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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1203-1208

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1203.)
© 2000 American Heart Association, Inc.

Vascular Biology

Simultaneously Increased TxA2 Activity in Isolated Arterioles and Platelets of Rats With Hyperhomocysteinemia

Zoltan Ungvari; Eszter Sarkadi-Nagy; Zsolt Bagi; Lajos Szollár; Akos Koller

From the Institute of Pathophysiology (Z.U., E.S.-N., Z.B., L.S., A.K.), Semmelweis University of Medicine, Budapest, Hungary, and the Department of Physiology (A.K.), New York Medical College, Valhalla, NY.

Correspondence to Akos Koller MD, PhD, Department of Physiology, New York Medical College, Valhalla, New York 10595. E-mail kolako{at}net.sote.hu


*    Abstract
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 *Abstract
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Abstract—We aimed to elucidate the effect of hyperhomocysteinemia (HHcy) on the synthesis of prostaglandins in rat skeletal muscle arterioles and platelets. Male Wistar rats were divided into 2 groups: (1) control rats, with plasma Hcy levels of 6.5±0.5 µmol/L (n=50) and (2) rats with HHcy, induced by daily intake of 1 g/kg body weight methionine in the drinking water for 4 weeks (plasma Hcy levels were 20.6±3.0 µmol/L, P<0.01 versus controls; n=50). Arterioles (diameter {approx}130 µm) were isolated from the gracilis muscle, cannulated, and pressurized (at 80 mm Hg), and changes in their diameters were followed by video microscopy. Constrictions to bradykinin (BK; 10-10 to 10-7 mol/L) were significantly greater in HHcy than in control rat arterioles (at 10-9 mol/L BK, changes were 11±3% in control and 41±9% in HHcy rats). The cyclooxygenase inhibitor indomethacin (10-5 mol/L), the prostaglandin H2/thromboxane A2 (PGH2/TxA2) receptor antagonist SQ 29,548 (10-6 mol/L), or the TxA2 synthase inhibitor furegrelate (5x10-6 mol/L) significantly decreased constrictions to BK in both groups but more so in HHcy arterioles, thus eliminating the difference between responses of HHcy and control arterioles. Constrictions to U46619 (a TxA2 analogue) were significantly greater in HHcy than in control arterioles (at 10-8 mol/L U46619, values for controls were 33±2% and 54±3% for HHcy). Endothelium removal or indomethacin treatment attenuated constrictions to U46619 in HHcy arterioles and eliminated the difference in responses. Also, aggregation of platelets from HHcy rats to collagen and ADP was significantly enhanced compared with controls (with 5 µg/mL collagen: controls, 23±5%; HHcy, 49±5%; with 10-7 mol/L ADP: controls, 25±3%; HHcy, 35±3%). Indomethacin or SQ 29,548 caused greater inhibition of aggregation of HHcy platelets compared with controls, thereby eliminating the differences between the 2 groups. Thus, HHcy enhances TxA2 synthesis both in the arteriolar endothelium and platelets. By promoting vascular constriction and platelet aggregation simultaneously, these alterations are likely to contribute to the atherothrombotic vascular diseases described in HHcy.


Key Words: homocysteine • methionine • endothelium • arterioles • platelet aggregation


*    Introduction
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 *Introduction
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 down arrowResults
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Several epidemiological studies have shown an association between elevated plasma homocysteine (Hcy) concentrations and atherothrombotic vascular diseases.1 2 3 4 In the general human population, mild hyperhomocysteinemia (HHcy) is rather common (1:70) and is found in {approx}30% of individuals with coronary, cerebrovascular, or peripheral atherothrombotic disease.3 4 Hcy is a thiol-containing amino acid that is formed from the essential amino acid methionine. Plasma Hcy concentration may increase in different pathophysiological conditions, including deficiency of vitamins such as folic acid, cyanocobalamin, and pyridoxal phosphate; and in the presence of various enzyme abnormalities (cystathionine ß-synthetase, temperature-sensitive methylenetetrahydrofolate reductase; for further information, see Reference 2 ), all of which participate in the metabolism of methionine and Hcy.

The mechanism(s) by which elevated Hcy promotes atherothrombotic vascular diseases is still not clearly elucidated and is likely to be multifactorial.5 6 It has been shown that HHcy stimulates smooth muscle proliferation7 8 and promotes LDL oxidation.9 There are reports demonstrating that HHcy impairs the vasoactive function of the endothelium as well. In experimental animals, severe HHcy resulted in endothelial cell loss.10 The adverse effect of Hcy was also confirmed by in vitro studies on cultured endothelial cells.11 12 In conduit arteries of patients with HHcy, vasodilation associated with reactive hyperemia is impaired,13 14 15 16 whereas reduced acetylcholine (ACh)-induced dilation was demonstrated in carotid arteries isolated from HHcy monkeys.17 18

Less is known, however, of how the function of microvessels is altered in HHcy. We have recently demonstrated that elevated levels of Hcy in rats interfere with skeletal muscle arteriolar responses, which are mediated or modulated by endothelium-derived NO.19 Alterations of NO synthesis/release are frequently accompanied by enhanced constrictor responses due to changes in other endothelial pathways, such as an increased synthesis of prostaglandin H2/thromboxane A2 (PGH2/TxA2).20 Interestingly, previous studies found an enhanced platelet activation to infusion of collagen, which caused a marked reduction of hindlimb blood flow in HHcy compared with control monkeys.17 The underlying mechanism for this alteration could be due to increased TxA2 synthesis of platelets in HHcy.21 Thus, it is likely that in HHcy, there are changes in vascular arachidonic acid metabolism as well, yet the effect of HHcy on endothelial prostaglandin synthesis in arterioles has not been elucidated.

On the basis of these findings, we hypothesized that in HHcy, the synthesis of PGH2 and/or TxA2 is simultaneously upregulated in vascular tissue and platelets. To test this hypothesis, we measured the arteriolar responses of control and HHcy rats to bradykinin (BK), an agent that is known to initiate the synthesis of arachidonic acid metabolites, such as PGI2 and TxA2, in the endothelium.22 23 24 BK has pathological implications as well, since it is an important mediator of various inflammatory processes that are also associated with atherosclerotic vascular diseases.25 Also, we investigated the arteriolar responses to intraluminal pressure and U46619, a stable TxA2 analogue, and the aggregation of platelets from the same animals in response to ADP and collagen, which are known to depend, at least in part, on TxA2 synthesis.


*    Methods
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 *Methods
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In male Wistar rats weighing {approx}150 g (purchased from Charles River Co), moderate HHcy was induced by administration of L-methionine (1 g · kg body weight-1 · d-1) and succinylsulfathiazole (0.1 g · kg body weight-1 · d-1) in the drinking water for a period of 4 weeks (n=50), as described previously.17 18 19 Succinylsulfathiazole was used to avoid bacterial proliferation and subsequent folate neoproduction. The doses administered were based on average daily fluid intake. Control animals (n=50) had free access to water. Animals were housed separately, fed standard rat chow, and were weighed at the start and end of the 4-week period.

Determination of Serum Hcy
Blood was collected from the aortas of fasted rats. It was immediately cooled on ice and centrifuged at 3000g for 20 minutes at 4°C to limit the release of Hcy from blood cells. Serum was then stored at -20°C until assayed. Total Hcy concentrations were measured by a high-performance liquid chromatography technique with fluorometric detection.19 26 In brief, 240 µL of serum and 60 µL of internal standard (N-acetyl-L-cysteine, 50 µmol/L final concentration) were reduced for 30 minutes at 4°C with 30 µL of tri-n-butylphosphine (10%). Deproteinization was performed with 300 µL of 10% trichloroacetic acid. After centrifugation, 100 µL of clear supernatant was mixed with 20 µL of 1.55 mol/L NaOH, 250 µL of 0.125 mol/L borate buffer (pH 9.5), and 50 µL of 1 mg/mL 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. After derivatization at 60°C (1 hour), the sample was analyzed on a high-performance liquid chromatograph (JASCO International Co Ltd) equipped with a fluorescence detector (LC 1255, GBC Scientific Equipment Pty Ltd). Separation was carried out on a 200x4.6-mmx5-µm Nucleosil C18 column. The eluent was 0.1 mol/L acetate buffer (pH 4.0) containing 2% methanol. The fluorescence intensities were measured with excitation at 386 nm and emission at 516 nm.

Analysis of Plasma Fatty Acid Content, Lipid Peroxidation Products, and Lipoprotein Susceptibility to Oxidation
Plasma lipids were extracted27 and the extracts were transmethylated with BF3/methanol. The fatty acid methyl esters were separated by capillary gas chromatography (Chrompack CP 9000 column: wall-coated open tubular fused-silica 50-mx0.25-mm CP-SIL88). Plasma lipid peroxidation was measured as thiobarbituric acid–reactive substances and expressed in malondialdehyde equivalents.27 In brief, each plasma sample (200 µmol/L) was mixed with thiobarbituric acid reagent (1 mL), heated (100°C, 20 minutes), and centrifuged, and the absorbance of the supernatant fraction was measured at 532 nm. LDL and VLDL were isolated by ultracentrifugation and were tested for their susceptibility to in vitro copper-induced oxidation.27 In brief, after isolation, lipoproteins were dialyzed for 20 hours at 4°C in the dark. against PBS (pH 7.4, purged with N2). The kinetics of LDL oxidation to CuCl2 (15 µmol/L) was determined by monitoring the change in diene absorption at 234 nm on a UV spectrophotometer.

Isolation of Arterioles
Experiments were conducted on isolated arterioles ({approx}130 µm active and {approx}180 µm passive diameters at 80 mm Hg) of rat gracilis muscle as described previously.19 20 28 In brief, on the fourth week, the rats were fasted overnight and then anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). The gracilis muscle was exposed and isolated from surrounding tissues. The muscle then was dissected out; placed in a silicone-lined Petri dish containing cold (0°C to 4°C) physiological saline composed of (in mmol/L) 110 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.0 KH2PO4, 10.0 dextrose, and 24.0 NaHCO3; and equilibrated with a gas mixture of 10% O2 and 5% CO2, balanced with N2, at pH 7.4. With the use of microsurgery instruments and an operating microscope, a 1.5- mm-long segment of the first-order arteriole running intramuscularly was isolated and transferred into an organ chamber containing 2 glass micropipettes filled with physiological saline solution. From a reservoir, the vessel chamber (15 mL) was continuously supplied with physiological saline solution at a rate of 20 mL/min. After the vessel had been mounted on the proximal micropipette and was secured with sutures, the perfusion pressure was raised to 20 mm Hg to clear the red blood cells from the lumen. Then the other end of the vessel was mounted on the distal pipette. Both micropipettes were connected with silicone tubing to an adjustable physiological saline solution reservoir. Pressures on both sides were measured by electromanometers. The perfusion pressure was slowly (over {approx}1 minute) increased to 80 mm Hg. The temperature was set at 37°C by a temperature controller (Grant Instruments), and the vessel was allowed to equilibrate for {approx}1 hour.

Experimental Protocols
After the equilibration period, changes in the diameter of arterioles in response to increases in perfusion pressure (from 10 to 20 and from 20 to 140 mm Hg in 20-mm Hg steps) were measured under zero-flow conditions.29 The pressure was maintained for 5 minutes at each pressure step to allow the vessel to reach a steady-state diameter. At the conclusion of each experiment, the suffusion solution was changed to a Ca2+-free physiological saline solution, which contained sodium nitroprusside (SNP, 10-4 mol/L) and EGTA (1.0 mmol/L); the vessel was incubated for 10 minutes and the pressure steps were repeated to obtain the maximum passive diameter at each pressure value (pressure–passive diameter relationship). The diameter was measured with a microangiometer and recorded on a chart recorder (Radelkis).

In preliminary studies, we selected doses of BK that elicited constrictions of gracilis arterioles. Responses of arterioles of control and HHcy rats to BK (10-9 to 10-7 mol/L) were compared in the absence and presence of indomethacin (10-5 mol/L), an inhibitor of prostaglandin synthesis; the specific TxA2 receptor antagonist SQ 29,548 (10-6 mol/L); or the specific TxA2 synthase inhibitor furegrelate30 (U63557A, 5x10-6 mol/L).

In separate experiments, the constrictor responses of arterioles of control and HHcy rats to the stable TxA2 analogue U46619 (10-11 to 5x10-6 mol/L) were compared before and after endothelium removal. The endothelium of the arteriole was removed by perfusion of the vessel with air for {approx}1 minute at a perfusion pressure of 20 mm Hg. The arteriole was then perfused with physiological saline solution to clear the debris. The perfusion pressure was then raised to 80 mm Hg for 30 minutes to establish a stable tone. The efficacy of endothelial denudation was ascertained by arteriolar responses to ACh (10-7 mol/L, an endothelium-dependent agent) and SNP (10-7 mol/L, an endothelium-independent agent) before and after administration of the air bolus.29 The infusion of air resulted in loss of function of the endothelium, as indicated by the absence of dilation to ACh, whereas dilation to SNP remained intact.

In further experiments, the constrictor responses of arterioles of control and HHcy rats to U46619 were compared in the presence and absence of SQ 29,548 (10-8, 2x10-8, 10-7, and 10-6 mol/L). The U46619 (10-10 to 10-6 mol/L) and SQ 29,548 (10-8 and 2x10-8 mol/L) interaction being apparently competitive, the negative logarithm (pA2) of the equilibrium dissociation constant (Ke) was calculated for the antagonist.31 32 The Ke was calculated from the equation Ke=a/(DR -1), where a is the molar concentration of antagonist, and DR, the dose ratio, is the measure of the rightward shift of the agonist dose-response curve. In separate experiments, the constrictor responses of arterioles of control and HHcy rats to U46619 (10-11 to 5x10-6 mol/L) were compared in the absence and presence of indomethacin (10-5 mol/L) or furegrelate (5x10-6 mol/L).

All drugs were added to the vessel chamber, and final concentrations are reported. After responses to each drug subsided, the system was flushed with physiological saline solution. Changes in diameter were expressed as a percentage of baseline.

Platelet Aggregation Studies
Aggregation of platelets was studied as described previously.21 33 34 In brief, heparinized blood was removed from the aorta of the same rats that were used to study isolated arterioles. Platelet-rich plasma (PRP) was obtained by centrifugation of blood at 250g for 10 minutes. Platelet-poor plasma (PPP) was prepared from the remaining volume of blood by centrifugation at 1600g for 10 minutes.33 The concentration of platelets in PRP was determined by light microscopy in a counting chamber and was adjusted to 0.3x109/mL by diluting it with PPP. Both PRP and PPP were kept at 22°C and used within 1.5 hours of blood collection. Cuvettes containing 0.5 mL of PRP were placed in a turbidimetric aggregometer (Mikron M304) under stirring (1000 rpm at 37°C). Aggregation was started by addition of collagen (2.5 to 20 µg/mL) or ADP (10-8 to 3x10-6 mol/L). Where indicated, indomethacin (5x10-5 mol/L) or SQ 29,548 (10-5 mol/L) was given 6 minutes before the start of platelet activation. The extent of aggregation was expressed as a percentage of the maximal change in optical density represented by autologous PPP.

U46619 was obtained from Cayman Chemical Co, and SQ 29,548 was obtained from Bristol-Myers Squibb; all other salts and chemicals were obtained from Sigma-Aldrich Co. Solutions were prepared on the day of the experiment. Data are expressed as mean±SEM. Statistical analyses were performed by 2-way ANOVA for repeated measures followed by Tukey’s post hoc test or Student’s t test, as appropriate. P<0.05 was considered statistically significant.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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The methionine-rich diet elicited a significantly greater concentration of Hcy in the plasma of HHcy compared with control rats (20.6±3.0 and 6.5±0.5 µmol/L, respectively; P<0.01). There was no difference in the body weight between control rats and methionine-fed rats (382±10 and 365±15 g, respectively; NS).

Plasma Fatty Acid Composition and Lipid Peroxidation Products
The 20:4(n-6) arachidonic acid content was significantly increased in plasma lipids of HHcy (n=6) compared with control (n=6) rats (24.6±2.5% and 16.3±1.7%, respectively; P<0.05). The lipoprotein oxidation rate was 17.0±4.0 and 17.5±8.0 nmol · min-1 · mg LDL protein-1; NS), and the thiobarbituric acid–reactive substances content was 12.5±1.5 and 11.8±2.0 nmol/mL (NS) in control and HHcy rats, respectively.

Pressure-Diameter Relationships of Arterioles
Isolated arterioles of gracilis muscle from both control and HHcy rats developed active tone in response to step increases in intraluminal pressure (10 to 140 mm Hg) without the use of any vasoactive agent (Figure 1Down). Initially, the diameter of these vessels increased from {approx}100 to {approx}150 µm in response to an increase in intravascular pressure from 10 to 40 mm Hg. Beyond this point, further increases in pressure resulted in constrictions of arterioles. There was no significant difference in pressure-induced responses of arterioles from the 2 groups of rats. In the absence of Ca2+ and in the presence of 10-4 mol/L SNP, the pressure–passive diameter relationship in each arteriole was also obtained (Figure 1Down). In this condition, step increases in pressure elicited increases in diameter of arterioles, reaching a plateau at 100 mm Hg pressure.



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  		Figure 1. Diameters of isolated skeletal muscle arterioles from control (C, n=10) and HHcy (n=10) rats as a function of perfusion pressure in the presence (circles) and absence (passive; triangles) of Ca2+ in the superfusion solution. Data are mean±SEM.

Arteriolar Responses to BK and U46619
In a dose-dependent manner, BK (10-9 to 10-7 mol/L) elicited significantly greater constrictions in arterioles from HHcy than those from control rats (Figure 2ADown). To elucidate the nature of mediators released in response to BK, we tested arteriolar responses in the presence of the prostaglandin synthesis inhibitor indomethacin and the specific PGH2/TxA2 receptor inhibitor SQ 29,548. Incubation with indomethacin or SQ 29,548 elicited significantly greater inhibition of BK-induced constrictions in arterioles of HHcy than of control rats and eliminated the difference between the responses in the 2 groups (Figures 2BDown and 2CDown). To further identify the constrictor prostanoid released in response to BK, we utilized the specific TxA2 synthase inhibitor furegrelate. Furegrelate also elicited significantly greater inhibition of BK-induced constrictions in arterioles of HHcy than of control rats and eliminated the difference between the responses in the 2 groups (Figure 2DDown).



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  		Figure 2. Constrictions to BK in arterioles of control (C) and HHcy rats under control conditions (A) or in the presence of the cyclooxygenase inhibitor indomethacin (10-5 mol/L; B), the TxA2 receptor antagonist SQ 29,548 (10-6 mol/L; C), or the TxA2 synthase inhibitor furegrelate (5x10-6 mol/L; D). Data are mean±SEM (n=5 to 10). *Significant (P<0.05) difference from control values.

In a dose-dependent manner, the stable TxA2 analogue U46619 (10-9 to 10-6 mol/L) elicited significantly greater constrictions of arterioles from HHcy than those from control rats (Figure 3ADown). Increasing doses of SQ 29,548 (10-8 to 10-7 mol/L) elicited gradual decreases in arteriolar constrictions to U46619, whereas 10-6 mol/L abolished the responses. A selected dose of SQ 29,548 (10-8 mol/L) caused a parallel rightward shift of U46619 dose-response curves in both control and HHcy arterioles (Figure 3BDown). From the parallel shift and assuming a competitive interaction, a pA2 of 8.69±0.18 and 8.56±0.17 (in control and HHcy arterioles, respectively; NS) was calculated.



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  		Figure 3. Constrictions to the stable TxA2 analogue U46619 in arterioles of control (C) and HHcy rats under control conditions (A) or in the presence of the TxA2 receptor antagonist SQ 29,548 (10-8 mol/L; B) or the cyclooxygenase inhibitor indomethacin (Indo, 10-5 mol/L; C) or in the absence of the endothelium ( -Endo; D). Data are mean±SEM (n=5 to 20). *Significant (P<0.05) difference from control values.

Indomethacin significantly decreased constrictions to U46619 in arterioles of HHcy rats. In the presence of indomethacin, constrictions to U46619 were not significantly different between control and HHcy arterioles (Figure 3CUp). To elucidate the cellular source of enhanced release of TxA2, the endothelium of arterioles was removed. Endothelium removal significantly attenuated constrictions to U46619 in arterioles of HHcy rats. In the absence of the endothelium, there was no significant difference between responses to U46619 in arterioles from control and HHcy rats (Figure 3DUp).

Platelet Aggregation Studies
Next we investigated the effect of collagen on aggregation of platelets isolated from the blood of HHcy and control rats. We found that collagen-induced aggregation of platelets from HHcy rats was significantly enhanced compared with that of controls (at 5 µg/mL, controls=23±5% and HHcy=49±5%; Figure 4ADown). Similarly, we found that ADP elicited significantly greater aggregation in HHcy than in control platelets (at 10-7 mol/L, controls=25±3% and HHcy=35±3%; Figure 4BDown). Indomethacin or SQ 29,548 caused greater inhibition of collagen- and ADP-induced aggregation of HHcy platelets compared with controls (Figure 5Down).



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  		Figure 4. Collagen- and ADP-induced (A and B, respectively) aggregation of platelets from control (C) and HHcy rats. Data are mean±SEM (n=6 to 19). *Significant (P<0.05) difference from control values.



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  		Figure 5. Collagen- and ADP-induced aggregation of platelets from control (C) and HHcy rats in the absence or presence of indomethacin (Indo, 5x10-5 mol/L) or SQ 29,548 (10-5 mol/L). Data are mean±SEM (n=8 to 10). *P<0.05 HHcy versus C; #P<0.05 before versus after treatment.


*    Discussion
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 *Discussion
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The salient findings of this study are that elevated plasma Hcy concentration in rats resulted in enhanced arteriolar constrictions to BK and the stable TxA2 analogue U46619 and an augmented collagen- and ADP-induced aggregation of platelets. The underlying mechanism for these alterations is likely an increased TxA2 synthesis in the arteriolar endothelium and platelets in HHcy.

Epidemiological studies have revealed that an elevated level of Hcy is an independent risk factor of human atherosclerosis.1 2 3 4 Previous studies suggested an impaired endothelium-associated dilation in conduit arteries of patients with HHcy.13 14 15 16 In addition, the endothelium-dependent relaxation of carotid artery and an increase in hindlimb circulation to ACh were shown to be reduced in monkeys with diet-induced HHcy.18 The endothelium is important in the local control of blood flow,20 29 35 and in several cardiovascular diseases, such as hypertension20 and atherosclerosis,29 an impaired endothelial regulation of microvascular tone has been documented. Recently, we have demonstrated that NO-mediated endothelial responses are impaired in skeletal muscle arterioles of HHcy rats,19 suggesting an important alteration in endothelial regulation of arteriolar tone.

Interestingly, impairment of endothelial NO synthesis is often associated with alterations of vascular synthesis of prostaglandins.36 37 For example, in hypertension, decreased NO synthesis and increased PGH2/TxA2 production are present simultaneously in skeletal muscle arterioles.20 38 Thus, we hypothesized that the impaired endothelial NO bioavailability in skeletal muscle arterioles would also lead to enhanced PGH2/TxA2 synthesis in HHcy. The methionine diet utilized in the present study19 21 34 39 increased plasma Hcy levels by {approx}3-fold, reaching a concentration similar to what was shown to be associated with an increased risk of vascular disease in humans.1 2 3 4

In the present experiments, we found no significant differences between the pressure-diameter curves of the 2 groups of arterioles (Figure 1Up), suggesting that the myogenic tone and the contractile activity of arteriolar smooth muscle are not affected in general by this model of HHcy, and that the observed alterations in arteriolar responses are not due to changes in the function of smooth muscle.

To test the role of PGH2/TxA2 in the impaired responses of arterioles, we investigated responses to BK, which are known to be mediated by multiple pathways, including eicosanoids. Previous studies in isolated porcine iliac arteries23 and renal afferent arterioles24 demonstrated that 10-9 to 10-7 mol/L BK elicited constriction, primarily due to the release of cyclooxygenase products from the endothelium,23 24 whereas higher concentrations (>10-7 mol/L) of BK exerted a direct effect on the vascular smooth muscle.40 We confirmed these findings by demonstrating that in rat gracilis muscle arterioles, BK-induced constrictions were mediated primarily by constrictor prostanoids, since both inhibition of prostaglandin synthesis with indomethacin and blocking the PGH2/TxA2 receptors with SQ29,548 (Figures 2BUp and 2CUp) inhibited these responses. We found that HHcy significantly enhanced BK-induced constrictions of arterioles (Figure 2AUp), which were likely due to an increased synthesis of PGH2/TxA2, as both indomethacin and SQ 29,548 substantially inhibited the responses, thereby eliminating the difference between HHcy and control arterioles (Figures 2BUp and 2CUp). Furthermore, the findings that the specific TxA2 synthase inhibitor furegrelate30 inhibited BK-induced constrictions and also eliminated the difference between responses in control and HHcy arterioles (Figure 2DUp) indicate that TxA2 is the primary constrictor prostaglandin synthesized to BK in arterioles of HHcy rats.

We also found that constrictions to the stable TxA2 analogue U46619 were enhanced in HHcy (Figure 3AUp). Because SQ 29,548 caused a similar rightward shift in the dose-response curves of control and HHcy arterioles to U46619 (as reflected by the calculated pA2 values; Figure 3BUp), we assumed that an altered TxA2 receptor sensitivity was unlikely to have contributed to the increased constriction to BK and U46619 in HHcy. Previously, it had been shown that vascular responses to U46619 were modulated by a further release of endothelial factors, such as TxA2.41 42 Thus, the increased constriction to U46619, as well as to BK in HHcy, is likely due to an additional release of TxA2 from the arteriolar endothelium. Indeed, the findings that indomethacin or removal of the endothelium decreased constrictions to U46619 in HHcy but not in control arterioles support this hypothesis (Figures 3CUp and 3DUp). Thus, arteriolar release of TxA2 may contribute to the increased urinary excretion of TxB2, the metabolite of TxA2, that has been demonstrated in patients with genetic HHcy.43 44 45 In the present study, we aimed to elucidate whether alterations in platelet function were also related to increased TxA2 activity in HHcy, independent of vascular mechanisms.

To that purpose, platelets were isolated from the blood of control and HHcy rats used for vascular studies, and agonist-induced platelet aggregation in the 2 groups was compared. Collagen and ADP are known to elicit platelet aggregation by a mechanism that involves TxA2 synthesis/release.33 46 We found that collagen- and ADP-induced platelet aggregation was significantly enhanced in HHcy rats (Figure 5Up) and that either inhibition of cyclooxygenase or blocking of the TxA2 receptors (Figure 5Up) eliminated the difference between aggregation of platelets from HHcy and control rats. These results indicate that the increased platelet aggregation in HHcy is most likely due to an enhanced formation of TxA2 in platelets. Earlier studies21 also demonstrated an increased ADP-induced aggregation of platelets associated with an enhanced TxA2 biosynthesis in rats with HHcy. Collectively, these studies suggest that simultaneous elevation of TxA2 synthesis in endothelial cells and platelets is responsible for the increased urinary excretion of TxB2 demonstrated in human HHcy.43 44 45

One of the mechanisms that might favor the formation of TxA2 is an increased formation of arachidonic acid. Indeed, we confirmed earlier findings21 by showing an increased level of arachidonic acid in the plasma of HHcy rats. This could be due to an elevated level of reactive oxygen species shown to be present in HHcy.21 Homocysteine, by the autooxidation of the sulfhydryl group, may promote the generation of oxygen free radicals, resulting in oxidative stress.12 In addition, homocysteine may decrease the intracellular level of glutathione and glutathione peroxidase, which are responsible for the elimination of oxygen free radicals.47 48 An enhanced level of reactive oxygen species is known to interfere with NO, a mechanism that may be responsible for the impaired arteriolar responses to ACh and histamine in HHcy.19 Also, NO and superoxide can form peroxynitrite, which may interfere with PGI2 synthase promoting the elevation of TxA2,49 especially when the level of its precursor arachidonic acid is elevated.

In conclusion, in HHcy, the reduced endothelial release of NO19 together with the simultaneously increased TxA2 synthesis in arterioles and platelets could interfere with endothelial regulation of blood flow and enhance platelet aggregation, thereby predisposing the circulatory system to atherothrombotic alterations.


*    Acknowledgments
 
This study was supported by grants from the Hungarian National Scientific Research Foundation (OTKA T-023863 and T-033117), the Health Science Council (ETT/524), and the National Heart, Lung, and Blood Institute, Bethesda, Md (HL-46813). The authors are indebted to Katalin Rischák for homocysteine measurements.

Received June 25, 1999; accepted February 7, 2000.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Malinow MR, Kang SS, Taylor LM, Wong PWK, Coull B, Inahara T, Mukerjee D, Serton G, Upson B. Prevalence of hyperhomocysteinemia in patients with peripheral arterial occlusive disease. Circulation. 1989;79:1180–1188.[Abstract/Free Full Text]
  2. Ueland PM, Refsum H. Plasma homocysteine, a risk factor for vascular disease: plasma levels in health, disease, and drug therapy. J Lab Clin Med. 1989;114:473–501.[Medline] [Order article via Infotrieve]
  3. Clarke R, Daly L, Robinson K, Naughten E, Cahalene S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med. 1991;313:709–715.[Abstract]
  4. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med. 1997;337:230–236.[Abstract/Free Full Text]
  5. Lentz SR. Mechanisms of thrombosis in hyperhomocysteinemia. Curr Opin Hematol. 1998;5:343–349.[Medline] [Order article via Infotrieve]
  6. Lentz SR. Homocysteine and vascular dysfunction. Life Sci. 1997;61:1205–1215.[Medline] [Order article via Infotrieve]
  7. Harker LA, Ross R, Slichter S, Scott RC. Homocystine-induced arteriosclerosis: the role of endothelial cell injury and platelet response in its genesis. J Clin Invest. 1976;58:731–741.
  8. Nishio E, Watanabe Y. Homocysteine as a modulator of platelet-derived growth factor in vascular smooth muscle cells: a possible role for hydrogen peroxide. Br J Pharmacol. 1997;122:269–274.[Medline] [Order article via Infotrieve]
  9. Toborek M, Kopieczna-Grzebieniak E, Drozdz M, Wieczorek M. Increased lipid peroxidation as a mechanism of methionine-induced atherosclerosis in rabbits. Atherosclerosis. 1995;115:217–224.[Medline] [Order article via Infotrieve]
  10. Hladovec J. Experimental homocysteinemia, endothelial lesions and thrombosis. Blood Vessels. 1979;16:202–205.[Medline] [Order article via Infotrieve]
  11. Wall RT, Harlan JM, Harker LA, Striker GE. Homocysteine-induced endothelial cell injury in vitro: a model for the study of vascular injury. Thromb Res. 1980;18:113–121.[Medline] [Order article via Infotrieve]
  12. Starkebaum G, Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest. 1986;77:1370–1376.
  13. Celermajer DS, Sorensen K, Ryalls M, Robinson J, Thomas O, Leonard JV, Deanfield JE. Impaired endothelial function occurs in the systemic arteries of children with homozygous homocystinuria but not their heterozygous parents. J Am Coll Cardiol. 1993;22:854–858.[Abstract]
  14. Tawakol A, Omland T, Gerhard M, Wu JT, Creager MA. Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation. 1997;95:1119–1121.[Abstract/Free Full Text]
  15. Wo KS, Chook P, Lolin YI, Cheung ASP, Chan LT, Sun YY, Sanderson JE, Metreweli C, Celermajer DS. Hyperhomocyst(e)inemia is a risk factor for arterial endothelial dysfunction in humans. Circulation. 1997;96:2542–2544.[Abstract/Free Full Text]
  16. Bellamy MF, McDowell IF, Ramsey MW, Brownlee M, Bones C, Newcombe RG, Lewis MJ. Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation. 1998;98:1848–1852.[Abstract/Free Full Text]
  17. Lentz SR, Sobey CG, Piegors DJ, Bhopatkar MY, Faraci FM, Malinow RM, Heistad DD. Vascular dysfunction in monkeys with diet-induced hyperhomocyst(e)inemia. J Clin Invest. 1996;98:24–29.[Medline] [Order article via Infotrieve]
  18. Lentz SR. Consequences of hyperhomocyst(e)inemia on vascular function in atherosclerotic monkeys. Arterioscler Thromb Vasc Biol. 1997;17:2930–2934.[Abstract/Free Full Text]
  19. Ungvari Z, Pacher P, Rischak K, Szollar L, Koller A. Dysfunction of nitric oxide mediation in isolated rat arterioles with methionine diet-induced hyperhomocysteinemia. Arterioscler Thromb Vasc Biol. 1999;19:1899–1904.[Abstract/Free Full Text]
  20. Huang A, Koller A. Both nitric oxide and prostaglandin-mediated responses are impaired in skeletal muscle arterioles of hypertensive rats. J Hypertens. 1996;14:887–895.[Medline] [Order article via Infotrieve]
  21. Durand P, Lussier-Cacan S, Blache D. Acute methionine load-induced hyperhomocysteinemia enhances platelet aggregation, thromboxane biosynthesis, and macrophage-derived tissue factor activity in rats. FASEB J. 1997;11:1157–1168.[Abstract]
  22. Fasciolo JC, Vargas L, Lama MC, Nolly H. Bradykinin-induced vasoconstriction of rat mesenteric arteries precontracted with noradrenaline. Br J Pharmacol. 1990;101:344–348.[Medline] [Order article via Infotrieve]
  23. Yu H, Carratero OA, Juncos LA, Garvin JL. Biphasic effect of bradykinin on rabbit afferent arterioles. Hypertension. 1998;32:287–292.[Abstract/Free Full Text]
  24. Persson K, Andersson RGG. Biphasic effect of bradykinin in isolated porcine iliac arteries is mediated by bradykinin B1 and B2 receptors. J Cardiovasc Pharmacol. 1998;31:306–313.[Medline] [Order article via Infotrieve]
  25. Raidoo DM, Ramsaroop R, Naidoo S, Muller-Esterl W, Bhoola KD. Kinin receptors in human vascular tissue: their role in atheromatous disease. Immunopharmacology. 1997;36:153–160.[Medline] [Order article via Infotrieve]
  26. Ulbink JB, Vermak WJH, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in humans serum. J Chromatogr Biomed Appl. 1991;565:441–446.
  27. Durand P, Prost M, Blache D. Folic acid deficiency enhances oral contraceptive-induced platelet hyperactivity. Arterioscler Thromb Vasc Biol. 1997;17:1939–1946.[Abstract/Free Full Text]
  28. Sun D, Kaley G, Koller A. Characteristics and origin of myogenic response in isolated gracilis muscle arterioles. Am J Physiol. 1994;266:H1177–H1183.[Abstract/Free Full Text]
  29. Kuo L, Davis MJ, Cannon MS, Chilian WM. Pathophysiological consequences of atherosclerosis extend into the coronary microcirculation: restoration of endothelium-dependent responses by L-arginine. Circ Res. 1992;70:465–476.[Abstract/Free Full Text]
  30. Lach E, Trifilieff A, Mousli M, Landry Y, Gies JP. Bradykinin-induced contraction of guinea pig lung in vitro. Naunyn Schmiedebergs Arch Pharmacol. 1994;350:201–208.[Medline] [Order article via Infotrieve]
  31. Arunlakshana O, Schild HO. Some quantitative uses of drug antagonists. Br J Pharmacol. 1959;14:48–58.[Medline] [Order article via Infotrieve]
  32. Kosterlitz HW, Watt AJ. Kinetic parameters of narcotic agonists and antagonists, with particular reference to N-allylnoroxymorphone (naloxone). Br J Pharmacol. 1968;33:266–276.[Medline] [Order article via Infotrieve]
  33. McNicol A. Platelet preparation and estimation of functional responses. In: Watson SP, Authi KS, eds. Platelets: A Practical Approach. Oxford, Great Britain: Oxford University Press; 1996:1–25.
  34. Durand P, Fortin LJ, Lussier-Cacan S, Davignon J, Blache D. Hyperhomocysteinemia induced by folic acid deficiency and methionine load: applications of a modified HPLC method. Clin Chim Acta. 1996;252:83–93.[Medline] [Order article via Infotrieve]
  35. Koller A, Messina EJ, Wolin MS, Kaley G. Endothelial impairment inhibits prostaglandin and EDRF-mediated arteriolar dilation in vivo. Am J Physiol. 1989;257:H1966–H1970.[Abstract/Free Full Text]
  36. Tesfamariam B. Selective impairment of endothelium-dependent relaxations by prostaglandin endoperoxide. J Hypertens. 1994;12:41–47.[Medline] [Order article via Infotrieve]
  37. Koller A, Sun D, Messina EJ, Kaley G. L-Arginine analogues blunt prostaglandin-related dilation of arterioles. Am J Physiol. 1993;26:H1194–H1199.
  38. Huang A, Sun D, Koller A. Endothelial dysfunction augments myogenic arteriolar constriction in hypertension. Hypertension. 1993;22:913–921.[Abstract/Free Full Text]
  39. Matthias D, Becker CH, Riezler R, Kindling PH. Homocysteine induced arteriosclerosis-like alterations of the aorta in normotensive and hypertensive rats following application of high doses of methionine. Atherosclerosis. 1996;122:201–216.[Medline] [Order article via Infotrieve]
  40. Briner VA, Tsai P, Schrier RW. Bradykinin: potential for vascular constriction in the presence of endothelial injury. Am J Physiol. 1993;264:F322–F327.[Abstract/Free Full Text]
  41. Weiner CP, Thompson LP, Liu KZ, Herrig JE. Endothelium-derived relaxing factor and indomethacin-sensitive contracting factor alter arterial contractile responses to thromboxane during pregnancy. Am J Obstet Gynecol. 1992;166:1171–1181.[Medline] [Order article via Infotrieve]
  42. Folger WH, Lawson D, Wilcox CS, Mehta JL. Response of rat thoracic aortic rings to thromboxane mimetic U46619: roles of endothelium-derived relaxing factor and thromboxane A2 release. J Pharmacol Exp Ther. 1991;258:669–675.[Abstract/Free Full Text]
  43. Coppola A, Albisinni R, Madonna P, Pagano A, Cerbone AM, Di-Minno G. Platelet and monocyte variables in homocystinuria due to cystathione-ß-synthase deficiency. Hematologica. 1997;82:189–190.[Abstract/Free Full Text]
  44. Di Minno G, Davi G, Margaglione M, Cirillo F, Grandone E, Ciabattoni G, Catalano I, Strisciuglio P, Andria G, Patrono C, Mancini M. Abnormally high thromboxane biosynthesis in homozygous homocystinuria: evidence for platelet involvement and probucol-sensitive mechanism. J Clin Invest. 1993;92:1400–1406.
  45. Vesterqvist O, Green K. Urinary excretion of 2,3-dinor-thromboxane B2 in man under normal conditions, following drugs and during some pathological conditions. Prostaglandins. 1984;27:627–644.[Medline] [Order article via Infotrieve]
  46. Blockmans D, Deckmyn H, Vermylen J. Platelet activation. Blood Rev. 1995;9:143–156.[Medline] [Order article via Infotrieve]
  47. Upchurch GR Jr, Welch GN, Fabian AJ, Pigazzi A, Keaney JF Jr, Loscalzo J. Stimulation of endothelial nitric oxide production by homocyst(e)ine. Atherosclerosis. 1997;132:177–185.[Medline] [Order article via Infotrieve]
  48. Outinen PA, Sood SK, Liaw PC, Sarge KD, Maeda N, Hirsh J, Ribau J, Podor TJ, Weitz JI, Austin RC. Characterization of the stress-inducing effects of homocysteine. Biochem J. 1998;332:213–221.
  49. Zou M, Jendral M, Ullrich V. Prostaglandin endoperoxide-dependent vasospasm in bovine coronary arteries after nitration of prostacyclin synthase. Br J Pharmacol. 1999;126:1283–1292.[Medline] [Order article via Infotrieve]



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