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Intimal hyperplasia, resulting from a complex cascade of events involving platelets, leukocytes, and smooth muscle cells, may be inhibited by the HMG-CoA reductase inhibitor pravastatin, which demonstrates inhibition of platelet activity and leukocyte adhesion and may be associated with inhibition of vascular smooth muscle cell proliferation and migration. Clopidogrel, an adenosine diphosphate (ADP) receptor inhibitor, was shown to decrease platelet activity and aggregation but not intimal hyperplasia (IH). We postulated that the combination of both pravastatin and clopidogrel would significantly decrease IH in a rat carotid endarterectomy model. Male Sprague-Dawley rats (n = 18) divided by treatment regimen underwent treatment for 2 weeks both before and after an open carotid endarterectomy. Serum collected at the time of harvest was measured for C-reactive protein (CRP), platelet activity, and total serum cholesterol; carotid arteries were removed and processed for IH determination. Control rats (n = 7) received oral vehicle daily before and following endarterectomy. Pravastatin-alone rats (n = 6) received oral pravastatin (10 mg/kg/day) before and after endarterectomy. Pravastatin plus clopidogrel rats (n = 5) received oral pravastatin (10 mg/kg/day) plus a preendarterectomy bolus of oral clopidogrel (4.3 mg/kg) before endarterectomy and resumed pravastatin (10 mg/kg/day) plus oral clopidogrel (1 mg/kg/day) postendarterectomy. Pravastatin alone and pravastatin plus clopidogrel significantly decreased CRP compared to controls (120.2 +/-11.2 and 134.1 +/- 9.9 vs 191.1 +/- 9.2 microg/mL, respectively p = 0.003 and p =0.0024). CRP levels were not different between pravastatin alone and pravastatin plus clopidogrel (p = 0.35). Platelet activity was significantly decreased by pravastatin alone and pravastatin plus clopidogrel in comparison to controls (7.3 +/- 2.2 and 6.6 +/- 2.8 vs 19.2 +/- 6.1 platelet reactive units (PRU), respectively p = 0.048 and p = 0.045). No significant difference was noted in platelet activity between pravastatin alone and pravastatin plus clopidogrel (p = 0.89). Pravastatin plus clopidogrel significantly reduced serum cholesterol compared to control and pravastatin alone (84.0 +/- 6.6 vs 110.4 +/- 7.4 and 117.0 +/- 8.8 mg/dL, respectively p = 0.03 and p = 0.01). Pravastatin alone did not decrease serum cholesterol compared to controls (p = 0.54). IH was not reduced by pravastatin alone compared to controls (p = 0.61) but was significantly decreased by pravastatin plus clopidogrel in comparison to control and pravastatin alone (3.0 +/- 1.1 vs 46.3 +/- 13.7 and 37.4 +/- 14.6% luminal stenosis, respectively p = 0.01 and p = 0.05). Pravastatin plus clopidogrel significantly decreased CRP, platelet activity, total serum cholesterol, and IH while pravastatin alone decreased only CRP and platelet activity. Intimal hyperplasia reduction may therefore be dependent on other contributors, possibly growth factors, cytokines, and oxidative stress. The combination of pravastatin plus clopidogrel may have synergistic or even additional inhibitory effects on IH. Pravastatin plus clopidogrel was effective in decreasing IH in a rat carotid endarterectomy model and may prove a useful therapy for IH reduction in the clinical setting.

Intimal hyperplasia results in significant morbidity and mortality following vascular intervention. Both platelets and elevated homocysteine have been implicated in the development of intimal hyperplasia. We previously demonstrated that a locally applied antiplatelet agent decreases the development of intimal hyperplasia. We were therefore interested in a systemic antiplatelet agent, clopidogrel. We hypothesized that clopidogrel would decrease platelet aggregation and activity and intimal hyperplasia. Male Sprague-Dawley rats underwent carotid endarterectomy (CEA) and treatment with either placebo or varying regimens of clopidogrel, including chronic, pre-CEA bolus, chronic plus pre-CEA bolus, and chronic plus post-CEA bolus; a homocystine diet was used to elevate both plasma homocysteine and the degree of intimal hyperplasia. Platelet aggregation, platelet activity, and intimal hyperplasia were then assessed. Platelet aggregation was not decreased with chronic clopidogrel; however, it was decreased with pre-CEA bolus clopidogrel. Similarly, platelet activity was not inhibited by chronic clopidogrel but was inhibited by pre-CEA and chronic plus pre-CEA bolus clopidogrel. Neither chronic, pre-CEA bolus, chronic plus pre-CEA bolus, nor chronic plus post-CEA bolus clopidogrel resulted in a decrease in intimal hyperplasia. Although pre-CEA bolus clopidogrel resulted in a decrease in both platelet aggregation and activity, it was unable to decrease the development of intimal hyperplasia at any dose. Additional factors must therefore contribute to the pathologic development of intimal hyperplasia.

Background: This study investigated Saratin's (Merck KGaA, Darmstadt, Germany) prevention of platelet adhesion and intimal hyperplasia at different doses and in the hyperhomocystinemia rat carotid endarterectomy (CEA) model., Methods: Rats were divided into two groups: (1) platelet adhesion or (2) luminal stenosis because of intimal hyperplasia. At CEA, rats received 0, 0.5, 5.0, 10.0, or 20.0 microg Saratin on the artery. Post-CEA platelet aggregation was evaluated by standard error of the mean. Intimal hyperplasia group received either (1) control or (2) 4.5 g/kg DL-homocystine diets for two weeks followed by CEA and treated with diluent or 5.0 microg Saratin. Endpoints included platelet adhesion, intimal hyperplasia, plasma homocysteine (HCys), and its metabolic enzymes cystathionine beta-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR)., Results: Platelet adhesion: post-CEA, platelet adhesion was reduced by 63%, 67%, and 67% in Saratin doses > or =5.0 microg. Intimal hyperplasia: 5.0 microg Saratin in the HCys group decreased intimal hyperplasia by 45% compared with the non-Saratin-treated HCys group. Plasma HCys levels were not altered with Saratin treatment in the HCys groups nor were CBS or MTHFR., Conclusions: Saratin significantly inhibited platelet adhesion at > or =5.0 microg, and Saratin at 5.0 microg attenuated luminal stenosis in a hyperhomocysteinemic rat CEA model.

Background: Homocysteine and smoking are independent risks for CVD; however their importance in post-CEA intimal hyperplasia is unclear. We performed a CEA in rats exposed to cigarette smoke with the hypothesis that smoking would increase intimal hyperplasia that may be associated with an elevated serum homocysteine. Folic acid (FA) and the homocysteine metabolic enzymes MTHFR and CBS were used to test for the significance of homocysteine elevation., Materials and Methods: Rats underwent an open CEA. N = 13 rats received smoke exposure 2 weeks prior, and 2 weeks post-CEA and N = 12 received no smoke. Each group was divided into either control or an FA-added diet resulting in four groups. Rats were sacrificed at 2 weeks post-CEA; liver, urine, blood, and carotid arteries samples were obtained., Results: Smoked rats had increased urinary peak and trough cotinine levels versus non-smoke rats, which decreased with FA. Smoke exposure increased intimal hyperplasia versus non-smoke controls by nearly 120% (57.8 +/- 6.2 versus 26.8 +/- 5.4% luminal stenosis, P = 0.005). Smoke-exposed rats had an increased serum homocysteine versus non-smoke controls (8.3 +/- 0.8 versus 5.7 +/- 0.8 microm, P = 0.014). Smoked rats given FA had decreased serum homocysteine compared to the smoke group. Along with reductions in homocysteine, FA eliminated the increase in intimal hyperplasia seen with smoke exposure (33.5 +/- 6.1 versus 57.8 +/- 6.2% luminal stenosis, P = 0.03). CBS activity decreased in smoked rats by nearly 20% versus non-smoke rats. FA supplementation in smoked rats both (1) increased CBS activity and (2) decreased MTHFR compared to control non-smoke-exposure levels., Conclusion: Smoking increases plasma homocysteine and post-CEA intimal hyperplasia. This suggests homocysteine has an etiological role in the intimal hyperplasia increase observed with smoking, since both were negated with FA.

Hyperhomocysteinemia, a known risk factor for cardiovascular disease, results in an elevation of intimal hyperplasia (IH) following a carotid endarterectomy (CEA) in a rat model. An exaggerated IH response following CEA has been observed in rats with dietary induced hyperhomocysteinemia. Type 2 diabetics often present with hyperhomocysteinemia and are at higher risk for developing vascular blockage following surgical procedures. To determine if insulin resistance increases IH risks following endarterectomy, the 3 goals of this study were: (1) to establish plasma homocysteine concentrations in dietary induced insulin-resistant rats and their controls, (2) to investigate whether a positive correlation of IH and plasma homocysteine response occurs following CEA in the insulin-resistant rat model, and (3) if so, to attempt to decrease IH by supplementation with folic acid, a known enzymatic cofactor in the homocysteine metabolic pathway. To achieve these aims, male rats (275 to 300 g) were fed 1 of 4 diets for a 4-month period: (1) high-fat sucrose (HFS), (2) low-fat complex carbohydrate (LFCC), (3) HFS + 25 mg/kg folic acid (HFS+F), or (4) LFCC + 25 mg/kg folic acid (LFCC+F). At the end of the 4-month period the rats underwent an open (non-balloon) unilateral CEA. Two weeks post-endarterectomy, blood, liver and carotid tissue were removed to measure plasma insulin, folic acid, and homocysteine, 2 key enzymes of homocysteine metabolism-methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS)-and percent lumenal stenosis (IH%). Computer-assisted morphometric analysis was used to measure the percentage of IH in the carotid artery. Plasma homocysteine was significantly higher in the HFS group when compared with the LFCC group (11.3+/-1.3 micromol/L v 7.4+/-0.6 mircomol/L, P=.008) as was post-endarterectomy IH producing lumenal stenosis (30.7%+/-4.2% v 14.0%+/-4.3%, P=.008). Plasma insulin in the HFS group was higher than the LFCC (control) group and was significant (36.3+/-3.0 microU/mL v 21.1+/-0.8 microU/mL, P=.0004). Folic acid supplementation in the HFS group resulted in reductions of plasma homocysteine (HFS v HFS+F, 11.3+/-1.3 micromol/L v 7.95+/-1.0 micromol/L, P=.02) and post-endarterectomy IH (HFS v HFS+F, 30.7%+/-4.2 % v 10.4%+/-1.6%, P=.0001). The control or LFCC group was not statistically different from the HFS+F group in homocysteine or IH. Folate supplementation did not decrease insulin concentrations in the HFS+F group compared to the LFCC group. We conclude that the HFS diet produced an insulin-resistant state with an elevated plasma homocysteine and an exaggerated IH response following carotid endarterectomy in this rat model. Dietary folate supplementation reduced plasma homocysteine concentrations in the HFS diet, which implicates hyperhomocysteinemia as an etiologic factor in the development of post-CEA IH in this insulin-resistant rat model.

Prosthetic dialysis access thrombosis and/or stenosis is the most common cause of graft impairment or loss and is primarily attributed to venous outflow stenosis due to intimal hyperplasia. Intimal hyperplasia is thought to result from interactions between areas of exposed subendothelial collagen in an injured vessel and platelets, resulting in platelet adhesion. Saratin, an inhibitor of the vWF-dependent binding of platelet to collagen interaction, has been shown in vitro to reduce the adhesion of platelets to collagen. In the current study, the authors investigated the effects of topical saratin administration in a canine dialysis access model in regard to intimal hyperplasia development at the venous anastomosis. Fourteen female mongrel dogs underwent placement of a femoral polytetrafluoroethylene (PTFE) dialysis access graft and were placed into 1 of 2 groups: 1) control or 2) experimental with topical saratin application. The experimental group had 600 microg of saratin (1 microg/microL) applied for 5 minutes directly onto the venous anastomosis before restoration of blood flow;control groups received vehicle control. At 4 weeks postoperative, a portion of the graft was removed along with a segment of the outflow vein. Veins were subsequently processed, sectioned, and analyzed along the length of the excised segment and divided into blocks that included the area of the graft toe, midanastomotic region and heel, and blocks A-E. Intimal hyperplasia was assessed by a computer-assisted morphometric analysis. Platelet counts and bleeding times were also measured. Vein segments in the control group (n=7) showed pronounced intimal hyperplasia in blocks B, C, and D as compared to the saratin group (n=6). Distribution of intimal hyperplasia by blocks between control and saratin groups were as follows: block [A] 8.6 +/- 1.9 vs 9.7 +/- 3.0% (p=NS), [B] 32.7 +/- 6.3 vs 10.7 +/- 3.5% (p=0.01), [C] 44.8 +/- 6.2% vs 10.3 +/- 1.5% (p=0.0004), [D] 40.8 +/- 11.0 vs 9.1 +/- 4.2% (p=0.02), [E] 7.5 +/- 5.5 vs 2.7 +/- 0.4% (p=NS). Intimal hyperplasia normalized to vein wall thickness also showed a significant reduction with saratin application. Bleeding times and platelet counts obtained at different time points during the experiment showed no difference between control and saratin groups. In a canine dialysis access model using PTFE grafts, topical application of saratin at the venous anastomosis decreased intimal hyperplasia development by as much as 77% when compared with control animals. Saratin provides for a method of substantially reducing intimal hyperplasia by direct local application without systemic side effects.

Objective: Hyperhomocysteinemia has been implicated as a causative factor in intimal hyperplasia development. The addition of dietary folate in a hyperhomocysteinemia, carotid endarterectomy rat model is postulated to decrease plasma homocysteine levels and, in turn, reduce post-carotid endarterectomy intimal hyperplasia., Methods: Each rat was fed one of six diets: (1) lab chow with no folate (n = 7), (2) lab chow with 10 mg/kg folate added (n = 3), (3) lab chow with 25 mg/kg folate added (n = 3), (4) a homocysteine diet with no folate (n = 7), (5) a homocysteine diet with 10 mg/kg folate added (n = 5), or (6) homocysteine diet with 25 mg/kg folate added (n = 5). Each rat then underwent an open carotid endarterectomy. In 2 weeks, intimal hyperplasia in the carotid artery was measured. Plasma homocysteine and folate levels were measured., Results: Plasma folate levels rose with folate administration. Plasma homocysteine in the lab chow group was 5.4 +/- 0.5 micromol/L and did not change with the addition of folate. In the homocysteine diet group, plasma homocysteine rose 10-fold over the lab chow group (51.9 +/- 6.5 vs 5.4 +/- 0.5, micromol/L, P <.0001). In the group fed a homocysteine diet with 10 mg/kg folate added, a significant decrease in plasma homocysteine was observed (17.5 +/- 8.5 vs 51.9 +/- 6.5, micromol/L, P =.0003). In the group fed a homocysteine diet with 25 mg/kg folate added, plasma homocysteine levels were further reduced to levels seen in the lab chow group (12.6 +/- 2.6 vs 5.4 +/- 0.5, micromol/L, P = not significant). The relationship between plasma folate and homocysteine was inverse (R = 0.39, P =.0036). Luminal stenosis due to intimal hyperplasia was minimal in lab chow groups and unaffected by folate. The homocysteine diet group demonstrated post-carotid endarterectomy luminal stenosis due to intimal hyperplasia (60.9% +/- 9.2%). In the group fed a homocysteine diet with 10 mg/kg folate added, intimal hyperplasia was reduced, compared with the homocysteine diet group (32.6% +/- 7.4% vs 60.9% +/- 9.2%, P =.009). In the group fed a homocysteine diet with 25 mg/kg folate added, intimal hyperplasia was reduced to lab chow group levels (10.8% +/- 0.8% vs 4.8% +/- 1.0%, P = not significant) and was reduced, compared with the group fed a homocysteine diet with 10 mg/kg folate added., Conclusion: The use of folate in this hyperhomocysteinemia carotid endarterectomy model and the resultant attenuation of plasma homocysteine elevation and intimal hyperplasia development lend strong support to homocysteine being an independent etiologic factor in post-carotid endarterectomy intimal hyperplasia.

Purpose: This preliminary study investigated the ability to elevate the serum homocysteine (H[e]) levels and investigated the increases in postoperative neointimal hyperplasia (IH) in an environment with hyperhomocysteinemia and the resultant restenosis in a rat carotid endarterectomy (CEA) model., Method: The 9 rats for the control group were fed rat chow, and the 8 rats for the H(e) group were fed H(e)-supplemented rat chow for 2 weeks before and after CEA. The animals underwent anesthesia, and a left common CEA was performed. After 14 days, the serum H(e) levels were measured and the left carotid artery was harvested and elastin stained. Morphometric measurements were used to calculate the area of stenosis of the lumen. The mean and the standard deviation of the mean were determined. The 2 groups were compared with the Mann-Whitney test and a linear regression model. Three additional rats per group were studied, with carotid artery sectioning with double immunohistochemical staining for 5-bromodeoxyuridine (BrdU) and alpha-smooth muscle (alpha-SM) actin., Results: The serum H(e) level in the H(e) group was 36.32 micromol/L +/- 15.28, and in the control group the level was 5.53 micromol/L +/- 2.06 (P =.0007). IH presented as percent lumen stenosis was 21.89% +/- 4.82% in the H(e) group and 4.82% +/- 1.64% in the control group (P =.0007). The linear regression model of the serum H(e) levels and the percent stenosis showed a linear relationship (r2 =.72). The alpha-SM actin staining revealed that nearly all of the cells in the IH area were of smooth muscle or myofibroblast origin and that 10.1% +/- 2.6% of the cells were stained for BrdU in the control group versus 23% +/- 7.1% in the H(e) group. Also, 9.3% +/- 2.6% of the cells in the IH area were stained for BrdU and for alpha-SM actin versus 19.1% +/- 5. 6% stained for both BrdU and alpha-SM actin in the H(e) group., Conclusion: This is the first study to examine IH after CEA and hyperhomocysteinemia in rats. The study shows that the elevation of serum H(e) levels can be obtained by feeding rats modified diets with added H(e). The consistent elevation of serum H(e) levels was associated with more than 4 times the amount of IH after a CEA in a rat model.