Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.     

miR-152/LIN28B axis modulates high-glucose-induced angiogenesis in human retinal endothelial cells via VEGF signaling

Diabetic retinopathy (DR) is a serious complication of diabetes contributing to blindness in patients. Inhibiting retinal neovascularization is a potent strategy for diabetic retinopathy treatment. Reportedly, the stable expression of lin-28 homolog B (LIN28B), a member of the highly conserved RNA-binding protein LIN28 family, could promote vascular endothelial growth factor (VEGF) expression; herein, we investigated the role and mechanism of LIN28B in diabetic retinopathy progression from the perspective of microRNA (miRNA) regulation. We identified miR-152 as a miRNA that may target the LIN28B 3′-untranslated region and can be significantly downregulated under high-glucose (HG) condition. The expression of miR-152 was remarkably suppressed, whereas the expression of LIN28B was significantly increased under HG condition within both human retinal endothelial cells (hRECs) and retinal microvascular endothelial cell line (hRMECs). miR-152 overexpression significantly suppressed, while LIN28B overexpression promoted the angiogenesis and the protein levels of proangiogenesis factors in both hRECs and hRMECs. More importantly, LIN28B overexpression could remarkably attenuate the effect of miR-152 overexpression. In summary, miR-152 overexpression could inhibit HG-induced angiogenesis in both hRECs and hRMECs via targeting LIN28B and suppressing VEGF signaling. Further, in vivo experiments are needed for the application of miR-152/LIN28B axis in the treatment for diabetic retinopathy.     

Insulin and β-adrenergic Receptors Inhibit Retinal Endothelial Cell Apoptosis Through Independent Pathways

Diabetic retinopathy results from altered insulin receptor signaling. Based on previous studies demonstrating an interaction between β-adrenergic receptors and insulin signaling in hyperglycemic conditions, we hypothesized that β-adrenergic receptor stimulation and insulin stimulation would act synergistically to inhibit one of the hallmarks of diabetic retinopathy, namely retinal endothelial cell apoptosis. To test this hypothesis, human retinal endothelial cells were grown in high glucose (25 mM) medium and treated with a β-1-adrenergic receptor agonist (xamoterol, 10 μM) alone, insulin alone (10 nM) or xamoterol + insulin. We then assessed changes in the levels of insulin receptor, insulin-like growth factor (IGF-1) receptor, and Akt phosphorylation, as well as cleaved caspase 3. Xamoterol alone significantly decreased insulin receptor, IGF-1 receptor and Akt phosphorylation, whereas insulin alone increased insulin receptor, IGF-1 receptor, and Akt phosphorylation. Xamoterol significantly decreased apoptosis of retinal endothelial cells. This data suggests that both β-adrenergic receptors and insulin can inhibit retinal endothelial cell apoptosis in hyperglycemic conditions, but inhibition occurs through independent pathways. These findings have implications for treatments of diabetic retinopathy.     

TNFα Inhibits IGFBP-3 through Activation of p38α and Casein Kinase 2 in Human Retinal Endothelial Cells

We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment.     

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-κB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-κB and caspase-3 by about 30–40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-κB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.     

Inhibition of the adrenomedullin/nitric oxide signaling pathway in early diabetic retinopathy

The nitric oxide (NO) signaling pathway is integrally involved in visual processing and changes in the NO pathway are measurable in eyes of diabetic patients. The small peptide adrenomedullin (ADM) can activate a signaling pathway to increase the enzyme activity of neuronal nitric oxide synthase (nNOS). ADM levels are elevated in eyes of diabetic patients and therefore, ADM may play a role in the pathology of diabetic retinopathy. The goal of this research was to test the effects of inhibiting the ADM/NO signaling pathway in early diabetic retinopathy. Inhibition of this pathway decreased NO production in high-glucose retinal cultures. Treating diabetic mice with the PKC β inhibitor ruboxistaurin for 5 weeks lowered ADM mRNA levels and ADM-like immunoreactivity and preserved retinal function as assessed by electroretinography. The results of this study indicate that inhibiting the ADM/NO signaling pathway prevents neuronal pathology and functional losses in early diabetic retinopathy.     

Notch signaling protects retina from nuclear factor-κB- and poly-ADP-ribose-polymerase-mediated apoptosis under high-glucose stimulation

Proliferative diabetic retinopathy, the primary cause of vision loss in adults, is one of serious microvascular complications caused by diabetes. Both poly-ADP-ribose-polymerase (PARP) and nuclear factor (NF)-κB signaling are involved in the injury process. Injury activates PARP, which in turn potentiates NF-κB activation and causes cell apoptosis. Like the NF-κB pathway, Notch1 signaling plays a key role in the regulation of cell proliferation, differentiation, and apoptosis. However, the connections between these signaling pathways are not well understood. In this study, we used both streptozotocin (STZ)-induced diabetic mice and human retinal vascular endothelial cells (HRVECs) cultured in high glucose to detect these relationships. We found that apoptosis was increased in both STZ-induced diabetic mice and high-glucose-treated HRVECs, which was due to increased activation of PARP, cleaved caspase3, and reduced expression of Notch1 and p-Akt. The results of Notch1 overexpression and knockdown indicated that Notch1 signaling participated in the interaction of PARP and p50, and inhibited PARP- and p50-mediated apoptosis directly. These phenomena could be blocked by pretreatment with the PI3K inhibitor wortmannin via reducing p-Akt levels. Thus, our study demonstrated that Notch1 signaling protects cells from PARP- and NF-κB-induced apoptosis under high glucose through the activation of Akt.     

Effect of Diabetes/Hyperglycemia on the Rat Retinal Adenosinergic System

The early stages of diabetic retinopathy (DR) are characterized by alterations similar to neurodegenerative and inflammatory conditions such as increased neural apoptosis, microglial cell activation and amplified production of pro-inflammatory cytokines. Adenosine regulates several physiological functions by stimulating four subtypes of receptors, A₁AR, A_2AAR, A_2BAR, and A₃AR. Although the adenosinergic signaling system is affected by diabetes in several tissues, it is unknown whether diabetic conditions in the retina can also affect it. Adenosine delivers potent suppressive effects on virtually all cells of the immune system, but its potential role in the context of DR has yet to be studied in full. In this study, we used primary mixed cultures of rat retinal cells exposed to high glucose conditions, to mimic hyperglycemia, and a streptozotocin rat model of type 1 diabetes to determine the effect diabetes/hyperglycemia have on the expression and protein levels of adenosine receptors and of the enzymes adenosine deaminase and adenosine kinase. We found elevated mRNA and protein levels of A₁AR and A_2AAR, in retinal cell cultures under high glucose conditions and a transient increase in the levels of the same receptors in diabetic retinas. Adenosine deaminase and adenosine kinase expression and protein levels showed a significant decrease in diabetic retinas 30 days after diabetes induction. An enzymatic assay performed in retinal cell cultures revealed a marked decrease in the activity of adenosine deaminase under high glucose conditions. We also found an increase in extracellular adenosine levels accompanied by a decrease in intracellular levels when retinal cells were subjected to high glucose conditions. In conclusion, this study shows that several components of the retinal adenosinergic system are affected by diabetes and high glucose conditions, and the modulation observed may uncover a possible mechanism for the alleviation of the inflammatory and excitotoxic conditions observed in diabetic retinas.     

Regulation of angiogenesis by glycogen synthase kinase-3beta

Glycogen synthase kinase-3beta (GSK3beta) plays important roles in metabolism, embryonic development, and tumorigenesis. Here we investigated the role of GSK3beta signaling in vascular biology by examining its function in endothelial cells (ECs). In EC, the regulatory phosphorylation of GSK3beta was found to be under the control of phosphoinositide 3-kinase-, MAPK-, and protein kinase A-dependent signaling pathways. The transduction of a nonphosphorylatable constitutively active mutant of GSKbeta promoted apoptosis under the conditions of prolonged serum deprivation or the disruption of cell-matrix attachments. Conversely, the transduction of catalytically inactive GSK3beta promoted EC survival under the conditions of cellular stress. Under normal cell culture conditions, the activation of GSK3beta signaling inhibited the migration of EC to vascular endothelial growth factor or basic fibroblast growth factor. Angiogenesis was inhibited by GSK3beta activation in an in vivo Matrigel plug assay, whereas the inhibition of GSK3beta signaling enhanced capillary formation. These data suggest that GSK3beta functions at the nodal point of converging signaling pathways in EC to regulate vessel growth through its control of vascular cell migration and survival.     

An in vitro cell model to study microglia activation in diabetic retinopathy

Microglial activation has been studied extensively in diabetic retinopathy. We have previously detected activation and migration of microglia in 8-week-old diabetic rat retinas. It is widely acknowledged that microglia-mediated inflammation contributes to the progression of diabetic retinopathy. However, existing cell models do not explore the role of activated microglia in vitro. In this study, microglia were subject to various conditions mimicking diabetic retinopathy, including high glucose, glyoxal, and hypoxia. Under high glucose or glyoxal treatment, microglia demonstrated only partially functional changes, while under hypoxia, microglia became fully activated showing enlarged cell bodies, enhanced migration and phagocytosis as well as increased production of pro-inflammatory factors such as cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS). The data indicate that hypoxia-treated microglia is an optimal in vitro model for exploration of microglia activation in diabetic retinopathy.     

Diabetic retinopathy and signaling mechanism for activation of matrix metalloproteinase-9

In the pathogenesis of diabetic retinopathy, H-Ras (a small molecular weight G-protein) and matrix metalloproteinase-9 (MMP9) act as pro-apoptotic, accelerating the apoptosis of retinal capillary cells, a phenomenon that predicts its development and the activation of MMP9 is under the control of H-Ras. The goal of this study is to elucidate the cellular mechanism by which H-Ras activates MMP9 culminating in the development of diabetic retinopathy. Using isolated retinal endothelial cells, the effect of regulation of H-Ras downstream signaling cascade, Raf-1, MEK, and ERK, was investigated on glucose-induced activation of MMP9. In vitro results were confirmed in the retina obtained from diabetic mice manipulated for MMP9 gene, and also in the retinal microvasculature obtained from human donors with diabetic retinopathy. Regulation of Raf-1/MEK/ERK by their specific siRNAs and pharmacologic inhibitors prevented glucose-induced activation of MMP9 in retinal endothelial cells. In MMP9-KO mice, diabetes had no effect on retinal MMP9 activation, and H-Ras/Raf-1/MEK signaling cascade remained normal. Similarly, donors with diabetic retinopathy had increased MMP9 activity in their retinal microvessels, the site of histopathology associated with diabetic retinopathy, and this was accompanied by activated H-Ras signaling pathway (Raf-1/ERK). Collectively, these results suggest that Ras/Raf-1/MEK/ERK cascade has an important role in the activation of retinal MMP9 resulting in the apoptosis of its capillary cells. Understanding the upstream mechanism responsible for the activation of MMP9 should help identify novel molecular targets for future pharmacological interventions to inhibit the development/progression of diabetic retinopathy.     

Endothelins: regulators of extracellular matrix protein production in diabetes

Fibronectin (FN), a key extracellular matrix protein, is upregulated in target organs of diabetic angiopathy and in cultured cells exposed to high levels of glucose. FN has also been reported to undergo alternative splicing to produce the extra domain-B (ED-B) containing isoform, which is exclusively expressed during embryogenesis, tissue repair, and tumoral angiogenesis. The present study was aimed at elucidating the role and mechanism of endothelins (ETs) in FN and ED-B FN expression in diabetes. We investigated vitreous samples for ED-B FN expression from patients undergoing vitrectomy for proliferative diabetic retinopathy. Our results show increased FN and ED-B FN expression in the vitreous of diabetic patients in association with augmented ET-1. Using an antibody specific to the ED-B segment of FN, we show an increase in serum ED-B FN levels in patients with diabetic retinopathy and nephropathy. We further examined retinal tissues, as well as renal and cardiac tissues, from streptozotocin-induced diabetic rats. Diabetes increased FN and ED-B FN in all three organs, which was prevented by ET antagonist bosentan. To provide insight into the mechanism of glucose-induced and ET-mediated ED-B FN upregulation, we assayed endothelial cells (ECs). Inhibition of mitogen-activated protein kinase with pharmacological inhibitors and protein kinase B with dominant negative transfections prevented glucose- and ET-1-mediated FN and ED-B FN expression. Furthermore, treatment of cells exposed to high levels of glucose with ET antagonist prevented the activation of all signaling pathways studied and normalized glucose-induced ED-B FN expression. We then determined the functional significance of ED-B in ECs and show that ED-B FN is involved in vascular endothelial growth factor expression and cellular proliferation. These studies show that glucose-induced and ET-mediated FN and ED-B FN expressions involve complex interplays between signaling pathways and that ET may represent an ideal target for therapy in chronic diabetic complications.     

Prolonged high-glucose exposure decreased SREBP-1/FASN/ACC in Schwann cells of diabetic mice via blocking PI3K/Akt pathway

Abnormal lipid metabolism and SREBP-1 downregulation are reported to be involved in the pathogenesis of diabetic peripheral neuropathy (DPN). In the current study, the relationship between PI3K/Akt signaling pathway and SREBP-1 expression was explored in Schwann cells of DPN. The phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression were inhibited in the sciatic nerves of diabetic mice versus those of normal mice, accompanied with the atrophy of nerve fiber and the irregular myelin sheath. High concentration glucose suppressed phospho-Akt (Ser 473), phospho-Akt (Thr 308), and SREBP-1 expression in cultured Schwann cell (RSC96 cell) in vitro, and 25 mmol/L glucose was enough to lead to the maximum inhibitory effect. The time-course effect of high glucose showed that Akt phosphorylation gradually decreased with the extension of stimulation time. Somewhat differently, short-term high-glucose exposure enhanced SREBP-1 expression and prolonged high-glucose stimulation reduced the SREBP-1 expression in RSC96 cells. Similarly, prolonged high-glucose stimulation also downregulated FASN messenger RNA (mRNA), ACC mRNA, intracellular triglyceride, and cholesterol. LY294002 suppressed Akt activation followed by the decreased SREBP-1, FASN, ACC, triglyceride, and cholesterol. Contrarily, the PI3K/Akt signaling pathway agonist insulin pretreatment avoided prolonged high-glucose stimulation-blocked Akt activation and increased SREBP-1, FASN, and ACC expression in the levels of protein and mRNA in RSC96 cells. The knockdown of SREBP-1 by shRNA prevented insulin-induced enhanced FASN, ACC mRNA expression, triglyceride, and cholesterol in high-glucose-treated RSC96 cells. In conclusion, prolonged high-glucose exposure inhibits the SREBP-1/FASN/ACC expression in the Schwann cells of DPN via the blockage of the PI3K/Akt signaling pathway.     

Sestrin2 regulates monocyte activation through AMPK-mTOR nexus under high-glucose and dyslipidemic conditions

The vicious cycle between hyperinsulinemia and insulin resistance results in the progression of atherosclerosis in the vessel wall. The complex interaction between hyperglycemia and lipoprotein abnormalities promotes the development of atherogenesis. In the early phase of atherosclerosis, macrophage-derived foam cells play an important role in vascular remodeling. Mechanistic target of rapamycin (mTOR) signaling pathway has been identified to play an essential role in the initiation, progression, and complication of atherosclerosis. Recently sestrin2, an antioxidant, was shown to modulate TOR activity and thereby regulating glucose and lipid metabolism. But the role of sestrin2 in monocyte activation is still not clearly understood. Hence, this study is focussed on investigating the role of sestrin2 in monocyte activation under hyperglycemic and dyslipidemic conditions. High-glucose and oxidized low-density lipoprotein (LDL) treatments mediated proinflammatory cytokine production (M1) with a concomitant decrease in the anti-inflammatory cytokine (M2) levels in human monocytic THP1 cells. Both glucose and oxidized LDL (OxLDL) in a dose and time-dependent manner increased the mTOR activation with a marked reduction in the levels of pAMPK and sestrin2 expression. Both high-glucose and OxLDL treatment increased foam cell formation and adhesion of THP1 cells to endothelial cells. Experiments employing activator or inhibitor of adenosine monophosphate kinase (AMPK) as well as overexpression or silencing of sestrin2 indicated that high-glucose mediated monocyte polarization and adhesion of monocytes to the endothelial cells were appeared to be programmed via sestrin2-AMPK-mTOR nexus. Our results evidently suggest that sestrin2 plays a major role in regulating monocyte activation via the AMPK–mTOR-pathway under diabetic and dyslipidemic conditions and also AMPK regulates sestrin2 in a feedback mechanism.     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

Thioredoxin interacting protein (TXNIP) induces inflammation through chromatin modification in retinal capillary endothelial cells under diabetic conditions

Chronic hyperglycemia and activation of receptor for advanced glycation end products (RAGE) are known risk factors for microvascular disease development in diabetic retinopathy. Thioredoxin‐interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin (TRX), plays a causative role in diabetes and its vascular complications. Herein we investigate whether HG and RAGE induce inflammation in rat retinal endothelial cells (EC) under diabetic conditions in culture through TXNIP activation and whether epigenetic mechanisms play a role in inflammatory gene expression. We show that RAGE activation by its ligand S100B or HG treatment of retinal EC induces the expression of TXNIP and inflammatory genes such as Cox2, VEGF‐A, and ICAM1. TXNIP silencing by siRNA impedes RAGE and HG effects while stable over‐expression of a cDNA for human TXNIP in EC elevates inflammation. p38 MAPK‐NF‐κB signaling pathway and histone H3 lysine (K) nine modifications are involved in TXNIP‐induced inflammation. Chromatin immunoprecipitation (ChIP) assays reveal that TXNIP over‐expression in EC abolishes H3K9 tri‐methylation, a marker for gene inactivation, and increases H3K9 acetylation, an indicator of gene induction, at proximal Cox2 promoter bearing the NF‐κB‐binding site. These findings have important implications toward understanding the molecular mechanisms of ocular inflammation and endothelial dysfunction in diabetic retinopathy. J. Cell. Physiol. 221: 262–272, 2009. © 2009 Wiley‐Liss, Inc     

Adiponectin inhibits high glucose-induced angiogenesis via inhibiting autophagy in RF/6A cells

Adiponectin, one of the adipose-derived hormone with metabolic activity, has been reported to conversely affect angiogenesis of endothelial cells in vitro. The previous study in animal models has demonstrated that adiponectin has a protective role in retinal vascular injury following pathological stimuli. However, clinical research regarding the relationship between plasma adiponectin level and diabetic retinopathy (DR) are inconclusive. The aim of this study was to investigate the effect of adiponectin on high glucose-induced retinal angiogenesis and its association with autophagy by using rhesus choroid-retinal endothelial (RF-6A) cells as a model. We found that cell vitality decreased and cell migration and tube formation increased in the high-glucose group. Treatment with adiponectin or 3-methyladenine (3-MA, an autophagy inhibitor) increased cell viability and inhibited cell migration and tube formation. In the high-glucose group, the protein expression of Bax and apoptosis rate of cells increased and the expression of Bcl-2 decreased, whereas treatment with adiponectin or 3-MA reversed these results. Autophagy was activated in the high-glucose group to present as more LC3B fluorescent dots and higher expressions of LC3B, Atg5 proteins as well as lower expression of p62. Treatment with adiponectin or 3-MA inhibited autophagy by promoting the expression of p-PI3K, p-AKT, and p-mTOR when compared with the high-glucose group. The results of this study suggested that adiponectin inhibits high glucose-induced angiogenesis of RF/6A cells by inhibiting autophagy, and promotion of the PI3K/AKT/mTOR pathway might be involved in the anti-autophagy activities of adiponectin.     

bFGF Promotes the Migration of Human Dermal Fibroblasts under Diabetic Conditions through Reactive Oxygen Species Production via the PI3K/Akt-Rac1- JNK Pathways

Fibroblasts play a pivotal role in the process of cutaneous wound repair, whereas their migratory ability under diabetic conditions is markedly reduced. In this study, we investigated the effect of basic fibroblast growth factor (bFGF) on human dermal fibroblast migration in a high-glucose environment. bFGF significantly increased dermal fibroblast migration by increasing the percentage of fibroblasts with a high polarity index and reorganizing F-actin. A significant increase in intracellular reactive oxygen species (ROS) was observed in dermal fibroblasts under diabetic conditions following bFGF treatment. The blockage of bFGF-induced ROS production by either the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) almost completely neutralized the increased migration rate of dermal fibroblasts promoted by bFGF. Akt, Rac1 and JNK were rapidly activated by bFGF in dermal fibroblasts, and bFGF-induced ROS production and promoted dermal fibroblast migration were significantly attenuated when suppressed respectively. In addition, bFGF-induced increase in ROS production was indispensable for the activation of focal adhesion kinase (FAK) and paxillin. Therefore, our data suggested that bFGF promotes the migration of human dermal fibroblasts under diabetic conditions through increased ROS production via the PI3K/Akt-Rac1-JNK pathways.