Cilostazol Inhibits Accumulation of Triglyceride in Aorta and Platelet Aggregation in Cholesterol-Fed Rabbits

Cilostazol is clinically used for the treatment of ischemic symptoms in patients with chronic peripheral arterial obstruction and for the secondary prevention of brain infarction. Recently, it has been reported that cilostazol has preventive effects on atherogenesis and decreased serum triglyceride in rodent models. There are, however, few reports on the evaluation of cilostazol using atherosclerotic rabbits, which have similar lipid metabolism to humans, and are used for investigating the lipid content in aorta and platelet aggregation under conditions of hyperlipidemia. Therefore, we evaluated the effect of cilostazol on the atherosclerosis and platelet aggregation in rabbits fed a normal diet or a cholesterol-containing diet supplemented with or without cilostazol. We evaluated the effects of cilostazol on the atherogenesis by measuring serum and aortic lipid content, and the lesion area after a 10-week treatment and the effect on platelet aggregation after 1- and 10-week treatment. From the lipid analyses, cilostazol significantly reduced the total cholesterol, triglyceride and phospholipids in serum, and moreover, the triglyceride content in the atherosclerotic aorta. Cilostazol significantly reduced the intimal atherosclerotic area. Platelet aggregation was enhanced in cholesterol-fed rabbits. Cilostazol significantly inhibited the platelet aggregation in rabbits fed both a normal diet and a high cholesterol diet. Cilostazol showed anti-atherosclerotic and anti-platelet effects in cholesterol-fed rabbits possibly due to the improvement of lipid metabolism and the attenuation of platelet activation. The results suggest that cilostazol is useful for prevention and treatment of atherothrombotic diseases with the lipid abnormalities

Serum TG analysis by HPLC.

<p>Lipoproteins in 10 week treatment serum samples were analyzed by HPLC. The mean value of the control group is shown by a red line and the cilostazol group by the blue line and the normal diet group by the green line. Control [n = 5], cilostazol [n = 5], normal diet [n = 3].</p

Changes of serum lipids and biomarkers.

<p>Concentrations of serum lipids and biomarkers were measured before and 2, 4, 6, 8, 10 weeks after drug administration. Data represent means ± S.D. Control [n = 5], cilostazol [n = 5]. *: p<0.05 (group*time), †: p<0.05 (group) versus control by repeated measures ANOVA. TC: total cholesterol, TG: triglyceride, FC: free cholesterol, HDL-C: high density lipoprotein cholesterol, PL: phospholipid, GOT: glutamate oxaloacetate transaminase, GPT: glutamate pyruvate transaminase, ALP: alkaline phosphatase, and CRP: C-reactive protein. TC, TG, FC, HDL, PL is shown at mg/dL, GOT, GPT, ALP at U/L and CRP at ng/mL.</p

Comparison of atherosclerotic areas in aorta.

<p>Plaques areas in the aorta of a typical animal in each group are shown by black areas (A). Individual values in the control group are shown by closed triangles (previous study) and closed circles (present study) and those in the cilostazol group by opened triangles (previous study) and opened circles (present study) and the horizontal bars in each group represent the mean and the vertical bars are the SD (B). Control [n = 10], cilostazol [n = 10]. *: p<0.05, **: p<0.01 versus control by a two-way ANOVA.</p

Platelet aggregation in rabbits.

<p>Comparison of platelet aggregation between normal and hypercholesterolemic rabbits (A). Effect of cilostazol on platelet aggregation in normal rabbits (B) or hypercholesterolemic rabbits (C). The control group are shown by closed circles and the cilostazol group by open circles. Correlation between serum TC and PATI values (D). Normal rabbits are shown by closed triangles and hypercholesterolemic rabbits by open triangles. Data represent means ± S.D. Normal rabbit study: control [n = 8], cilostazol [n = 8], hypercholesterolemic rabbit study: control [n = 5], cilostazol [n = 5]. **: p<0.01 versus control by t-test. PATI: Platelet aggregatory threshold index, TC: total cholesterol, R: correlative rate.</p

Immunohistochemical staining of macrophages in the proximal ascending aorta.

<p>Cross sections of the proximal ascending aorta were stained by an anti-macrophage RAM11 antibody. The brown part represents the macrophage-positive area. Typical images are shown for the control and cilostazol groups. Two of the five samples in the cilostazol group were not stained by the RAM11 antibody (the right image of cilostazol), although all samples were stained in the control group.</p

Add-on effect of probucol in atherosclerotic, cholesterol-fed rabbits treated with atorvastatin.

OBJECTIVE: Lowering the blood concentration of low-density lipoprotein (LDL) cholesterol is the primary strategy employed in treating atherosclerotic disorders; however, most commonly prescribed statins prevent cardiovascular events in just 30% to 40% of treated patients. Therefore, additional treatment is required for patients in whom statins have been ineffective. In this study of atherosclerosis in rabbits, we examined the effect of probucol, a lipid-lowering drug with potent antioxidative effects, added to treatment with atorvastatin. METHODS AND RESULTS: Atherosclerosis was induced by feeding rabbits chow containing 0.5% cholesterol for 8 weeks. Probucol 0.1%, atorvastatin 0.001%, and atorvastatin 0.003% were administered solely or in combination for 6 weeks, beginning 2 weeks after the start of atherosclerosis induction. Atorvastatin decreased the plasma concentration of non-high-density lipoprotein cholesterol (non-HDLC) dose-dependently; atorvastatin 0.003% decreased the plasma concentration of non-HDLC by 25% and the area of atherosclerotic lesions by 21%. Probucol decreased the plasma concentration of non-HDLC to the same extent as atorvastatin (i.e., by 22%) and the area of atherosclerotic lesions by 41%. Probucol with 0.003% atorvastatin decreased the plasma concentration of non-HDLC by 38% and the area of atherosclerotic lesions by 61%. Co-administration of probucol with atorvastatin did not affect the antioxidative effects of probucol, which were not evident on treatment with atorvastatin alone, such as prevention of in vitro LDL-oxidation, increase in paraoxonase-1 activity of HDL, and decreases in plasma and plaque levels of oxidized-LDL in vivo. CONCLUSIONS: Probucol has significant add-on anti-atherosclerotic effects when combined with atorvastatin treatment; suggesting that this combination might be beneficial for treatment of atherosclerosis

An Atherogenic Paigen-Diet Aggravates Nephropathy in Type 2 Diabetic OLETF Rats

<div><p>Diabetic nephropathy develops in association with hyperglycemia, is aggravated by atherogenic factors such as dyslipidemia, and is sometimes initiated before obvious hyperglycemia is seen. However, the precise mechanisms of progression are still unclear. In this study, we investigated the influence of an atherogenic Paigen diet (PD) on the progression of nephropathy in spontaneous type 2 diabetic OLETF rats. Feeding PD to male OLETF rats for 12 weeks caused an extensive increase in excretion of urinary albumin and markers of tubular injury such as KIM-1 and L-FABP, accompanied by mesangial expansion and tubular atrophy. PD significantly increased plasma total cholesterol concentration, which correlates well with increases in urine albumin excretion and mesangial expansion. Conversely, PD did not change plasma glucose and free fatty acid concentrations. PD enhanced renal levels of mRNA for inflammatory molecules such as KIM-1, MCP-1, TLR4 and TNF-α and promoted macrophage infiltration and lipid accumulation in the tubulointerstitium and glomeruli in OLETF rats. Intriguingly, PD had little effect on urine albumin excretion and renal morphology in normal control LETO rats. This model may be useful in studying the complex mechanisms that aggravate diabetic nephropathy in an atherogenic environment.</p></div

An atherogenic Paigen-diet worsens the nephropathy of OLETF rats.

<p>Time-dependent changes in urine albumin excretion (A), semi-quantitative analyses of glomerular sclerosis (B) and plasma lipids and glucose concentrations (C) 12 weeks after starting to feed chow or an atherogenic diet, presented as mean ± SD. There were five animals in the following groups: LETO rats fed normal chow (LETO-NC) and Paigen diet (LETO-PD), and OLETF rats fed NC (OLETF-NC) and Western diet (OLETF-WD); and nine in the OLETF rats fed PD (OLETF-PD) groups. *p < 0.05; **p < 0.01; ns, not significant vs OLETF-NC group among three OLETF groups by Dunnett’s test. #p < 0.05; ##p < 0.01; NS, not significant by unpaired t-test. There were no significant differences between LETO-NC and LETO-PD or LETO-NC and OLETF-NC groups in urine albumin (unpaired t-test). TC, total cholesterol; TG, triacylglycerol.</p