Clinical review 145: Pleiotropic effects of statins: lipid reduction and beyond.

There is accumulating evidence that statins have beneficial effects that are independent of their classical actions on lipoproteins. These effects include reductions in inflammation in the vasculature, kidney, and bone. Potential beneficial effects of these agents include enhancement of nitric oxide production in vasculature and the kidney. These agents appear to reduce bone fractures and may improve insulin sensitivity and reduce the likelihood of persons progressing from impaired glucose tolerance to type II diabetes. Potential beneficial pleiotropic effects of statins are covered in this review.

Role of PI3-kinase in isoproterenol and IGF-1 induced ecNOS activity.

Phosphatidylinositol 3-kinase (PI3-K) has been shown to mediate insulin and insulin-like growth factor-1 (IGF-1)-induced nitric oxide (NO) generation and, thus, vascular tone. A role for PI3-K in G-protein-coupled receptor signal transduction has been reported. As beta (beta2)-adrenergic vascular actions are partly dependent on NO, we have investigated the role of PI3-K in isoproterenol (Iso) and IGF-1 induced endothelial NO synthase (ecNOS) activity in rat aortic endothelial cells (RAEC). Cell lysates of RAEC, exposed to Iso (10 micromol/L) for 5 min and 6 h, and to IGF-1 (100 nM) for 10 min and 6 h, or pretreated with PI3-K inhibitor Wortmannin (WT), were used for measuring PI3-K activity, p85kDa regulatory protein, and citrulline production. Results show that Iso and IGF-1 increased a p85 subunit and citrulline production, and also enhanced 32P incorporation into PIP3. Pretreatment with WT inhibited Iso-stimulated ecNOS, as well as, PI3-K activity. Iso enhanced association of ecNOS with the triton X-100-insoluble fraction of RAEC. These data indicate that the endothelial cell PI3-K pathway mediates, in part, the release of NO and subsequent vasorelaxation in response to this beta-agonist, as well as, IGF-1.

Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells.

Inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs) is upregulated in arterial injury and plays a role in regulating VSMC proliferation and restenosis. Inflammatory cytokines [e.g., interleukin-1beta (IL-1beta)] released during vascular injury induce iNOS. Small GTP-binding proteins of the Ras superfamily play a major role in IL-1beta-dependent signaling pathways. In this study, we examined the role of Rho GTPases in regulating iNOS expression in VSMCs. Treatment of VSMCs with mevastatin, which inhibits isoprenylation of Rho and other small GTP-binding proteins, produced significantly higher amounts of IL-1beta-evoked NO and iNOS protein compared with control. Similarly, bacterial toxins [Toxin B from Clostridium difficile and C3 ADP-ribosyl transferase (C3) toxin from Clostridium botulinium] that specifically inactivate Rho proteins increased NOS products (NO and citrulline) and iNOS expression. Toxin B increased the activity of iNOS promoter-reporter construct in VSMCs. Both toxins enhanced IL-1beta-stimulated iNOS expression and NO production. These data demonstrate for the first time that inhibition of Rho induces iNOS and suggest a role for Rho protein in IL-1beta-stimulated NO production in VSMCs.

Interleukin-1beta-induced nitric oxide production in rat aortic endothelial cells: inhibition by estradiol in normal and high glucose cultures.

Expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide (NO) production are associated with septic shock, atherosclerosis, and cytokine-induced vascular injury. Estrogen is known to impact vascular injury and vascular tone, in part through regulation of NO production. In the current study, we examined the effect of physiological concentrations of estradiol on interleukin-1beta (IL-1beta)-induced NO production in rat aortic endothelial cells (RAECs). 17Beta-estradiol significantly decreased IL-1beta-induced iNOS protein levels and reduced NO production in RAECs. High glucose (25 mM) elevated the increase in IL-1beta-induced iNOS protein and NO production. Nevertheless, estradiol still inhibited IL-1beta-induced iNOS and NO production even in the presence of high glucose. These data suggest that estradiol may exert its beneficial effects in part by inhibiting induction of endothelial iNOS, a possible mechanism for the protective effect of estradiol against diabetes-associated cardiovascular complications.

Calcium and protein kinase C mediate high-glucose-induced inhibition of inducible nitric oxide synthase in vascular smooth muscle cells.

Abnormal vascular smooth muscle (VSMC) proliferation is a key feature in diabetes-associated atherosclerotic disease. Since nitric oxide inhibits VSMC tone, migration, adhesion, and proliferation, we examined the effects of high glucose on IL-1beta-induced NO release from VSMCs in culture. Confluent smooth muscle cells, preincubated with either 5 mmol/L (mM) or 20 mmol/L (mM) glucose for 48 hours, were stimulated with IL-1beta. Nitrite was measured in the culture medium after 24 hours. IL-1beta-induced a 15-fold increase in NO production in normal glucose medium. Glucose (10 to 30 mmol/L (mM)) significantly reduced the response to IL-1beta. High glucose (20 mmol/L (mM)) inhibited IL-1beta-evoked NO production by approximately 50%. IL-1beta-stimulated [3H] citrulline-forming activity of the nitric oxide synthase (NOS) was also significantly lower in high-glucose-exposed cells, and this was reflected in diminished cellular levels of NOS protein. To assess the role of protein kinase C (PKC), membrane PKC activity was measured, and glucose (20 mmol/L (mM)) significantly increased it. Immunoblotting of the membranes revealed a glucose-induced increase in the PKC betaII isoform. 1,2-Dioctanoyl-glycerol, a PKC activator, mimicked the high-glucose effect on IL-1beta-induced NO release, while staurosporine, a PKC inhibitor, reversed it. The role of calcium in the glucose-mediated inhibition of cytokine-induced NO release was determined by treatment with BAPTA, an intracellular chelator of calcium. BAPTA partially reversed the inhibitory effects of glucose. Increasing intracellular calcium by A23187, an ionophore or thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, significantly decreased IL-1beta-induced NO release and NOS expression. These results indicate that glucose-induced inhibition of IL-1beta-stimulated NO release and NOS expression may be mediated by PKC activation and increased intracellular calcium.

IGF-I regulation of Na(+)-K(+)-ATPase in rat arterial smooth muscle.

Insulin-like growth factor I (IGF-I) is vasodilatory and mitogenic for vascular smooth muscle cells (VSMC). Alteration in VSMC Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity is hypothesized to underlie abnormal vascular tone and growth in hypertension and diabetes. Therefore, we investigated effects of IGF-I on Na(+)-K(+)-ATPase activity in rat aortic VSMC. IGF-I increases pump activity in a dose- and time-dependent manner: the minimal dose required was 10(-10) M, and the minimal time required was 20 min (at 10(-8) M) to increase activity. Similar effects persisted through 12 h. In Na(+)-loaded cells, IGF-I does not further stimulate activity. Blockade of Na+/H+ exchange attenuates IGF-I-induced increases in activity after 30 min but has no effect after 12 h. Northern blot analyses reveal that expression of the alpha 1- and the alpha 2-subunits of the pump were unaffected by IGF-I. Plasma membrane alpha 1- and alpha 2-protein were also unaffected, suggesting translocation of preformed pools was not responsible for the increases. Inhibitors revealed that neither tyrosine kinase activity, RNA transcription, protein synthesis, nitric oxide synthase activity, or protein kinase C activity mediated this IGF-I effect. Therefore, IGF-I regulates Na pump activity in the short term by an Na+/H+ exchange-dependent but transcription/translocation-independent mechanism. These data suggest that IGF-I, known to be produced by VSMC, may regulate tone and growth responses abnormal in disease states such as hypertension and diabetes.

Insulin like growth factor 1 increases vascular smooth muscle nitric oxide production.

Insulin like growth factor 1 (IGF-1) vasodilates, increases blood flow and lowers blood pressure; nitric oxide (NO) has been suggested to mediate some of these effects. We studied the role of IGF-1 in the regulation of NO production in vascular smooth muscle cells (VSMC). IGF-1 induced a concentration and time-dependent increase in NO release from endothelium-denuded aortic rings. Pre-incubation with cycloheximide or aminoguanidine blocked IGF-1-stimulated NO release. In addition, a six-fold increase in NO production by VSMC was seen upon incubation with IGF-1. These results suggest that IGF-1 induces NO release in VSMC through a process that involves new protein synthesis and the inducible isoform of nitric oxide synthase.

Insulin-like growth factor I diminishes in vivo and in vitro vascular contractility: role of vascular nitric oxide.

Although most insulin-like growth factor I (IGF-I) in the circulation is generated by the liver, the hormone is also produced locally by the vasculature, suggesting its potential importance in regulation of regional blood flow. Accordingly, we studied the effects of in vivo exposure to IGF-I (5.1 nmol, i.v.) as well as in vitro incubation (100 nM) on endothelium-intact rat tail artery contractile responses to KCl and norepinephrine (NE). Systemic administration of IGF-I resulted in transient lowering of blood pressure, with maximal reduction occurring at 15 min and a return to baseline by 60 min. Maximal contractility of rings removed from animals 90 min after a bolus injection of IGF-I, when blood pressure had returned to normal, was significantly reduced for both KCl (58%) and NE (51%) without a change in sensitivity. Similar data were obtained when rings from untreated animals were preincubated in vitro for 90 min; maximal contractility in response to KCl was decreased by 31% and that to NE by 22%. L-Nitroarginine methyl ester, an inhibitor of nitric oxide (NO) production, administered in vivo before IGF-I or added to the bath buffer reversed the attenuation. The nearly identical in vivo and in vitro results suggest that the observed diminution in contractility is a direct effect of IGF-I on the vasculature, probably mediated in large part by the release of NO. This idea is supported by our observation that IGF-I stimulates NO production in intact vessels. Further, the latency required indicates that rather complex mechanisms involving actions common on both receptor- and nonreceptor-mediated events are initiated by IGF-I and/or NO.