Reduced thrombogenicity of surface-treated Nitinol implants steered by altered protein adsorption.

Blood-contacting medical implants made of Nitinol and other titanium alloys, such as neurovascular flow diverters and peripheral stents, have the disadvantage of being highly thrombogenic. This makes the use of systemic (dual) anti-platelet/anticoagulant therapies inevitable with related risks of device thrombosis, bleeding and other complications. Meeting the urgent clinical demand for a less thrombogenic Nitinol surface, we describe here a simple treatment of standard, commercially available Nitinol that renders its surface ultra-hydrophilic and functionalized with phosphate ions. The efficacy of this treatment was assessed by comparing standard and surface-treated Nitinol disks and braids, equivalent to flow diverters. Static and dynamic (Chandler loop) blood incubation tests showed a drastic reduction of thrombus formation on treated devices. Surface chemistry and proteomic analysis indicated a key role of phosphate and calcium ions in steering blood protein adsorption and avoiding coagulation cascade activation and platelet adhesion. A good endothelialization of the surface confirmed the biocompatibility of the treated surface. STATEMENT OF SIGNIFICANCE: Titanium alloys such as Nitinol are biocompatible and show favorable mechanical properties, which led to their widespread use in medical implants. However, in contact with blood their surface triggers the activation of the intrinsic coagulation cascade, which may result in catastrophic thrombotic events. The presented results showed that a phosphate functionalization of the titanium oxide surface suppresses the activation of both coagulation cascade and platelets, avoiding the subsequent formation of a blood clot. This novel approach has therefore a great potential for mitigating the risks associated to either thrombosis or bleeding complications (due to systemic anticoagulation) in patients with cardiovascular implants.

Minor role of mature adipocyte mineralocorticoid receptor in high fat induced obesity.

Obesity is a major risk factor that contribute to the development of cardiovascular disease and type 2 diabetes. Mineralocorticoid receptor (MR) expression is increased in the adipose tissue of obese patients and several studies provide evidence that MR pharmacological antagonism improves glucose metabolism in genetic and diet-induced mouse models of obesity. In order to investigate whether the lack of adipocyte MR is sufficient to explain these beneficial metabolic effects, we generated a mouse model with inducible adipocyte-specific deletion of Nr3c2 gene encoding MR (adipo-MRKO). We observed a significant, yet not complete, reduction of Nr3c2 transcript and MR protein expression in subcutaneous and visceral adipose depots of adipo-MRKO mice. Notably, only mature adipocyte fraction lacks MR, whereas the stromal vascular fraction maintains normal MR expression in our mouse model. Adipo-MRKO mice fed a 45% high fat diet for 14 weeks did not show any significant difference in body weight and fat mass compared to control littermates. Glucose and insulin tolerance tests revealed that mature adipocyte MR deficiency did not improve insulin sensitivity in response to a metabolic homeostatic challenge. Accordingly, no significant changes were observed in gene expression profile of adipogenic and inflammatory markers in adipose tissue of adipo-MRKO mice. Moreover, pharmacological MR antagonism in mature primary murine adipocytes, which differentiated ex vivo from wild-type mice, did not display any effect on adipokine expression. Taken together, these data demonstrate that the depletion of MR in mature adipocytes displays a minor role in diet-induced obesity and metabolic dysfunctions.

The endogenous cannabinoid system stimulates glucose uptake in human fat cells via phosphatidylinositol 3-kinase and calcium-dependent mechanisms.

BACKGROUND: The endogenous cannabinoid system participates in the regulation of energy balance, and its dysregulation may be implicated in the pathogenesis of obesity. Adipose tissue endocannabinoids may produce metabolic and endocrine effects, but very few data are available in human adipose tissue and in primary human fat cells. EXPERIMENTAL DESIGN: We measured expression of type 1 and type 2 cannabinoid receptors (CNR), enzymes of cannabinoids synthesis and degradation in human omental, sc abdominal, and gluteal adipose tissue from lean and obese subjects. Furthermore, we assessed the effect of CNR1 stimulation on glucose uptake and intracellular transduction mechanisms in primary human adipocytes. Then we assessed the reciprocal regulation between CNR1 and peroxisome proliferator-activated receptor-gamma (PPARgamma). Finally, we tested whether leptin and adiponectin are regulated by CNR1 in human adipocytes. RESULTS: We found that most genes of the endocannabinoid system are down-regulated in gluteal fat and up-regulated in visceral and sc abdominal adipose tissue of obese patients. Treatment of adipocytes with rosiglitazone markedly down-regulated CNR1 expression, whereas Win 55,212 up-regulated PPARgamma. Win 55,212 increased (+50%) glucose uptake, the translocation of glucose transporter 4, and intracellular calcium in fat cells. All these effects were inhibited by SR141716 and wortmannin and by removing extracellular calcium. Win 55,212 and SR141716 had no effect on expression of adiponectin and leptin. CONCLUSIONS: These results indicate a role for the local endocannabinoids in the regulation of glucose metabolism in human adipocytes and suggest a role in channeling excess energy fuels to adipose tissue in obese humans.

Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors.

We previously showed a decreased expression of vitamin D receptor (VDR) mRNA/protein in a small group of adrenocortical carcinoma (ACC) tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5' regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas) were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesis.

Adipocyte Mineralocorticoid Receptor Activation Leads to Metabolic Syndrome and Induction of Prostaglandin D2 Synthase.

Metabolic syndrome is a major risk factor for the development of diabetes mellitus and cardiovascular diseases. Pharmacological antagonism of the mineralocorticoid receptor (MR), a ligand-activated transcription factor, limits metabolic syndrome in preclinical models, but mechanistic studies are lacking to delineate the role of MR activation in adipose tissue. In this study, we report that MR expression is increased in visceral adipose tissue in a preclinical mouse model of metabolic syndrome and in obese patients. In vivo conditional upregulation of MR in mouse adipocytes led to increased weight and fat mass, insulin resistance, and metabolic syndrome features without affecting blood pressure. We identified prostaglandin D2 synthase as a novel MR target gene in adipocytes and AT56, a specific inhibitor of prostaglandin D2 synthase enzymatic activity, blunted adipogenic aldosterone effects. Moreover, translational studies showed that expression of MR and prostaglandin D2 synthase is strongly correlated in adipose tissues from obese patients.

1α,25-Dihydroxyvitamin D3 inhibits the human H295R cell proliferation by cell cycle arrest: a model for a protective role of vitamin D receptor against adrenocortical cancer.

UNLABELLED: Using the human H295R adrenocortical carcinoma cell line as a model, we analyzed the role of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3)]--vitamin D receptor (VDR) axis in the growth of adrenocortical cancer (ACC). The presence of VDR in various adrenocortical tissues, including ACC, was also investigated. DNA synthesis was evaluated by [³H]thymidine cell incorporation after treatment with 1α,25(OH)2D3 at increasing doses. The effect of 1α,25(OH)2D3 on cell cycle and apoptosis was analyzed with a flow cytometer. Cyclin-dependent kinase 4 (CDK4) expression, a molecular marker of G1-S cell cycle transition phase, was evaluated in cells treated with 1α,25(OH)2D3 before and after VDR gene silencing. 1α,25(OH)2D3 treatment inhibited cell proliferation by 20% at a dose of 1 nM, in parallel with steroid secretion decrease. A cell cycle arrest in G1, with no change in apoptotic cell proportion, was observed after 10 nM 1α,25(OH)2D3 cell exposure. CDK4 activation was reduced by 10 nM 1α,25(OH)2D3 but was not affected by 1α,25(OH)2D3 after VDR gene silencing. Expression of VDR mRNA was lower in ACC than in benign adrenocortical tumors. VDR immunostaining was evident in benign tumors but it was weak in ACC tissues. CONCLUSIONS: Slightly supra-physiological concentrations of 1α,25(OH)2D3 have a moderate anti-proliferative effect on H295R cells. Anti-proliferative effect was due to cell cycle arrest in G1 phase, without inducing apoptosis. The low mRNA expression levels at qRT-PCR as well as the weak immunohistochemical expression of VDR in ACC, suggests a protective role of VDR against malignant adrenocortical growth.

Genes implicated in insulin resistance are down-regulated in primary aldosteronism patients.

Primary aldosteronism (PA) patients display an increased incidence of insulin resistance. Herein we demonstrate the decreased gene expression of lipid metabolism genes PCK1, PLIN, ADIPOQ and PPARG in the visceral adipose tissue (VAT) of PA patients compared to age-, sex- and BMI-matched controls. In VAT, the expression of PCK1, PLIN, ADIPOQ and PPARG was inversely correlated with aldosterone levels; furthermore, PLIN and ADIPOQ gene expression was correlated with potassium levels. Therefore, raised aldosterone and low potassium may contribute to the reduced expression of these genes in PA patients. Finally, incubation of primary cultures of human adipocytes with aldosterone resulted in a decrease in the expression of PCK1, PLIN and ADIPOQ and this effect was blocked by eplerenone. Therefore, the characteristic aldosterone excess of PA patients may mediate the down-regulation of PCK1, PLIN and ADIPOQ in VAT that in turn may contribute to the insulin resistance observed in PA patients.

Analysis of insulin sensitivity in adipose tissue of patients with primary aldosteronism.

OBJECTIVE: The objective of the study was to assess the effect of high aldosterone levels on insulin sensitivity of adipose tissue in humans. METHODS: Visceral adipose tissue (VAT) was obtained from patients with aldosterone-producing adenoma (APA; n=14) and, as controls, nonfunctioning adenoma (NFA; n=14) undergoing laparoscopic adrenalectomy. Homeostasis model assessment index was higher and potassium was lower in APA than NFA (P<0.05). Immunohistochemistry, Western blotting, and real-time PCR were used to detect and quantify mineralocorticoid receptor (MR) expression. Transcript levels of peroxisome proliferative-activated receptor-gamma, insulin receptor, glucose transporter 4, insulin receptor substrate-1 and -2, leptin, adiponectin, IL-6, monocyte chemoattractant protein-1, glucocorticoid receptor (GR)-alpha, 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1, and HSD11B2 were quantified. The effect of increasing aldosterone concentrations on 2-deoxy-[3H]d-glucose uptake was tested in human sc abdominal adipocytes. RESULTS: Expression of MR was demonstrated in VAT, with no difference between APA and NFA as to mRNA levels of MR, GRalpha, HSD11B1, and glucose metabolism and inflammation factors. In cultured adipocytes, basal and insulin-stimulated glucose uptake were unaffected by 1-100 nM (normal/hyperaldosteronism) and impaired only by much higher, up to 10 microM, aldosterone concentrations. The impairment was prevented by RU486 but not by eplerenone. CONCLUSIONS: Gene expression of insulin signaling/inflammatory molecules was similar in VAT of APA and NFA patients, not supporting an effect of aldosterone excess on insulin sensitivity of adipose tissues. Only at pharmacological concentrations and through GR activation, aldosterone reduced glucose uptake in adipocytes. Insulin resistance in primary aldosteronism might occur in compartments other than fat and/or depend on concurrent environmental factors.