Study of garlic extracts and fractions on cholesterol plasma levels and vascular reactivity in cholesterol-fed rats.

Garlic is known for its pharmacologic and nutritional properties. In previous studies, garlic elicited a reduction in plasma levels of lipids by inhibiting hepatic cholesterol synthesis. The aim of this study was to investigate in an in vivo model the effects of garlic extract and some fractions on cholesterol levels and vascular reactivity in cholesterol-fed rats. Rats were fed a cholesterol-enriched diet for 16 wk and were divided into 10 groups as follows: control and hypercholesterolemic diet groups, 4 groups fed frozen garlic fractions and 4 groups fed raw garlic fractions with different doses. Blood samples were obtained to analyze HDL and LDL cholesterol levels. After treatment, rats were killed. The heart, liver and kidneys were weighed; the aorta was isolated, mounted in organ chambers and vascular reactivity was tested. Plasma concentration of cholesterol was 58 mg/dL (100%) at the beginning of the study and increased to 102 mg/dL (153%; hypercholesterolemic group) at the end of the treatment. Plasma total cholesterol decreased in all groups treated with garlic; moreover, this effect was higher in rats fed raw garlic fractions and extracts. LDL decreased significantly with respect to the hypercholesterolemic group in all groups treated with garlic fractions and extracts (P: < 0.01); however, an increase in HDL was found in those treated with frozen fractions and extracts. The liver:body weight ratio decreased in all treated groups. The relaxing effect of acetylcholine (ACh) was enhanced in arteries contracted with noradrenaline (NE). These data suggest that garlic fractions could prevent diet-induced hypercholesterolemia and vascular alterations in the endothelium-dependent relaxation associated with atherosclerosis.

Growth inhibitory activity of indapamide on vascular smooth muscle cells.

Abnormal vascular smooth muscle cell proliferation has a fundamental role in the pathogenesis of vascular diseases. Indapamide is an oral diuretic antihypertensive drug effective for patients with mild or moderate essential hypertension. We now investigated the effects of indapamide on the growth of aortic vascular smooth muscle cells (A10 cell line). Indapamide inhibited cell proliferation as measured by the tetrazolium salt XTT (sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) test. The increase in cell number was significantly reduced in the presence of indapamide 10(-6) and 5 x 10(-4) M (P < 0.05 n = 3 and P < 0.01, n = 3, respectively). Serum-induced DNA synthesis, determined as the incorporation of 5-bromo-2'-deoxyuridine (BrdU), was concentration-dependently inhibited by indapamide. BrdU incorporation was 47.2+/-1.6% (10% foetal calf serum). Indapamide treatment markedly prevented BrdU incorporation (37.2+/-2.1%, 29.2+/-4.8%, 15.0+/-1.8%, 8.7+/-2.1%) indapamide 10(-6), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. Cell-cycle progression was also evaluated. Flow cytometry analysis of DNA content in synchronised cells revealed blocking of the serum-inducible cell-cycle progression by indapamide. This inhibition was abolished when the drug was added 2 h after serum repletion, indicating that indapamide must act at the early events of a cell cycle to be fully effective against DNA synthesis. In addition, serum-induced intracellular Ca2+ movements and also p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation were studied in the presence or absence of indapamide. Indapamide 10(-5) and 5 x 10(-5) M decreased significantly cytosolic free calcium, and the p44/p42 mitogen-activated protein kinase phosphorylation (5 x 10(-5) M) stimulated by 10% foetal calf serum. In accordance with this finding, indapamide (5 x 10(-4) M) caused a 95% to 99% decrease in the early elevation of c-fos expression as evaluated by northern blot analysis of mRNA induced after serum addition. In conclusion, our results indicate that indapamide reduces vascular smooth muscle cell proliferation by a mechanism which involves a decrease in the intracellular Ca2+ movements that might link with the mitogen-activated protein kinase (MAPK) pathway, altering cell-cycle progression.

Effect of trandolapril on vascular responsiveness in cholesterol-fed rabbit-isolated arteries.

According to the World Health Organisation, cardiovascular disorders are one of the main causes of morbi/mortality in the western world. The effect of trandolapril (0.3 mg kg(-1) day(-1)), a non-sulphydryl angiotensin-converting enzyme (ACE) inhibitor, on the vascular responsiveness in aorta isolated from hypercholesterolemic rabbits was examined. Three groups of rabbits (n=30) were used: Group 0 (control group); Group 1 (hypercholesterolemic group, 0.5% (wt/wt) cholesterol-enriched diet) and Group 2 (hypercholesterolemic+trandolapril 0.3 mg kg(-1) day(-1)). After 18 weeks of treatment, the rabbits were killed and the thoracic aorta, proximal coronary and mesenteric (5th branch) arteries were isolated, cleaned off and mounted in an organ bath. Trandolapril had no significant effect on plasma cholesterol, high density lipoprotein (HDL) or low density lipoprotein (LDL). Despite the lack of effect of the drug on the above-mentioned parameters, treatment with trandolapril improved endothelium-dependent relaxation induced by acetylcholine in aortic and mesenteric rings from hypercholesterolemic rabbits treated with trandolapril. The relaxation induced by 10(-5) M acetylcholine were 65.0+/-4.0%; 24. 0+/-9.4% (P<0.01, n=10) and 51.3+/-7.0% (P<0.01, n=10) in aortic rings from Groups 0, 1 and 2, respectively, and 50.0+/-12.0%; 10. 1+/-10.0% (P<0.01, n=10); 61.0+/-9.7% (P<0.01, n=10) in small mesenteric rings from Groups 0, 1 and 2, respectively. In addition, trandolapril treatment improved the increase in serotonin-induced contraction in proximal coronary arteries with respect to the hypercholesterolemic group. On the other hand, we did not find any differences among the group in endothelium-independent relaxation induced by sodium nitroprusside. These results provide evidence that trandolapril restores endothelium-dependent relaxation in hypercholesterolemic rabbit-isolated arteries. These data suggest that trandolapril might have beneficial action in the prevention of vascular alteration involved in atherosclerosis.

L-Citrulline, the by-product of nitric oxide synthesis, decreases vascular smooth muscle cell proliferation.

Endothelium injury plays an important role in atherosclerosis. Damage to the endothelium results in vascular smooth muscle cell proliferation. Natriuretic peptides present a potent antimitogenic action, mediating their biological effects via the binding of guanylate cyclase-linked atrial natriuretic peptide (ANP) receptor and the production of cyclic GMP. In a previous study, we demonstrated that L-citrulline, the by-product of nitric oxide synthesis, could relax rabbit aortic rings by stimulating the guanylate cyclase-linked ANP receptor. In this work, we investigated the effect of L-citrulline on vascular smooth muscle cell proliferation. L-Citrulline (10(-8) M) significantly decreased rat aortic (A10 cell line) vascular smooth muscle proliferation. The percentage of inhibition exerted by L-citrulline on days 3, 5, and 7 of the proliferation curve was 20.0 +/- 0.5%, 37.5 +/- 8.3%, and 28. 5 +/- 7.2%, respectively. In addition, L-citrulline also inhibited serum-induced DNA synthesis, measured as 5-bromo-2'-deoxyuridine incorporation. 5-Bromo-2'-deoxyuridine incorporation into nuclei of vehicle-treated cells was 40.5 +/- 2.4%, whereas in L-citrulline-treated cells the percentage decreased to 36.0 +/- 4.1%, 29.1 +/- 2.0% (P <.01, n = 4), 30.5 +/- 2.4% (P <.05, n = 4), and 23.1 +/- 0.5% (P <.001, n = 4) for 10(-10), 10(-9), 10(-8), and 10(-7) M, respectively. Zaprinast, a phosphodiesterase type V inhibitor, enhanced 5-bromo-2'-deoxyuridine incorporation in serum-stimulated cells. Moreover, L-citrulline inhibition of serum-stimulated DNA synthesis was abolished by HS-142-1 (10(-5) M), an ANP receptor antagonist. In another group of experiments, L-citrulline was shown to increase intracellular cyclic GMP levels from 2.1 +/- 0.2 pmol of cGMP/mg protein to 4.1 +/- 0.1 for L-citrulline (10(-8) M) (P <.001, n = 3). These findings suggest that L-citrulline decreases vascular smooth muscle cell proliferation in the A10 cell line by acting on DNA synthesis by mechanisms that involve the ANP receptor.

Study of Calcium Dobesilate in Diabetic Rats.

According to the World Health Organization (WHO) 74% of diabetic patients die of vascular complications. Previous reports have shown that endothelium-dependent relaxation of diabetic vasculature is more sensitive to free radical-induced injury. Calcium dobesilate (DOBE) has been successfully used in the treatment of diabetic retinopathy. The aims of this study were to investigate the in vivo and ex vitro effects of DOBE on both contractile and relaxing responses in isolated diabetic rat aorta. Four groups of rats were used: Wistar rats (Group 0); spontaneously diabetic rats (BB/wor rats) (Group 1); BB/wor rats treated with DOBE 50 mg/kg/day (Group 2); and BB/wor rats treated with 500 mg/kg/day (Group 3). At 180 days after the development of diabetes, the animals were killed and the thoracic aorta were isolated, cleaned off, and mounted in an organ chamber. Two groups of experiments were carried out. In the first group (in vitro), incubation with DOBE 10(-4) in aortic rings isolated from BB/wor rats decreased the contraction induced by noradrenaline (NA) 10(-6) M (1.21 +/- 0.11 g vs 0.67 +/- 0.01 g P < 0.01, n = 8 in diabetic rings with or without the presence of DOBE 10(-4) M, respectively), and this decrease was prevented by propranolol 10(-6) M (1.20 +/- 0.6 g). DOBE 10(-5) and 10(-4) M increased the endothelium-dependent relaxation induced by ACh in BB/wor rats [the maximal relaxation with ACh 10(-5) M was 50.0 +/- 5.1 vs 72.0 +/- 11.0 (p < 0.05, n = 8) and 69.0 +/- 7.8 (p < 0.05, n = 8) in BB/wor rats and after the incubation with DOBE 10(-5) and 10(-4) M, respectively], however, incubation with DOBE did not modify the endothelium-independent relaxation in these rats. In the second part of the study (ex vitro), we found an increase in the endothelium-dependent relaxation in arteries from diabetic rats treated with DOBE (Groups 2) compared with Group 1 (BB/wor rats) although we did not find any improvement in the endothelium-independent relaxation. Thus, in spontaneously diabetic rats, DOBE restored endothelium-dependent, but not independent, relaxation to normal and also decreased the contractile responses induced by NA through a mechanism that involves beta-adrenergic receptors.

In vitro effects of calcium dobesilate on the responsiveness of spontaneously diabetic rat aorta.

We tested the effect of calcium dobesilate (DOBE) in aorta from spontaneously diabetic (BB/wor) rats. The contraction induced by 10(-6) M noradrenaline (NA) in BB/wor rats was smaller than that induced in control rats (1.21+/-0.11 vs 0.82+/-0.02 g, P<0.01, n=8, respectively) in arteries with intact endothelium. Incubation with DOBE (10(-4) M) impaired the contractions induced by NA in BB/wor rats (1.21+/-0.11 vs 0.67+/-0.01 g, P<0.01, n=8). The effect of DOBE was reversed by 10(-6) M propranolol (0.67+/-0.01 vs 1.20+/-0.60g, P<0.001, n=8, with 10(-4) M DOBE and 10(-4)M DOBE plus 10(-6) M propranolol, respectively). DOBE increased the endothelium-dependent relaxation in arteries from diabetic rats. These findings suggest that DOBE might improve vascular reactivity in BB/wor rats.