Biomedical subjects

Ruth B Caldwell

Publications and source records attributed to Ruth B Caldwell.

At least 19 recordsLinked to original sources

PubMed2006

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Animals↗

PubMed2006

Protection against myocardial ischemia/reperfusion injury by short-term diabetes: enhancement of VEGF formation, capillary density, and activation of cell survival signaling.

The aims of this study were to determine effects of diabetes duration on myocardial ischemia/reperfusion (I/R) injury and test whether time-dependent differences in sensitivity of the streptozotocin diabetic rat heart to I/R are related to differences in vascular density, levels of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS) expression, NO formation, activation of Akt, and/or oxidative stress. After 2 or 6 weeks of streptozotocin-induced diabetes, I/R injury was induced by occlusion (30 min) and reperfusion of the left descending coronary artery. After 2 weeks of diabetes, infarct size and cleavage of caspase-3, a proapoptosis signal, were decreased as compared with normoglycemic controls or rats that had been diabetic for 6 weeks, whereas capillary density and levels of VEGF and eNOS protein and cardiac NO(x) levels were all increased. Phosphorylation of Akt, a prosurvival signal, was also significantly increased after 2 weeks of diabetes. Cardiac lipid peroxidation was comparable to controls after 2 weeks of diabetes, whereas levels of nitrotyrosine, a peroxynitrite biomarker, were reduced. After 6 weeks of diabetes, lipid peroxidation was increased and levels of VEGF and plasma NO were reduced as compared with controls or rats diabetic for 2 weeks. Our results indicate endogenous cardioprotective mechanisms become transiently activated in this early stage of diabetes and that this may protect the heart from I/R injury through enhancement of VEGF and eNOS expression, NO formation, activation of cell survival signals, and decreased oxidative stress.

Animals↗

PubMed2006

Simvastatin improves diabetes-induced coronary endothelial dysfunction.

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors decrease cardiovascular morbidity in diabetic patients, but the mechanism is unclear. We studied the actions of simvastatin (SIM) in enhancing NO bioavailability and reducing oxidative stress in coronary vessels from diabetic rats and in rat coronary artery endothelial cells (RCAEC) exposed to high glucose. Coronary arteries isolated from diabetic rats showed decreases in acetylcholine (ACh)-mediated maximal relaxation from 81.0 +/- 4.5% in controls to 43.5 +/- 7.6% at 4 weeks and 22.3 +/- 0.6% at 10 weeks of diabetes. This effect was associated with oxidative stress in coronary vessels as shown by dichlorofluorescein (DCF) imaging and nitrotyrosine labeling. Diabetes also reduced trans-coronary uptake of [(3)H]l-arginine. Supplemental l-arginine (50 mg/kg/day p.o.) did not improve coronary vasorelaxation to ACh. However, SIM treatment (5 mg/kg/day subcutaneously) improved maximal ACh relaxation to 65.8 +/- 5.1% at 4 weeks and 47.1 +/- 3.9% at 10 weeks. Coronary arteries from rats treated with both SIM and l-arginine demonstrated the same maximal relaxation to ACh (66.1 +/- 3%) as SIM alone. Mevalonate and l-NAME (N(omega)-nitro-l-arginine methyl ester hydrochloride) inhibited the response to ACh in SIM-treated diabetic rats. Coronary arteries from all groups relaxed similarly to sodium nitroprusside. SIM increased endothelial NO synthase protein levels and blocked diabetes-induced increases in DCF and nitrotyrosine labeling in diabetic coronary vessels. SIM treatment restored normal NO levels in media from high-glucose-treated RCAEC and plasma of diabetic rat. Treatment with SIM or the NADPH oxidase inhibitor apocynin also blocked high-glucose-induced increases in reactive oxygen species and superoxide formation in RCAEC. Taken together, these data suggest that SIM improves diabetes-induced coronary dysfunction by reducing oxidative stress and increasing NO bioavailability.

Animals↗

PubMed2006

Neuroprotective and blood-retinal barrier-preserving effects of cannabidiol in experimental diabetes.

Diabetic retinopathy is characterized by blood-retinal barrier (BRB) breakdown and neurotoxicity. These pathologies have been associated with oxidative stress and proinflammatory cytokines, which may operate by activating their downstream target p38 MAP kinase. In the present study, the protective effects of a nonpsychotropic cannabinoid, cannabidiol (CBD), were examined in streptozotocin-induced diabetic rats after 1, 2, or 4 weeks. Retinal cell death was determined by terminal dUTP nick-end labeling assay; BRB function by quantifying extravasation of bovine serum albumin-fluorescein; and oxidative stress by assays for lipid peroxidation, dichlorofluorescein fluorescence, and tyrosine nitration. Experimental diabetes induced significant increases in oxidative stress, retinal neuronal cell death, and vascular permeability. These effects were associated with increased levels of tumor necrosis factor-alpha, vascular endothelial growth factor, and intercellular adhesion molecule-1 and activation of p38 MAP kinase, as assessed by enzyme-linked immunosorbent assay, immunohistochemistry, and/or Western blot. CBD treatment significantly reduced oxidative stress; decreased the levels of tumor necrosis factor-alpha, vascular endothelial growth factor, and intercellular adhesion molecule-1; and prevented retinal cell death and vascular hyperpermeability in the diabetic retina. Consistent with these effects, CBD treatment also significantly inhibited p38 MAP kinase in the diabetic retina. These results demonstrate that CBD treatment reduces neurotoxicity, inflammation, and BRB breakdown in diabetic animals through activities that may involve inhibition of p38 MAP kinase.

Animals↗

PubMed2005

Peroxynitrite increases VEGF expression in vascular endothelial cells via STAT3.

Increased expression of vascular endothelial growth factor (VEGF) has been correlated with increased oxidative stress and formation of peroxynitrite in numerous disease conditions, including diabetic microangiopathy, tumor angiogenesis, and atherosclerosis. In this study we tested the hypothesis that peroxynitrite stimulates VEGF expression. Treatment of microvascular endothelial cells with exogenous peroxynitrite induced a time- and dose-dependent increase in VEGF mRNA, which peaked within 1 h of treatment at a concentration of 100 muM. The increase in VEGF mRNA was followed by a significant increase in VEGF protein. To define the molecular mechanisms involved, the effect of peroxynitrite was determined on the activation of two transcription factors known to regulate VEGF expression during hypoxia and tumor angiogenesis-signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor-1 (HIF-1). Peroxynitrite caused activation and nuclear translocation of STAT3, but not HIF-1. Moreover, transduction of endothelial cells with dominant-negative STAT3 abrogated the peroxynitrite-induced increase in VEGF mRNA. The increase in VEGF mRNA was also blocked by inhibitors of transcription and was unaffected by the inhibition of protein synthesis. These results indicate that peroxynitrite causes increased expression of VEGF in vascular endothelial cells by a process that requires the activation of STAT3.

Active Transport, Cell Nucleus↗

PubMed2005

Hyperglycemia and reactive oxygen species mediate apoptosis in aortic endothelial cells through Janus kinase 2.

The generation of reactive oxygen species (ROS) has been implicated in the perturbation of endothelial function and cell death. However, the specific signaling pathways which mediate and modifying this response have not been fully elucidated. Therefore, in this study we tested the hypothesis that activation of JAK2 is involved in the aortic endothelial cell (EC) response to ROS. When ECs were exposed to HG (25 mM) for 6 h or ROS (i.e., H(2)O(2) (100 microM)) for 1 h and returned to normal medium we found a decrease in cell density and morphologic signs of apoptosis. Furthermore, incubation of ECs with HG and H(2)O(2) also resulted in the tyrosine phosphorylation of JAK2. In addition, pretreatment of ECs with AG-490, an inhibitor of JAK2, prevented nuclear fragmentation, whereas inhibitors of Jun kinase (SP 600125), MAP kinase (PD 98059), Src kinase (PP2) or PI-3 kinase (wortmannin) were without effect. Finally, immunoblot analysis of caspase-3 and PARP cleavage confirmed a role for activation of JAK2 in both HG- or ROS-induced apoptosis, based on inhibition by either AG-490 or adenoviral transfection with a dominant-negative JAK2 mutant. In conclusion the activation of JAK2 plays a pivotal role in oxidant stress-induced commitment of ECs to apoptosis, based on studies with HG and H(2)O(2).

Adenoviridae↗

PubMed2005

Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration.

In diabetic retinopathy, endothelial cell apoptosis is paradoxically increased despite upregulation of the potent pro-survival factor VEGF. We tested the hypothesis that elevated glucose levels disrupt VEGF's pro-survival function via peroxynitrite-mediated alteration of the Akt-1/p38 MAP kinase signaling pathway by studies of retinal endothelial cells in vitro. High glucose or exogenous peroxynitrite caused significant increases in apoptosis in the presence or absence of VEGF. Treatment with a peroxynitrite decomposition catalyst blocked these effects, implying a causal role of peroxynitrite. Peroxynitrite or high glucose treatment also increased phosphorylation of p38 MAP kinase, whereas phosphorylation of Akt-1 was significantly decreased in basal or VEGF-stimulated conditions. High glucose- or peroxynitrite-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI 3-kinase that blocked PI 3-kinase and Akt-1 kinase activity. Inhibiting peroxynitrite formation or blocking tyrosine nitration of p85 restored the activity of PI 3-kinase and Akt-1 kinase, blocked phosphorylation of p38 MAP kinase and normalized pro-survival function. Transfecting the cells with constitutively active Akt-1 or inhibiting activity of p38 MAP kinase completely masked the pro-apoptotic effects of high glucose and exogenous peroxynitrite, suggesting an interaction between the Akt-1 and p38 MAP kinase pathways. In conclusion, high glucose treatment blocks the pro-survival effect of VEGF and causes accelerated endothelial cell apoptosis via the action of peroxynitrite in causing tyrosine nitration of PI 3-kinase, inhibiting activity of Akt-1 kinase and increasing the activity of p38 MAP kinase.

Adenoviridae↗

PubMed2005

Inhibition of NAD(P)H oxidase activity blocks vascular endothelial growth factor overexpression and neovascularization during ischemic retinopathy.

Because oxidative stress has been strongly implicated in up-regulation of vascular endothelial growth factor (VEGF) expression in ischemic retinopathy, we evaluated the role of NAD(P)H oxidase in causing VEGF overexpression and retinal neovascularization. Dihydroethidium imaging analyses showed increased superoxide formation in areas of retinal neovascularization associated with relative retinal hypoxia in a mouse model for oxygen-induced retinopathy. The effect of hypoxia in stimulating superoxide formation in retinal vascular endothelial cells was confirmed by in vitro chemiluminescence assays. The superoxide formation was blocked by specific inhibitors of NAD(P)H oxidase activity (apocynin, gp91ds-tat) indicating that NAD(P)H oxidase is a major source of superoxide formation. Western blot and immunolocalization analyses showed that retinal ischemia increased expression of the NAD(P)H oxidase catalytic subunit gp91phox, which localized primarily within vascular endothelial cells. Treatment of mice with apocynin blocked ischemia-induced increases in oxidative stress, normalized VEGF expression, and prevented retinal neovascularization. Apocynin and gp91ds-tat also blocked the action of hypoxia in causing increased VEGF expression in vitro, confirming the specific role of NAD(P)H oxidase in hypoxia-induced increases in VEGF expression. In conclusion, NAD(P)H oxidase activity is required for hypoxia-stimulated increases in VEGF expression and retinal neovascularization. Inhibition of NAD(P)H oxidase offers a new therapeutic target for the treatment of retinopathy.

Acetophenones↗

PubMed2005

Vascular endothelial growth factor and diabetic retinopathy: role of oxidative stress.

Retinal neovascularization and macular edema are central features of diabetic retinopathy, a major cause of blindness in working age adults. The currently established treatment for diabetic retinopathy targets the vascular pathology by laser photocoagulation. This approach is associated with significant adverse effects due the destruction of neural tissue and is not always effective. Characterization of the molecular and cellular processes involved in vascular growth and hyperpermeability has led to the recognition that the angiogenic growth factor and vascular permeability factor VEGF (vascular endothelial growth factor) play a pivotal role in the retinal microvascular complications of diabetes. Thus, VEGF represents an important target for therapeutic intervention in diabetic retinopathy. Agents that directly inhibit the actions of VEGF and its receptors show considerable promise, but have not proven to be completely effective in blocking pathological angiogenesis. Therefore, a better understanding of the molecular events that control VEGF expression and mediate its downstream actions is important to define more precise therapeutic targets for intervention in diabetic retinopathy. This review highlights the current understanding of the process by which VEGF gene expression is regulated and how VEGF's biological effects are altered during diabetes. In particular, cellular and molecular alterations seen in diabetic models are considered in the context of high glucose-mediated oxidative stress effects on VEGF expression and action. Potential therapeutic strategies for preventing VEGF overexpression or blocking its pathological actions in the diabetic retina are considered.

Adrenal Cortex Hormones↗

PubMed2004

Alternative splicing of the APC gene in the neural retina and retinal pigment epithelium.

PURPOSE: Hypertrophy and hyperplasia of the retinal pigment epithelium (RPE) is associated with an inherited predisposition to human familial adenomatous polyposis coli, suggesting that expression of the adenomatous polyposis coli (APC) tumor suppressor may regulate RPE proliferation/differentiation. Distinctive APC isoforms exist in different cell types due to alternative splicing of the APC transcripts. We hypothesize that differences in expression patterns of APC protein isoforms are critical to RPE proliferation/differentiation. METHODS: To investigate these relationships, APC gene expression was characterized in the retinas and RPE from fetal and adult human and mouse, and in the epiretinal membranes (ERM) from 5 patients with proliferative vitreoretinopathy (PVR). Expression patterns of alternative splice-forms of APC transcripts were evaluated by comparative quantitative RT-PCR. Exon 1 of APC encodes a heptad repeat that confers the ability of APC to homodimerize. APC protein isoforms containing or lacking this heptad were characterized by western blot analysis and immunohistochemistry. RESULTS: Comparative quantitative RT-PCR demonstrated a predominant exon 1 containing, conventional APC splice-form in the early developing fetal RPE and retina, and in all the tested ERM samples from patients with PVR. This method also demonstrated an increased level of exon 1 lacking APC splice-form in the mature RPE and retina. Western blot analysis and immunofluorescence microscopy demonstrated the conventional APC only in the RPE, and the APC isoform without the first heptad repeat in both the retina and RPE. Immunofluorescence microscopy also demonstrated only the conventional APC in the ERM samples tested. CONCLUSIONS: These results suggest that alternative splicing of APC leads to differential APC expression with potentially unique functions. APC isoform without the first heptad repeat may play a role in cell cycle cessation in the adult retina and RPE, and the down regulation of this APC isoform may contribute to the potential of RPE to migrate and proliferate.

Adenomatous Polyposis Coli Protein↗

PubMed2003

Normal vascular development in mice deficient in endothelial NO synthase: possible role of neuronal NO synthase.

PURPOSE: Nitric oxide formation by nitric oxide synthase (NOS) has been implicated in vascular injury and retinal neovascularization during oxygen-induced retinopathy. However, the role of NOS in normal retinal vascular development and growth has not been studied. The purpose of these experiments was to characterize the expression of NOS in relation to vascular development and to determine the effect of deleting endothelial NOS (eNOS) on this process. METHODS: Retinal vascular development was analyzed in 150 eNOS+/+ and eNOS-/- mice ranging from 1 day to 6 months old by using a combination of morphometric and biochemical approaches. The pattern of vascular development was analyzed in retinal tissue sections and whole-mount preparations labeled with fluorescein-conjugated Griffonia simplicifolia lectin. Analysis of vascular density and arterial diameter were performed with the lectin-labeled whole-mounts using computer-assisted morphometry. NO production was quantified by measuring retinal levels of nitrate/nitrite accumulation using the Greiss reaction. Western blotting techniques with isoform-specific NOS antibodies were used to evaluate differences in levels of NOS protein expression. Retinal distribution of nNOS was characterized using nNOS immunocytochemistry and NADPH diaphorase histochemistry. RESULTS: These analyses showed that the rate and pattern of retinal vascular development in eNOS-/- mice were comparable with those in wild-type control mice. Measurement of vascular density showed no significant differences between the two strains. The amount of NO production in the eNOS-/- retina was also equivalent to that in the eNOS+/+ retina. Analysis of nNOS expression within the eNOS+/+ and eNOS-/- mice showed similar levels of total nNOS protein in the two strains. Inducible NOS was not detected in either strain. Studies of nNOS distribution showed intense labeling of the deep capillary plexus in the eNOS-/- retina. This was not seen in the wild-type retinas. The number of neuronal cells showing NADPH-diaphorase activity was also significantly increased in the eNOS-/- mice. CONCLUSIONS: Development of the retinal vasculature occurs normally without eNOS. The observations of similar levels of NO production, perivascular redistribution of nNOS and increased numbers of NADPH-diaphorase reactive neurons in the eNOS-/- retinas suggest that increases in vascular-associated nNOS activity compensate for the eNOS deficiency in the developing mutant retina.

Animals↗

PubMed2003

VEGF differentially activates STAT3 in microvascular endothelial cells.

Increased VEGF expression is found in several pathologies characterized by abnormal angiogenesis. Previous studies have shown that the transcription factor STAT3 mediates VEGF gene transcription and its activation. In this study, Western analysis and confocal immunocytochemistry were used to examine STAT3 activation in retinal microvascular endothelial cells (BREC). We found that VEGF rapidly induces STAT3 tyrosine phosphorylation and nuclear translocation. Immunoprecipitation studies also showed that VEGF forms a complex with VEGFR2 only in BREC and not in aortic macrovascular endothelial cells (BAEC). In addition, quantitative real-time RT-PCR analysis of VEGF-induced VEGF expression showed a significant increase in specific mRNA formation only in BREC and not in BAEC, and this effect was significantly reduced by antisense-mediated reduction of STAT3 expression. Furthermore, studies conducted in human dermal microvascular endothelial cells (HDMEC) showed that, in this endothelial cell type, VEGF autocrine expression is also accompanied by STAT3 activation as in BREC. In this study we showed that VEGF can differentially induce STAT3 activation in micro- versus macro-vascular endothelial cells and that this effect is linked to VEGFR2/STAT3 complex formation, which correlates with VEGF autocrine ability to stimulate its own gene expression.

Active Transport, Cell Nucleus↗

PubMed2003

Hyperoxia induces retinal vascular endothelial cell apoptosis through formation of peroxynitrite.

Hyperoxia exposure induces capillary endothelial cell apoptosis in the developing retina, leading to vaso-obliteration followed by proliferative retinopathy. Previous in vivo studies have shown that endothelial nitric oxide synthase (NOS3) and peroxynitrite are important mediators of the vaso-obliteration. Now we have investigated the relationship between hyperoxia, NOS3, peroxynitrite, and endothelial cell apoptosis by in vitro experiments using bovine retinal endothelial cells (BREC). We found that BREC exposed to 40% oxygen (hyperoxia) for 48 h underwent apoptosis associated with activation of caspase-3 and cleavage of the caspase substrate poly(ADP-ribose) polymerase. Hyperoxia-induced apoptosis was associated with increased formation of nitric oxide, peroxynitrite, and superoxide anion and was blocked by treatment with uric acid, nitro-L-arginine methyl ester, or superoxide dismutase. Analyses of the phosphatidylinositol 3-kinase/Akt kinase survival pathway in cells directly treated with peroxynitrite revealed inhibition of VEGF- and basic FGF-induced activation of Akt kinase. These results suggest that hyperoxia-induced formation of peroxynitrite induces BREC apoptosis by crippling key survival pathways and that blocking peroxynitrite formation prevents apoptosis. These data may have important clinical implications for infants at risk of retinopathy of prematurity.

Animals↗

PubMed2003

VEGF-induced paracellular permeability in cultured endothelial cells involves urokinase and its receptor.

Vascular endothelial growth factor/vascular permeability factor (VEGF) has been implicated in blood/tissue barrier dysfunctions associated with pathological angiogenesis, but the mechanisms of VEGF-induced permeability increase are poorly understood. Here, the role of VEGF-induced extracellular proteolytic activities on the endothelial cell permeability increase is evaluated. Confluent monolayers of bovine retinal microvascular endothelial (BRE) cells grown on porous membrane were treated with VEGF or urokinase plasminogen activator (uPA), and permeability changes were analyzed. uPA-induced permeability was rapid and sustained, but VEGF-induced permeability showed a biphasic pattern: a rapid and transient phase (1-2 h) followed by delayed and sustained phase (6-24 h). The delayed, but not the early phase of VEGF-induced permeability, was blocked by anti-uPA or anti-uPAR (uPA receptor) antibodies and was accompanied by reduced transendothelial electrical resistance, indicating the paracellular route of permeability. Confocal microscopy and Western blotting showed that VEGF treatment increased free cytosolic beta-catenin, which was followed by beta-catenin nuclear translocation, upregulation of uPAR, and downregulation of occludin. Membrane-bound occludin was released immediately after uPA treatment, but with a long delay after VEGF treatment, suggesting a requirement for uPAR gene expression. In conclusion, VEGF induces a sustained paracellular permeability in capillary endothelial cells that is mediated by activation of the uPA/uPAR system.

Animals↗

PubMed2003

Neuroprotective effect of (-)Delta9-tetrahydrocannabinol and cannabidiol in N-methyl-D-aspartate-induced retinal neurotoxicity: involvement of peroxynitrite.

In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic Delta9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL,a superoxide dismutase-mimetic), N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a dose- and time-dependent accumulation of nitrite/nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma.

Animals↗

PubMed2003

Experimental diabetes causes breakdown of the blood-retina barrier by a mechanism involving tyrosine nitration and increases in expression of vascular endothelial growth factor and urokinase plasminogen activator receptor.

The purpose of these experiments was to determine the specific role of reactive oxygen species (ROS) in the blood-retinal barrier (BRB) breakdown that characterizes the early stages of vascular dysfunction in diabetes. Based on our data showing that high glucose increases nitric oxide, superoxide, and nitrotyrosine formation in retinal endothelial cells, we hypothesized that excess formation of ROS causes BRB breakdown in diabetes. Because ROS are known to induce increases in expression of the well-known endothelial mitogen and permeability factor vascular endothelial growth factor (VEGF) we also examined their influence on the expression of VEGF and its downstream target urokinase plasminogen activator receptor (uPAR). After 2 weeks of streptozotocin-induced diabetes, analysis of albumin leakage confirmed a prominent breakdown of the BRB. This permeability defect was correlated with significant increases in the formation of nitric oxide, lipid peroxides, and the peroxynitrite biomarker nitrotyrosine as well as with increases in the expression of VEGF and uPAR. Treatment with a nitric oxide synthase inhibitor (N-omega-nitro-L-arginine methyl ester, 50 mg/kg/day) or peroxynitrite scavenger (uric acid, 160 mg/kg/day) blocked the breakdown in the BRB and prevented the increases in formation of lipid peroxides and tyrosine nitration as well as the increases in expression of VEGF and uPAR. Taken together, these data indicate that early diabetes causes breakdown of the BRB by a mechanism involving the action of reactive nitrogen species in promoting expression of VEGF and uPAR.

Animals↗

PubMed2003

High glucose-induced tyrosine nitration in endothelial cells: role of eNOS uncoupling and aldose reductase activation.

PURPOSE: Analyses in diabetic rats have shown that breakdown of the blood-retina barrier is associated with increased formation of peroxynitrite, nitric oxide, and lipid peroxidation. The permeability increase is blocked by treatments that also prevent the increases in reactive oxygen species, suggesting their causal role in vascular dysfunction. The purpose of this study was to determine the specific effects of high glucose and high osmolarity on the formation of nitrotyrosine, nitric oxide, and superoxide anion in retinal vascular endothelial cells and to evaluate the metabolic pathways involved. METHODS: Cultured retinal endothelial cells were maintained for 5 days in media with different concentrations of glucose or osmotic control reagents and tested for effects on protein tyrosine nitration and nitric oxide synthase (NOS) expression, using immunoblot techniques. NOS activity was determined by assays for nitrite formation and conversion of arginine to citrulline. Superoxide anion formation was assayed by hydroethidine staining. RESULTS: Increased concentrations of glucose or 3-methyL-o-glucose stimulated formation of nitric oxide (NO) and superoxide induced protein nitration on tyrosine and increased expression and activity of endothelial nitric oxide synthase (eNOS). The effects of glucose were more potent: Inhibiting NOS or aldose reductase (AR), scavenging superoxide or peroxynitrite, or supplementing the NOS substrate L-arginine or cofactor tetrahydrobiopterin blocked the formation of reactive oxygen species and prevented protein tyrosine nitration. CONCLUSIONS: Increases in glucose levels and osmotic stress similar to those in diabetic patients increase the formation of nitrotyrosine in retinal endothelial cells because of their actions increasing NOS activity and causing superoxide formation due to eNOS uncoupling and AR activation.

Aldehyde Reductase↗

PubMed2002

HGF regulation of RPE proliferation in an IL-1beta/retinal hole-induced rabbit model of PVR.

PURPOSE: To understand molecular events that lead to retinal pigment epithelial (RPE) cell proliferation and migration during the early phases of proliferative vitreoretinopathy (PVR) in a rabbit model. METHODS: Retinal holes were created and interleukin-1beta(IL-1beta) was injected intravitreally. Eyes were examined by indirect ophthalmoscopy and eyecup pieces containing retinal holes were analyzed at different times after the surgery up to 4 weeks. RPE proliferation and migration were examined by immunohistochemistry. Tyrosine phosphorylation of extracellular signal regulated kinase (ERK) and hepatocyte growth factor receptor (HGFR or c-met) was determined by immunoprecipitation and western blot analysis. Tyrosine phosphorylation of c-met and morphological studies was performed on vitreous treated ARPE-19 cells. Expression of c-jun was determined by Northern blot analysis. Matrix metalloproteinase (MMP) content in vitreous was assessed by zymography. RESULTS: Indirect ophthalmoscopy identified formation of epiretinal membrane and immunohistochemistry identified proliferative and migratory RPE and other cells in the posterior segment containing retinal holes at 4 weeks post-surgery. Tyrosine phosphorylation of ERK and c-met occurred in this segment within 30 min of surgery. ARPE-19 cells treated with vitreous from the 24 h post-surgical eyes, but not with control vitreous or IL-1beta, showed morphological changes and tyrosine phosphorylation of c-met. Northern blot analysis in this segment identified upregulation of c-jun within 30 min of surgery and the expression peaked at 72 h. Zymographic analysis of vitreous identified MMP-9 in 12-72 h post-surgery. CONCLUSIONS: These data suggest that the presence of retinal holes and IL-1beta may lead to activation of HGF, mitogen activated protein kinases (MAPK), c-jun and extracellular matrix remodeling, resulting in proliferative and migratory cells in the wounded retina.

Animals↗