Serum osteopontin predicts glycaemic profile improvement in metabolic syndrome: A pilot study.

Prediabetes is often observed in patients with Metabolic Syndrome (MetS) and might be associated with metabolic and inflammatory alterations. Here, we investigated whether the inflammatory molecule osteopontin (OPN) might have a prognostic impact in a cohort of MetS patients (n = 85) with baseline normal glycaemia or impaired fasting glucose (IFG) over one year of recommended pharmacological treatments and Mediterranean diet. Patients were then followed up for 12 months with intermediate evaluation after 6 months. At all time points, anthropometric and clinical data were recorded, alongside with haematological and biochemical profiles, including serum concentrations of OPN. As expected, Mediterranean diet improves glycaemic profile in patients with IFG. Baseline serum OPN failed to be associated with baseline anthropometric or biochemical variables. At baseline, higher levels of OPN were shown in patients with IFG as compared to normal glycaemia. Two distinct subgroups of patients in whom OPN decreased or remained stable/increased at follow-up were identified. When higher serum OPN levels were observed at baseline, greater reduction was observed at 1-year follow-up. Reduction in circulating OPN levels was associated with metabolic improvement in terms of blood pressure, LDL-c, HDL-c, and glycaemia. At both univariate and adjusted logistic regression analyses, serum OPN emerged as an independent predictor of glycaemic profile improvement at 1-year follow-up (adjOR 1.05 [1.00-1.10]; P = .041). In conclusion, pharmacological and dietetic interventions improved glycaemic profile in patients with MetS. In particular, glycaemic improvement was demonstrated in patients who also reduce circulating OPN levels. Higher OPN levels at baseline predict normalization of glycaemic profile.

Dipeptidyl peptidase-4 inhibitors do not alter GH/IGF-I axis in adult diabetic patients.

PURPOSE: Incretin-based therapies have been introduced in clinical practice for type 2 diabetes mellitus (T2DM) treatment in the last few years. Current available medications of this class include glucagon-like peptide 1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors. In addition to GLP-1, DPP-4 is able to inactivate many others peptides as hypothalamic growth hormone-releasing hormone (GHRH). The aim of this exploratory study was to evaluate, on adult diabetic patients, the impact of therapy with incretins, particularly DPP-4 inhibitors on GH/IGF-I axis. METHODS: 60 patients with T2DM were included in the study and they were divided into three groups (age and sex comparable) on the basis of their hypoglycemic drugs in the last 4 months: group 1 (17 patients, exenatide or liraglutide + metformin), group 2 (18 patients, sitagliptin or vildagliptin + metformin), group 3 (25 patients, metformin). Anthropometric data, glycemia, glycosylated hemoglobin (HbA1c), IGF-I and acid-labile subunit (ALS) were collected in all patients. RESULTS: Weight, waist circumference and BMI of group 1 were significantly higher (P < 0.05) compared to the other groups. Fasting plasma glucose and HbA1c of the group 1 were similar compared to those of group 3 (P ns) and higher compared to those of group 2 (P < 0.05). IGF-I absolute values, IGF-I SDS were not significantly different in the three groups. CONCLUSIONS: Our data evidence that DPP-4 inhibition does not influence significantly GH/IGF-I system, confirming what was observed in animal models. Further studies are needed to better characterize the properties of these molecules on endocrine system.

Baseline hs-CRP predicts hypertension remission in metabolic syndrome.

BACKGROUND: Inflammation, overweight and other cardiovascular risk factors might negatively impact on hypertension remission in metabolic syndrome (MetS), independently of the pharmacological treatment. Here, the potential influence of systemic inflammation (assessed by serum high-sensitivity C-reactive protein [hs-CRP]) on hypertension remission will be investigated in a cohort of hypertensive patients with MetS. MATERIAL AND METHODS: Hypertensive patients with MetS (n = 100) were enrolled, treated under current behavior/dietary/pharmacological recommendations and followed up for 12 months. All patients received medications and nutritional advice based on Mediterranean-like dietary pattern in addition to psychological and physical activity counselling. At baseline (T0), 6 (T1) and 12 (T2) months of follow-up, clinical data, haematological and biochemical profiles and serum hs-CRP were measured. RESULTS: As compared to T0, at T2 patients displayed improvements in anthropometric and metabolic profiles. At T2, the hypertension remission rate was 13.0%. Serum hs-CRP did not change overtime in the overall cohort. Surprisingly, patients who experienced hypertension remission were less treated with antihypertensive drugs, but developed a weak improvement in anthropometric measures during follow-up. The hypertension remission group had lower baseline levels of hs-CRP as compared to non-remission. Low baseline hs-CRP (<2 µg/mL, cut-off value identified by ROC curve) predicted hypertension remission, independently of antihypertensive treatment implementation, baseline systolic blood pressure and waist circumference improvement. CONCLUSIONS: Remission of hypertension in MetS is independently associated with baseline low CRP levels, which might suggest a critical role for inflammation in sustaining high blood pressure levels.

Response to anti-VEGF-A treatment of endothelial cells in vitro.

This study was conducted to compare the effects of two anti-VEGF-A drugs, Ranibizumab and Aflibercept, on the expression and secretion of VEGFs family members, and on their influence in proliferation and migration of endothelial cells (HECV) in vitro. HECV cells were exposed 24 h (T1), 4 days (T2) and 6 days (T3) to Ranibizumab or Aflibercept at pharmacodynamically relevant concentrations (Ranibizumab: 12.5 µg/ml and 125 µg/ml; Aflibercept: 50 µg/ml and 500 µg/ml). Cell viability and then expression and secretion of VEGF-A, VEGF-B, VEGF-C and PlGF were evaluated respectively by Real Time-PCR and ELISA. Intracellular signaling activated by VEGF-A and VEGF-C was investigated evaluating phosphorylation of VEGFR2. Influence in would healing was evaluated through scratch assay. In general no differences were observed among the tested concentrations of anti-vegf drugs. Ranibizumab and Aflibercept did not affect HECV cell viability in all experimental times. At T1, Ranibizumab decreased mRNA levels of VEGF-A, induced VEGF-C secretion, abrogated phosphorylation of VEGFR2 stimulated by VEGF-A, and impaired ability of HECV cells to repair wound healing. Aflibercept decreased mRNA levels of VEGF-A, -B and PlGF; slightly increased basal level of phVEGFR2, and completely abrogated phosphorylation stimulated by VEGF-A and VEGF-C. No effects on secretion of VEGF-B and on would healing were observed after exposure to Aflibercept. Prolonged exposure to anti-VEGFs decreased expression and secretion of VEGF-A and VEGF-B, up-regulated VEGF-C mRNA levels and its secretion, and increased basal phosphorylation of VEGFR2. Acute treatment with Ranibizumab or Aflibercept evoked different responses on endothelial cells, however these differences were lost after prolonged exposure. Scratch test results suggest that treatment with Ranibizumab may be more effective than Aflibercept in reducing angiogenic potential of endothelial cells in vitro.

Glucagon-like peptide-1 secreting cell function as well as production of inflammatory reactive oxygen species is differently regulated by glycated serum and high levels of glucose.

Glucagon-like peptide-1 (GLP-1), an intestinal hormone contributing to glucose homeostasis, is synthesized by proglucagon and secreted from intestinal neuroendocrine cells in response to nutrients. GLP-1 secretion is impaired in type 2 diabetes patients. Here, we aimed at investigating whether diabetic toxic products (glycated serum (GS) or high levels of glucose (HG)) may affect viability, function, and insulin sensitivity of the GLP-1 secreting cell line GLUTag. Cells were cultured for 5 days in presence or absence of different dilutions of GS or HG. GS and HG (alone or in combination) increased reactive oxygen species (ROS) production and upregulated proglucagon mRNA expression as compared to control medium. Only HG increased total production and release of active GLP-1, while GS alone abrogated secretion of active GLP-1. HG-mediated effects were associated with the increased cell content of the prohormone convertase 1/3 (PC 1/3), while GS alone downregulated this enzyme. HG upregulated Glucokinase (GK) and downregulated SYNTHAXIN-1. GS abrogated SYNTHAXIN-1 and SNAP-25. Finally, high doses of GS alone or in combination with HG reduced insulin-mediated IRS-1 phosphorylation. In conclusion, we showed that GS and HG might regulate different pathways of GLP-1 production in diabetes, directly altering the function of neuroendocrine cells secreting this hormone.

Evidence for the gut microbiota short-chain fatty acids as key pathophysiological molecules improving diabetes.

In type 2 diabetes, hyperglycemia, insulin resistance, increased inflammation, and oxidative stress were shown to be associated with the progressive deterioration of beta-cell function and mass. Short-chain fatty acids (SCFAs) are organic fatty acids produced in the distal gut by bacterial fermentation of macrofibrous material that might improve type 2 diabetes features. Their main beneficial activities were identified in the decrease of serum levels of glucose, insulin resistance as well as inflammation, and increase in protective Glucagon-like peptide-1 (GLP-1) secretion. In this review, we updated evidence on the effects of SCFAs potentially improving metabolic control in type 2 diabetes.

Advanced glycation end products play adverse proinflammatory activities in osteoporosis.

Osteoporosis is a major public health burden that is expected to further increase as the global population ages. In the last twenty years, advanced glycation end products (AGEs) have been shown to be critical mediators both in the pathogenesis and development of osteoporosis and other chronic degenerative diseases related to aging. The accumulation of AGEs within the bone induces the formation of covalent cross-links with collagen and other bone proteins which affects the mechanical properties of tissue and disturbs bone remodelling and deterioration, underlying osteoporosis. On the other hand, the gradual deterioration of the immune system during aging (defined as immunosenescence) is also characterized by the generation of a high level of oxidants and AGEs. The synthesis and accumulation of AGEs (both localized within the bone or in the systemic circulation) might trigger a vicious circle (in which inflammation and aging merged in the word "Inflammaging") which can establish and sustain the development of osteoporosis. This narrative review will update the molecular mechanisms/pathways by which AGEs induce the functional and structural bone impairment typical of osteoporosis.

Autonomous artificial intelligence versus teleophthalmology for diabetic retinopathy.

Purpose: To assess the role of artificial intelligence (AI) based automated software for detection of Diabetic Retinopathy (DR) compared with the evaluation of digital retinography by two double masked retina specialists. Methods: Two-hundred one patients (mean age 65 ± 13 years) with type 1 diabetes mellitus or type 2 diabetes mellitus were included. All patients were undergoing a retinography and spectral domain optical coherence tomography (SD-OCT, DRI 3D OCT-2000, Topcon) of the macula. The retinal photographs were graded using two validated AI DR screening software (Eye Art TM and IDx-DR) designed to identify more than mild DR. Results: Retinal images of 201 patients were graded. DR (more than mild DR) was detected by the ophthalmologists in 38 (18.9%) patients and by the AI-algorithms in 36 patients (with 30 eyes diagnosed by both algorithms). Ungradable patients by the AI software were 13 (6.5%) and 16 (8%) for the Eye Art and IDx-DR, respectively. Both AI software strategies showed a high sensitivity and specificity for detecting any more than mild DR without showing any statistically significant difference between them. Conclusions: The comparison between the diagnosis provided by artificial intelligence based automated software and the reference clinical diagnosis showed that they can work at a level of sensitivity that is similar to that achieved by experts.

Retinal pigment epithelial cells express a functional receptor for glucagon-like peptide-1 (GLP-1).

Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that has been shown to improve glucose homeostasis in type 2 diabetes. The biological effects of GLP-1 are mediated by its specific receptor GLP-1R that is expressed in a wide range of tissues, where it is responsible of the extra-pancreatic effects of GLP-1. Since the retinal pigment epithelium (RPE), that forms the outer retinal barrier, has a key role in protecting from diabetic retinopathy (DR), we investigated the potential expression and function of GLP-1R in a RPE cell line. ARPE-19 cells were cultured in DMEM/F12 supplemented with 10% FBS. The expression of GLP-1R was evaluated at both mRNA and protein levels. Then, the activation postreceptor intracellular signal transduction pathways (extracellular signal-regulated kinases 1 and 2 [ERK1/2] and protein kinase B [PKB]) were assessed by western blot in normal cells or silenced for GLP-1R in the presence or absence of 10 nmol/L GLP-1. The potential connections between intracellular signalling pathways triggered by GLP-1 stimulation were performed before incubating cells with kinase pharmacological inhibitors of mitogen-activated protein kinase (MEK)1/2, phosphatydilinositol-3kinase (PI3K), or epidermal growth factor receptor (EGFR). The results showed that GLP1R is expressed at both mRNA and protein level in ARPE-19 cells. Stimulation with GLP-1 strongly activated PKB and ERK1/2 phosphorylation till 40 min of exposure. GLP-1-mediated activation of both kinases was dependent on the upstream activation of PI3K and EGFR. Finally, treatment with GLP-1 did not affect the spontaneous release of VEGF-A from ARPE-19 cells. In conclusion, this paper showed that the presence of functional GLP-1R is expressed in RPE cells. These data might represent the rationale to further investigate the potential direct beneficial effects of GLP-1 treatment against DR.

An emerging role of glucagon-like peptide-1 in preventing advanced-glycation-end-product-mediated damages in diabetes.

Glucagon-like peptide-1 (GLP-1) is a gut hormone produced in the intestinal epithelial endocrine L cells by differential processing of the proglucagon gene. Released in response to the nutrient ingestion, GLP-1 plays an important role in maintaining glucose homeostasis. GLP-1 has been shown to regulate blood glucose levels by stimulating glucose-dependent insulin secretion and inhibiting glucagon secretion, gastric emptying, and food intake. These antidiabetic activities highlight GLP-1 as a potential therapeutic molecule in the clinical management of type 2 diabetes, (a disease characterized by progressive decline of beta-cell function and mass, increased insulin resistance, and final hyperglycemia). Since chronic hyperglycemia contributed to the acceleration of the formation of Advanced Glycation End-Products (AGEs, a heterogeneous group of compounds derived from the nonenzymatic reaction of reducing sugars with free amino groups of proteins implicated in vascular diabetic complications), the administration of GLP-1 might directly counteract diabetes pathophysiological processes (such as pancreatic ß-cell dysfunction). This paper outlines evidence on the protective role of GLP-1 in preventing the deleterious effects mediated by AGEs in type 2 diabetes.

Glucagon-like peptide-1 triggers protective pathways in pancreatic beta-cells exposed to glycated serum.

Advanced glycation end products (AGEs) might play a pathophysiological role in the development of diabetes and its complications. AGEs negatively affect pancreatic beta-cell function and the expression of transcriptional factors regulating insulin gene. Glucagon-like peptide-1 (GLP-1), an incretin hormone that regulates glucose homeostasis, might counteract the harmful effects of AGEs on the beta cells in culture. The aim of this study was to identify the intracellular mechanisms underlying GLP-1-mediated protection from AGE-induced detrimental activities in pancreatic beta cells. HIT-T15 cells were cultured for 5 days with glycated serum (GS, consisting in a pool of AGEs), in the presence or absence of 10 nmol/L GLP-1. After evaluation of oxidative stress, we determined the expression and subcellular localization of proteins involved in maintaining redox balance and insulin gene expression, such as nuclear factor erythroid-derived 2 (Nrf2), glutathione reductase, PDX-1, and MafA. Then, we investigated proinsulin production. The results showed that GS increased oxidative stress, reduced protein expression of all investigated factors through proteasome activation, and decreased proinsulin content. Furthermore, GS reduced ability of PDX-1 and MafA to bind DNA. Coincubation with GLP-1 reversed these GS-mediated detrimental effects. In conclusion, GLP-1, protecting cells against oxidants, triggers protective intercellular pathways in HIT-T15 cells exposed to GS.

Update on the protective molecular pathways improving pancreatic beta-cell dysfunction.

The primary function of pancreatic beta-cells is to produce and release insulin in response to increment in extracellular glucose concentrations, thus maintaining glucose homeostasis. Deficient beta-cell function can have profound metabolic consequences, leading to the development of hyperglycemia and, ultimately, diabetes mellitus. Therefore, strategies targeting the maintenance of the normal function and protecting pancreatic beta-cells from injury or death might be crucial in the treatment of diabetes. This narrative review will update evidence from the recently identified molecular regulators preserving beta-cell mass and function recovery in order to suggest potential therapeutic targets against diabetes. This review will also highlight the relevance for novel molecular pathways potentially improving beta-cell dysfunction.

The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques.

AIMS: The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. METHODS AND RESULTS: The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. CONCLUSION: Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

Vascular endothelial growth factor-C secretion is increased by advanced glycation end-products: possible implication in ocular neovascularization.

PURPOSE: Neovascularization is a common complication of many degenerative and vascular diseases of the retina. Advanced glycation end-products (AGEs) have a pathologic role in the development of retinal neovascularization, mainly for their ability in upregulating vascular endothelial growth factor-A (VEGF-A) secretion. The aim of this study was to investigate whether AGEs are able to modulate the secretion of VEGF-C, another angiogenic factor that increases the effect of VEGF-A. METHODS: A human retinal pigment epithelial cell line (ARPE-19) and human endothelial vascular cell line (HECV) cells were cultured for 24 h in presence of AGEs, and then mRNA expression of VEGF-C was analyzed with reverse transcription-polymerase chain reaction (RT-PCR). To verify whether AGEs-induced VEGF secretion is mediated by RAGE (Receptor for AGEs), RAGE expression was depleted using the small interfering RNA method. To investigate whether VEGF-A is involved in upregulating VEGF-C secretion, the cells were cultured for 24 h in the presence of bevacizumab, a monoclonal antibody against VEGF-A, alone or in combination with AGEs. VEGF-A and VEGF-C levels in the supernatants of the treated cells were evaluated with enzyme-linked immunosorbent assay. RESULTS: Exposure to AGEs significantly increased VEGF-C gene expression in ARPE-19 cells. AGEs-induced VEGF-C secretion was upregulated in retinal pigment epithelium (RPE) and endothelial cells. Downregulation of RAGE expression decreased VEGF-A secretion in cell models, and increased VEGF-C secretion in ARPE-19 cells. Adding bevacizumab to the culture medium upregulated constitutive VEGF-C secretion but did not affect AGEs-induced VEGF-C secretion. CONCLUSIONS: These findings suggest that AGEs take part in the onset of retinal neovascularization, not only by modulating VEGF-A but also by increasing VEGF-C secretion. In addition, our results suggest that VEGF-C may compensate for treatments that reduce VEGF-A.

Pathophysiological role of inflammatory molecules in paediatric ischaemic brain injury.

Ischaemic stroke is one of the major causes of death and lifelong disability also in the paediatric population. Strong scientific effort has been put to clarify the pathophysiology of this disease in adults. However, only few studies have been performed in children. Preliminary results indicate that pathophysiological processes might differently affect the poststroke neuronal injury in neonates as compared to children. During the neural development, selective molecular mechanisms might be differently triggered by an ischaemic insult, thus potentially resulting in defined postischaemic clinical outcomes. Basic research studies in neonatal animal models of cerebral ischaemia have recently shown a potential role of soluble inflammatory molecules (such as cytokines, chemokines and oxidants) as pivotal players of neuronal injury in both perinatal and childhood ischaemic stroke. Although larger clinical trials are still needed to confirm these preliminary results, the potential benefits of selective treatments targeting inflammation in perinatal asphyxia encephalopathy might represent a promising investigation field in the near future. In this review, we will update evidence on the pathophysiological role of soluble inflammatory mediators in neonatal and childhood ischaemic stroke. Recent evidence on potential anti-inflammatory treatments to improve paediatric stroke prognosis will be discussed.

Lipoprotein(a) Modulates Carotid Atherosclerosis in Metabolic Syndrome.

Background and Aim: High lipoprotein(a) [Lp(a)] is a well-established cardiovascular (CV) risk factor, but the effect of mildly elevated Lp(a) on CV health is largely unknown. Our aim was to evaluate if Lp(a) is associated with the severity of carotid atherosclerosis (CA) in the specific subset of metabolic syndrome (MetS). Patients and Methods: Subjects with diagnosed MetS and ultrasound-assessed CA were enrolled. Those patients were categorized according to the severity of CA (moderate vs. severe), and the circulating levels of Lp(a) alongside with clinical, anthropometric, and biochemical data were collected. Results: Sixty-five patients were finally included: twenty-five with moderate and forty with severe CA (all with asymptomatic disease). Intergroup comparison showed Lp(a) as the only significantly different variable [6 (2-12) mg/dl vs. 11.5 (6-29.5) mg/dl; p = 0.018]. Circulating levels of Lp(a) were also confirmed as the only variable independently associated with severity of CA at logistic regression analysis [OR 2.9 (95% CI 1.1-7.8); p = 0.040]. ROC curve analysis for Lp(a) confirmed a serum level of 10 mg/dl as the best cut-off value [AUC 0.675 (95% CI 0.548-0.786)]. Although sensitivity and specificity were suboptimal (69.0 and 70.4%, respectively)-likely due to the small sample size-this result is in line with those previously reported in the literature. Conclusion: Lp(a) is independently associated with severity of CA in the subgroup of MetS patients.

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome.

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 µM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.

miR-126 Mimic Counteracts the Increased Secretion of VEGF-A Induced by High Glucose in ARPE-19 Cells.

Vascular endothelial growth factor-A (VEGF-A) has a pathologic role in microvascular diabetic complication, such as diabetic retinopathy (DR). miR-126 plays an important role in vascular development and angiogenesis by regulating the expression of VEGF-A. Since levels of miR-126 have been found downregulated in diabetes, this study is aimed at investigating whether hyperglycemia affects expression of miR-126 in a retinal pigment epithelium cell line. ARPE-19 cells were transfected with miR-126 inhibitor or with miR-126 mimic and the respective scramble negative control. After 24 hours, medium was replaced and cells were cultured for 24 hours in normal (CTR) or diabetic condition (HG). Then, we analyzed mRNA levels of miR-126, VEGF-A, PI3KR2, and SPRED1. We also evaluated protein amount of HIF-1α, PI3KR2, and SPRED1 and VEGF-A secretion. The results showed that exposure of ARPE-19 cells to HG significantly decreased miR-126 levels; mRNA levels of VEGF-A and PI3KR2 were inversely correlated with those of miR-126. Overexpression of miR-126 under HG restored HIF-1α expression and VEGF-A secretion to the level of CTR cells. These results indicate that reduced levels of miR-126 may contribute to DR progression by increasing expression of VEGF-A in RPE cells. In addition, we provide evidence that upregulation of miR-126 in RPE cells counteracts the rise of VEGF-A secretion induced by hyperglycemia. In conclusion, our data support a role of miR-126 mimic-approach in counteracting proangiogenic effects of hyperglycemia.

Advanced Glycation End-Products and Hyperglycemia Increase Angiopoietin-2 Production by Impairing Angiopoietin-1-Tie-2 System.

The angiopoietin-Tie-2 system plays a crucial role in the maintenance of endothelial integrity. Hyperglycemia and advanced glycation end-products (AGEs) are involved in endothelial cell dysfunction responsible of the pathogenesis of microvascular complications of diabetes. Here, we investigated whether glycated serum (GS) or hyperglycemia (HG) affect the angiopoietin-Tie-2 system in the microvascular endothelial cells HMEC-1. We found that culture for 5 days in the presence of AGEs and HG (alone or in combination) decreased cell proliferation, increased reactive oxygen species (ROS) production, and reduced ratio between the oxidized and the reduced form of glutathione. Since angiopoietin-1 (Ang-1) signaling regulates angiopoietin-2 (Ang-2) expression through inactivation of the forkhead transcription factor FoxO1, we investigated intracellular signaling of Ang-1 and expression of Ang-2. HG and AGEs reduced phosphorylation of Akt and abrogated phosphorylation of FoxO1 induced by Ang-1 without affecting neither Tie-2 expression nor its activation. Furthermore, AGEs and/or HG induced nuclear translocation of FoxO1 and increased Ang-2 production. In conclusion, we demonstrated that both hyperglycemia and AGEs affect the angiopoietin-Tie-2 system by impairing Ang-1/Tie-2 signaling and by increasing Ang-2 expression. These results suggest that therapeutic strategies useful in preventing or delaying the onset of diabetic vascular complications should be aimed to preserve Ang-1 signaling.

Glycated fetal calf serum affects the viability of an insulin-secreting cell line in vitro.

The purpose of the present study was to evaluate the direct effects of advanced glycation end products (AGEs) on beta-cells by their exposure to a glycated serum to estimate the cellular viability and the related insulin secretion. Glycation of fetal calf serum was obtained by incubation with 50 mol/L ribose at 37 degrees C for 7 days; at the end of this incubation period, the pentosidine content ranged between 15 and 16 x 10(5) pmol/L. HIT-T15 cells, a pancreatic islet cell line, were grown and cultured for 5 days in Roswell Park Memorial Institute (RPMI) medium containing either not glycated (NGS) or glycated (GS) fetal calf serum. Cellular oxidative stress (ie, thiobarbituric acid-reactive substances) was assessed by high-performance liquid chromatography. Cellular viability was evaluated by detection of proliferation, cell necrosis, and cell apoptosis rate. The insulin secretion and the related intracellular content were evaluated by enzyme-linked immunosorbent assay. The present study reported, after 5 days of exposure to the glycation environment, a moderately reduced cellular proliferation (-20.44% +/- 2.92%) with a corresponding increase of cell necrosis (+67.7% +/- 1.56%) and cell apoptosis (+39.83% +/- 2.92%) rate in comparison with the untreated cells. Oxidative intracellular stress was higher in GS conditions compared with the NGS ones (+293.3% +/- 87.53%). Insulin release from GS-treated HIT-T15 cells was lower than that of NGS-treated cells both when cells were stimulated with low glucose concentration (2.8 mmol/L, -30.3% +/- 4.91%) or when they were challenged with high glucose concentration (16.7 mmol/L, -29.2% +/- 5.82%). Incubation of HIT-T15 cells with glycated serum also caused a significant decrease of insulin intracellular content (-44.47% +/- 9.98%). Thus, AGEs were shown to exert toxic effects on insulin-secreting cells. Chronically high intracellular oxidative stress, due to accumulation of AGEs, affects the insulin secretion machinery. The present data suggest a pivotal role of the non-enzymatic glycation process in the onset and progression of diabetes during aging and a direct adverse effect of a glycated environment on the pancreatic islet cells.