Pro-inflammatory gene expression profile in obese adults with high plasma GIP levels.

BACKGROUND: Glucose-dependent insulinotropic peptide (GIP) provides a novel link between the immune system and the gut, although results from different experimental and observational studies are contradictory, ranging from anti-inflammatory, through neutral to pro-inflammatory action of GIP. Thus, the aim of this study was to analyze inflammatory pathways on the level of gene expression and circulating inflammatory markers in relation to plasma GIP level. SUBJECTS/METHODS: The study included 128 obese adults. Two groups of obese subjects were created according to fasting GIP levels, with cutoff point at the 66th percentile and compared in respect with molecular and circulating markers of inflammation. GIP, interleukin (IL)-6 and adipokines: leptin, adiponectin, visfatin were measured by enzyme-linked immunosorbent assay. Inflammatory markers: monocyte chemoattractant protein-1 (MCP-1), sE-Selectin, sVCAM-1, sPECAM-1 were studied at fasting and after nutrient challenges. Gene expression in blood cells was determined by human gene microarray. RESULTS: Obese patients with high GIP levels had elevated fasting glucose (Q2 (Q1-Q3): 5.6 (5.0-6.0) vs 5.0 (4.8-5.4), P<0.001), homeostasis model assessment of insulin resistance (Q2 (Q1-Q3): 3.68 (2.72-5.42) vs 2.70 (2.13-4.33), P=0.021), thus increased markers of insulin resistance as well as elevated inflammatory markers Il-6 (Q2 (Q1-Q3): 1.34 (1.0-2.04) vs 1.12 (0.76-1.64), P=0.045), MCP-1 (Q2 (Q1-Q3): 363 (287-447) vs 323 (263-389), P=0.026). Leptin to adiponectin ratio was significantly associated with fasting plasma GIP levels (ß (95% CI): 0.84 (0.10-1.59)) independently of glucose levels. sE-Selectin was found to be a factor influencing GIP response to oral glucose intake (ß (95% CI): 0.47 (0.14-0.81)) and sVCAM was found to be a factor influencing GIP response to high-fat meal intake (ß (95% CI): 0.19 (0.01-0.37)). We identified 32 genes of inflammatory pathways differentially expressed in subjects with a high plasma GIP level compared to low GIP. Most upregulated genes play a role in leukocyte chemotaxis and tissue infiltration. CONCLUSIONS: These findings support the hypothesis that increased GIP signaling has a role in chronic low-grade inflammation.

A gene variation (rs12691) in the CCAT/enhancer binding protein α modulates glucose metabolism in metabolic syndrome.

BACKGROUND AND AIMS: CCAAT/enhancer-binding protein alpha (CEBPA) is a transcription factor involved in adipogenesis and energy homeostasis. Caloric restriction reduces CEBPA protein expression in patients with metabolic syndrome (MetS). A previous report linked rs12691 SNP in CEBPA to altered concentration of fasting triglycerides. Our objective was to assess the effects of rs12691 in glucose metabolism in Metabolic Syndrome (MetS) patients. METHODS AND RESULTS: Glucose metabolism was assessed by static (glucose, insulin, adiponectin, leptin and resistin plasma concentrations) and dynamic (disposition index, insulin sensitivity index, HOMA-IR and acute insulin response to glucose) indices, performed at baseline and after 12 weeks of 4 dietary interventions (high saturated fatty acid (SFA), high monounsaturated fatty acid (MUFA), low-fat and low-fat-high-n3 polyunsaturated fatty acid (PUFA)) in 486 subjects with MetS. Carriers of the minor A allele of rs12691 had altered disposition index (p = 0.0003), lower acute insulin response (p = 0.005) and a lower insulin sensitivity index (p = 0.025) indicating a lower insulin sensitivity and a lower insulin secretion, at baseline and at the end of the diets. Furthermore, A allele carriers displayed lower HDL concentration. CONCLUSION: The presence of the A allele of rs12691 influences glucose metabolism of MetS patients.

Effects of dietary fat modification on insulin sensitivity and on other risk factors of the metabolic syndrome--LIPGENE: a European randomized dietary intervention study.

BACKGROUND: Excessive energy intake and obesity lead to the metabolic syndrome (MetS). Dietary saturated fatty acids (SFAs) may be particularly detrimental on insulin sensitivity (SI) and on other components of the MetS. OBJECTIVE: This study determined the relative efficacy of reducing dietary SFA, by isoenergetic alteration of the quality and quantity of dietary fat, on risk factors associated with MetS. DESIGN: A free-living, single-blinded dietary intervention study. SUBJECTS AND METHODS: MetS subjects (n = 417) from eight European countries completed the randomized dietary intervention study with four isoenergetic diets distinct in fat quantity and quality: high-SFA; high-monounsaturated fatty acids and two low-fat, high-complex carbohydrate (LFHCC) diets, supplemented with long chain n-3 polyunsaturated fatty acids (LC n-3 PUFAs) (1.2 g per day) or placebo for 12 weeks. SI estimated from an intravenous glucose tolerance test (IVGTT) was the primary outcome measure. Lipid and inflammatory markers associated with MetS were also determined. RESULTS: In weight-stable subjects, reducing dietary SFA intake had no effect on SI, total and low-density lipoprotein cholesterol concentration, inflammation or blood pressure in the entire cohort. The LFHCC n-3 PUFA diet reduced plasma triacylglycerol (TAG) and non-esterified fatty acid concentrations (P < 0.01), particularly in men. CONCLUSION: There was no effect of reducing SFA on SI in weight-stable obese MetS subjects. LC n-3 PUFA supplementation, in association with a low-fat diet, improved TAG-related MetS risk profiles.

The human immunodeficiency virus (HIV1) protease inhibitor sanquinavir activates autophagy and removes lipids deposited in lipid droplets.

Reduction in mortality and increased average life span of the human immunodeficiency virus (HIV)-infected patients treated with antiretroviral therapy (ART) are associated with the risk of unwanted effects, such as insulin resistance and dyslipidemia with cardiovascular complications. Antiretroviral therapy may also be associated with lipodystrophy characterized as peripheral lipoatrophy with central fat accumulation. Understanding the molecular mechanisms of lipodystrophy caused by ART is important for therapeutic strategy and the prediction of side-effects. Influence of protease inhibitor saquinavir (SQV) on preadipocyte differentiation was analyzed in in vitro human Chub-S7 cell line model. For measurement of the effects of SQV the drug was added to differentiated or non-differentiated cells. The influence of SQV on changes in the profile of gene expression was verified by microarray and changes in lipid species content were analyzed using GC-MS/MS. Results were confirmed by real-time PCR and analysis of autophagy. Addition of SQV to differentiated Chub-S7 cells lead to removal of lipids deposited in lipid droplets, down-regulation of expression of transcription factors and markers of adipocyte differentiation. Antiviral activity of SQV based on its non-selective inhibition of proteases resulted in proteasome inhibition, induction of endoplasmic reticulum stress and induction of macroautophagy. This activity was accompanied by an increase in PI, PEPL, PC lipid species especially with MUFA and PUFA. Additionally up-regulation of miR-100-3p, miR-222-5p, miR-483-5p were found, which correlated with obesity, insulin resistance, increasing insulin secretion and activation of lipolysis. Our results indicated that SQV, by inhibition of proteasome protein degradation, activated the unfolded protein response resulting in autophagic breakdown of lipids deposited in adipose tissue causing lipodystrophy.

Proangiogenic activity of beta-carotene is coupled with the activation of endothelial cell chemotaxis.

Endothelial cells play an important role in angiogenesis (formation of new vessels from preexisting ones), which is essential for organogenesis, tissue remodeling but also inflammatory response, carcinogenesis in all periods of our life. Beta-carotene (BC) in non-toxic concentrations (up to 3 microM) had no detectable effect on HUVECs (human umbilical vein endothelial cells) proliferation or apoptosis, despite significant changes of the expression patterns of pro- and anti-apoptotic genes. However beta-carotene did not change the tubulogenic activity of HUVEC in the in vitro angiogenesis model, it potently accelerated the bFGF-induced development of microcapillaries, as well as the migration of endothelial cells, in matrigel plug injected subcutaneously to mice. Potent activation of endothelial cell migration in the in vitro model of chemotaxis was also observed. According to the microarray data, genes involved in cell/cell and cell/matrix adhesion, matrix reorganization, activation of chemotaxis, the G-protein regulated intracellular signaling as well as genes involved in the rapid remodeling of protein cytoskeleton were the most affected by BC in HUVEC. We conclude that beta-carotene in the physiological concentration range stimulates early steps of angiogenesis by the activation of cellular migration as well as matrix reorganization and decrease of cell adhesion.

Assessment of incretins in oral glucose and lipid tolerance tests may be indicative in the diagnosis of metabolic syndrome aggravation.

Incretins stimulated by oral meals are claimed to be protective for the pancreatic beta cells, to increase insulin secretion, to inhibit glucagon release, slow gastric emptying (glucagon-like peptide-1) and suppress appetite. Recently it has however been suggested that glucagon-like peptide-1 (GLP-1) is putative early biomarker of metabolic consequences of the obesity associated proinflammatory state. The study was aimed to compare the release of incretins and some of early markers of inflammation at the fasting and postprandial period induced by functional oral glucose as well as lipid load in healthy controls and patients with metabolic syndrome (MS) to see if functional tests may be helpful in searching for the inflammatory status of patients. Fifty patients with MS and 20 healthy volunteers (C) participated in this study. The 3-hour oral glucose (OGTT) and the 8-hour oral lipid (OLTT) tolerance tests were performed. At fasting leptin and adiponectin, as well as every 30 minutes of OGTT and every 2 hours of OLTT blood concentration of GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucose, insulin, triglycerides, free fatty acids, glutathione peroxidase, interleukin-6, sE-selectin, monocyte chemoattractant protein-1 (MCP1) and visfatin were measured. At fasting and during both OGTT and OLTT the level of incretins did not differ between the MS and the C group. Both glucose and lipids reach food activated incretins secretion. Glucose was the main GLP-1 release activator, while the lipid load activated evidently GIP secretion. A significantly larger AUC-GIP after the lipid-rich meal over the carbohydrate meal was observed, while statistically bigger value of AUC-GLP-1 was noticed in OGTT than in OLTT (P < 0.001) within each of the investigated groups. In patients with the highest fasting plasma GIP concentration (3(rd) tertile), IL-6, MCP-1, sE-selectin and visfatin blood levels were increased and correlated with glutathione peroxydase, leptin/adiponectin ratio, higher visfatin and interleukin-6 levels. The fat containing meals stimulate the long-lasting release of incretins, mainly GIP, parallel to the increase of the markers of low grade inflammation associating obesity in metabolic syndrome. The possibility of use of the postprandial (OLTT) GIP release measurement for the low grade inflammation progress in MS patients is suggested.

Ambulatory blood pressure, left ventricular mass and vascular phenotypes in relation to the endothelial nitric oxide synthase gene Glu298Asp and intron 4 polymorphisms in a population-based family study.

Reduced nitric oxide production is associated with pathological changes in the cardiovascular system. In a study of randomly chosen families, we analysed the relationship between two polymorphisms (Glu298Asp and intron 4) of the endothelial nitric oxide synthase (eNOS) and ambulatory blood pressure (ABP), left ventricular mass index (LVMI) and vascular phenotypes. The study population consisted of 127 parents and 167 offspring. All subjects underwent 24 h ABP monitoring using a SpaceLabs 90207 device. 2D and M-mode echocardiograms were obtained. Pulse wave velocity between the common carotid and femoral artery was measured with the Complior device, and the carotid intima-media thickness (IMT) was assessed by ultrasound. For statistical analysis, covariables and correlations between relatives were taken into account. The frequency of genotypes was as follows: for Glu298Asp: 55.1%-Glu/Glu, 40.1%-Glu/Asp and 4.8%-Asp/Asp; for intron 4: 65.0%-4 b/b, 33.3%-4 b/a and 1.7%-4 a/a, being in Hardy-Weinberg equilibrium (P > or = 0.29). There was no relationship between the eNOS gene polymorphisms and ABP or LVMI either in parents or their offspring. Among parents, carriers of the 298Asp allele had higher IMT values as compared with Glu/Glu homozygotes (0.94 vs 0.70 mm; P = 0.007). Among offspring, there was a similar tendency (0.60 vs 0.53 mm; P = 0.10), which was confirmed by transmission disequilibrium tests for quantitative variables (P > or = 0.07). Our findings indicate that the Glu298Asp polymorphism of eNOS identifies patients with larger carotid IMT, also in younger subjects.

The selected pathophysiological aspects of PPARs activation.

Peroxisome proliferator activated receptors (PPARs) belong to a subfamily of transcription nuclear factors. Three isoforms of PPARs have been identified: alpha, beta/delta and gamma, encoded by different genes and distributed in various tissues. They play important roles in metabolic processes like regulation of glucose and lipid redistribution. They also have anti-atherogenic, anti-inflammatory as well as anti-hypertensive functions. In hypertension-induced cardiac hypertrophy, both PPARa and PPARg activation reveal cardio-protective effect. Despite these beneficial functions, several recent experimental reports point to the possibille unfavorable effects of PPARs activation in lipid metabolism (lipotoxicity) in cardiomyocytes, which can lead to pathologic cardiac hypertrophy in such diseases as diabetes type 2, metabolic syndrome or obesity. This paper reviews evidences and hypotheses about the new pathophysiological aspects of PPARs activation.

Relationships between obesity, bipolar spectrum features, and personality traits: a case-control study.

OBJECTIVE: Recently there has been widening stream of research on the relationships between obesity and mental disorders. Patients with obesity seem to be prone to developing bipolar spectrum disorders and they present with specific personality traits. The aim of this study was to analyze the associations between obesity, bipolarity features, and personality traits. PATIENTS AND METHODS: A nested case-control study was performed. Patients with obesity constituted the sample of cases (N = 90), and healthy individuals were ascribed to the control group (N = 70). The lifetime presence of bipolarity features was analyzed with the Mood Disorder Questionnaire (MDQ), while personality traits were assessed with the NEO-Five Factor Inventory (NEO-FFI). RESULTS: Bipolarity features were more prevalent in the patients with obesity, as compared to healthy individuals. Patients with obesity had both higher mean value of MDQ score (p = 0.01) and a higher proportion of subjects with MDQ score ≥ 7 points (p = 0.012) as well as lower score on the NEO-FFI openness to experience (p > 0.001), compared to control subjects. Using multivariate model, in patients with obesity, a significant positive correlation between bipolarity and neuroticism, and negative with agreeableness and conscientiousness was established. Such relationship was not observed in control subjects. CONCLUSIONS: In the population of patients with obesity, there is a specific combination between bipolarity and personality traits (high-trait neuroticism, low-trait conscientiousness, and low-trait agreeableness). This may have some consequences for both pharmacological and psychological management of such patients.

Prostacyclin-dependent differences in TXA2 formation by platelets from normal and atherosclerotic rabbits.

Intact platelets from atherosclerotic rabbits release more thromboxane B2 (TXB2) and 12-hydroxyeicosatetraenoic acid (HETE) than platelets from normal rabbits following incubation with [14C]arachidonic acid (AA). In contrast, no difference is found between homogenates of platelets from normal and atherosclerotic animals. PGI2 stimulates cAMP accumulation and inhibits TXB2 formation more potently in intact platelets from atherosclerotic than from normal rabbits, but has no effect on AA metabolism in homogenised platelets.

Effects of dipyridamole in experimental atherosclerosis. Action on PGI2, platelet aggregation and atherosclerotic plaque formation.

Experimental atherosclerosis in rabbits induced by feeding a standard atherogenic diet for 4 months resulted in an increased sensitivity of platelets to the proaggregatory action of collagen and ADP. Treatment with dipyridamole (3 x 10 mg/day i.m.) for 4 weeks normalized platelet loss in atherosclerotic rabbits and abolished the increased sensitivity to proaggregatory collagen, but not to ADP. Dipyridamole treatment lowered basal as well as PGI2-induced cAMP levels below values seen in platelets from normal rabbits, but the stimulation by PGI2 relative to basal cAMP levels was not affected or even increased by dipyridamole treatment. Dipyridamole did not affect the increased sensitivity of platelets from atherosclerotic rabbits to the antiaggregatory action of PGI2, indicating that dipyridamole decreased absolute cAMP levels, probably due to reduction of the adenine nucleotide pool in platelets without affecting the adenylate cyclase function. Dipyridamole enhanced atherosclerotic plaque formation in arterial walls. Basal as well as PGI2-stimulated cAMP content was lower in homogenates from atherosclerotic than from normal aortic tissue. Dipyridamole-treated animals showed a further decrease in basal as well as PGI2-stimulated cAMP content of the aortic tissue, suggesting that this decrease in cAMP content may be linked to the enhanced proliferative activity seen in artherosclerotic plaque formation.

On the mechanism of thrombolytic action of thromboxane synthetase inhibitors.

Using our in vivo model for studying drugs which prevent deposition of thrombi or dissipate thrombi formed in extra-corporeal circulation over a collagen strip superfused with arterial blood of anaesthetized and heparinized cats, we have found that dazoxiben--a thromboxane synthetase inhibitor--possesses not only antithrombotic but also thrombolytic potency in vivo (ED50 = 3.8 mg/kg i.v.). The thrombolytic potency of dazoxiben was antagonized by aspirin at a dose of 50 mg/kg i.v. Moreover, dazoxiben stimulated the generation of prostacyclin in isolated rat aortic slices incubated in platelet rich plasma, but not in platelet poor plasma. It is suggested that the thrombolytic potency of thromboxane synthetase inhibitors after their systemic administration is associated with the release of prostacyclin and/or prostacyclin-stable metabolites by the vascular endothelium owing to feeding of prostacyclin synthetase with prostaglandin endoperoxides accumulated in platelets following the inhibition of thromboxane synthetase.

Effect of carbenoxolone on the biological activity of nitric oxide: relation to gastroprotection.

1. The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aortic strips precontracted by phenylephrine and on protection of rat gastric mucosa against ethanol-induced injury. 2. Carbenoxolone (100-300 microM) caused a significant and concentration-dependent potentiation of rat peritoneal neutrophil (RPN)- 3-morpholino-syndnonimine (SIN-1)- or iloprost-induced inhibition of platelet aggregation. Higher concentrations (500 microM) of carbenoxolone alone markedly inhibited platelet aggregation. Pretreatment with carbenoxolone (100-300 microM) antagonized the reversal of the RPN- or SIN-1-induced antiaggregatory effect by oxyhaemoglobin (10 microM). 3. Rat aortic strips with intact endothelium precontracted by phenylephrine (0.1-0.3 microM) were relaxed by carbenoxolone (100-300 microM) in a concentration-dependent manner. Relaxations were abolished by mechanical removal of the endothelium or by incubation with methylene blue (10 microM) or NG-nitro-L-arginine (L-NNA, 100 microM). Sodium nitroprusside (10 nM)-induced relaxations of endothelium-denuded rat aortic strips were potentiated by carbenoxolone (100 microM). . The carbenoxolone (200 mg kg-1, p.o.)-induced gastroprotection against ethanol was antagonized by L-NNA (5-40 mg kg-1) in a dose-dependent manner. Pretreatment of rats with indomethacin (10 mg kg-1, s.c.) increased the effect of L-NNA. 5. The results suggest that the activity of carbenoxolone in the experimental systems tested is due to phosphodiesterase inhibition, although radical scavenging properties of the drug could contribute to some of the effects observed. In the rat gastric mucosa both increased prostaglandin levels and effects on the NO system could contribute to the protective action of carbenoxolone.

Release and vasoconstrictor effect of leukotriene C-like immunoreactive material in the anaphylactic guinea-pig mesenteric vascular bed.

Antigen challenge of sensitized isolated guinea-pig mesentery perfused under constant pressure resulted in a significant flow reduction, which was paralleled by the release of leukotriene (LT)C4-like immunoreactivity into the perfusates. The lipoxygenase inhibitor quercetin inhibited the release of immunoreactive LTC4 and simultaneously abolished the antigen-induced mesenteric flow reduction. Mesenteric flow reduction was also observed after injection of exogenous LTC4. The results demonstrate that LTC4 is a constrictor of the mesenteric vascular bed and that endogenous LTC4-like immunoreactive material is a mediator of anaphylactic vasoconstriction in the guinea-pig mesentery.

Formation of leukotriene C4-like material by rat brain tissue.

Pieces of rat brain incubated in oxygenated Tyrode solution were stimulated with the divalent cation ionophore A 23187. Incubation media were assayed for leukotriene (LT) C4-like immunoreactivity as well as for prostaglandin (PG) D2, 6-keto-PGF1 alpha and thromboxane (TX) B2 using specific and sensitive radioimmunoassays. Spontaneous release of the cyclooxygenase products but only of trace amounts of LTC4-like immunoreactivity was detected. Stimulation with ionophore A 23187 increased the level of cyclooxygenase products and to a smaller extent the level of LTC4-like immunoreactivity. Inhibition of cyclooxygenase by indomethacin was paralleled by a significantly increased release of LTC4-like material under basal conditions as well as in the presence of ionophore A 23187. On the other hand, the synthesis of LTC4-like material was significantly decreased by the lipoxygenase inhibitors quercetin and nordihydroguaiaretic acid. The presence of LT-like material in rat brain incubates was confirmed by bioassay. The capacity of brain tissue to synthesize LT-like material may be important for cerebral blood flow as well as cellular functions in the central nervous system.

Ticlopidine and platelet function in healthy volunteers.

The influence of a 4-weeks therapy with 500 mg ticlopidine daily on platelet function parameters was examined in 10 male healthy volunteers aged 20-33 years in order to extend the knowledge on the antiplatelet activity of this substance. Ticlopidine significantly (p less than 0.01) affected ex-vivo platelet aggregation induced by ADP and increased platelet sensitivity to the antiaggregatory action of PGI2. Generation of TXB2 from endogenous substrate during spontaneous clotting of blood (serum-TXB2), conversion of exogenous radiolabelled arachidonic acid into TXB2 and MDA-formation in isolated platelets were unaffected by the treatment. The TXB2-level in plasma of volunteers, however, was decreased, after administration of the drug. The diminished alpha-granule content liberation (beta-thromboglobulin: p less than 0.01; PDGF: p less than 0.01; PF4 not significant) indicates that ticlopidine induces a decrease in platelet activity. The beneficial effect on release reaction is not associated with a decrease in TXA2-formation. Our results demonstrate that ticlopidine inhibits platelet activity, especially the PDGF-release. These results confirm the value of this drug in the prevention of atherosclerosis and its thromboembolic complications.

Beneficial results of pentoxifylline ('Trental') therapy in arteriosclerosis obliterans: possible mechanism of action.

A study was carried out in 17 patients with arteriosclerosis obliterans to assess the effectiveness of treatment with pentoxifylline ('Trental') and to investigate its possible mechanism of action. Patients received pentoxifylline on a combined intravenous/oral dosage regimen for 3 weeks and then were maintained on 800 mg orally for a further 2 weeks. The results showed that there was clinical improvement in 16 patients which was evident as a significant increase in the pain-free walking distance and blood flow in ischaemic legs, and the disappearance of rest pain. These changes were seen as soon as 1 week after the start of treatment. At the same time, an increase was observed in platelet sensitivity to the anti-aggregatory action of endogenous PGI2, as well as an activation of fibrinolysis in blood. After 5 weeks of treatment, further improvement was observed; however, platelets were no more hypersensitive to PGI2, and fibrinolytic activity of blood returned to the previous value. In experiments ex vivo, no release of a disaggregatory substance into blood was observed after a single bolus intravenous injection of pentoxifylline in patients with arteriosclerosis obliterans. It is concluded that the beneficial results of pentoxifylline therapy in such patients may be explained partially by a potentiation of the action of endogenous PGI2.

Ligands of peroxisome proliferator-activated receptor-gamma increase the generation of vascular endothelial growth factor in vascular smooth muscle cells and in macrophages.

Peroxisome proliferator-activated receptors-gamma (PPARgamma) are ligand-inducible transcription factors of the nuclear hormone receptor superfamily. We examined the effect of PPARgamma activation on the generation of vascular endothelial growth factor (VEGF), one of the major angiogenic agents. Rat vascular smooth muscle cells (VSMC) and murine macrophages RAW264.7 were incubated for 24 h with PPARgamma activators: prostaglandin J2 and ciglitazone. PPARgamma were expressed in VSMC and RAW cells and their activity was upregulated in the presence of PGJ2 and ciglitazone. Incubation of the cells with PPARgamma activators significantly augmented the release of VEGF protein into the media, both in resting and in IL-1beta- or LPS-stimulated cultures. The higher protein generation was connected with the increased expression of mRNA and transcriptional activation of VEGF promoter. We conclude that the activation of PPARgamma upregulates the generation of VEGF and may be involved in the regulation of angiogenesis.

Nitric oxide mediates the mitogenic effects of insulin and vascular endothelial growth factor but not of leptin in endothelial cells.

The regulation of vascular wall homeostasis by nitric oxide (NO) generated by endothelium is being intensively studied. In the present paper, the involvement of NO in the vascular endothelial growth factor (VEGF), insulin or leptin-stimulated proliferation of human endothelial cells (HUVEC) was measured by [3H]thymidine or bromodeoxyuridine incorporation. VEGF and insulin, but not leptin, increased NO generation in HUVEC, as detected with ISO-NO electrode. Proliferation of HUVEC induced by leptin was not changed or was higher in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase (NOS) inhibitor. In contrast, L-NAME blunted the proproliferative effect of VEGF and insulin. Furthermore, we demonstrated that, in human arterial smooth muscle cells (hASMC) transfected with endothelial NOS (eNOS) gene, the generation of biologically active VEGF protein was NO-dependent. Inhibition of NO generation by L-NAME decreased the synthesis of VEGF protein and attenuated HUVEC proliferation induced by conditioned media from transfected hASMC. Endothelium-derived NO seems to participate in VEGF and insulin, but not leptin, mitogenic activity. Additionally, the small amounts of NO released from endothelial cells, as mimicked by eNOS transfection into hASMC, may activate generation of VEGF in sub-endothelial smooth muscle cells, leading to increased synthesis of VEGF protein necessary for turnover and restitution of endothelial cells.