Importance of intracellular angiotensin II in vascular smooth muscle cell apoptosis: inhibition by the angiotensin AT1 receptor antagonist irbesartan.

The intracellular uptake of Angiotensin II has been described, although its physiological role is not yet understood. We aimed to study the role of Angiotensin II internalization in Angiotensin II-induced apoptosis. Vascular smooth muscle cells were cultured from male Wistar-Kyoto rats and treated with Angiotensin II (1 microM, 48 h). Apoptosis was assessed by DNA fragmentation, cell cytometry and caspase-3 activity. The Angiotensin AT(1) receptor antagonist irbesartan (0.1-10 microM) and the inhibitors of Angiotensin II internalization phenylarsine oxide (PAO, 20 microM), but not the AT(2) receptor antagonist PD123319 (S-(+)-1-[(4-(Dimethylamino)-3-methylphenyl)methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid di(trifluoroacetate) salt), decreased Angiotensin II-mediated apoptosis. Pre-treatment with irbesartan, but not with PD123319, blocked Angiotensin II internalization. We found a strong correlation between intracellular Angiotensin II staining and Angiotensin II-induced apoptosis for all compared groups. We therefore conclude that internalization of Angiotensin II is involved in apoptosis of vascular smooth muscle cells induced by this peptide.

Role of TGF-beta1 in vascular smooth muscle cell apoptosis induced by angiotensin II.

Both Angiotensin II and transforming growth factor beta-1 (TGF-beta1) are important mediators of vascular smooth muscle cell function and have been reported to mediate the balance between proliferation and apoptosis. Some crosstalk between Angiotensin II and TGF-beta1 in end-organ hypertension has been established. However, whether TGF-beta1 is able to mediate Angiotensin II-induced vascular cell damage remains unknown. Vascular smooth muscle cells were obtained from rat thoracic aorta and cultured in 10% foetal calf serum. In all experiments, medium was changed to a low-serum (0.4% foetal calf serum) or serum-free one with or without Angiotensin II. Apoptosis was assessed by DNA fragmentation, DNA synthesis was measured as bromo-deoxyuridine uptake. TGF-beta1 production was determined by Enzyme-linked Immunosorbent Assay (ELISA) from cell conditioned media, RT-PCR from cell lysates and confocal immunostaining of fixed cells. Angiotensin II induced apoptosis in the absence of DNA synthesis when coincubated at 1 microM. Neither the specific anti-TGF-beta1 monoclonal antibody (50 microg/ml) nor the novel activin-like kinase (ALK)-4/5/7 synthetic inhibitor SB-431542 (4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide) at 10 microM were able to inhibit this effect. Angiotensin II induced expression of TGF-beta1 without further secretion of this cytokine. This effect was not affected by incubation with the AT1 inhibitor irbesartan (10 microM). A pharmacological approach to TGF-beta1 inhibition would be unable to reverse the apoptotic effect of Angiotensin II on vascular smooth muscle cells.

Expresión de COX-2 en arterias y células vasculares de pacientes diabéticos y no diabéticos / COX-2 expression in arteries and vascular cells from diabetic and nondiabetic patients

Introducción. El objetivo de este trabajo fue estudiar la expresión de ciclooxigenasa 2 (COX-2) en arterias y células de músculo liso vascular provenientes de pacientes diabéticos y no diabéticos, así como su inducción tras incubar células de pacientes no diabéticos con un preparado de triglicéridos. Material y métodos. Se utilizaron segmentos de arterias mamarias internas usadas en cirugía de revascularización coronaria, y se procedió a homogeneizarlos, fijarlos o cultivarlos para obtener células de músculo liso vascular. La expresión de COX-2 se analizó por Western blot y microscopia confocal. Para inducir in vitro la expresión de COX-2, se incubaron las células de pacientes no diabéticos con Intralipid®. Resultados. Todas las medidas indicaron una mayor expresión de COX-2 tanto en arterias como en células de pacientes diabéticos. La expresión de COX-2 se correlacionó con el índice EuroSCORE, como índice pronóstico de mortalidad precoz y de complicaciones mayores en la cirugía coronaria, menos favorable. Asimismo, la expresión de COX-2 se incrementó de forma significativa con concentraciones crecientes de Intralipid®. Conclusiones. La expresión de COX-2 en arterias mamarias internas se relaciona con la diabetes mellitus y un índice EuroSCORE más desfavorable. También es posible aumentar la expresión de COX-2 incubando las células de pacientes no diabéticos con triglicéridos, lo que refuerza la asociación entre hipertrigliceridemia, diabetes tipo 2, inflamación vascular y un peor pronóstico clínico (AU)

Introduction. The aim of this study was to investigate cyclooxygenase (COX)-2 expression in arteries and vascular smooth muscle cells from diabetic and nondiabetic patients. A further aim was to analyze COX-2 induction after cells from nondiabetic patients were incubated with a triglyceride-rich solution. Matherial and methods. Segments from internal mammary arteries discarded after coronary artery bypass grafting were used. Arterial segments were homogenized, fixed, or prepared for vascular smooth muscle cell cultures. COX-2 expression was assessed by both Western blot and confocal microscopy. For in vitro induction of COX-2 expression, cells from nondiabetic patients were incubated with Intralipid®. Results. All measurements showed increased COX-2 expression in the arteries and cells from diabetic patients. COX-2 expression was correlated with a poorer prognosis measured by the EuroSCORE scale. In addition, COX-2 expression was significantly enhanced when cells were incubated with increasing Intralipid® concentrations. Conclusions. COX-2 expression in internal mammary arteries is related to type 2 diabetes and a poorer EuroSCORE. In addition, COX-2 expression was experimentally increased when cells from nondiabetic patients were incubated with triglycerides, which enhances the associations among hypertriglyceridemia, type 2 diabetes, vascular inflammation, and a poorer clinical prognosis (AU)

A cholesterol-enriched diet induces ultrastructural changes in retinal and macroglial rabbit cells.

The purpose of this study was to ascertain whether the excess of cholesterol in rabbits induces ultrastructural retinal changes similar to those observed in human age-related macular degeneration (AMD). New Zealand rabbits were divided into two groups: Control (GO; n=10), fed standard diet for 8 months; hypercholesterolemic (G1; n=10), fed with 0.5% cholesterol-enriched diet for 8 months. Eyes were processed for transmission electron microscopy (TEM) and immunohistochemistry (anti-glial fibrillary acidic protein, GPAP). In comparison with GO, G1 exhibited alterations in all the retinal layers that were more intense in areas overlying altered retinal pigment epithelium (RPE). RPE changes showed no preferential location. In G1, Bruch's membrane was thicker as a result particle build-up in the collagen layers; the cytoplasm of RPE showed dense bodies, debris from cell membranes, vacuoles and numerous clumps of lipids; necrosis and apoptosis were detected in different retinal layers; Müller cells and astrocytes were reactive with instances of apoptosis and necrosis; some Müller cells filled up the empty spaces left by degenerated neurons in all retinal layers; some Müller cell nuclei were displaced to the nerve-fiber layer (NFL); epiretinal perivascular astrocytes contained drops of lipids; the NFL had very few astrocytes and the basal membranes of capillaries in the NFL was thicker. Excess cholesterol induces ultrastructural changes in the rabbit retina similar to those in human AMD. Given that lipid intake is most dependent on food composition, dietary regimen could help induce or prevent retinal disease.

Human vascular smooth muscle cells from diabetic patients are resistant to induced apoptosis due to high Bcl-2 expression.

An emerging body of evidence suggests that vascular remodeling in diabetic patients involves a perturbation of the balance between cell proliferation and cell death. Our aim was to study whether arteries and vascular smooth muscle cells (VSMCs) isolated from diabetic patients exhibit resistance to apoptosis induced by several stimuli. Internal mammary arteries (IMAs) were obtained from patients who had undergone coronary artery bypass graft surgery. Arteries from diabetic patients showed increasing levels of Bcl-2 expression in the media layer, measured by immunofluorescence and by Western blotting. Human IMA VSMCs from diabetic patients showed resistance to apoptosis, measured as DNA fragmentation and caspase-3 activation, induced by C-reactive protein (CRP) and other stimuli, such as hydrogen peroxide and 7beta-hydroxycholesterol. The diabetic cells also exhibited overexpression of Bcl-2. Knockdown of Bcl-2 expression with Bcl-2 siRNA in cells from diabetic patients reversed the resistance to induced apoptosis. Consistent with the above, we found that pretreatment of nondiabetic VSMCs with high glucose abolished the degradation of Bcl-2 induced by CRP. Moreover, cell proliferation was increased in diabetic compared with nondiabetic cells. This differential effect was potentiated by glucose. We conclude that the data provide strong evidence that arterial remodeling in diabetic patients results from a combination of decreased apoptosis and increased proliferation.

Kaempferol inhibits apoptosis in vascular smooth muscle induced by a component of oxidized LDL.

Antioxidants such as flavonoids afford protection against oxysterols-induced toxicity. We have investigated the effect of kaempferol and rutin, active components of red wine, in the apoptosis induced by 7beta-hydroxycholesterol in rat vascular smooth muscle cells. 7beta-Hydroxycholesterol induced apoptosis in vascular smooth muscle which include BcL-x(L) degradation, caspase-3 activation and DNA fragmentation. The apoptosis induced by 7beta-hydroxycholesterol was prevented by pretreatment with kaempferol (10-30 microM), but not with rutin. Interestingly preincubation with the estrogen receptor alpha antagonist ICI 182,780 (1 microM) prior to kaempferol partially reverted the antiapoptotic effect of this flavonoid on caspase-3 activation and DNA fragmentation induced by 7beta-hydroxycholesterol. In conclusion, the flavonoid kaempferol, unlike rutin, diminished the apoptosis induced by a component of oxidized low-density lipproteins (oxLDL). This effect was partially mediated by the estrogen receptor alpha.

Compromised arterial function in human type 2 diabetic patients.

Diabetes is associated with a perturbation of signaling pathways in vascular tissue, which causes vasomotor dysfunction such as hypertension and accelerated atherosclerosis. In the present study, the mechanisms of vasomotor dysfunction, Akt (Thr308 and Ser473) phosphorylation and expression of endothelial NO (nitric oxide) synthase, and inducible NO synthase were investigated in human diabetic internal mammary arteries. The phospho-Akt (Thr308) level in arteries from diabetic patients was reduced to about one-half of the level in nondiabetic patients, suggesting impaired insulin signaling in human diabetic vascular tissue. Augmented vasoconstriction was observed in diabetic arteries, due in part to deficiency of basal and stimulated NO production. This correlated with decreased endothelial NO synthase expression and activity in diabetic vessels. The sensitivity of diabetic vessels to the NO donor, sodium nitroprusside, was reduced as well, suggesting that NO breakdown and/or decreased sensitivity of smooth muscle to NO are also responsible for abnormal vasoconstriction. In addition, the abnormal vasoconstriction in diabetic vessels was not completely abolished in the presence of Nomega-nitro-L-arginine methyl ester, revealing that NO-independent mechanisms also contribute to vasomotor dysfunction in diabetes. In conclusion, diabetes downregulates the Akt-signaling pathway and compromises human arterial function through a decrease in NO availability as well as through NO-independent mechanisms.

Relationship between vasodilation capacity and phenolic content of Spanish wines.

We aimed to determine: 1) the concentration of polyphenols in Spanish red wines, 2) the vasodilatory properties of those wines in relation with their polyphenol concentrations and 3) the vasodilation induced by some of these polyphenols in rat aortic rings. In the wines studied the concentration of rutin and kaempferol was high compared with other polyphenols. All wines relaxed precontracted rat aortic rings and this effect was directly related with the concentration of myricetin and kaempferol in the wines. Kaempferol and rutin also induced endothelium-dependent and independent relaxation, kaempferol was more potent. This relaxation was not inhibited by the estrogen receptor alpha antagonist ICI 182,760. Kaempferol also potentiated the endothelium-dependent relaxation induced by acetylcholine, which was reversed by Nw-nitro-l-arginine methyl ester (l-NAME). These findings show a good correlation between the concentration of polyphenols (especially kaempferol) of Spanish red wines and the vasodilatory effect, which may confer on them unique features in the prevention of cardiovascular disease.

Pioglitazone induces vascular smooth muscle cell apoptosis through a peroxisome proliferator-activated receptor-gamma, transforming growth factor-beta1, and a Smad2-dependent mechanism.

Thiazolidinediones, such as pioglitazone, seem to exert direct antiatherosclerotic and antirestenotic effects on type 2 diabetes, in part due to an induction of vascular smooth muscle cell (VSMC) apoptosis. We aimed to study the role of transforming growth factor (TGF)-beta in rat aortic VSMC. Pioglitazone at 100 micromol/l increased apoptosis without affecting DNA synthesis, and this effect was reversed by an anti-TGF-beta1 antibody. Extracellular TGF-beta1 levels were rapidly increased after treatment with pioglitazone in a peroxisome proliferator-activated receptor (PPAR)-gamma-dependent mechanism because this secretion was blocked by the PPAR-gamma inhibitor GW9662. Pioglitazone subsequently increased the nuclear recruitment of phospho-Smad2, without any effect on protein expression. According to our results, we propose that the apoptotic effect of pioglitazone on VSMC depends on the following sequence: PPAR-gamma activation, TGF-beta1 release, and selective phospho-Smad2 nuclear recruitment. Management of Smad signaling on VSMC might provide future clinical benefits in vascular diseases.

Calcium dobesilate attenuates vascular injury and the progression of diabetic retinopathy in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetic retinopathy (DR) is a highly specific vascular complication of type 1 and type 2 diabetes mellitus. Calcium dobesilate (DOBE) has been tested in the treatment of diabetic retinopathy showing a slowdown of the progression of the disease after long-term oral treatment. The aim of this study was to determine the effects of DOBE on vascular and diabetic retinopathy in streptozotocin (STZ) diabetic rats. METHODS: Diabetes was induced in wistar rats by the administration of STZ (60 mg/kg, i.p.). Rats were divided into three groups (n = 30). Group 0 (GO): nondiabetic rats. Group 1 (G1): 14 months of insulin treatment after diabetes development. Group 2 (G2): 14 months of insulin treatment after diabetes development plus DOBE (500 mg/kg/day). At the end of the treatment, vascular reactivity was tested. The study of the vascularization of the retina was performed on wholemounts of trypsin retinal digest preparations and retinal sections. RESULTS: Relaxation induced by acetylcholine decreased in the aorta arteries from diabetic rats but it was restored to control values in the DOBE-treated group (71.8 +/- 4.5%, 53.3 +/- 0.5%, 67.4 +/- 4.6% in group 0, 1 and 2 respectively). DOBE treatment also restored noradrenaline (1.08 +/- 0.05 g, 1.70 +/- 0.08 g, 1.13 +/- 0.05 g in group 0, 1 and 2 respectively) and caffeine-induced contractions. Diabetic state did not cause any alteration in mesenteric arteries. The analysis of the retinal digests showed vascular tortuosity, acellular capillaries, focal accumulations of capillaries and reduction of the number of pericytes in G1. The vascular changes observed in G2 seem to be intermediate between the control and the diabetic rats. CONCLUSIONS: We showed that long-term treatment with DOBE attenuated the progression of diabetic retinopathy and the alterations in vascular reactivity in streptozotocin-induced diabetic rats.

An in vitro study of different extracts and fractions of Allium sativum (garlic): vascular reactivity.

The aim of this study was to investigate the effect of different novel extracts and fractions obtained from Allium sativum (garlic) on in vitro vessel contraction in order to deepen our knowledge of their mechanism of action on vascular reactivity. The contraction induced by noradrenaline (NE, 10(-6) or 10(-5) M) or KCl (80 mM) was relaxed with all the extracts and fractions studied, but this effect was higher with RG 20-100 (raw garlic fraction) and FG 20-100 (frozen garlic fraction). To increase our understanding of the mechanisms of action of RG 200-100 and FG 200-100, we found their inhibitory actions were retained in the absence of endothelium, whereas inhibition of the entry of extracellular calcium and mobilization of intracellular calcium may play an instrumental role.

Effect of somatostatin on rabbit isolated coronary arteries.

Somatostatin analogues are capable of inhibiting vascular smooth muscle and endothelial cell proliferation. However, little is known about the effect of somatostatin on vascular responses in endothelium-denuded coronary arteries in vitro. The aim of this work was to determine whether or not somatostatin prevented the contractile response induced by 5-hydroxytryptamine and acetylcholine in endothelium-denuded rabbit coronary arteries. Somatostatin attenuated the contraction produced by 5-hydroxytryptamine in both proximal (PC) and distal coronary (DC) arteries (contraction induced by 10(-4) M 5-hydroxytryptamine was inhibited by 10(-6) M somatostatin by 90.8 +/- 11.0% (P < 0.001, n = 9) and by 46.2 +/- 14.0% (P < 0.05, n = 9) in DC and PC, respectively), but concentration-dependently decreased the contraction induced by U46619 (11alpha-epoxy-methanoprostaglandin F2alpha) only in PC arteries, suggesting that the response of PC and DC arteries to somatostatin were qualitatively different. Furthermore, we suggest that somatostatin may enhance acetylcholine-induced relaxation by combination of increasing endothelium-dependent relaxation (by a NO-dependent mechanism) and blocking contraction at the muscle level.