Gabriella Doronzo

Insulin activates vascular endothelial growth factor in vascular smooth muscle cells: influence of nitric oxide and of insulin resistance

Background  We aimed to evaluate whether insulin influences vascular endothelial growth factor (VEGF) synthesis and secretion in cultured vascular smooth muscle cells (VSMCs) via nitric oxide (NO) and whether these putative effects are lost in insulin‐resistant states.

Adipocytokines in Atherothrombosis: Focus on Platelets and Vascular Smooth Muscle Cells

Visceral obesity is a relevant pathological condition closely associated with high risk of atherosclerotic vascular disease including myocardial infarction and stroke. The increased vascular risk is related also to peculiar dysfunction in the endocrine activity of adipose tissue responsible of vascular impairment (including endothelial dysfunction), prothrombotic tendency, and low-grade chronic inflammation. In particular, increased synthesis and release of different cytokines, including interleukins and tumor necrosis factor-α (TNF-α), and adipokines—such as leptin—have been reported as associated with future cardiovascular events. Since vascular cell dysfunction plays a major role in the atherothrombotic complications in central obesity, this paper aims at focusing, in particular, on the relationship between platelets and vascular smooth muscle cells, and the impaired secretory pattern of adipose tissue.

In central obesity, weight loss restores platelet sensitivity to nitric oxide and prostacyclin.

Central obesity shows impaired platelet responses to the antiaggregating effects of nitric oxide (NO), prostacyclin, and their effectors—guanosine 3′,5′‐cyclic monophosphate (cGMP) and adenosine 3′,5′‐cyclic monophosphate (cAMP). The influence of weight loss on these alterations is not known. To evaluate whether a diet‐induced body‐weight reduction restores platelet sensitivity to the physiological antiaggregating agents and reduces platelet activation in subjects affected by central obesity, we studied 20 centrally obese subjects before and after a 6‐month diet intervention aiming at reducing body weight by 10%, by measuring (i) insulin sensitivity (homeostasis model assessment of insulin resistance (HOMAIR)); (ii) plasma lipids; (iii) circulating markers of inflammation of adipose tissue and endothelial dysfunction, and of platelet activation (i.e., soluble CD‐40 ligand (sCD‐40L) and soluble P‐selectin (sP‐selectin)); (iv) ability of the NO donor sodium nitroprusside (SNP), the prostacyclin analog Iloprost and the cyclic nucleotide analogs 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) to reduce platelet aggregation in response to adenosine‐5‐diphosphate (ADP); and (v) ability of SNP and Iloprost to increase cGMP and cAMP. The 10 subjects who reached the body‐weight target showed significant reductions of insulin resistance, adipose tissue, endothelial dysfunction, and platelet activation, and a significant increase of the ability of SNP, Iloprost, 8‐Br‐cGMP, and 8‐Br‐cAMP to reduce ADP‐induced platelet aggregation and of the ability of SNP and Iloprost to increase cyclic nucleotide concentrations. No change was observed in the 10 subjects who did not reach the body‐weight target. Changes of platelet function correlated with changes of HOMAIR. Thus, in central obesity, diet‐induced weight loss reduces platelet activation and restores the sensitivity to the physiological antiaggregating agents, with a correlation with improvements in insulin sensitivity.

Impaired synthesis and action of antiaggregating cyclic nucleotides in platelets from obese subjects: possible role in platelet hyperactivation in obesity.

Background  Subjects with central obesity exhibit platelet hyperactivity, which is involved in the atherosclerotic process and therefore can account for the increased risk of cardiovascular morbidity and mortality. The aim of the study was to evaluate whether alterations of platelet function in obesity involve synthesis and/or action of the two antiaggregating cyclic nucleotides adenosine 3′,5′‐cyclic monophosphate (cAMP) and guanosine 3′,5′‐cyclic monophosphate (cGMP).

Nitric oxide activates PI3-K and MAPK signalling pathways in human and rat vascular smooth muscle cells: Influence of insulin resistance and oxidative stress

OBJECTIVE Vascular smooth muscle cells (VSMCs) from the animal model of insulin resistance obese Zucker rats (OZR) show impaired ability of nitric oxide (NO) to increase cGMP and of cGMP to activate its specific kinase PKG, these defects being attributable to oxidative stress. We aimed to investigate the intracellular signalling downstream PKG in human and rat VSMC, and to clarify whether it is modified by insulin resistance and oxidative stress. METHODS In aortic VSMC from humans, lean Zucker rats (LZR) and OZR, we measured by Western blots the activation induced by NO and cGMP of signalling molecules of PI3-K and MAPK pathways, with or without PKG inhibition, hydrogen peroxide and antioxidants. We explored the mechanism of the increased oxidative stress in VSMC from OZR by measuring superoxide anion concentrations (luminescence method) with or without inhibition of NADPH oxidase, xanthine oxidase, and mitochondrial electron transport chain complex and by measuring superoxide dismutase (SOD) expression (Western blot) and activity. RESULTS In VSMC from humans and LZR, the NO/cGMP/PKG pathway activates both PI3-K (Akt, mTOR) and MAPK (ERK-1/2, p38MAPK) signalling. This effect is attenuated in VSMC from OZR, in which the greater oxidative stress is mediated by NADPH oxidase and mitochondrial complex and by a reduced synthesis/activity of SOD. Impairment of the NO/cGMP/PKG signalling is reproduced in VSMC from LZR by hydrogen peroxide and reverted in VSMC from OZR by antioxidants. CONCLUSIONS In VSMC from an animal model of insulin resistance the NO/cGMP/PKG intracellular signalling is impaired due to an increased oxidative stress.

Homocysteine rapidly increases matrix metalloproteinase-2 expression and activity in cultured human vascular smooth muscle cells Role of phosphatidyl inositol 3-kinase and mitogen activated protein kinase pathways

In this study we aimed to test the hypothesis that in human vascular smooth muscle cells (VSMC) homocysteine influences synthesis and release of matrix metalloproteinase-2 (MMP-2), which is deeply involved in vascular remodeling and atherosclerotic plaque instabilization. Experiments were carried out in cultured human VSMC exposed to 50-500 micromol/l homocysteine after a 24-hour culture with MEM containing 0.1% BSA. Both in supernatants and cell lysates we evaluated MMP-2 activity (gelatin zimography), MMP-2 andTIMP-2 protein synthesis (Western immunoblotting). Homocysteine effects were investigated also after cell exposure to i) specific MEK inhibitor PD98059 (30 micromol/l) to evaluate the involvement of Mitogen-Activated Protein Kinase (MAPK) and ii) specific phosphatidylinositol 3-kinase (P13-K) inhibitor LY294002 (100 micromol/l) to evaluate the involvement of P13-K pathway. Gelatin zimography evidenced that MMP-2 activity is increased both in conditioned media and in cell lysates starting from 8-hour incubation with 100 micromol/l homocysteine. Western blot analysis evidenced increased MMP-2 levels in both conditioned media and cell lysates. Cell exposure to PD98059 and LY294002 prevented homocysteine effects on MMP-2 synthesis. Homocysteine, at concentrations associated with increased risk of cardiovascular events, increases MMP-2 activity, synthesis and secretion in VSMC through a mechanism involving the activation of MAPK and P13-K pathways. These data suggest that homocysteine is directly involved in mechanisms leading to remodelling and instabilization of atherosclerotic plaques.

Role of NMDA receptor in homocysteine-induced activation of Mitogen-Activated Protein Kinase and Phosphatidyl Inositol 3-Kinase pathways in cultured human vascular smooth muscle cells

INTRODUCTION Exposure of vascular smooth muscle cells (VSMC) to homocysteine, at concentrations associated with an increased risk of cardiovascular events, enhances synthesis and secretion of Matrix Metalloproteinase-2 (MMP-2), which is involved in atherosclerotic plaque instabilization. This effect was prevented by inhibitors of Mitogen Activated Protein Kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3-K) pathways, allowing to hypothesize that homocysteine activates both these pathways, likely via a receptor-mediated mechanism. One possible receptor is N-methyl-D-aspartate receptor (NMDAr), which is expressed in VSMC and is involved in homocysteine effects in other cell types. MATERIALS AND METHODS VSMC exposed to DL-homocysteine or NMDA (100 micromol/L for both; 5 min-8 hours), were investigated by measuring: i) phosphorylation of ERK1/2, p38MAPK (signaling molecules of MAPK pathway) and Akt and p70S6K (signaling molecules of PI3-K pathway) by western blot; ii) synthesis and secretion of MMP-2 (western blot); iii) activation of MMP-2 (gelatin zimography). To evaluate NMDAr involvement in the homocysteine effects, the experiments were repeated in the presence of a non-competitive NMDAr-antagonist MK-801 (50 micromol/L) or L-glycine (10 micromol/L), which inhibits NMDAr function by promoting its internalization. RESULTS DL-homocysteine and NMDA time-dependently increased: i) the phosphorylation of ERK1/2, p38 MAPK, Akt and p70S6K (ANOVA, p<0.0001); ii) the synthesis, secretion and activation of MMP-2. DL-homocysteine and NMDA effects were prevented by VSMC pre-incubation with MK-801 or high L-glycine concentrations. CONCLUSIONS In human VSMC homocysteine-at concentrations associated with increased cardiovascular risk- activates MAPK and PI3-K pathways and MMP-2 synthesis and secretion through NMDA receptor, a potential mechanism involved in intracellular signaling in response to homocysteine in VSMC.

Contribution of insulin resistance to vascular dysfunction

Insulin is a vascular hormone, able to influence vascular cell responses. In this review, we consider the insulin actions on vascular endothelium and on vascular smooth muscle cells (VSMC) both in physiological conditions and in the presence of insulin resistance. In particular, we focus the relationships between activation of insulin signalling pathways of phosphatidylinositol-3 kinase (PI3-K) and mitogen-activated protein kinase (MAPK) and the different vascular actions of insulin, with a particular attention to the insulin ability to activate the pathway nitric oxide (NO)/cyclic GMP/PKG via PI3-K, owing to the peculiar relevance of NO in vascular biology. We also discuss the insulin actions mediated by the MAPK pathway (such as endothelin-1 synthesis and secretion and VSMC proliferation and migration) and by the interactions between the two pathways, both in insulin-sensitive and in insulin-resistant states. Finally, we consider the influence of free fatty acids, cytokines and endothelin on vascular insulin resistance.

Leptin and Vascular Smooth Muscle Cells

This review concerns the influence of leptin on vascular smooth muscle cells (VSMC). VSMC express different isoforms of the leptin receptor (Ob-R) able to activate a wide range of intracellular signalling pathways, mediating many relevant biological actions. In particular, leptin promotes processes deeply involved in atherogenesis, such as VSMC migration, hypertrophy, proliferation, osteogenic differentiation and metalloproteinase expression. The intracellular signalling molecules involved are JAK/STAT, PI3K/Akt, ERK 1/2, p38 MAPK, mTOR, and RhoA/ROCK. Some of these leptin actions are particularly evident in stretching conditions; others are mediated by the NAD(P)H-induced increase of Reactive Oxygen Species. A leptin-induced activation of angiotensin and endothelin systems, with up-regulation of the synthesis of the two hormones and of their receptors, has also been demonstrated. Other studies, however, showed that leptin increases in VSMC the nitric oxide production by activating the inducible nitric oxide synthase, and, via nitric oxide, exerts a vasodilating effect and impairs the proliferative and vasoconstricting actions of angiotensin II. Both the potentially harmful and the potentially beneficial effects exerted by leptin in VSMC are lost in VSMC from animal models of genetically determined leptinresistance. Cultured VSMC from leptin-resistant animals are also resistant to insulin and to nitric oxide. It is not known, however, whether in human obesity, a condition characterized by hypothalamic leptin resistance and by compensatory hyperleptinemia, VSMC are sensitive or resistant to leptin: only in the first case the predictive role of circulating leptin on cardiovascular events could support a pathogenetic influence of the hormone on atherosclerosis.

High Glucose Inhibits the Aspirin-Induced Activation of the Nitric Oxide/cGMP/cGMP-Dependent Protein Kinase Pathway and Does Not Affect the Aspirin-Induced Inhibition of Thromboxane Synthesis in Human Platelets

Since hyperglycemia is involved in the “aspirin resistance” occurring in diabetes, we aimed at evaluating whether high glucose interferes with the aspirin-induced inhibition of thromboxane synthesis and/or activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway in platelets. For this purpose, in platelets from 60 healthy volunteers incubated for 60 min with 5–25 mmol/L d-glucose or iso-osmolar mannitol, we evaluated the influence of a 30-min incubation with lysine acetylsalicylate (L-ASA; 1–300 μmol/L) on 1) platelet function under shear stress; 2) aggregation induced by sodium arachidonate or ADP; 3) agonist-induced thromboxane production; and 4) NO production, cGMP synthesis, and PKG-induced vasodilator-stimulated phosphoprotein phosphorylation. Experiments were repeated in the presence of the antioxidant agent amifostine. We observed that platelet exposure to 25 mmol/L d-glucose, but not to iso-osmolar mannitol, 1) reduced the ability of L-ASA to inhibit platelet responses to agonists; 2) did not modify the L-ASA–induced inhibition of thromboxane synthesis; and 3) prevented the L-ASA–induced activation of the NO/cGMP/PKG pathway. Preincubation with amifostine reversed the high-glucose effects. Thus, high glucose acutely reduces the antiaggregating effect of aspirin, does not modify the aspirin-induced inhibition of thromboxane synthesis, and inhibits the aspirin-induced activation of the NO/cGMP/PKG pathway. These results identify a mechanism by which high glucose interferes with the aspirin action.