgenic sickle mice

In sickle cell disease, inflammatory activation of vascular endothelium and increased leukocyte-endothelium interaction may play an important role in the occurrence of vasoocclusion. In sickle mouse models, inflammatory stimuli (e.g., hypoxia-reoxygenation and cytokines) result in increased leukocyte recruitment and can initiate vasoocclusion, suggesting that anti-inflammatory therapy could be beneficial in management of this disease. We have tested the hypothesis that inhibition of endothelial activation in a transgenic mouse model by anti-inflammatory agents would lead to reduced leukocyte recruitment and improved microvascular blood flow in vivo. In transgenic sickle mice, hypoxia-reoxygenation resulted in greater endothelial oxidant production than in control mice. This exaggerated inflammatory response in transgenic mice, characterized by increased leukocyte recruitment and microvascular flow abnormalities, was significantly attenuated by antioxidants (allopurinol, SOD, and catalase). In contrast, control mice exhibited a muted response to antioxidant treatment. In addition, hypoxia-reoxygenation induced activation of NF-kappaB in transgenic sickle mice but not in control mice. In transgenic sickle mice, sulfasalazine, an inhibitor of NF-kappaB activation and endothelial activation, attenuated endothelial oxidant generation, as well as NF-kappaB activation, accompanied by a marked decrease in leukocyte adhesion and improved microvascular blood flow. Thus targeting oxidant generation and/or NF-kappaB activation may constitute promising therapeutic approaches in sickle cell disease.     

Lack of nitric oxide synthases increases lipoprotein immune complex deposition in the aorta and elevates plasma sphingolipid levels in lupus

Systemic lupus erythematosus (SLE) patients display impaired endothelial nitric oxide synthase (eNOS) function required for normal vasodilatation. SLE patients express increased compensatory activity of inducible nitric oxide synthase (iNOS) generating excess nitric oxide that may result in inflammation. We examined the effects of genetic deletion of NOS2 and NOS3, encoding iNOS and eNOS respectively, on accelerated vascular disease in MRL/lpr lupus mouse model. NOS2 and NOS3 knockout (KO) MRL/lpr mice had higher plasma levels of triglycerides (23% and 35%, respectively), ceramide (45% and 21%, respectively), and sphingosine 1-phosphate (S1P) (21%) compared to counterpart MRL/lpr controls. Plasma levels of the anti-inflammatory cytokine interleukin 10 (IL-10) in NOS2 and NOS3 KO MRL/lpr mice were lower (53% and 80%, respectively) than counterpart controls. Nodule-like lesions in the adventitia were detected in aortas from both NOS2 and NOS3 KO MRL/lpr mice. Immunohistochemical evaluation of the lesions revealed activated endothelial cells and lipid-laden macrophages (foam cells), elevated sphingosine kinase 1 expression, and oxidized low-density lipoprotein immune complexes (oxLDL-IC). The findings suggest that advanced vascular disease in NOS2 and NOS3 KO MRL/lpr mice maybe mediated by increased plasma triglycerides, ceramide and S1P; decreased plasma IL-10; and accumulation of oxLDL-IC in the vessel wall. The results expose possible new targets to mitigate lupus-associated complications.     

Regulation of vascular tone in animals overexpressing the sarcolemmal calcium pump

The mechanisms governing vascular smooth muscle tone are incompletely understood. In particular, the role of the sarcolemmal calcium pump PMCA (plasma membrane calmodulin-dependent calcium ATPase), which extrudes Ca2+ from the cytosol, and its importance compared with the sodium/calcium exchanger remain speculative. To test whether the PMCA is a regulator of vascular tone, we generated transgenic mice overexpressing the human PMCA4b under control of the arterial smooth muscle-specific SM22alpha promoter. This resulted in an elevated systolic blood pressure compared with littermate controls. In PMCA-overexpressing mice, endothelium-dependent relaxation of norepinephrine-preconstricted aortic rings to acetylcholine did not differ from wild type controls (76 +/- 8% versus 79 +/- 8% of maximum relaxation; n = 12, n.s.). De-endothelialized aortas of transgenic mice exhibited stronger maximum contraction to KCl (100 mmol/liter) compared with controls (86 +/- 6% versus 68 +/- 7% of reference KCl contraction at the beginning of the experiment; p <0.05). Preincubation of de-endothelialized vessels with the nitric oxide synthase (NOS) inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) (10-5 mol/liter) resulted in a stronger contraction to KCl (p <0.05 versus without l-NAME), thus unmasking vasodilatory effects of inherent NO production. Maximum contraction to KCl after preincubation with l-NAME did not differ between PMCA mice and controls. In analogy to the results in PMCA-overexpressing mice, contractions of de-endothelialized aortas of neuronal NOS-deficient mice to KCl were significantly increased compared with controls (151 +/- 5% versus 131 +/- 6% of reference KCl contraction; p <0.05). In conclusion, our data suggest a model in which the sarcolemmal Ca2+ pump down-regulates activity of the vascular smooth muscle Ca2+/calmodulin-dependent neuronal NOS by a functionally relevant interaction. Therefore, the PMCA represents a novel regulator of vascular tone.     

Overexpression of arginase in the aged mouse penis impairs erectile function and decreases eNOS activity: influence of in vivo gene therapy of anti-arginase

Since both increased nitric oxide (NO) synthase (NOS) abundance and diminished NO signaling have been reported in the aging penis, the role of NO in the adaptations of aging remains controversial. Here we tested the hypothesis that arginase, an enzyme that competes with NOS for the substrate l-arginine, contributes to erectile dysfunction with advanced age in the B6/129 mouse strain. Arginase protein abundance, mRNA expression, and enzyme activity were elevated in aged compared with young penile endothelial cells. In addition, endothelial NOS (NOS3) protein abundance was greater in aged versus young penile endothelial cells, whereas NOS activity and cGMP levels were reduced. Calcium-dependent l-arginine-to-l-citrulline conversion and cGMP formation increased significantly in aged mouse penes in the presence of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). However, there was no effect on l-arginine-to-l-citrulline conversion or cGMP accumulation in the endothelium from young mouse penes. To assess the functional role of arginase in the inhibition of NOS pathway responsiveness in the penis, we evaluated the effects of ABH and an adeno-associated virus encoding an antisense sequence to arginase I (AAVanti-arginase) on erectile function in vivo. ABH and AAVanti-arginase enhanced endothelium-dependent erectile responses in the aged mice without altering endothelium-independent responses. Paralleling our in vitro observations, ABH or AAVanti-arginase did not affect vascular responses in the young mice. Inhibition of the arginase pathway improves endothelial function in the aging penile circulation, suggesting that the arginase pathway may be exploited to improve erectile dysfunction associated with aging.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury

C-reactive protein (CRP) is a risk marker and a potential modulator of vascular disease. Whether CRP modulates nitric oxide (NO) synthase (NOS) activity and NO metabolism remains unclear. We studied the effect of CRP on NO metabolism in transgenic mice that express human CRP (CRPtg). CRPtg and wild-type mice were subjected to controlled femoral artery wire injury. CRP serum levels at baseline and 6 and 24 h after injury were 12.4 +/- 9, 18.6 +/- 6.9, and 58.4 +/- 13 mg/l, respectively, in CRPtg mice but were undetectable at all time points in wild-type mice. Endothelial NOS protein and mRNA expression were significantly suppressed in the injured arteries of CRPtg mice (n = 5, P < 0.05). A similar reduction in eNOS expression was observed in the distant lung and heart. NO release after injury was significantly lower in CRPtg mice, as measured by nitrate and nitrite breakdown products, with a concomitant suppression of cGMP NO signaling after injury. Endothelial NOS and NO expression after vascular injury are locally and systemically suppressed in mice that express human CRP. These in vivo observations support the hypothesis that CRP modulates NO metabolism and may have implications regarding the mechanisms by which CRP modulates vascular disease.     

Divergent contractile and structural responses of the murine PKC-epsilon null pulmonary circulation to chronic hypoxia

Loss of PKC-epsilon limits the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) in the mouse. Therefore, we hypothesized that loss of PKC-epsilon would decrease the contractile and/or structural response of the murine pulmonary circulation to chronic hypoxia (Hx). However, the pattern of lung vascular responses to chronic Hx may or may not be predicted by the acute HPV response. Adult PKC-epsilon wild-type (PKC-epsilon(+/+)), heterozygous null, and homozygous null (PKC-epsilon(-/-)) mice were exposed to normoxia or Hx for 5 wk. PKC-epsilon(-/-) mice actually had a greater increase in right ventricular (RV) systolic pressure, RV mass, and hematocrit in response to chronic Hx than PKC-epsilon(+/+) mice. In contrast to the augmented PA pressure and RV hypertrophy, pulmonary vascular remodeling was increased less than expected (i.e., equal to PKC-epsilon(+/+) mice) in both the proximal and distal PKC-epsilon(-/-) pulmonary vasculature. The contribution of increased vascular tone to this pulmonary hypertension (PHTN) was assessed by measuring the acute vasodilator response to nitric oxide (NO). Acute inhalation of NO reversed the increased PA pressure in hypoxic PKC-epsilon(-/-) mice, implying that the exaggerated PHTN may be due to a relative deficiency in nitric oxide synthase (NOS). Despite the higher PA pressure, chronic Hx stimulated less of an increase in lung endothelial (e) and inducible (i) NOS expression in PKC-epsilon(-/-) than PKC-epsilon(+/+) mice. In contrast, expression of nNOS in PKC-epsilon(+/+) mice decreased in response to chronic Hx, while lung levels in PKC-epsilon(-/-) mice remained unchanged. In summary, loss of PKC-epsilon results in increased vascular tone, but not pulmonary vascular remodeling in response to chronic Hx. Blunting of Hx-induced eNOS and iNOS expression may contribute to the increased vascular tone. PKC-epsilon appears to be an important signaling intermediate in the hypoxic regulation of each NOS isoform.     

Upregulation of endothelial nitric oxide synthase in rat aorta after ingestion of fish oil-rich diet

A previous study with aortic segments isolated from rats fed a fish oil-rich diet indicated an increase in acetylcholine-induced nitric oxide (.NO)-mediated relaxation. However, it remained to be elucidated whether a fish oil-rich diet affects the vascular activity per se and the point of the.NO-cGMP pathway at which fish oil acts. For this purpose, two groups of Sprague-Dawley rats were fed a semipurified diet containing 5% lipids, either corn oil (CO) or menhaden oil (MO), for 8 wk. We studied the mRNA and protein levels of endothelial NO synthase (eNOS) and NOS activity. The bioavailability of vascular.NO was assessed directly by electron spin resonance spectroscopy. The levels of cGMP, l-arginine, and l-citrulline were also evaluated in homogenates. Superoxide anion (O(2)(-).) production and related antioxidant activities were also studied in aortic segments. The aortic content of eNOS mRNA was increased in rats fed the MO-rich diet. This resulted in increases in both eNOS protein levels (70% relative to the rats fed the CO-rich diet) and NOS activity (102%);.NO production increased by 90%, cGMP levels increased by 100%, and l-arginine decreased by 30%. No change in aortic O(2)(-). production was caused by dietary MO. The upregulation of the eNOS-cGMP pathway induced by dietary MO may contribute to the maintenance of vascular homeostasis and explain its beneficial effect in the prevention of arterial diseases.     

Effect of the Peroxisome Proliferator Ciprofibrate on Lipid Peroxidation and 8-Hydroxydeoxyguanosine Formation in Transgenic Mice with Elevated Hepatic Catalase Activity

Peroxisome proliferators are a group of non-genotoxic hepatic carcinogens which have been proposed to act by increasing oxidative damage in the liver. To test this hypothesis, we have produced a transgenic mouse line that has elevated catalase activity specifically in the liver. In this study, we have examined if catalase overexpression influences the induction of lipid peroxidation or oxidative DNA damage, two mechanisms which have been hypothesized to be important in the carcinogenesis by peroxisome proliferators. Transgenic mice or non-transgenic litter mates were fed either 0.01% ciprofibrate or a control diet for 21 days. The activities of fatty acyl CoA oxidase and lauric acid hydroxylase were not significantly affected by catalase overexpression, although the ratio of fatty acyl CoA oxidase to catalase was significantly decreased in transgenic animals. Hepatic lipid peroxidation was estimated by quantifying the concentrations of malondialdehyde and conjugated dienes. Ciprofibrate treatment did not affect either endpoint, but catalase overexpression increased the concentrations of malondialdehyde (in untreated mice only) and conjugated dienes (in both untreated and ciprofibrate-fed mice). Oxidative DNA damage was estimated by quantifying 8-hydroxydeoxyguanosine (8-OHdG) by high-performance liquid chromatography/electrochemical detection. Ciprofibrate treatment significantly increased hepatic 8-OHdG concentrations, in agreement with several previous studies, but catalase overexpression did not significantly affect them, although 8-OHdG concentrations were decreased 50% in untreated mice. These results imply that the metabolism of hydrogen peroxide by catalase is not an important factor in the development of hepatic lipid peroxidation. The decrease in hepatic 8-OHdG in untreated transgenic mice and the increase seen after ciprofibrate administration imply that hydrogen peroxide is important in the formation of 8-OHdG. While the lack of decreased 8-OHdG levels in ciprofibrate-treated transgenic mice does not support this conclusion, it is possible that catalase levels were not sufficiently high to affect this endpoint. Transgenic mice with higher hepatic catalase activities may be required to resolve this issue.     

Introduction and expression of the human Bs-globin gene in transgenic mice.

Owing to the episodic and unpredictable nature of the sickling crisis, many aspects of the disease sickle cell anemia have resisted in vivo analysis. The lack of an animal model has hindered the pathophysiological investigation of this disease, as well as deterred the development of pharmacological therapies. The transgenic mouse system offers a new means for creating animals that make a specified mutant gene product, and we have used this system to create a series of mice that contain the human beta s-globin gene. These animals express this gene in the appropriate tissues and at the same point in development as the adult mouse globin genes are expressed. We have crossed the human beta s-containing transgenic mice with a beta-thalassemic mouse line and examined the hemoglobins produced by these mice. Their red cells contain 10% mouse alpha/human beta s hybrid hemoglobin, which partially corrects the thalassemic phenotype of the homozygous beta-thalassemic animals. Though the red cells do not sickle, other properties of the human beta s gene in these mice indicate the potential for the eventual development of a transgenic animal model for sickle cell anemia.     

Synergistic effect of angiotensin II and nitric oxide synthase inhibitor in increasing aortic stiffness in mice

Although they are implicated on their own as risk factors for cardiovascular disease, the potential link between nitric oxide (NO) deficiency, ANG II, and vascular stiffening has not been tested before. We evaluated the role of chronic ANG II treatment and NO deficiency, alone and in combination, on aortic stiffness in mice and tested parameters contributing to increases in active or passive components of vascular stiffness, including blood pressure, vascular smooth muscle contractility, and extracellular matrix components. Untreated (control) mice and mice treated with a NO synthase (NOS) inhibitor [N(omega)-nitro-L-arginine methyl ester (L-NAME), 0.5 g/l] were implanted with osmotic minipumps delivering ANG II (500 ng.kg(-1).min(-1)) for 28 days. Aortic stiffness was then measured in vivo by pulse wave velocity (PWV) and ex vivo by load-strain analysis to obtain values of maximal passive stiffness (MPS). Blood pressure and aortic contractility ex vivo were measured. ANG II treatment or NOS inhibition with L-NAME did not independently increase vascular stiffness; however, the combined treatments worked synergistically to increase PWV and MPS. The combined treatments of ANG II + L-NAME also significantly increased aortic wall collagen content while decreasing elastin. These novel results suggest that NO deficiency and ANG II act synergistically to increase aortic stiffness in mice predominantly via changes in aortic wall collagen/elastin ratio.     

Chronic-arginine supplementation improves endothelial cell vasoactive functions in hypercholesterolemic and atherosclerotic monkeys

Chronic exposure to l-arginine results in regression of atherosclerotic lesions and reversal of endothelial dysfunction. We investigated whether chronic l-arginine supplementation induces regression of atherosclerotic lesions and reversal of endothelial dysfunction in atherogenic rhesus monkeys and the mechanism which leads to these effects. About 12 male rhesus monkeys were fed 1% cholesterol and 18 g butter for 6 months to create an experimental model of hypercholesterolaemia and atherosclerosis (Group I) and 12 monkeys were fed standard stock diet for 6 months (Group II). After, 6 months these two groups were further divided into 2 sub-groups which in addition to their respective diets were fed 2.5% l-arginine in drinking water for additional 6 months (Group III and Group IV). Systemic nitric oxide (NO) formation was assessed as plasma nitrite and cGMP formation every 3 months. Oxygen free radical (OFR) generation and malondialdehyde production as an index of lipid peroxidation were determined. Changes in isometric tension were compared in isolated ring segments of thoracic aorta from normal and hypercholesterolemic animals.Cholesterol feeding progressively reduced plasma nitrite and cGMP generation (p<0.05). Dietary l-arginine partly restored the levels of plasma nitrite and cGMP (p<0.05) but did not change plasma cholesterol levels. l-arginine significantly reduced aortic intimal thickening, blocked the production of carotid and coronary intimal plaques and completely preserved endothelium-dependent vasodilator function. Further, l-arginine significantly inhibited generation of the reactive oxygen species (ROS) generation and lipid peroxidation.Chronic oral supplementation with l-arginine blocks the progression of plaques via restoration of nitric oxide synthase substrate availability and reduction of vascular oxidative stress. (Mol Cell Biochem 269: 1–11, 2005)     

Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD.

In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta-globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S-Antilles-D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2-globin gene, each linked to the beta-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.     

Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human alpha- and beta(S)-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E(2) (PGE(2)) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.