Endothelial glycocalyx in retina, hyperglycemia, and diabetic retinopathy.

The endothelial glycocalyx (EG) is a meshlike network present on the apical surface of the endothelium. Membrane-bound proteoglycans, the major backbone molecules of the EG, consist of glycosaminoglycans attached to core proteins. In addition to maintaining the integrity of the endothelial barrier, the EG regulates inflammation and perfusion and acts as a mechanosensor. The loss of the EG can cause endothelial dysfunction and drive the progression of vascular diseases including diabetic retinopathy. Therefore, the EG presents a novel therapeutic target for treatment of vascular complications. In this review article, we provide an overview of the structure and function of the EG in the retina. Our particular focus is on hyperglycemia-induced perturbations in the glycocalyx structure in the retina, potential underlying mechanisms, and clinical trials studying protective treatments against degradation of the EG.

Decreased retinal and choroidal endothelial surface molecules in spontaneously hypertensive rats.

Hypertension is associated with changes in the retina and choroid, with resulting consequences of increased vascular permeability and microhemorrhages. To date, very little information is available regarding the changes in the retinal and choroidal endothelial surface layer. In this study, we have examined changes in protein expression of several molecules including platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial cadherin (VE-cadherin), glypican-1, and syndecan-1, in spontaneously hypertensive rats (SHR) compared to control normotensive Wistar Kyoto (WKY) rats. In male SHR vs WKY rat retinas, decreases were found for VE-cadherin and syndecan-1; whereas in female retinas, decreases were found for PECAM-1, glypican-1, and syndecan-1. In male SHR vs WKY rat choroid, we found an increase in glypican-1, but choroidal syndecan-1 was decreased in SHR in both males and females. Therefore, decreases in SHR of both retinal and choroidal syndecan-1 were found in both males and females. These losses of syndecan-1 were accompanied by an increase in plasma levels of the proteoglycan, indicating possible systemic endothelial shedding. In contrast, plasma levels of glypican-1 decreased. Interestingly, in normotensive WKY rats, retinal levels of all four endothelial surface molecules were higher in females than in males, in some cases, by substantial amounts. In summary, a number of changes occur in endothelial surface molecules in SHR, with some changes being sex-dependent; it is possible that the loss of these molecules contributes to the vascular dysfunction that occurs in hypertensive retina and choroid.

Effect of high glucose on glycosaminoglycans in cultured retinal endothelial cells and rat retina.

INTRODUCTION: The endothelial glycocalyx regulates vascular permeability, inflammation, and coagulation, and acts as a mechanosensor. The loss of glycocalyx can cause endothelial injury and contribute to several microvascular complications and, therefore, may promote diabetic retinopathy. Studies have shown a partial loss of retinal glycocalyx in diabetes, but with few molecular details of the changes in glycosaminoglycan (GAG) composition. Therefore, the purpose of our study was to investigate the effect of hyperglycemia on GAGs of the retinal endothelial glycocalyx. METHODS: GAGs were isolated from rat retinal microvascular endothelial cells (RRMECs), media, and retinas, followed by liquid chromatography-mass spectrometry assays. Quantitative real-time polymerase chain reaction was used to study mRNA transcripts of the enzymes involved in GAG biosynthesis. RESULTS AND CONCLUSIONS: Hyperglycemia significantly increased the shedding of heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). There were no changes to the levels of HS in RRMEC monolayers grown in high-glucose media, but the levels of CS and HA decreased dramatically. Similarly, while HA decreased in the retinas of diabetic rats, the total GAG and CS levels increased. Hyperglycemia in RRMECs caused a significant increase in the mRNA levels of the enzymes involved in GAG biosynthesis (including EXTL-1,2,3, EXT-1,2, ChSY-1,3, and HAS-2,3), with these increases potentially being compensatory responses to overall glycocalyx loss. Both RRMECs and retinas of diabetic rats exhibited glucose-induced alterations in the disaccharide compositions and sulfation of HS and CS, with the changes in sulfation including N,6-O-sulfation on HS and 4-O-sulfation on CS.

The role of tumor necrosis factor-α and interferon-γ in the hyperglycemia-induced ubiquitination and loss of platelet endothelial cell adhesion molecule-1 in rat retinal endothelial cells.

OBJECTIVE: This study aimed to investigate the role of the hyperglycemia-induced increase in tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the ubiquitination and degradation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the diabetic retina. METHODS: Type I diabetes was induced in rats by the injection of streptozotocin, with age-matched non-diabetic rats as controls. Primary rat retinal microvascular endothelial cells were grown in normal or high glucose media for 6 days or in normal glucose media for 24 h with addition of TNF-α and/or IFN-γ. PECAM-1, TNF-α, IFN-γ, and ubiquitin levels were assessed using Western blotting, immunofluorescence, and immunoprecipitation assays. Additionally, proteasome activity was assessed both in vivo and in vitro. RESULTS: Under hyperglycemic conditions, total ubiquitination levels in the retina and RRMECs, and PECAM-1 ubiquitination levels in RRMECs, were significantly increased. Additionally, TNF-α and IFN-γ levels were significantly increased under hyperglycemic conditions. PECAM-1 levels in RRMECs treated with TNF-α and/or IFN-γ were significantly decreased. Moreover, there was a significant decrease in proteasome activity in the diabetic retina, hyperglycemic RRMECs, and RRMECs treated with TNF-α or IFN-γ. CONCLUSION: Tumor necrosis factor-α and IFN-γ may contribute to the hyperglycemia-induced loss of PECAM-1 in retinal endothelial cells, possibly by upregulating PECAM-1 ubiquitination.

Retinal hypoxia and angiogenesis with methamphetamine.

Central retinal artery occlusion, retinopathy, and retinal neovascularization have been reported in methamphetamine (METH) abusers. In the current study, we investigated whether METH induces retinal neovascularization in a mouse model, and if so, whether the neovascularization is associated with increased hypoxia, hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF). Mice were administrated METH by intraperitoneal injection over a 26-day period, or injected with saline as a vehicle control. The number of retinal arterioles and venules were counted using in vivo live imaging following infusion with fluorescein isothiocyanate-dextran. Excised retinas were stained with griffonia simplicifolia lectin I and flat mounted for a measurement of vascularity (length of vessels per tissue area) with AngioTool. Retinal hypoxia was examined by formation of pimonidazole adducts with an anti-pimonidazole antibody, and HIF-1α and VEGFa protein levels in the retina were detected by immunoblot. METH administration increased vascularity (including the number of arterioles) measured on Day 26. Retinal VEGFa protein level was not changed in METH-treated mice on Day 5, but was increased on Day 12 and Day 26. Hypoxia (pimonidazole adduct formation) was increased in retinas of METH-treated mice on Day 12 and Day 26, as were HIF-1α protein expression levels. These results indicate that METH administration induces hypoxia, HIF-1α, VEGFa, and angiogenesis in the retina.

Hyperglycemia-induced effects on glycocalyx components in the retina.

PURPOSE: Diabetic retinopathy is a vision-threatening complication of diabetes characterized by endothelial injury and vascular dysfunction. The loss of the endothelial glycocalyx, a dynamic layer lining all endothelial cells, contributes to several microvascular pathologies, including an increase in vascular permeability, leukocyte plugging, and capillary occlusion, and may drive the progression of retinopathy. Previously, a significant decrease in glycocalyx thickness has been observed in diabetic retinas. However, the effects of diabetes on specific components of the retinal glycocalyx have not yet been studied. Therefore, the aim of our study was to investigate changes in synthesis, expression, and shedding of retinal glycocalyx components induced by hyperglycemia, which could provide a novel therapeutic target for diabetic retinopathy. METHODS: Primary rat retinal microvascular endothelial cells (RRMECs) were grown under normal glucose (5 mM) or high-glucose (25 mM) conditions for 6 days. The mRNA and protein levels of the glycocalyx components were examined using qRT-PCR and Western blot analysis, respectively. Further, mass spectrometry was used to analyze protein intensities of core proteins. In addition, the streptozotocin-induced Type 1 diabetic rat model was used to study changes in the expression of the retinal glycocalyx in vivo. The shedding of the glycocalyx was studied in both culture medium and in plasma using Western blot analysis. RESULTS: A significant increase in the shedding of syndecan-1 and CD44 was observed both in vitro and in vivo under high-glucose conditions. The mRNA levels of syndecan-3 were significantly lower in the RRMECs grown under high glucose conditions, whereas those of syndecan-1, syndecan-2, syndecan-4, glypican-1, glypican-3, and CD44 were significantly higher. The protein expression of syndecan-3 and glypican-1 in RRMECs was reduced considerably following exposure to high glucose, whereas that of syndecan-1 and CD44 increased significantly. In addition, mass spectrometry data also suggests a significant increase in syndecan-4 and a significant decrease in glypican-3 protein levels with high glucose stimulation. In vivo, our data also suggest a significant decrease in the mRNA transcripts of syndecan-3 and an increase in mRNA levels of glypican-1 and CD44 in the retinas of diabetic rats. The diabetic rats exhibited a significant reduction in the retinal expression of syndecan-3 and CD44. However, the expression of syndecan-1 and glypican-1 increased significantly in the diabetic retina. CONCLUSIONS: One of the main findings of our study was the considerable diversity of glucose-induced changes in expression and shedding of various components of endothelial glycocalyx, for example, increased endothelial and retinal syndecan-1, but decreased endothelial and retinal syndecan-3. This indicates that the reported decrease in the retinal glycocalyx in diabetes in not a result of a non-specific shedding mechanism. Moreover, mRNA measurements indicated a similar diversity, with increases in endothelial and/or retinal levels of syndecan-1, glypican-1, and CD44, but a decrease for syndecan-3, with these increases in mRNA potentially a compensatory reaction to the overall loss of glycocalyx.

Hyperglycemia-induced ubiquitination and degradation of ß-catenin with the loss of platelet endothelial cell adhesion molecule-1 in retinal endothelial cells.

OBJECTIVE: Increased retinal vascular permeability is one of the earliest manifestations of diabetic retinopathy. The aim of this study was to investigate the role of hyperglycemia-induced platelet endothelial cell adhesion molecule-1 loss on retinal vascular permeability via the ß-catenin pathway. METHODS: Type I diabetes was induced in male Wistar rats using streptozotocin injections, with age-matched non-diabetic rats as controls. Rat retinal microvascular endothelial cells were grown under normal or high glucose conditions for 6 days. Small interfering Ribonucleic Acid was used to knock down platelet endothelial cell adhesion molecule-1 in rat retinal microvascular endothelial cells for loss-of-function studies. Retinas and rat retinal microvascular endothelial cells were subjected to Western blot, immunofluorescence labeling, and co-immunoprecipitation analyses to assess protein levels and interactions. A biotinylated gelatin and fluorescein isothiocyanate-avidin assay was used for retinal endothelial cell permeability studies. RESULTS: ß-catenin, ß-catenin/platelet endothelial cell adhesion molecule-1 interaction, active Src homology 2 domain-containing protein tyrosine phosphatase were significantly decreased, while ß-catenin ubiquitination levels and endothelial permeability were significantly increased, in hyperglycemic retinal endothelial cells. Similar results were observed with platelet endothelial cell adhesion molecule-1 partial knockdown, where ß-catenin and active Src homology 2 domain-containing protein tyrosine phosphatase levels were decreased, while phospho-ß-catenin and retinal endothelial cell permeability were increased. CONCLUSION: Platelet endothelial cell adhesion molecule-1 loss may contribute to increased retinal endothelial cell permeability by attenuating ß-catenin levels under hyperglycemic conditions.

Acute changes in the retina and central retinal artery with methamphetamine.

Methamphetamine (METH), an addictive stimulant of neurotransmitters, is associated with cardiovascular and neurological diseases. METH-induced ophthalmic complications are also present but have been insufficiently investigated. The purpose of this study is to investigate the retinal effects of METH. C57BL/6 mice were administrated progressively increasing doses of METH (0-6 mg/kg) by repetitive intraperitoneal injections for 5 days (4 times per day). Retinal degeneration was examined by morphological changes and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Norepinephrine levels were measured by ELISA, protein expression levels were determined by immunoblot and immunostaining, and gelatinase activity was examined by zymography. The thickness of the retina and the number of nuclei in the inner and outer nuclear layers were decreased by METH. Retinal cell death and astrocyte activation by METH treatment were confirmed by TUNEL assay and glial fibrillary acidic protein expression, respectively. Increased tumor necrosis factor-α protein in the retina and elevated norepinephrine levels in plasma were found in METH-treated mice. Platelet endothelial cell adhesion molecule-1 (PECAM-1) protein expression level was decreased in the retina and central retinal artery (CRA) by METH treatment, along with the endothelial proteoglycans glypican-1 and syndecan-1. Moreover, a regulator of the extracellular matrix, matrix metalloproteinase-14 (MMP-14) in the retina, and MMP-2 and MMP-9 in plasma, were increased by METH treatment. In conclusion, METH administration is involved in retinal degeneration with a vascular loss of PECAM-1 and the glycocalyx in the CRA and retina, and an increase of MMPs.

Effect of diabetes and hyaluronidase on the retinal endothelial glycocalyx in mice.

We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW = 155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW = 376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.

Diabetic retinopathy: Breaking the barrier.

Diabetic retinopathy (DR) remains a major complication of diabetes and a leading cause of blindness among adults worldwide. DR is a progressive disease affecting both type I and type II diabetic patients at any stage of the disease, and targets the retinal microvasculature. DR results from multiple biochemical, molecular and pathophysiological changes to the retinal vasculature, which affect both microcirculatory functions and ultimately photoreceptor function. Several neural, endothelial, and support cell (e.g., pericyte) mechanisms are altered in a pathological fashion in the hyperglycemic environment during diabetes that can disturb important cell surface components in the vasculature producing the features of progressive DR pathophysiology. These include loss of the glycocalyx, blood-retinal barrier dysfunction, increased expression of inflammatory cell markers and adhesion of blood leukocytes and platelets. Included in this review is a discussion of modifications that occur at or near the surface of the retinal vascular endothelial cells, and the consequences of these alterations on the integrity of the retina.

Anemia and retinal function in a mouse model of acute colitis.

Individuals with inflammatory bowel diseases (IBD) have an elevated risk of ocular inflammation. Both the anterior and posterior eye can be affected by IBD, although posterior eye dysfunction is more likely to go undetected. Little investigative attention has been directed toward the mechanisms of ocular dysfunction with IBD; however, given the prevalence of anemia in IBD and the effects of anemia on the retina, we examined the association between retinal function (electroretinography, ERG) and the anemia induced by experimental IBD, and we tested for a potential retinal benefit of acutely attenuating anemia (via red blood cell (RBC) infusion). Colitis was induced in mice in a model involving drinking water ingestion of dextran sodium sulfate (DSS), with untreated drinking water administered to controls. A subset of the DSS mice was infused with RBCs to attenuate the severity of the anemia induced by DSS. ERG signals (a-waves, b-waves, and oscillatory potential amplitudes and implicit times) were compared between the three groups of mice to evaluate retinal function. ERG amplitudes were significantly decreased in DSS mice compared to controls, with the amplitudes demonstrating a positive correlation with hematocrit, that is, the lowest ERG amplitudes were found with the most severe cases of anemia. An acute infusion of RBCs into DSS mice provided an improvement in the oscillatory potential implicit times, but no significant improvements in other ERG parameters. Despite the association between anemia and ERG signals in DSS-induced colitis, acute RBC infusion may only partially attenuate the associated retinal dysfunction.

High Glucose Induces Oxidative Stress That Alters Glycocalyx Proteoglycan Levels in Primary Rat Retinal Microvascular Endothelial Cells and in Isolated Ophthalmic Arteries.

Our purpose in this study was to identify the role played by oxidative stress in the changes to proteoglycans that occur under hyperglycemic conditions, using primary rat retinal microvascular endothelial cells (RRMEC) and cultured ophthalmic arteries. The cells and blood vessels obtained from rats were cultured in normal glucose (5.6 mM) and high glucose (25 mM) with or without N-acetylcysteine (NAC), an antioxidant. Intracellular oxidative stress was determined by measuring dihydroethidium (DHE) fluorescence and malondialdehyde (MDA)-modified protein levels. mRNA and protein levels were evaluated using quantitative real-time polymerase chain reaction and immunoblot, respectively. High glucose increased levels of glypican-1 mRNA and protein. The level of syndecan-1 mRNA also was increased, but its protein level was decreased, by high glucose. Evaluation of DHE and MDA showed that high glucose increased oxidative stress. These changes caused by high glucose were significantly reversed by NAC treatment. Matrix metalloproteinase-9 (MMP-9) levels, which increased under high glucose conditions, were suppressed by NAC treatment. Oxidative stress caused by hyperglycemia may be responsible for significant changes to the ocular endothelial glycocalyx.

Decreased retinal blood flow in experimental colitis; improvement by eye drop administration of losartan.

Patients with inflammatory bowel disease suffer not only from gut inflammation, but also from extraintestinal manifestations of the disease, including ocular pathology. The mechanisms causing ocular inflammation in these patients are unknown. The purpose of the current study was to investigate the possible vascular changes occurring in the retina using a mouse model of acute colitis, that is, ingestion of dextran sodium sulfate (DSS). Intravital microscopy of anesthetized mice revealed that DSS caused a significant 30-40% decrease in retinal red blood cell velocities, and a 45% decrease in total retinal blood flow, but no changes in intraocular pressure. To determine whether the decreases in retinal perfusion could be inhibited by an angiotensin II receptor antagonist, losartan was administered by eye drops in a subset of the mice prior to the intravital microscopy measurements. Topical losartan was able to largely attenuate the altered hemodynamics induced by DSS. We conclude that angiotensin II might be a possible target for reducing the vascular changes occurring distantly in the eye during colitis.

Retinal blood flow abnormalities following six months of hyperglycemia in the Ins2(Akita) mouse.

The aim of this study was to characterize the microvascular flow abnormalities and oxygenation changes that are present following six months of hyperglycemia in the diabetic Ins2(Akita) mouse. Previous studies have shown decreased retinal blood flow in the first several weeks of hyperglycemia in rodents, similar to the decreases seen in the early stages of human diabetes. However, whether this alteration in the mouse retina continues beyond the initial weeks of diabetes has yet to be determined, as are the potential consequences of the decreased flow on retinal oxygenation. In this study, male Ins2(Akita) and age-matched C57BL/6 (non-diabetic) mice were maintained for a period of six months, at which time intravital microscopy was used to measure retinal blood vessel diameters, blood cell velocity, vascular wall shear rates, blood flow rates, and transient capillary occlusions. In addition, the presence of hypoxia was assessed using the oxygen-sensitive probe pimonidazole. The diabetic retinal microvasculature displayed decreases in red blood cell velocity (30%, p<0.001), shear rate (25%, p<0.01), and flow rate (40%, p<0.001). Moreover, transient capillary stoppages in flow were observed in the diabetic mice, but rarely in the non-diabetic mice. However, no alterations were observed in retinal hypoxia as determined by a pimonidazole assay, suggesting the possibility that the decreases seen in retinal blood flow may be dictated by a decrease in retinal oxygen utilization.

The Endothelial Glycocalyx and Retinal Hemodynamics.

PURPOSE: Previous studies suggest that the endothelial glycocalyx adds to vascular resistance, inhibits thrombosis, and is critical for regulating homogeneous blood flow and ensuring uniform red blood cell (RBC) distribution. However, these functions and consequences of the glycocalyx have not been examined in the retina. We hypothesize that the endothelial glycocalyx is a critical regulator of retinal hemodynamics and perfusion and decreases the propensity for retinal thrombus formation. METHODS: Hyaluronidase and heparinase, which are endothelial glycocalyx-degrading enzymes, were infused into mice. Fluorescein isothiocyanate-dextran (2000 kDa) was injected to measure lumen diameter, while RBC velocity and distribution were measured using fluorescently labeled RBCs. The diameters and velocities were used to calculate retinal blood flow and shear rates. Mean circulation time was calculated by measuring the difference between arteriolar and venular mean transit times. Rose Bengal dye was infused, followed by illumination with a green light to induce thrombosis. RESULTS: The acute infusion of hyaluronidase and heparinase led to significant increases in both arteriolar (7%) and venular (16%) diameters in the retina, with a tendency towards increased arteriolar velocity. In addition, the degradation caused a significant decrease in the venular shear rate (14%). The enzyme infusion resulted in substantial increases in total retinal blood flow (26%) and retinal microhematocrit but no changes in the mean circulation time through the retina. We also observed an enhanced propensity for retinal thrombus formation with the removal of the glycocalyx. CONCLUSIONS: Our data suggest that acute degradation of the glycocalyx can cause significant changes in retinal hemodynamics, with increases in vessel diameter, blood flow, microhematocrit, pro-thrombotic conditions, and decreases in venular shear rate.

Inhibition of 20-HETE attenuates diabetes-induced decreases in retinal hemodynamics.

The mechanisms of early diabetes-induced decreases in retinal blood flow have yet to be fully determined. The aim of this study was to explore the hypothesis that 20-hydroxyeicosatetraenoic acid (20-HETE) plays a role in the early decrease of retinal hemodynamics in diabetic mice. 20-HETE has been implicated previously in the diabetes-enhanced vasoconstriction of mesenteric and renal vessels; however, its role in the diabetic retinal microcirculation has not been investigated. Diabetes was induced by multiple low-dose injections of streptozotocin (STZ; 50 mg/kg for 5 consecutive days), then ∼2 weeks later the mice were administered daily intraperitoneal injections with or without the 20-HETE inhibitor HET0016 (2.5 mg/kg/day) for the following 2 weeks. Non-diabetic age-matched mice were included as controls. Intravital microscopy was used to obtain measurements of retinal vascular diameters and red blood cell (RBC) velocities for the feed arterioles and draining venules extending out of and into the optic disk. From these values, wall shear rates and blood flow rates were calculated. Diabetes induced approximately 30-40% decreases in RBC velocity, wall shear rate, and blood flow rate. These decreases were attenuated to 5-10% in the mice given HET0016. In summary, the 20-HETE inhibitor HET0016 is able to attenuate the retinal hemodynamic changes induced by diabetes.

Increase in retinal hypoxia-inducible factor-2α, but not hypoxia, early in the progression of diabetes in the rat.

Hypoxia and the associated hypoxia-inducible factors (HIFs) may be influential in the progression of diabetic retinopathy. However, little is known of the extent of hypoxia and the levels of HIFs early in the progression of the disease. In the current study, we injected the oxygen-dependent probe pimonidazole (Hypoxyprobe™-1) into diabetic rats, and also performed immunohistochemistry to determine the retinal levels of HIF-1α and HIF-2α. The rats were made diabetic using a single injection of streptozotocin (STZ; 60 mg/kg), with vehicle-injected rats used as non-diabetic controls. The measurements of hypoxia and HIF levels were obtained three weeks following STZ injection, at which time we have previously found significant decreases in retinal blood flow in the same model. In the current experiments, no increases in either HIF-1α or hypoxia were observed in the diabetic rats (compared with controls), and there was even a tendency for hypoxia levels to be decreased (tissue more highly oxygenated). However, we did observe an increase in HIF-2α in the retinas of the diabetic rats. Therefore, we conclude that early diabetes-induced increases in HIF-2α occur independently of hypoxia.

Thromboxane-prostanoid receptor expression and antagonism in dextran-sodium sulfate-induced colitis.

OBJECTIVE: In the current study of murine colitis, the potential roles of thromboxane and the thromboxane-prostanoid (TP) receptor were investigated, in as much as thromboxane signaling has been implicated in human inflammatory bowel disease. METHODS: Colitis was induced in C57BL/6 mice via ingestion of dextran sodium sulfate (DSS), with or without co-administration of the thromboxane synthase inhibitor ozagrel (25 mg/kg/day) or the TP receptor antagonist vapiprost (2.5 mg/kg/day). RESULTS: Immunohistochemistry of colonic tissue demonstrated a DSS-induced increase in TP receptor expression, but not of thromboxane synthase. Moreover, tissue levels of the metabolite thromboxane B(2) were unchanged by DSS. Vapiprost, but not ozagrel, partially attenuated histologic signs of inflammation induced by DSS, with vapiprost allowing a smaller increase in colon weight per unit length than ozagrel. Vapiprost also tended to attenuate DSS-induced alterations in intestinal transit. CONCLUSIONS: In summary, TP receptor antagonism was more effective than thromboxane synthase inhibition in alleviating DSS-induced colitis in mice.

Candesartan Normalizes Changes in Retinal Blood Flow and p22phox in the Diabetic Rat Retina.

Angiotensin II has been implicated in the progression of diabetic retinopathy, which is characterized by altered microvasculature, oxidative stress, and neuronal dysfunction. The signaling induced by angiotensin II can occur not only via receptor-mediated calcium release that causes vascular constriction, but also through a pathway whereby angiotensin II activates NADPH oxidase to elicit the formation of reactive oxygen species (ROS). In the current study, we administered the angiotensin II receptor antagonist candesartan (or vehicle, in untreated animals) in a rat model of type 1 diabetes in which hyperglycemia was induced by injection of streptozotocin (STZ). Eight weeks after the STZ injection, untreated diabetic rats were found to have a significant increase in tissue levels of angiotensin converting enzyme (ACE; p < 0.05) compared to non-diabetic controls, a 33% decrease in retinal blood flow rate (p < 0.001), and a dramatic increase in p22phox (a subunit of the NADPH oxidase). The decrease in retinal blood flow, and the increases in retinal ACE and p22phox in the diabetic rats, were all significantly attenuated (p < 0.05) by the administration of candesartan in drinking water within one week. Neither STZ nor candesartan induced any changes in tissue levels of superoxide dismutase (SOD-1), 4-hydroxynonenal (4-HNE), or nitrotyrosine. We conclude that one additional benefit of candesartan (and other angiotensin II antagonists) may be to normalize retinal blood flow, which may have clinical benefits in diabetic retinopathy.

Attenuation of streptozotocin-induced microvascular changes in the mouse retina with the endothelin receptor A antagonist atrasentan.

Hyperglycemia mediates endothelial cell dysfunction through a number of potential mechanisms that could result in the decrease of retinal blood flow early in diabetes. The aim of this study was to explore the role of endothelin receptor A (ET(A)) in the early decrease of retinal blood flow in diabetic mice. Diabetes was induced by streptozotocin, then ∼1 wk later the mice were administered drinking water with or without the ET(A) receptor antagonist atrasentan (7.5mg/kg/day) for the following 3 weeks. Non-diabetic age-matched mice with or without atrasentan were included as controls. For each mouse, measurements of retinal vascular diameters and red blood cell (RBC) velocities were obtained via intravital microscopy for the 5-7 feed arterioles (and draining venules) extending out of (and into) the optic disk, and from these values, flow rates and wall shear rates were calculated. Additionally, the number of retinal capillaries was counted by fluorescent immunostaining of platelet-endothelial cell adhesion molecule-1 (PECAM-1). Diabetes induced statistically significant decreases in RBC velocity, flow rate, and wall shear rate, with these alterations partially inhibited by atrasentan. No changes were observed in PECAM-1 expression among groups. The changes induced by diabetes, and the attenuation provided by atrasentan, were greater in the smaller retinal arterioles. In summary, ET(A) appears to play a role in the early decreases in retinal blood flow in a mouse model of diabetes.