Advanced glycation end products increase,through a protein kinase C-dependent pathway,vascular endothelial growth factor expression in retinal endothelial cells. Inhibitory effect of gliclazide

Accumulating evidence points to a causal role for advanced glycation end products (AGEs) in the development of diabetic vascular complications, including retinopathy. Possible pathogenic mechanisms linking AGEs to diabetic retinopathy include protein kinase C (PKC) activation, oxidative stress, and vascular endothelial growth factor (VEGF) expression. In the present study, we investigated the effect of AGEs on VEGF expression in bovine retinal endothelial cells (BRECs) and determined the role of PKC and oxidative stress in this effect. Incubation of BRECs with AGEs led to enhanced VEGF mRNA and protein expression. This treatment also induced PKC translocation in these cells. The AGE-induced increases in VEGF expression and PKC activation were inhibited by the pan-specific PKC inhibitor, calphostin C, and by the antioxidant drug and compounds, gliclazide, N-acetylcysteine, and vitamin E. In contrast, glyburide which does not exhibit antioxidant properties, did not affect the AGE-induced VEGF expression. Exposure of BRECs to AGEs resulted in a significant increase of nuclear protein binding to the NF-kappa B consensus sequence of the VEGF promoter region. Induction of DNA binding activity for NF-kappa B by AGEs was prevented by gliclazide. Treatment of BRECs with AGEs also increased the proliferation of these cells. This effect was abrogated by incubating the cells with an anti-VEGF antibody and was inhibited in the presence of gliclazide. Overall, these data demonstrate that AGEs increase VEGF expression in retinal endothelial cells through generation of oxidative stress and downstream activation of the PKC pathway. Targeting VEGF expression with specific pharmacological agents, such as antioxidants and PKC inhibitors, may prove efficacious for the treatment of diabetic retinopathy.     

Activation of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase by advanced glycation end products links oxidative stress to altered retinal vascular endothelial growth factor expression

Increasing evidence indicates that advanced glycation end products (AGEs) promote retinal alterations through oxidative stress. However, the pathways involved in AGE-induced generation of reactive oxygen species (ROS) in retinal cells are poorly defined. In the present study, we investigated the role of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase in AGE-induced ROS intracellular generation and vascular endothelial growth factor (VEGF) expression in bovine retinal endothelial cells (BRECs). Incubation of BRECs with 100 microg/mL AGEs increased ROS generation and VEGF expression in these cells. Treatment of the cells with the NADPH oxidase inhibitors, apocynin and diphenylene iodonium, inhibited these effects. In retinal endothelial cells exposed to AGEs, translocation of protein kinase C (PKC)-beta2 and p47phox was observed. Inhibition of PKC by treatment of the cells with calphostin C, GF10923X, and LY379196 totally suppressed AGE-mediated p47phox translocation and ROS generation. Incubation of BRECs with gliclazide inhibited AGE-induced PKC-beta2 and p47phox translocation and totally abrogated AGE-mediated ROS generation and VEGF expression. Overall, these results demonstrate that AGEs induce intracellular ROS generation and VEGF expression in retinal endothelial cells through a PKC-dependent activation of NADPH oxidase. Inhibition of retinal NADPH oxidase expression and ROS generated by this system provides a new potential mechanism by which gliclazide may affect retinal VEGF expression and exert a beneficial effect on diabetic retinopathy.     

TGF-β effects on airway smooth muscle cell proliferation, VEGF release and signal transduction pathways

Background and objective:   Airway smooth muscle (ASM) cell hyperplasia is a key feature of airway remodelling. Mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) are key components in signal transduction associated with cell proliferation; MAPK consists of the extracellular signal-regulated kinase (ERK), p38MAPK and c-Jun NH₂-terminal kinase (JNK). The effect of transforming growth factor (TGF)-β on the proliferation of ASM cells, the release of vascular endothelial growth factor (VEGF) by ASM cells and relevant signal transduction pathways were investigated.
Methods:   ASM cells were growth-arrested for 48 h then stimulated with platelet-derived growth factor (PDGF), TGF-β and dexamethasone. ASM cells were also treated with specific inhibitors of MAPK (PD98059), PI3K (wortmannin) and JNK (SP600125). Cell proliferation and VEGF concentrations were measured.
Results:   TGF-β neither augmented ASM cell proliferation nor showed a synergistic effect on PDGF-mediated ASM cell proliferation. Dexamethasone did not suppress ASM cell proliferation. VEGF release was augmented by TGF-β stimulation in a time-dependent manner, and was further enhanced by co-stimulation with PDGF and TGF-β. Dexamethasone suppressed VEGF release significantly. TGF-β enhanced PI3K phosphorylation, while PDGF augmented both ERK and PI3K phosphorylation. Wortmannin inhibited both TGF-β- and PDGF-stimulated VEGF release.
Conclusions:   TGF-β may facilitate airway remodelling by promoting VEGF release through the PI3K pathway, rather than via ASM cell proliferation.     

Ethanol Upregulates iNOS Expression in Colon Through Activation of Nuclear Factor-kappa B in Rats

Background:  Alcohol inhibits colonic motility but the mechanism is unknown. The goal of this study was to test the possibility that nuclear factor-kappa B (NF-κB) is involved in the upregulation of inducible nitric oxide synthase (iNOS) expression induced by ethanol in colon.
Methods:  The isometric contraction of longitudinal muscle strips of proximal colon (LP) was monitored by polygraph. Western blot analysis was used to measure the amount of iNOS and I-κB in the cytoplasm and P65 in the nucleus. Immunohistochemistry was applied to locate iNOS in colon.
Results:  Ethanol (87mM) inhibited the contraction of LP. Pretreatment of S-methylisothioure (SMT) (1 mM), a specific iNOS inhibitor, Pyrrolidine dithiocarbamate (PDTC) (10 mM) and BAY11-7082(10 mM), specific inhibitors of NF-κB significantly reversed the inhibitory effect of ethanol on LP contraction. Ethanol increased the amount of iNOS and content of NO in colon, and these effects were attenuated by pretreatment of PDTC. Following ethanol administration, the amount of I-κB in the cytoplasm decreased, but that of P65, the subunit of NF-κB in the nucleus, increased. The iNOS was located in the cell body of myenteric plexus in colon.
Conclusion:  Ethanol inhibited the contraction of LP in colon mainly through activation of NF-κB, the subsequent upregulation of iNOS expression and increase of NO release in myenteric plexus.     

Augmentation of Ethanol-Induced Cell Damage and Activation of Nuclear Factor-κB by Annexin IV in Cultured Cells

Background: We previously reported that the amount of annexin IV expression was increased in culture cells by exposure to ethanol. Here, to investigate the physiologic role of annexin IV, we analyzed ethanol-induced cytotoxicity and nuclear factor (NF)-κB activity by using annexin IV-overexpressed cells.
Methods: Annexin IV overexpression was performed by transfection of expression vector, in which annexin IV complementary DNA was ligated, to culture cells (rat glioma C6 cells and human neuroblastoma SH-SY5Y cells). Ethanol-induced cytotoxicity was assayed by measuring the mitochondrial enzyme (dehydrogenase) activity or trypan blue exclusion. NF-κB activity was measured by electrophoretic mobility shift assay with a κB-oligonucleotide probe.
Results: Ethanol-induced cytotoxicity was increased by overexpression of annexin IV in both C6 cells and SH-SY5Y cells. Annexin IV overexpression augmented ethanol-induced NF-κB activation.
Conclusions: Ethanol-induced increase in annexin IV expression might amplify ethanol-induced cytotoxicity via NF-κB activation.     

Role of Endotoxin in NF-κB Activation by Ethanol in Rat Hepatocytes

Background Endotoxin plays an important role in the progression of alcoholic liver injury. However, the role of endotoxin in acute activation of NF-κB by ethanol remains unclear.
Methods In primary rat hepatocyte cultures treated with ethanol and/or endotoxin, the DNA-binding activity of NF-κB in the nuclear extract was estimated by electrophoretic mobility shift assay. After pretreatment of a CYP2E1 inhibitor, 4-methyl pyrazole or diallyl sulfide, NF-κB activity was also measured in the same manner.
Results Ethanol or endotoxin caused activation of NF-κB in primary rat hepatocytes. Taking 50 mM of ethanol and endotoxin together raised the rapid increase in NF-κB activation, but both 100 mM of ethanol and endotoxin treatment reduced the increase. Addition of diallyl sulfide decreased the activity at rapid phase but increased it after 60 min, whereas addition of 4-methyl pyrazole caused no change of the activity at rapid phase but raised it after 60 min. Pretreatment with both endotoxin and a CYP2E1 inhibitor raised the activation of NF-κB constantly after ethanol addition. These findings suggest that endotoxin plays a critical role in the metabolism-independent activation of NF-κB by ethanol.
Conclusions Endotoxin raised metabolism-independent activation of NF-κB by high ethanol in rat hepatocytes.     

Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways

Objective  Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play divergent roles in the process of atherosclerosis. We investigated the effects of AGE-human serum albumin (AGE-HSA) on receptor expression for AGEs (RAGE) and EPCs apoptosis. Methods  The human mononuclear cells were obtained by Ficoll density gradient centrifugation and cultured in M199 medium containing rh-VEGF (30 ng/ml), rh-b-FGF(6 ng/ml) and 20% NBCS for 8 days. The adhesive EPCs were sequentially harvested after 24 h synchronization and challenged with AGE-HSA (concentration range from 0 to 300 μg/ml) for 24 h and 200 μg/ml AGE-HSA (time range from 0 to 36 h). EPCs apoptosis and migration were determined, expressions of RAGE, phosphorylated ERK1/2, JNK and p38 mitogen-activated protein kinase (MAPK) of EPCs were quantified by fluorescent quantitation RT-PCR and Western-blot, effect of AGE-HSA on NF-κB activtiy was determined by EMSA (electrophoretic mobility shift assay) in the presence and absence of special MAPK pathways pathway inhibitors. Results  AGE-HSA upregulated the expression of RAGE, this effect could be significantly inhibited by p38 MAPK and ERK MAPK inhibitor, but not by JNK MAPK inhibitor. AGE-HSA also promoted EPCs apoptosis and inhibited EPCs migration and increased NF-κB activity, these effects could be significantly attenuated by the anti-RAGE neutralizing antibody as well as by p38 and ERK MAPK inhibitors. Conclusion  AGE-HSA could promote atherosclerosis by upregulating EPCs RAGE expressions and promoting EPCs apoptosis via p38, ERK MAPK pathways, activation of NF-κB might also play a role in this process. C. Sun and C. Liang contributed equally to this work. Returned for 1. Revision: 13 December 2007 1. Revision received: 20 February 2008 Returned for 2. Revision: 7 March 2008 2. Revision received: 9 June 2008     

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in mantle cell lymphoma

Objectives:  Mantle cell lymphoma (MCL) is an incurable B cell lymphoma, and novel treatment strategies are urgently needed. We evaluated the effects of combined treatment with the proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) on MCL. Bortezomib acts by targeting the proteasome, and – among other mechanisms – results in a reduced nuclear factor-kappa B (NF-κB) activity. HDACi promote histone acetylation, and also interfere with NF-κB signaling.
Methods:  Human MCL cell lines (JeKo-1, Granta-519 and Hbl-2) were exposed to bortezomib and/or SAHA. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. Reactive oxygen species (ROS) were analyzed using the fluorophore H₂DCFDA. In addition, activated caspases, proteasome- and NF-κB activity were quantified.
Results:  Combined incubation with bortezomib and SAHA resulted in synergistic cytotoxic effects, as indicated by combination index values <1 using the median effect method of Chou and Talalay. The combination of both inhibitors led to a strong increase in apoptosis as compared to single agents and was accompanied by enhanced ROS generation, while each agent alone only modestly induced ROS. The free radical scavenger N -acetyl- l -cysteine blocked the ROS generation and reduced the apoptosis significantly. In addition, coexposure of bortezomib and SAHA led to increased caspase-3, -8 and -9 activity, marked reduction of proteasome activity and decrease of NF-κB activity.
Conclusions:  This is the first report giving evidence that SAHA and bortezomib synergistically induce apoptosis in MCL cells. These data build the framework for clinical trials using combined proteasome and histone deacetylase inhibition in the treatment of MCL.     

Glimepiride upregulates eNOS activity and inhibits cytokine-induced NF-κB activation through a phosphoinoside 3-kinase–Akt-dependent pathway

Aims:  Several studies suggest increased mortality postcoronary angioplasty in patients on sulphonylureas. However, a theoretical reduction in cardiac risk has been suggested with the newer sulphonylurea agents, which differ from the first-generation agents. In the present study, we investigated whether a third generation of sulphonylurea, glimepiride, might stimulate nitric oxide (NO) production and thereby inhibit cytokine-induced nuclear factor (NF)-κB activation in endothelial cells compared with the classical sulphonylurea glibenclamide.
Methods and Results:  We demonstrated that glimepiride, but not glibenclamide, induces NO production in human umbilical vein endothelial cells (HUVEC). A significant increase in endothelial NO synthase (eNOS) activity, measured in terms of citrulline production, was observed with glimepiride treatment. Akt phosphorylation followed by phosphorylation of eNOS (Ser1177) was observed with glimepiride treatment in HUVEC. Moreover, two phosphoinoside 3-kinase inhibitors, wortmannin and LY294002, significantly inhibited glimepiride-induced NO production. We also demonstrated inhibition of tumour necrosis factor-alpha (TNFα)-induced NF-κB activation in HUVEC treated with glimepiride, which was attenuated by pretreatment with N^ω-nitro- l -arginine methyl ester. We also demonstrated a marked increase in p65 in nuclear extracts from untreated HUVEC following stimulation with TNFα, which was dose dependently inhibited by glimepiride, but not by glibenclimide in association with NF-κB levels.
Conclusion:  These data suggest that glimepiride might be a preferable sulphonylurea agent in the setting of type 2 diabetes and vascular disease because it may have protective effects on vascular endothelial cells.     

Pioglitazone attenuates fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells

Aims:  Thiazolidinediones (TZDs), ligands for peroxisome proliferator–activated receptor γ, are antidiabetic agents that improve hyperglycemia by decreasing insulin resistance in obese diabetic animal models and patients with type 2 diabetes. We have studied whether pioglitazone, a TZD, can exert a direct effect against pancreatic β-cell lipoapoptosis.
Methods:  MIN6 cells were cultured in medium containing either 5.6 (low glucose) or 25 mM glucose (high glucose) in the presence or absence of 0.5 mM palmitate for 48 h. We examined the effect of 10 μM pioglitazone on MIN6 cells on glucose-stimulated insulin secretion, cellular ATP, uncoupling protein-2 (UCP-2) mRNA expression, intracellular triglyceride content, reactive oxygen species production, the number of apoptotic cells and nuclear factor-κB (NF-κB) activity.
Results:  Pioglitazone recovered partly impaired glucose-stimulated insulin secretion and cellular ATP in MIN6 cell exposed to high glucose with 0.5 mM palmitate. Pioglitazone suppressed intracellular triglyceride accumulation in cells exposed to high glucose with 0.5 mM palmitate. Palmitate-induced upregulation of UCP-2 mRNA levels was suppressed by pioglitazone in a dose-dependent manner. Pioglitazone decreased palmitate-induced reactive oxygen species production in MIN6 cells by 24% and in mouse islet cells by 53%. Pioglitazone also decreased palmitate-induced NF-κB activity by 40% and protected β-cells from palmitate-induced apoptosis by 22% in MIN6 cell.
Conclusions:  Pioglitazone attenuated fatty acid–induced oxidative stress and apoptosis in pancreatic β-cells. TZDs might be used as a mean for maintaining β-cell survival and preserving capacity of insulin secretion in patients with diabetes mellitus.     

Inhibitors of protein kinase C sensitise multiple myeloma cells to common genotoxic drugs

Objectives:  Despite high dose treatment regimes multiple myeloma (MM) disease is still not curable. Patients become resistant to cytotoxic drugs and die of disease progression. Therefore, besides new cytotoxic compounds drug sensitisers are urgently needed.
Methods:  The MM cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the common anti-myeloma drugs like melphalan together with protein kinase C (PKC) inhibitors. Growth inhibition was measured using the water-soluble tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1 assay), and apoptosis was determined by flow cytometry after staining with fluorescein isothiocyanate-labeled annexin V (annexin-V-FITC) and propidium iodide. Intracellular signalling was shown by western blotting.
Results:  In this study we show that the combination of melphalan or doxorubicin with a PKC inhibitor, Gö6976 or enzastaurin, strongly increases cell toxicity. Increase of cytotoxicity is shown to be due to increased induction of apoptosis. Furthermore, we show that the protective effect of human bone marrow stromal cells (hBMSC) is abrogated by the PKC inhibitors. Finally, western blotting experiments revealed that incubation of myeloma cells with cytotoxic drugs like melphalan or doxorubicin leads to increased phosphorylation and therefore degradation of inhibitor of nuclear factor kappa B (IκB) and release of nuclear factor kappa B (NFκB). In contrast, enzastaurin inhibits phosphorylation of IκB.
Conclusions:  We conclude that the combination of conventional drugs and PKC inhibitors might be very effective and represents a new strategy in the treatment of MM.     

Effect of vascular endothelial growth factor on cultured endothelial cell monolayer transport properties

Vascular endothelial growth factor (VEGF) is a potent enhancer of microvascular permeability in vivo. To date, its effects on hydraulic conductivity (L(p)) and diffusive albumin permeability (P(e)) of endothelial monolayers have not been thoroughly assessed in vitro. We hypothesized that VEGF affects endothelial transport properties differently depending on vessel location and endothelial phenotype. Using three well-established endothelial cell culture models-human umbilical vein endothelial cells (HUVECs), bovine aortic endothelial cells (BAECs), and bovine retinal microvascular cells (BRECs)-grown on porous, polycarbonate filters we were able to produce baseline transport properties characteristic of restrictive barriers. Our results show 3.1-fold and 5.7-fold increases in endothelial L(p) for BAEC and BREC monolayers, respectively, at the end of 3 h of VEGF (100 ng/ml) exposure. HUVECs, however, showed no significant alteration in L(p) after 3 h (100 ng/ml) or 24 h (25 ng/ml) of incubation with VEGF even though they were responsive to the inflammatory mediators, thrombin (1 U/ml; 27-fold increase in L(p) in 25 min) and bradykinin (10 microM; 4-fold increase in L(p) in 20 min). Protein kinase C (PKC) and nitric oxide (NO) are downstream effectors of VEGF signaling. BAEC L(p) was responsive to activation of NO (SNAP) and PKC (PMA), whereas these agents had no effect in altering HUVEC L(p). Moreover, BAECs exposed to the PKC inhibitor, staurosporine (50 ng/ml), exhibited significant attenuation of VEGF-induced increase in L(p), but inhibition of nitric oxide synthase (NOS) with L-NMMA (100 microM) had no effect in altering the VEGF-induced increase in L(p). These data provide strong evidence that in BAECs, the VEGF-induced increase in L(p) is mediated by a PKC-dependent mechanism. Regarding diffusive albumin P(e), at the end of 3 h, BAECs and BRECs showed 6.0-fold and 9. 9-fold increases in P(e) in response to VEGF (100 ng/ml), whereas VEGF had no significant effect after 3 h (100 ng/ml) or 24 h (25 ng/ml) in changing HUVEC P(e). In summary, these data indicate that VEGF affects endothelial transport properties differently depending on the vessel type and that differences in cell signaling pathways underlie the differences in VEGF responsiveness.     

Antioxidants attenuate hyperglycaemia-mediated brain endothelial cell dysfunction and blood–brain barrier hyperpermeability

Aims:  Hyperglycaemia (HG), in stroke patients, is associated with worse neurological outcome by compromising endothelial cell function and the blood–brain barrier (BBB) integrity. We have studied the contribution of HG-mediated generation of oxidative stress to these pathologies and examined whether antioxidants as well as normalization of glucose levels following hyperglycaemic insult reverse these phenomena.
Methods:  Human brain microvascular endothelial cell (HBMEC) and human astrocyte co-cultures were used to simulate the human BBB. The integrity of the BBB was measured by transendothelial electrical resistance using STX electrodes and an EVOM resistance meter, while enzyme activities were measured by specific spectrophotometric assays.
Results:  After 5 days of hyperglycaemic insult, there was a significant increase in BBB permeability that was reversed by glucose normalization. Co-treatment of cells with HG and a number of antioxidants including vitamin C, free radical scavengers and antioxidant enzymes including catalase and superoxide dismutase mimetics attenuated the detrimental effects of HG. Inhibition of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C but not phosphoinositide 3 kinase (PI3 kinase) also reversed HG-induced BBB hyperpermeability. In HBMEC, HG enhanced pro-oxidant (NAD(P)H oxidase) enzyme activity and expression that were normalized by reverting to normoglycaemia.
Conclusions:  HG impairs brain microvascular endothelial function through involvements of oxidative stress and several signal transduction pathways.     

RETRACTED ARTICLE: Gold nanoparticles inhibit vascular endothelial growth factor-induced angiogenesis and vascular permeability via Src dependent pathway in retinal endothelial cells

The purpose of this study was to investigate the effect of gold nanoparticles on the signaling cascade related to angiogenesis and vascular permeability induced by Vascular Endothelial Growth Factor (VEGF) in Bovine retinal endothelial cells (BRECs). The effect of VEGF and gold nanoparticles on cell viability, migration and tubule formation was assessed. PP2 (Src Tyrosine Kinase inhibitor) was used as the positive control and the inhibitor assay was performed to compare the effect of AuNPs on VEGF induced angiogenesis. The transient transfection assay was performed to study the VEGFR2/Src activity during experimental conditions and was confirmed using western blot analysis. Treatment of BRECs with VEGF significantly increased the cell proliferation, migration and tube formation. Furthermore, gold nanoparticles (500 nM) significantly inhibited the proliferation, migration and tube formation, in the presence of VEGF in BRECs. The gold nanoparticles also inhibited VEGF induced Src phosphorylation through which their mode of action in inhibiting angiogenic pathways is revealed. The fate of the gold nanoparticles within the cells is being analyzed using the TEM images obtained. The potential of AuNPs to inhibit the VEGF165-induced VEGFR-2 phosphorylation is also being confirmed through the receptor assay which elucidates one of the possible mechanism by which AuNPs inhibit VEGF induced angiogenesis. These results indicate that gold nanoparticles can block VEGF activation of important signaling pathways, specifically Src in BRECs and hence modulation of these pathways may contribute to gold nanoparticles ability to block VEGF-induced retinal neovascularization.     

Phosphatidylethanol in high density lipoproteins increases the vascular endothelial growth factor in smooth muscle cells

To study whether qualitative changes in high density lipoprotein (HDL) phospholipids mediate part of the advantageous effects of ethanol on atherosclerosis, we investigated whether HDL associated phosphatidylethanol (PEth) affects the secretion of vascular endothelial growth factor (VEGF) from cultured human smooth muscle cells. Serum-starved human umbilical vein HUVS-112D smooth muscle cells were incubated in the presence of PEth–HDL, HDL, or buffer. The phosphorylation of protein kinase C (PKC) and mitogen activated protein kinase (p44/42 MAPK) was determined by specific antibodies against phosphorylated and total proteins. VEGF concentrations were measured from cell culture medium of the cells. PEth increased the secretion of VEGF into the culture medium of HUVS cells. PEth–HDL increased the PKC phosphorylation by 2.1-fold and p44/42 MAPK phosphorylation by 3.3-fold compared with HDL, indicating that PEth-containing HDL particles influence vascular smooth muscle cells by PKC and p44/42 MAPK signalling. This may mediate the effects of ethanol on vascular wall by increasing the VEGF secretion from smooth muscle cells. The secreted VEGF may inhibit the formation of neointima and in doing so helps prevent atherosclerosis.