Effect of Mthfr genotype on diet-induced hyperhomocysteinemia and vascular function in mice

Deficiency of methylenetetrahydrofolate reductase (MTHFR) predisposes to hyperhomocysteinemia and vascular disease. We tested the hypothesis that heterozygous disruption of the Mthfr gene sensitizes mice to diet-induced hyperhomocysteinemia and endothelial dysfunction. Mthfr(+/-) and Mthfr(+/+) mice were fed 1 of 4 diets: control, high methionine (HM), low folate (LF), or high methionine/low folate (HM/LF). Plasma total homocysteine (tHcy) was higher with the LF and HM/LF diets than the control (P<.01) or HM (P<.05) diets, and Mthfr(+/-) mice had higher tHcy than Mthfr(+/+) mice (P<.05). With the control diet, the S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratio was lower in the liver and brain of Mthfr(+/-) mice than Mthfr(+/+) mice (P<.05). SAM/SAH ratios decreased further in Mthfr(+/+) or Mthfr(+/-) mice fed LF or LF/HM diets (P<.05). In cerebral arterioles, endothelium-dependent dilation to 1 or 10 microM acetylcholine was markedly and selectively impaired with the HM/LF diet compared with the control diet for both Mthfr(+/+) (maximum dilation 5% +/- 2% versus 21% +/- 4%; P<.01) and Mthfr(+/-) (6% +/- 2% versus 21% +/- 3%; P<.01) mice. These findings demonstrate that the Mthfr(+/-) genotype sensitizes mice to diet-induced hyperhomocysteinemia and that hyperhomocysteinemia alters tissue methylation capacity and impairs endothelial function in cerebral microvessels.     

Paradoxical absence of a prothrombotic phenotype in a mouse model of severe hyperhomocysteinemia

Hyperhomocysteinemia confers a high risk for thrombotic vascular events, but homocysteine-lowering therapies have been ineffective in reducing the incidence of secondary vascular outcomes, raising questions regarding the role of homocysteine as a mediator of cardiovascular disease. Therefore, to determine the contribution of elevated homocysteine to thrombosis susceptibility, we studied Cbs(-/-) mice conditionally expressing a zinc-inducible mutated human CBS (I278T) transgene. Tg-I278T Cbs(-/-) mice exhibited severe hyperhomocysteinemia and endothelial dysfunction in cerebral arterioles. Surprisingly, however, these mice did not display increased susceptibility to arterial or venous thrombosis as measured by photochemical injury in the carotid artery, chemical injury in the carotid artery or mesenteric arterioles, or ligation of the inferior vena cava. A survey of hemostatic and hemodynamic parameters revealed no detectible differences between control and Tg-I278T Cbs(-/-) mice. Our data demonstrate that severe elevation in homocysteine leads to the development of vascular endothelial dysfunction but is not sufficient to promote thrombosis. These findings may provide insights into the failure of homocysteine-lowering trials in secondary prevention from thrombotic vascular events.     

Vitronectin inhibits the thrombotic response to arterial injury in mice

Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1) and integrins and may play an important role in the vascular response to injury by regulating fibrinolysis and cell migration. However, the role of VN in the earliest response to vascular injury, thrombosis, is not well characterized. The purpose of this study was to test the hypothesis that variation in vitronectin expression alters the thrombotic response to arterial injury in mice. Ferric chloride (FeCl3) injury was used to induce platelet-rich thrombi in mouse carotid arteries. Wild-type (VN +/+, n = 14) and VN-deficient (VN -/-, n = 15) mice, matched for age and gender, were studied. Time to occlusion after FeCl3 injury was determined by application of a Doppler flowprobe to the carotid artery. Occlusion times of VN -/- mice were significantly shorter than those of VN +/+ mice (6.0 +/- 1.2 minutes v 17.8 +/- 2.3 minutes, respectively, P < .001). Histologic analysis of injured arterial segments showed that thrombi from VN +/+ and VN -/- mice consisted of dense platelet aggregates. In vitro studies of murine VN +/+ and VN -/- platelets showed no significant differences in ADP-induced aggregation, but a trend towards increased thrombin-induced aggregation in VN -/- platelets. Purified, denatured VN inhibited thrombin-induced platelet aggregation, whereas native VN did not. Thrombin times of plasma from VN -/- mice (20.5 +/- 2.1 seconds, n = 4) were significantly shorter than those of VN +/+ mice (34.2 +/- 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN -/- plasma prolonged the thrombin time into the normal range, suggesting that VN inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an explanation for the accelerated thrombosis observed in VN -/- mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity.     

Bradykinin B2 receptor knockout mice are protected from thrombosis by increased nitric oxide and prostacyclin

Bradykinin (BK) liberates nitric oxide, prostacyclin, and tissue plasminogen activator from endothelial cells. We hypothesized that BK B2 receptor knockout (KO) mice (BKB2R(-/-)) have increased thrombosis risk. Paradoxically, the BKB2R(-/-) mice have long bleeding times and delayed carotid artery thrombosis, 78 +/- 6.7 minutes, versus 31 +/- 2.7 minutes in controls. The mechanism(s) for thrombosis protection was sought. In BKB2R(-/-) plasma coagulation, fibrinolysis and anticoagulant proteins are normal except for an increased prekallikrein and decreased factor XI. BKB2R(-/-) mice have elevated BK 1-5 (160 +/- 75 fmol/mL, vs 44 +/- 29 fmol/mL in controls) and angiotensin II (182 +/- 41 pg/mL, vs 49 +/- 7 pg/mL in controls). Ramipril treatment shortens vessel occlusion time. BKB2R(-/-) mice have elevated plasma 6-keto-PGF1alpha (666 +/- 232 ng/mL, vs 23 +/- 5.3 ng/mL in controls) and serum nitrate (61 +/- 5.3 microM, vs 24 +/- 1.8 microM in controls). Treatment with L-NAME (NG-mono-methyl-L-arginine ester) or nimesulide shortens the thrombosis time. BKB2R(-/-) mice have increased angiotensin receptor 2 (AT2R) mRNA and protein expression. Treatment with an AT2R antagonist, PD123 319, normalizes the thrombosis time and nitrate and 6-keto-PGF1alpha. The long bleeding times in BKB2R(-/-) mice also correct with L-NAME and nimesulide therapy. In BKB2R(-/-) mice, angiotensin II binding to an overexpressed AT2R promotes thromboprotection by elevating nitric oxide and prostacyclin. These investigations indicate a pathway for thrombosis risk reduction via the plasma kallikrein/kinin and renin angiotensin systems.     

Effect of hyperhomocysteinemia on protein C activation and activity

Hyperhomocysteinemia has been proposed to inhibit the protein C anticoagulant system through 2 mechanisms: decreased generation of activated protein C (APC) by thrombin, and resistance to APC caused by decreased inactivation of factor Va (FVa). We tested the hypotheses that generation of APC by thrombin is impaired in hyperhomocysteinemia in monkeys and that hyperhomocysteinemia produces resistance to APC in monkeys, mice, and humans. In a randomized crossover study, cynomolgus monkeys were fed either a control diet or a hyperhomocysteinemic diet for 4 weeks. Plasma total homocysteine (tHcy) was approximately 2-fold higher when monkeys were on the hyperhomocysteinemic diet than when they were on the control diet (9.8 +/- 2.0 microM versus 5.6 +/- 1.0 microM; P <.05). After infusion of human thrombin (25 microg/kg of body weight), the peak level of plasma APC was 136 +/- 16 U/mL in monkeys fed the control diet and 127 +/- 13 U/mL in monkeys fed the hyperhomocysteinemic diet (P >.05). The activated partial thromboplastin time was prolonged to a similar extent by infusion of thrombin in monkeys fed the control diet and in those fed the hyperhomocysteinemic diet. The sensitivity of plasma FV to human APC was identical in monkeys on control diet and those on hyperhomocysteinemic diet. We also did not detect resistance of plasma FV to APC in hyperhomocysteinemic mice deficient in cystathionine beta-synthase (plasma tHcy, 93 +/- 16 microM) or in human volunteers with acute hyperhomocysteinemia (plasma tHcy, 45 +/- 6 microM). Our findings indicate that activation of protein C by thrombin and inactivation of plasma FVa by APC are not impaired during moderate hyperhomocysteinemia in vivo.     

Structure of cerebral arterioles in cystathionine beta-synthase-deficient mice

We examined effects of hyperhomocysteinemia on structure and mechanics of cerebral arterioles. We measured plasma total homocysteine (tHcy) and pressure, diameter, and cross-sectional area of the vessel wall in maximally dilated cerebral arterioles in heterozygous cystathionine beta-synthase-deficient (CBS(+/-)) mice and wild-type (CBS(+/+)) littermates that were provided with drinking water that was unsupplemented (control diet) or supplemented with 0.5% L-methionine (high-methionine diet). Plasma tHcy was 5.0+/-1.1 micro mol/L in CBS(+/+) mice and 8.3+/-0.9 micro mol/L in CBS(+/-) mice (P<0.05 versus CBS(+/+) mice) fed the control diet. Plasma tHcy was 17.2+/-4.6 micro mol/L in CBS(+/+) mice and 21.2+/-3.9 micro mol/L in CBS(+/-) mice (P<0.05) fed the high-methionine diet. Cross-sectional area of the vessel wall was significantly increased in CBS(+/-) (437+/-22 micro m(2)) mice fed control diet and CBS(+/+) (442+/-36 micro m(2)) and CBS(+/-) (471+/-46 micro m(2)) mice fed high-methionine diet relative to CBS(+/+) (324+/-18 micro m(2)) mice fed control diet (P<0.05). During maximal dilatation, the stress-strain curves in cerebral arterioles of CBS(+/-) mice on control diet and CBS(+/+) and CBS(+/-) mice on high-methionine diet were shifted to the right of the curve in cerebral arterioles of CBS(+/+) mice on control diet, an indication that distensibility of cerebral arterioles was increased in mice with elevated levels of plasma tHcy. Thus, hyperhomocysteinemia in mice was associated with hypertrophy and an increase in distensibility of cerebral arterioles. These findings suggest that hyperhomocysteinemia promotes cerebral vascular hypertrophy and altered cerebral vascular mechanics, both of which may contribute to the increased incidence of stroke associated with hyperhomocysteinemia.     

M118, a novel low-molecular weight heparin with decreased polydispersity leads to enhanced anticoagulant activity and thrombotic occlusion in ApoE knockout mice

Heparin and low-molecular weight heparin (LMWH) are complex, heterogeneous polysaccharides used in the treatment of arterial and venous thrombosis. M118 is a novel LMWH with low polydispersity and pronounced anti-Xa and anti-thrombin (IIa) activity as compared to current LMWHs. To determine if M118 is effective in preventing thrombosis in the setting of a vascular plaque, apolipoprotein E knockout mice fed a high fat diet were injected with M118, enoxaparin, unfractionated heparin, or saline control and examined for arterial thrombosis using a rose bengal laser induced carotid artery injury model. M118 significantly increased the time to occlusion as compared to control and unfractionated heparin but not compared to enoxaparin although fewer M118 treated animals had any vascular occlusion present at the time of protocol completion. Platelet-neutrophil aggregates were studied by flow cytometry and were found to be decreased with M118 as compared to enoxaparin. This is the first published report examining M118, a novel LMWH designed to have low polydispersity and enhanced anticoagulant activity. In an animal model of vascular plaque, M118 is a potent inhibitor of arterial thrombosis and, despite lower in vivo anti-Xa and anti-IIa activity levels, M118 was superior to UFH in the prevention of arterial thrombosis.     

Effects of folate supplementation in hyperhomocysteinemic pigs.

OBJECTIVES: The aim of this study was to evaluate the therapeutic effects of folic acid in the pig model of hyperhomocysteinemia. BACKGROUND: We have previously shown that pigs fed a methionine-rich diet develop hyperhomocysteinemia, arterial lesions and thrombotic events. Elevated homocysteine level is an independent risk factor for atherosclerosis that can be markedly lowered with daily folic acid administration. However, it is not known whether this treatment can prevent arterial lesions. METHODS: Three groups of pigs were studied: 8 control subjects received a standard diet; 8 received a methionine-rich diet for four months; 8 received a methionine-rich diet for 1 month and then the methionine-rich diet + 5 mg/day folic acid for 3 months. At month 4 after hemodynamic investigation, all the pigs were sacrificed. RESULTS: Control animals developed few usual vascular streaks. All the pigs fed a methionine-rich diet without folic acid treatment developed hyperhomocysteinemia (10.3+/-1.3 micromol/liter at basal state, 18.2+/-2.5 micromol/liter at one month and 14.6+/-3.8 micromol/liter at four months), hemodynamic abnormalities and diffuse arterial lesions with smooth muscle cell hyperplasia, endothelial alterations and elastic lamina dislocation. In this group, one pig died of venous thromboembolism and one of myocardial infarction. The pigs fed a methionine-rich diet + folic acid displayed similar arterial lesions and two had thrombotic events (one myocardial infarction and one pulmonary embolism), despite normalization of homocysteine levels (10.9+/-1.3 micromol/liter at basal state, 19.5+/-2.5 micromol/liter at one month and 11.4+/-3.8 micromol/liter at four months). CONCLUSIONS: In the pig model of hyperhomocysteinemia, 5 mg/day folic acid did not prevent arterial lesions or thrombotic events.     

Effect of systemic nitric oxide synthase inhibition on arterial stiffness in humans.

Stiffening of large elastic arteries impairs the buffering function of the arterial system and contributes to cardiovascular disease. The aim of this study was to determine whether endothelium-derived nitric oxide (NO) modulates the stiffness of large elastic arteries in humans. Seven apparently healthy adults (60+/-3 years, 2 males and 5 females) underwent systemic alpha-adrenergic blockade (phentolamine) and systemic NO synthase inhibition using NG-monomethyl-L-arginine (L-NMMA) in sequence. Phentolamine was given first to isolate contribution of NO to arterial stiffness by preventing reflex changes in sympathetic tone that result from systemic NO synthase inhibition, and also to compare arterial stiffness at a similar mean arterial pressure. Mean arterial blood pressure decreased (p<0.05) after phentolamine infusion but returned to baseline levels after L-NMMA infusion. The carotid beta-stiffness index (via simultaneous ultrasound and applanation tonometry on the common carotid artery) did not change after the restraint of systemic alpha-adrenergic nerve activity (9.8+/-1.2 vs. 9.1+/-1.1 U) but increased (p<0.05) after NO synthase inhibition (12.6+/-2.0 U). These results suggest that NO appears to modulate central arterial stiffness in humans.     

Functional thrombomodulin deficiency causes enhanced thrombus growth in a murine model of carotid artery thrombosis

Abstract. Thrombomodulin (TM) bound thrombin initiates the protein C anticoagulant pathway and defects in TM result in enhanced coagulation. Recent studies suggest a role for TM in arterial vascular disease. In order to corroborate this association we studied arterial thrombus formation in mice with a functional TM defect. We used mice homozygous for a ⁴⁰⁴Glu-to-Pro mutation in the TM gene (TM^pro/pro) and compared these with wildtype littermates in a model of FeCl₃ induced carotid artery thrombosis. Time-to-occlusion (TTO) was assessed by arterial blood flow measurement, using a Doppler flow probe. Complete occlusion occurred in 8/10 (80%) TM^pro/pro mice and in 3/11 (27%) littermate controls. Mean time to occlusion (TTO) [± SE] was 767 ± 196 s in the F2-TM^pro/pro mice, versus 1507 ± 159 s in controls (p = 0.007, Mann Whitney U test). Histology and immunostaining for tissue factor did not reveal any differences in thrombus morphology or thrombogenicity between the two groups.These data confirm and extend the fiding that a functional deficiency in TM results in enhanced thrombus formation in a murine model of carotid artery thrombosis and support a role for TM defects in arterial thrombotic disease.     

Nitric oxide production decreases after salt loading but is not related to blood pressure changes or nitric oxide-mediated vascular responses

BACKGROUND: Nitric oxide production is a homeostatic mechanism that may regulate blood pressure during salt loading. Salt-sensitive hypertension in animal models and in humans is characterized by increased blood pressure and decreased nitric oxide production after salt loading. It is not known if this impaired nitric oxide production is the result of hypertension or is a mechanism contributing to the blood pressure response to salt. METHODS AND RESULTS: The effects of salt loading on blood pressure, nitric oxide-mediated vasodilation and nitric oxide production were measured in 25 normotensive subjects after 6 days on either a high (400 mmol/day) or low (10 mmol/day) sodium, low nitrate diet. Mean arterial pressure increased during the high-salt diet [4 +/- 1 mmHg (mean +/- SEM)] in 12 subjects and remained unchanged or decreased (-4 +/- 1 mmHg) in 13 subjects. Plasma nitrite and nitrate, a measure of nitric oxide production, decreased significantly from 39 +/- 3.3 micromol/l during the low-salt diet to 22.4 +/- 2.4 micromol/l during the high-salt diet (P = 0.0001). However, changes in mean arterial pressure from low- to high-salt diet did not correlate with changes in plasma nitrite and nitrate (r = 0.14, P = 0.51). Forearm blood flow increased significantly (P <0.0001) in response to mental stress, a nitric oxide-mediated response, but was not affected by sodium intake (from 7.8 +/- 0.9 to 11.2 +/- 1.4 ml/min per 100 ml during low salt versus 8.5 +/- 1.2 to 10.4 +/- 1.3 ml/min per 100 ml during high salt,P = 0.3). CONCLUSIONS: Salt loading results in a decrease in nitric oxide production in both salt-sensitive and salt-resistant normotensive subjects, which is independent of changes in blood pressure and does not affect the nitric oxide-mediated vascular response to mental stress. In contrast to salt-resistant animal models, salt loading in healthy subjects does not increase nitric oxide production. Therefore, the increased blood pressure response to salt loading may occur through mechanisms other than nitric oxide, or salt-sensitive individuals are more sensitive to the reduced nitric oxide production that occurs after salt loading in both salt-sensitive and salt-resistant subjects.     

Chronic blockade of nitric oxide does not produce hypertension in baroreceptor denervated rabbits

Although the vascular action of endothelium-derived nitric oxide in modulating arterial pressure is well established, nitric oxide can also act as a neurotransmitter in the central nervous system. In addition, there is evidence for an interaction between nitric oxide and baroreceptor afferent processing; thus, nitric oxide may regulate blood pressure through central modulation of arterial baroreflexes. To test this possible interaction of nitric oxide and baroreflexes in the long-term regulation of blood pressure, we measured arterial pressure and heart rate responses to nitric oxide blockade by using L-NAME (50 mg/kg per day in drinking water) over 7 days in baroreceptor intact and sinoaortic denervated conscious rabbits. In the baroreceptor intact animals, blockade of nitric oxide leads to a significant increase in mean arterial pressure (from 75+/-2 to 84+/-3 mm Hg) and decrease in heart rate (from 233+/-8 to 195+/-8 bpm) that was sustained over the 7 days of nitric oxide blockade. In the sinoaortic denervated animals, blockade of nitric oxide initially led to a similar increase in arterial pressure (82+/-3 mm Hg on the second day), but in all sinoaortic denervated animals this increase was not sustained and recovered back to pre-L-NAME levels. This finding indicates that baroreflexes play an important role in the long-term control of blood pressure, and, second, that one mediator of this control is nitric oxide.     

Increased salt-sensitivity in endothelial nitric oxide synthase-knockout mice

BACKGROUND: Although impaired nitric oxide production contributes importantly to salt-sensitivity, the role of the endothelial isoform of nitric oxide synthase (eNOS) has received little attention. In the present study we compared the effects of a high-salt diet on the blood pressure response of eNOS knockout (eNOS-/-) and control (eNOS+/+) mice. METHODS: Mean arterial pressure (MAP), heart rate, pulse pressure, and activity levels were recorded by telemetry in mice fed a regular-salt diet (0.7% NaCl) followed by 6 weeks on either a high-salt (8% NaCl) or regular-salt diet. RESULTS: The eNOS-/- mice exhibited a 15% increase in MAP and a 2- to 2.5-fold increase in salt-sensitivity relative to the control strain. Salt-induced increases in MAP were well sustained in eNOS-/-, whereas in eNOS+/+ the initial increase was biphasic. The effects of salt on MAP were particularly pronounced during locomotor activity, during the dark phase, and at the peak levels of MAP recorded over the course of the day. The high-salt diet also led to a transient increase in the proportion of time spent active. Levels of heart rate and pulse pressure were relatively unaffected by the high-salt diet. CONCLUSION: The eNOS-/- mice exhibit an increased blood pressure response to a high-salt diet. This finding suggests that eNOS normally provides an important contribution to the body's adaptation to a salt load and that reduced production of NO by eNOS may promote salt-sensitivity and salt-induced hypertension.     

Enhanced blood pressure variability in eNOS knockout mice.

It has been shown previously that endogenous nitric oxide can buffer arterial blood pressure variability in dogs and rats. In these former studies, all isoforms of the nitric oxide synthase were blocked pharmacologically and an increased blood pressure variability was observed. Thus the question as to which isoform of the nitric oxide synthase is responsible for the blood pressure buffering effect of endogenous nitric oxide remains unraveled. In the present study, we therefore compared blood pressure variability in knockout mice that lack specifically the gene for endothelial nitric oxide synthase with their respective wild-type controls. One day after carotid artery cannulation, blood pressure was recorded in these conscious mice. During resting conditions, blood pressure variability was markedly enhanced in knockout mice compared with wild-type mice (10.5+/-1.5 mm Hg2 vs 6.0+/-0.8 mm Hg2, P<0.05). Power spectral analysis revealed that this increase in blood pressure variability is manifested at low frequencies that range from 0.05 to 0.40 s-1 (Hz) (5.1+/-1.0 mm Hg2 vs 2.5+/-0.5 mm Hg2, P<0.05). On the basis of these results, we conclude that the blood pressure buffering effect of endogenous nitric oxide is mediated by the endothelial isoform of the nitric oxide synthase. In addition, endothelial nitric oxide is most effective in buffering blood pressure oscillations at frequencies that range from 0.05 to 0.40 s-1 (Hz) in conscious mice.     

Decreased arteriolar density in endothelial nitric oxide synthase knockout mice is due to hypertension, not to the constitutive defect in endothelial nitric oxide synthase enzyme

BACKGROUND : Hypertension in endothelial nitric oxide synthase knockout (eNOS-/-) mice is believed to be partly due to altered vasodilatation. However, nitric oxide (NO) is also known to play an important part in angiogenesis. OBJECTIVE : To investigate whether capillary and arteriolar density were impaired in eNOS-/- mice, as this could account for increased vascular resistance and hypertension. METHODS : Using immunohistochemistry with mouse monoclonal smooth muscle alpha-actin antibody to detect arterioles and rabbit polyclonal fibronectin antibody to detect capillaries, we quantified arteriolar and capillary density in the left ventricle and in the gracilis muscle from eNOS-/- mice compared with those in C57BL6J littermates (n = 6-8) in 8- and in 12-week-old mice. In a second set of experiments, we treated 8-week-old normotensive eNOS-/- mice with the antihypertensive vasodilator, hydralazine, for 1 month. RESULTS : Eight-week-old eNOS-/- mice were normotensive and presented similar arteriolar and capillary densities in cardiac and skeletal muscles compared with those in eNOS+/+ mice. Twelve-week-old eNOS/- mice were hypertensive (mean arterial pressure 118 +/- 21 mmHg compared with 64 +/- 2 mmHg; P < 0.05). Capillary densities were similar in eNOS-/- mice and eNOS+/+ mice in the heart (4154 +/- 123 and 4051 +/- 247/mm2, respectively) and in skeletal muscle (961 +/- 40 and 1025 +/- 41/mm2, respectively). Arteriolar densities were 15% lower in skeletal muscle and in the heart in eNOS-/- mice than in the eNOS+/+ control group (P < 0.05). Hydralazine prevented hypertension and arteriolar rarefaction in eNOS-/- mice, whereas capillary density was unaffected by treatment with the vasodilator. CONCLUSION : In young non-hypertensive eNOS-/- mice, the lack of eNOS did not affect microvascular densities in either of the muscles studied. In adult hypertensive eNOS-/- mice, we observed a lower arteriolar density, but a similar capillary density compared with controls. Hydralazine prevented hypertension and arteriolar rarefaction in adult mice, suggesting a non-NO-dependent pathway. Capillary density was not affected by hydralazine.     

Hyperhomocysteinemia evoked by folate depletion: effects on coronary and carotid arterial function

High circulating concentrations of homocysteine (ie, hyperhomocysteinemia [Hhcy]) impair the vascular function of peripheral conduit arteries and arterioles perfusing splanchnic and skeletal muscle regions. The effects of HHcy on coronary resistance vessel function and other indexes of vascular function, ie, arterial permeability and stiffening, are unclear. We tested the hypotheses that HHcy impairs coronary resistance vessel reactivity; increases carotid arterial permeability; and initiates arterial stiffening. Male rats that consumed folate-replete (CON, n=44) or folate-deplete (HHcy, n=48) chow for 4 to 5 weeks had total plasma homocysteine concentrations of 7+/-2 or 58+/-4 micromol/L, respectively. Maximal acetylcholine-evoked relaxation (approximately 40% vs approximately 60%) and tension development from baseline in response to nitric oxide synthase inhibition (approximately 20% vs approximately 40%) were lower (both P<0.05) in coronary resistance vessels (approximately 120 microm, internal diameter) isolated from HHcy versus CON animals, respectively, whereas sodium nitroprusside-evoked relaxation and contractile responses to serotonin and potassium chloride were similar between groups. Permeability to 4400 MW and 65 000 MW fluorescently labeled (TRITC) dextran reference macromolecules (quantitative fluorescence microscopy) was approximately 44% and approximately 24% greater (P<0.05), respectively, in carotid arteries from HHcy versus CON rats. Maximal strain, evaluated by using a vessel elastigraph, was less ( approximately 32% vs 42%, P<0.05) in carotid arterial segments from HHcy versus CON animals, respectively. Finally, estimates of oxidative (copper-zinc+manganese superoxide dismutase activity) and glycoxidative (pentosidine) stress were elevated (P<0.05) in arterial tissue from HHcy versus CON rats. These findings suggest that moderately severe HHcy evoked by folate-depletion impairs endothelium-dependent relaxation of coronary resistance vessels, increases carotid arterial permeability, and initiates arterial stiffening. HHcy may produce these effects by a mechanism associated with increased oxidative and glycoxidative stress.     

Direct demonstration of P-selectin- and VCAM-1-dependent mononuclear cell rolling in early atherosclerotic lesions of apolipoprotein E-deficient mice.

Apolipoprotein E-deficient (ApoE-/-) mice develop atherosclerotic lesions throughout the arterial tree, including the carotid bifurcation. Although the expression of adhesion molecules such as ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and P-selectin on endothelium that overlie atherosclerotic plaques has been implicated in monocyte recruitment to developing lesions, monocyte adhesion in atherosclerotic vessels has not been observed directly. To investigate which adhesion molecules may be important in monocyte adhesion to atherosclerotic lesions, an isolated mouse carotid artery preparation was developed and perfused with mononuclear cells. We show rolling and attachment of the human monocytic cell line U937 and the mouse monocyte-macrophage cell line P388D1 in carotid arteries from 10- to 12-week-old ApoE-/- and C57BL/6 wild-type mice fed a Western-type diet (21% fat wt/wt) for 4 to 5 weeks. No rolling was observed in carotid arteries from C57BL/6 or BALB/c wild-type mice fed a chow diet and little was observed in BALB/c mice fed a Western-type diet. This model represents early lesion development as shown by minimal macrophage infiltration in the intima of carotid arteries from ApoE-/- mice fed a Western-type diet. Rolling was observed at shear stresses that were characteristic of the low-shear recirculation zone near the carotid bifurcation. Mononuclear cell attachment and rolling were significantly inhibited by monoclonal antibody blockade of P-selectin or its leukocyte ligand P-selectin glycoprotein ligand-1. Rolling velocities increased after monoclonal antibody blockade of mononuclear cell alpha4-integrin or VCAM-1, which indicates that alpha4-integrin interacting with VCAM-1 stabilizes rolling interactions and prolongs monocyte transit times.     

Increased eNO and pulmonary iNOS expression in eNOS null mice.

Nitric oxide (NO) is a major regulatory molecule of the cardiovascular system; however, measurement of vascular NO synthesis in vivo represents a major challenge. NO stemming from the lower respiratory tract has been used as a marker of vascular endothelial function. Experimental evidence for this concept is lacking. Therefore, the aim of the present study was to investigate this relationship. Lower respiratory tract exhaled NO concentration, together with systemic and pulmonary artery pressure, was measured in endothelial nitric oxide synthase (NOS) (eNOS) null mice (eNOS-/-). Similar studies were performed in inducible NOS (iNOS) null mice (iNOS-/-). Defective endothelial NO synthesis in eNOS-/- mice (evidenced by systemic and pulmonary hypertension) was associated with augmented exhaled NO levels (12.5 +/- 1.9 versus 9.8 +/- 1.2 parts per billion (ppb), eNOS-/- versus wild type), whereas normal endothelial NO synthesis in iNOS-/- mice was associated with decreased exhaled NO levels (4.3 +/- 1.5 ppb). Augmented exhaled NO levels in eNOS-/- mice were associated with upregulation of iNOS expression in the lung. These results indicate that inducible nitric oxide synthase is a major determinant of gaseous nitric oxide production in the lung, and lower respiratory tract exhaled nitric oxide does not always represent a marker of vascular endothelial nitric oxide synthesis.     

Essential role of AT1A receptor in the development of 2K1C hypertension

The aims of this study were to delineate the relative contribution of angiotensin II (ANG II) subtype 1A (AT1A) and 1B (AT1B) receptors to the development of two-kidney, one-clip (2K1C) Goldblatt hypertension in mice, to examine if increased nitric oxide synthase (NOS) activity counteracts the vasoconstrictor influences of ANG II in 2K1C hypertensive mice, and to determine the role of ANG II type 2 (AT2) receptors in 2K1C hypertension in mice. AT(1A) ANG II receptor knockout (AT1A-/-) and wild-type (AT1A+/+) mice underwent clipping of the right renal artery. Systolic blood pressure (SBP) was significantly lower in AT1A-/- compared with AT1A+/+ mice, and neither clip placement nor AT2 receptor blockade with PD 123319 (PD) altered SBP in AT1A-/- mice. A significant and sustained rise in SBP from 119+/-5 to 163+/-6 mm Hg was observed in the 2K1C AT1A+/+ mice from day 10 to day 26. Chronic PD infusion did not alter the course of hypertension in 2K1C/AT1A+/+. Acute PD infusion did not alter mean arterial pressure (MAP) in AT1A+/+, PD/AT1A+/+, 2K1C/AT1A+/+, PD/2K1C/AT1A+/+, AT1A-/-, PD/AT1A-/-, and PD/2K1C/AT1A-/- mice compared with basal levels. In contrast, acute PD infusion caused significant increases in MAP in 2K1C/AT1A-/- mice. The subsequent acute NOS inhibition caused greater increases in MAP in 2K1C/AT1A+/+ and PD/2K1C/AT1A+/+ mice than in AT1A+/+ and PD/AT1A+/+ mice. These results support the essential role of AT1A receptors in mediating 2K1C hypertension and support the hypothesis that augmented NO production serves as a counteracting system in this model of hypertension.     

Hyperlipidemia promotes thrombosis after injury to atherosclerotic vessels in apolipoprotein E-deficient mice

The increased risk of hyperlipidemia on the development of complications of atherosclerosis is well established. Cholesterol-lowering therapies lead to a decrease in the incidence of vascular thrombotic events that is out of proportion to the reduction in plaque size. This suggests that the occurrence of acute thrombosis overlying a disrupted plaque is influenced by changes in lipid levels. The influence of acute hyperlipidemia on the development of thrombosis overlying an atherosclerotic plaque in vivo has not been extensively studied. We used a murine model of vascular injury induced by a photochemical reaction to elicit thrombus formation overlying an atherosclerotic plaque. Fifteen apolipoprotein E-deficient mice were maintained on normal chow until the age of 30 weeks. Five days before the induction of thrombosis, 6 mice were started on a high fat diet, and 9 mice were continued on normal chow. Mice then underwent photochemical injury to the common carotid artery immediately proximal to the carotid bifurcation, where an atherosclerotic plaque is consistently present. Mice maintained on normal chow developed occlusive thrombi, determined by cessation of blood flow, 44+/-5 minutes (mean+/-SEM) after photochemical injury, whereas mice fed a high fat chow developed occlusive thrombosis at 27+/-3 minutes (P<0.02). Histological analysis confirmed the presence of acute thrombus formation overlying an atherosclerotic plaque. These studies demonstrate a useful model for assessing the determinants of thrombosis in the setting of atherosclerosis and show that acute elevations in plasma cholesterol facilitate thrombus formation at sites of atherosclerosis after vascular injury.