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.

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.

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.

Response to anti-VEGF-A treatment of retinal pigment epithelial cells in vitro.

PURPOSE: The neovascular or wet form of age-related macular degeneration is characterized by the growth of abnormal blood vessels in the retina stimulated by vascular endothelial growth factors (VEGF). In the last decade, several anti-VEGF drugs have been developed for treating neovascular diseases of the eyes. This study was conducted to compare the effects of 2 anti-VEGF-A drugs, ranibizumab and aflibercept, on the expression and secretion of VEGF family members in retinal pigment epithelial cells (RPE) in vitro. METHODS: ARPE-19 cells were exposed for 24 hours to ranibizumab or aflibercept at clinical dose concentration. Cell viability and expression and secretion of VEGF-A, VEGF-B, VEGF-C, and placental growth factor (PlGF) were evaluated respectively by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Ranibizumab and aflibercept did not affect ARPE-19 cell viability after 24 hours of treatment. Ranibizumab increased expression of VEGF-A and PlGF. On the contrary, expression and secretion of VEGF-C was decreased by ranibizumab. PlGF secretion was not affected by ranibizumab. Aflibercept strongly increased VEGF-A and PlGF expression but reduced their detection on the culture media, and decreased expression and secretion of VEGF-C. No effect on expression and secretion of VEGF-B was observed after exposure to these drugs. CONCLUSIONS: Ranibizumab and aflibercept exert similar effects on VEGF expression and secretion, leading to establishing an antiangiogenic environment. Increased VEGF-A expression observed in RPE cells treated with these drugs suggests a compensatory response of the cells to the lack of VEGF-A.

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.

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.

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.

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.

Advanced glycation endproducts and diabetes. Beyond vascular complications.

Advanced Glycation Endproducts (AGEs) are a group of heterogeneous compounds formed by the non enzymatic reactions between aldehydic group of reducing sugars with proteins, lipids or nucleic acids. Formation and accumulation of AGEs is related with the aging process and is accelerated in diabetes. Type 2 diabetes, the most common form of diabetes, is characterized by hyperglycaemia and insulin resistance associated to a progressive deterioration of beta cell function and mass. The pathogenic role of AGEs in vascular diabetic complications is widely recognised. Recently other aspects of the detrimental effects of AGEs in type 2 diabetes are emerged: AGEs interfere with the complex molecular pathway of insulin signaling, leading to insulin resistance; AGEs modify the insulin molecule, and, consequently, its function; AGEs decrease insulin secretion and insulin content. In this article we review the role of AGEs in type 2 diabetes, beyond their involvement in vascular complications.

Lercanidipine in type II diabetic patients with mild to moderate arterial hypertension.

This study evaluates the effects of lercanidipine antihypertensive treatment on glucose homeostasis in patients with type II diabetes mellitus with mild to moderate hypertension. Forty patients were enrolled. After a 2-week wash-out period, they were randomly allocated to receive in double-blind manner either 10 mg or 20 mg in single daily administration for 8 weeks. Nonresponding patients after the initial 4 weeks, were titrated up to 20 mg and 30 mg lercanidipine, respectively. At the end of the double-blind treatment, all patients entered in single-blind 4 weeks placebo follow-up. Systolic and diastolic blood pressure significantly decreased in both groups of patients after 4 weeks of treatment, and decreased further during the following 4 weeks. In both groups, progressive and significant decrease in fasting blood glucose, glycosylated hemoglobin and area under the curve of the oral glucose tolerance test were detected during lercanidipine treatment. Similarly, a decrease in serum fructosamine values were also observed. All variables returned to towards baseline values during the placebo follow-up period. Adverse events (headache and mild asthenia) were limited to two patients and resolved spontaneously. These data indicate that lercanidipine is effective in lowering high blood pressure in hypertensive patients with type II diabetes mellitus and does not exert negative effects on glucose homeostasis.