P. Cutini

Testosterone modulates platelet aggregation and endothelial cell growth through nitric oxide pathway

The aim of the present study was to investigate the effect of testosterone on the modulation of cellular events associated with vascular homeostasis. In rat aortic strips, 5-20 min treatment with physiological concentrations of testosterone significantly increased nitric oxide (NO) production. The rapid action of the steroid was suppressed by the presence of an androgen receptor antagonist (flutamide). We obtained evidence that the enhancement in NO synthesis was dependent on the influx of calcium from extracellular medium, because in the presence of a calcium channel blocker (verapamil) the effect of testosterone was reduced. Using endothelial cell (EC) cultures, we demonstrated that androgen directly acts at the endothelial level. Chelerythrine or PD98059 compound completely suppressed the increase in NO production, suggesting that the mechanism of action of the steroid involves protein kinase C and mitogen-activated protein kinase pathways. It is known that endothelial NO released into the vascular lumen serves as an inhibitor of platelet activation and aggregation. We showed that testosterone inhibited platelet aggregation and this effect was dependent on endothelial NO synthesis. Indeed, the enhancement of NO production elicited by androgen was associated with EC growth. The steroid significantly increased DNA synthesis after 24 h of treatment, and this mitogenic action was blunted in the presence of NO synthase inhibitor N-nitro-l-arginine methyl ester. In summary, testosterone modulates vascular EC growth and platelet aggregation through its direct action on endothelial NO production.

The role of sex steroids on cellular events involved in vascular disease.

In this work we checked the hypothesis whether estrone, progesterone, and testosterone are able to modulate the interactions between platelets, monocytes, and endothelial cells either under basal or inflammatory conditions. Using adhesion assays we demonstrated that pretreatment of endothelial cells with estrone, progesterone, or testosterone prevented monocytes and platelets adhesion induced by the proinflammatory agent bacterial lipopolysaccharide. The hormones reduced the expression of mRNA of ICAM-1, VCAM-1, and P-selectin, endothelial surface proteins that mediate monocytes and platelets adhesion respectively. Integrins are the main leukocyte proteins that allow firm adhesion. Using flow cytometry we showed that estrone treatment of monocytes reduced CD11b and CD11c expression, either under basal or injury (lipopolysaccharide) conditions. The three steroids inhibited platelet aggregation in a nitric oxide dependent manner. Platelet function was not affected by the steroid treatment. The molecular mechanisms of action exerted by the steroids included the participation of the intracellular signaling pathways PKC, MAPK, and PI3K, which selectively and differentially mediate the stimulation of nitric oxide release. We evidence that estrone, progesterone, and testosterone modulate monocyte and platelet adhesion to endothelial cells, events that play a major role in the initiation and progression of vascular lesions. The steroid action was evidenced under basal or inflammatory conditions. The mechanisms of action exerted by the steroids included stimulation of nitric oxide production and the participation of PKC, MAPK, and PI3K systems.

The soyabean isoflavone genistein modulates endothelial cell behaviour.

The aim of the present study was to investigate the direct action of the phyto-oestrogen genistein (Gen) on vascular endothelial behaviour, either in the presence or absence of proinflammatory agents. In rat aortic endothelial cell (EC) cultures, 24 h of treatment with Gen significantly increased cell proliferation in a wide range of concentration (0.001-10 nm). This mitogenic action was prevented by the oestrogen receptor (ER) antagonist ICI 182780 or by the presence of the specific NO synthase inhibitor l-nitro-arginine methyl ester. When monocytes adhesion to EC was measured, Gen partially attenuated leucocyte adhesion not only under basal conditions, but also in the presence of bacterial lipopolysaccharides (LPS). The effect of the phyto-oestrogen on the expression of EC adhesion molecules was evaluated. Gen down-regulated the enhancement in mRNA levels of E-selectin, vascular cell adhesion molecule-1 and P-selectin elicited by the proinflammatory agent bacterial LPS. The regulation of EC programmed death induced by the isoflavone was also demonstrated. Incubation with 10 nm Gen prevented DNA fragmentation induced by the apoptosis inductor H2O2. The results presented suggest that Gen would exert a protective effect on vascular endothelium, due to its regulatory action on endothelial proliferation, apoptosis and leucocyte adhesion, events that play a critical role in vascular diseases. The molecular mechanism displayed by the phyto-oestrogen involved the participation of the ER and the activation of the NO pathway.

Cross-talk between rapid and long term effects of progesterone on vascular tissue

We tested the hypothesis whether; the non-genomic action of progesterone (Pg) on vascular tissue would be associated with hormonal long term effect on the modulation of cell growth. Using rat aortic strips, we showed that the stimulatory effect of Pg on nitric oxide synthesis involved both kinase and phosphatase pathways. The increase in the vasoactive production was prevented by the MAPK inhibitor (PD98059). In addition, preincubation with a phosphatase antagonist potentiated the hormonal effect. Pg increased PKC activity, but the inhibition of PKC did not alter the stimulatory action of the hormone on nitric oxide generation. In endothelial cell cultures (EC), 24h treatment with Pg significantly diminished cell proliferation. This antiproliferative effect was suppressed by the PKC inhibitor chelerythrine (chel) and l-NAME (nitric oxide synthase inhibitor). We also observed that Pg stimulates EC migration. In summary, the present findings provide evidence of an integration of genomic and non-genomic effects in the mechanism of action displayed by Pg in vascular tissue. The fast effects elicited by the hormone implies signal transduction activation required for the regulation of vasoactive production, but also necessary for the modulation of endothelial cells growth.

Role of progesterone on the regulation of vascular muscle cells proliferation, migration and apoptosis

The purpose of this study was to investigate the effect of progesterone (Pg) on cellular growth, migration, apoptosis, and the molecular mechanism of action displayed by the steroid. To that end, rat aortic vascular smooth muscle cell (VSMC) cultures were employed. Pg (10nM) significantly increased [(3)H]thymidine incorporation after 24h of treatment. The enhancement in DNA synthesis was blunted in the presence of an antagonist of Pg receptor (RU486 compound). The mitogenic action of the steroid was suppressed by the presence of the compounds PD98059 (MEK inhibitor), chelerythrine (PKC inhibitor), and indomethacin (cyclooxygenase antagonist) suggesting that the stimulation of DNA synthesis involves MAPK, PKC, and cyclooxygenase transduction pathways. The proliferative effect of the hormone depends on the presence of endothelial cells (EC). When muscle cells were incubated with conditioned media obtained of EC treated with Pg, the mitogenic action of the steroid declined. Wounding assays shows that 10nM Pg enhances VSMC migration and motility. The role of the steroid on programmed cell death was measured using DNA fragmentation technique. Four hours of treatment with 10nM Pg enhanced DNA laddering in a similarly extent to the apoptotic effect induced by the apoptogen hydrogen peroxide (H(2)O(2)). In summary the results presented provide evidence that Pg enhances cell proliferation, migration, and apoptosis of VSMC. The modulation of cell growth elicited by the steroid involves integration between genomic and signal transduction pathways activation.

Differential regulation of endothelium behavior by progesterone and medroxyprogesterone acetate

Medroxyprogesterone acetate (MPA) is a synthetic progestin commonly used in hormone replacement therapy (HRT). The aim of this research was to study and compare the effect of progesterone (Pg) and MPA on the regulation of cellular events associated with vascular homeostasis and disease. Platelet adhesion to endothelial cells (ECs), nitric oxide (NO) production, and cell migration were studied using murine ECs in vitro exposed to the progestins. After 7 min of treatment, MPA significantly inhibited NO synthesis with respect to control values; meanwhile, Pg markedly increased vasoactive production. In senile ECs, the stimulatory action of Pg decreases; meanwhile, MPA maintained its ability to inhibit NO synthesis. The presence of RU486 antagonized the action of each steroid. When ECs were preincubated with PD98059 (MAPK inhibitor) or chelerythrine (protein kinase C (PKC) inhibitor) before Pg or MPA treatment, the former totally suppressed the steroid action, but the PKC antagonist did not affect NO production. In the presence of a PI3K inhibitor (LY294002), a partial reduction in Pg effect and a reversal of MPA action were detected. Using indomethacin, the contribution of the cyclooxygenase (COX) pathway was also detected. On platelet adhesion assays, Pg inhibited and MPA stimulated platelet adhesion to ECs. Under inflammatory conditions, Pg prevented platelet adhesion induced by lipopolysaccharide (LPS); meanwhile, MPA potentiated the stimulatory action of LPS. Finally, although both steroids enhanced migration of ECs, MPA exhibited a greater effect. In conclusion, the data presented in this research provide evidence of a differential regulation of vascular function by Pg and MPA.

Vascular action of bisphosphonates: In vitro effect of alendronate on the regulation of cellular events involved in vessel pathogenesis

In this work we investigate whether, despite the procalcific action of alendronate on bone, the drug would be able to regulate in vitro the main cellular events that take part in atherosclerotic lesion generation. Using endothelial cell cultures we showed that Alendronate (1-50μM) acutely enhances nitric oxide production (10-30min). This stimulatory action of the bisphosphonate involves the participation of MAPK signaling transduction pathway. Under inflammatory stress, the drug reduces monocytes and platelets interactions with endothelial cells induced by lipopolysaccharide. Indeed the bisphophonate exhibits a significant inhibition of endothelial dependent platelet aggregation. The molecular mechanism of alendronate (ALN) on leukocyte adhesion depends on the regulation of the expression of cell adhesion related genes (VCAM-1; ICAM-1); meanwhile the antiplatelet activity is associated with the effect of the drug on nitric oxide production. On vascular smooth muscle cells, the drug exhibits ability to decrease osteogenic transdifferentiation and extracellular matrix mineralization. When vascular smooth muscle cells were cultured in osteogenic medium for 21days, they exhibited an upregulation of calcification markers (RUNX2 and TNAP), high alkaline phosphatase activity and a great amount of mineralization nodules. ALN treatment significantly down-regulates mRNA levels of osteoblasts markers; diminishes alkaline phosphatase activity and reduces the extracellular calcium deposition. The effect of ALN on vascular cells differs from its own bone action. On calvarial osteoblasts ALN induces cell proliferation, enhances alkaline phosphatase activity, and increases mineralization, but does not affect nitric oxide synthesis. Our results support the hypothesis that ALN is an active drug at vascular level that regulates key processes involved in vascular pathogenesis through a direct action on vessel cells.