Glucose variability and inner retinal sensory neuropathy in persons with type 1 diabetes mellitus.

PurposeTo quantify early neuroretinal alterations in patients with type 1 diabetes mellitus (T1DM) and to assess whether glycemic variability contributes to alterations in neuroretinal structure or function.MethodsThirty patients with T1DM and 51 controls underwent comprehensive ophthalmic examination and assessment of retinal function or structure with frequency doubling perimetry (FDP), contrast sensitivity, dark adaptation, fundus photography, and optical coherence tomography (OCT). Diabetic participants wore a subcutaneous continuous glucose monitor for 5 days, from which makers of glycemic variability including the low blood glucose index (LGBI) and area under the curve (AUC) for hypoglycemia were derived.ResultsSixteen patients had no diabetic retinopathy (DR), and 14 had mild or moderate DR. Log contrast sensitivity for the DM group was significantly reduced (mean±SD=1.63±0.06) compared with controls (1.77±0.13, P<0.001). OCT analysis revealed that the inner temporal inner nuclear layer (INL) was thinner in patients with T1DM (34.9±2.8 µm) compared with controls (36.5±2.9 µm) (P=0.023), although this effect lost statistical significance after application of the Bonferroni correction for multiple comparisons. Both markers of glycemic variability, the AUC for hypoglycemia (R=-0.458, P=0.006) and LGBI (R=-0.473, P=0.004), were negatively correlated with inner temporal INL thickness.ConclusionsPatients with T1DM and no to moderate DR exhibit alterations in inner retinal structure and function. Increased glycemic variability correlates with retinal thinning on OCT imaging, suggesting that fluctuations in blood glucose may contribute to neurodegeneration.

A multistep validation process of biomarkers for preclinical drug development.

Biomarkers that can be measured in preclinical models in a high-throughput, reproducible manner offer the potential to increase the speed and efficacy of drug development. Development of therapeutic agents for many conditions is hampered by the limited number of validated preclinical biomarkers available to gauge pharmacoefficacy and disease progression, but the validation process for preclinical biomarkers has received limited attention. This report defines a five-step preclinical biomarker validation process and applies the process to a case study of diabetic retinopathy. By showing that a gene expression panel is highly reproducible, coincides with disease manifestation, accurately classifies individual animals and identifies animals treated with a known therapeutic agent, a biomarker panel can be considered validated. This particular biomarker panel consisting of 14 genes (C1inh, C1s, Carhsp1, Chi3l1, Gat3, Gbp2, Hspb1, Icam1, Jak3, Kcne2, Lama5, Lgals3, Nppa, Timp1) can be used in diabetic retinopathy pharmacotherapeutic research, and the biomarker development process outlined here is applicable to drug development efforts for other diseases.

TELEMAM: a cluster randomised trial to assess the use of telemedicine in multi-disciplinary breast cancer decision making.

AIM: The TELEMAM trial aimed to assess the clinical effectiveness and costs of telemedicine in conducting breast cancer multi-disciplinary meetings (MDTs). METHODS: Over 12 months 473 MDT patient discussions in two district general hospitals (DGHs) were cluster randomised (2:1) to the intervention of telemedicine linkage to breast specialists in a cancer centre or to the control group of 'in-person' meetings. Primary endpoints were clinical effectiveness and costs. Economic analysis was based on a cost-minimisation approach. RESULTS: Levels of agreement of MDT members on a scale from 1 to 5 were high and similar in both the telemedicine and standard meetings for decision sharing (4.04 versus 4.17), consensus (4.06 versus 4.20) and confidence in the decision (4.16 versus 4.07). The threshold at which the telemedicine meetings became cheaper than standard MDTs was approximately 40 meetings per year. CONCLUSION: Telemedicine delivered breast cancer multi-disciplinary meetings have similar clinical effectiveness to standard 'in-person' meetings.

Energy sources for glutamate neurotransmission in the retina: absence of the aspartate/glutamate carrier produces reliance on glycolysis in glia.

The mitochondrial transporter, the aspartate/glutamate carrier (AGC), is a necessary component of the malate/aspartate cycle, which promotes the transfer into mitochondria of reducing equivalents generated in the cytosol during glycolysis. Without transfer of cytosolic reducing equivalents into mitochondria, neither glucose nor lactate can be completely oxidized. In the present study, immunohistochemistry was used to demonstrate the absence of AGC from retinal glia (Müller cells), but its presence in neurons and photoreceptor cells. To determine the influence of the absence of AGC on sources of ATP for glutamate neurotransmission, neurotransmission was estimated in both light- and dark-adapted retinas by measuring flux through the glutamate/glutamine cycle and the effect of light on ATP-generating reactions. Neurotransmission was 80% faster in the dark as expected, because photoreceptors become depolarized in the dark and this depolarization induces release of excitatory glutamate neurotransmitter. Oxidation of [U-14C]glucose, [1-14C]lactate, and [1-14C]pyruvate in light- and dark-adapted excised retinas was estimated by collecting 14CO2. Neither glucose nor lactate oxidation that require participation of the malate/aspartate shuttle increased in the dark, but pyruvate oxidation that does not require the malate/aspartate shuttle increased to 36% in the dark. Aerobic glycolysis was estimated by measuring the rate of lactate appearance. Glycolysis was 37% faster in the dark. It appears that in the retina, ATP consumed during glutamatergic neurotransmission is replenished by ATP generated glycolytically within the retinal Müller cells and that oxidation of glucose within the Müller cells does not occur or occurs only slowly.

Abnormal centrifugal axons in streptozotocin-diabetic rat retinas.

PURPOSE: To characterize the effects of diabetes on the expression of histidine decarboxylase mRNA and on the morphology of the histaminergic centrifugal axons in the rat retina. METHODS: Rats were made diabetic by streptozotocin. After 3 months, retinal histidine decarboxylase expression was analyzed by in situ hybridization in radial sections. Flatmount retinas from a second group of rats were labeled with an antiserum to histamine or an antibody to phosphorylated neurofilament protein. RESULTS: Histidine decarboxylase mRNA was expressed in cells in the inner and outer nuclear layers of diabetic retinas, but not in normal retinas. However, immunoreactive (IR) histamine was not localized to perikarya in either the normal or the diabetic retinas. Instead, a population of centrifugal axons was labeled. These axons emerged from the optic disc and had varicose terminal branches in the inner plexiform layer (IPL) of the peripheral retina. Some branches ended on large retinal blood vessels and others in dense clusters in the IPL. In rats with streptozotocin-induced diabetes, the centrifugal axon terminals developed many large swellings that contained neurofilament immunoreactivity; these swellings were rare in normal retinas. CONCLUSIONS: Diabetes perturbs the retinal histaminergic system, causing increases in histidine decarboxylase mRNA expression in neurons or glia and abnormal focal swellings on the centrifugal axons.

Excessive hexosamines block the neuroprotective effect of insulin and induce apoptosis in retinal neurons.

In addition to microvascular abnormalities, neuronal apoptosis occurs early in diabetic retinopathy, but the mechanism is unknown. Insulin may act as a neurotrophic factor in the retina via the phosphoinositide 3-kinase/Akt pathway. Excessive glucose flux through the hexosamine biosynthetic pathway (HBP) is implicated in the development of insulin resistance in peripheral tissues and diabetic complications such as nephropathy. We tested whether increased glucose flux through the HBP perturbs insulin action and induces apoptosis in retinal neuronal cells. Exposure of R28 cells, a model of retinal neurons, to 20 mm glucose for 24 h attenuated the ability of 10 nm insulin to rescue them from serum deprivation-induced apoptosis and to phosphorylate Akt compared with 5 mm glucose. Glucosamine not only impaired the neuroprotective effect of insulin but also induced apoptosis in R28 cells in a dose-dependent fashion. UDP-N-acetylhexosamines (UDP-HexNAc), end products of the HBP, were increased approximately 2- and 15-fold after a 24-h incubation in 20 mm glucose and 1.5 mm glucosamine, respectively. Azaserine, a glutamine:fructose-6-phosphate amidotransferase inhibitor, reversed the effect of 20 mm glucose, but not that of 1.5 mm glucosamine, on attenuation of the ability of insulin to promote cell survival and phosphorylate Akt as well as accumulation of UDP-HexNAc. Glucosamine also impaired insulin receptor processing in a dose-dependent manner but did not decrease ATP content. By contrast, in L6 muscle cells, glucosamine impaired insulin receptor processing but did not induce apoptosis. These results suggest that the excessive glucose flux through the HBP may direct retinal neurons to undergo apoptosis in a bimodal fashion; i.e. via perturbation of the neuroprotective effect of insulin mediated by Akt and via induction of apoptosis possibly by altered glycosylation of proteins. The HBP may be involved in retinal neurodegeneration in diabetes.

Insulin rescues retinal neurons from apoptosis by a phosphatidylinositol 3-kinase/Akt-mediated mechanism that reduces the activation of caspase-3.

The ability of insulin to protect neurons from apoptosis was examined in differentiated R28 cells, a neural cell line derived from the neonatal rat retina. Apoptosis was induced by serum deprivation, and the number of pyknotic cells was counted. p53 and Akt were examined by immunoblotting after serum deprivation and insulin treatment, and caspase-3 activation was examined by immunocytochemistry. Serum deprivation for 24 h caused approximately 20% of R28 cells to undergo apoptosis, detected by both pyknosis and activation of caspase-3. 10 nm insulin maximally reduced the amount of apoptosis with a similar potency as 1.3 nm (10 ng/ml) insulin-like growth factor 1, which acted as a positive control. Insulin induced serine phosphorylation of Akt, through the phosphatidylinositol (PI) 3-kinase pathway. Inhibition of PI 3-kinase with wortmannin or LY294002 blocked the ability of insulin to rescue the cells from apoptosis. SN50, a peptide inhibitor of NF-kappaB nuclear translocation, blocked the rescue effect of insulin, but neither insulin or serum deprivation induced phosphorylation of IkappaB. These results suggest that insulin is a survival factor for retinal neurons by activating the PI 3-kinase/Akt pathway and by reducing caspase-3 activation. The rescue effect of insulin does not appear to be mediated by NF-kappaB or p53. These data suggest that insulin provides trophic support for retinal neurons through a PI 3-kinase/Akt-dependent pathway.

Shear stress regulates occludin content and phosphorylation.

Previous studies determined that shear stress imposed on bovine aortic endothelial cell (BAEC) monolayers increased the hydraulic conductivity (L(P)); however, the mechanism by which shear stress increases L(P) remains unknown. This study tested the hypothesis that shear stress regulates paracellular transport by altering the expression and phosphorylation state of the tight junction protein occludin. The effect of shear stress on occludin content was examined by Western blot analysis. Ten dyn/cm(2) significantly reduced occludin content in a time-dependent manner such that after a 3 h exposure to shear, occludin content decreased to 44% of control. Twenty dyn/cm(2) decreased occludin content to 50% of control and increased L(P) by 4.7-fold after 3 h. Occludin expression and L(P) depend on tyrosine kinase activity because erbstatin A (10 microM) attenuated both the shear-induced decrease in occludin content and increase in L(P). Shear stress increased occludin phosphorylation after 5 min, 15 min, and 3 h exposures. The shear-induced increase in occludin phosphorylation was attenuated with dibutyryl (DB) cAMP (1 mM), a reagent previously shown to reverse the shear-induced increase in L(P). We conclude that shear stress rapidly (< or = 5 min) increases occludin phosphorylation and significantly decreases the expression of occludin over 1-4 h. Alterations in the occludin phosphorylation state and occludin total content are potential mechanisms by which shear stress increases L(P).

Effect of VEGF on retinal microvascular endothelial hydraulic conductivity: the role of NO.

PURPOSE: Vascular endothelial growth factor (VEGF) increases microvascular permeability in vivo and has been hypothesized to play a role in plasma leakage in diabetic retinopathy. Few controlled studies have been conducted to determine the mechanism underlying the effect of VEGF on transport properties (e.g., hydraulic conductivity [Lp]). This study was conducted to determine the effect of VEGF on bovine retinal microvascular endothelial LP and the role of nitric oxide (NO) and the guanylate cyclase/guanosine 3', 5'-cyclic monophosphate/protein kinase G (GC/cGMP/PKG) pathway downstream of NO in mediating the VEGF response. METHODS: Bovine retinal microvascular endothelial cells (BRECs) were grown on porous polycarbonate filters, and water flux across BREC monolayers in response to a pressure differential was measured to determine endothelial LP RESULTS: VEGF (100 ng/ml) increased endothelial LP: within 30 minutes of addition and by 13.8-fold at the end of 3 hours of exposure. VEGF stimulated endothelial monolayers to release NO and incubation of the BRECs with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA; 100 microM) significantly attenuated the VEGF-induced LP increase. It was observed that incubation of the monolayers with the GC inhibitor LY-83583 (10 microM) did not alter the VEGF-mediated LP: response. Addition of the cGMP analogue 8-br-cGMP (1 mM) did not change the baseline LP over 4 hours. Also, the PKG inhibitor KT5823 (1 microM) did not inhibit the response of BREC LP to VEGF. CONCLUSIONS: These experiments indicate that VEGF elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.

Altered expression of retinal occludin and glial fibrillary acidic protein in experimental diabetes. The Penn State Retina Research Group.

PURPOSE: To investigate how diabetes alters vascular endothelial cell tight junction protein and glial cell morphology at the blood-retinal barrier (BRB). METHODS: The distribution of the glial marker, glial fibrillary acidic protein (GFAP), and the endothelial cell tight junction protein occludin were explored by immunofluorescence histochemistry in flatmounted retinas of streptozotocin (STZ)-diabetic and age-matched control rats, and in BB/Wor diabetes-prone and age-matched diabetes-resistant rats. RESULTS: GFAP immunoreactivity was limited to astrocytes in control retinas. Two months of STZ-diabetes reduced GFAP immunoreactivity in astrocytes and increased GFAP immunoreactivity in small groups of Müller cells. After 4 months of STZ-induced diabetes, all Müller cells had intense GFAP immunoreactivity, whereas there was virtually none in the astrocytes. BB/Wor diabetic rats had similar changes in GFAP immunoreactivity. Occludin immunoreactivity in normal rats was greatest in the capillary bed of the outer plexiform layer and arterioles of the inner retina but much less intense in the postcapillary venules. Diabetes reduced occludin immunoreactivity in the capillaries and induced redistribution from continuous cell border to interrupted, punctate immunoreactivity in the arterioles. Forty-eight hours of insulin treatment reversed the pattern of GFAP and occludin immunoreactivity in the STZ-diabetic rats. CONCLUSIONS: Diabetes alters GFAP expression in retinal glial cells, accompanied by reduction and redistribution of occludin in endothelial cells. These changes are consistent with the concept that altered glial-endothelial cell interactions at the BRB contribute to diabetic retinopathy.

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.

Retinal neurodegeneration: early pathology in diabetes.

Normal vision depends on the normal function of retinal neurons, so vision loss in diabetes must ultimately be explained in terms of altered neuronal function. However to date relatively little attention has been paid to the impact of diabetes on the neural retina. Instead, the focus of most research has been primarily on retinal vascular changes, with the assumption that they cause altered neuronal function and consequently vision loss. An increasing body of evidence suggests that alterations in neuronal function and viability may contribute to the pathogenic mechanisms of diabetic retinopathy beginning shortly after the onset of diabetes. This view arises from neurophysiological, psychometric, histopathological and biochemical observations in humans and experimental animals. The collective evidence from past and recent studies supports the hypothesis that neurodegeneration, together with functional changes in the vasculature, is an important component of diabetic retinopathy. The authors invite other investigators to include the neural retina as a component of their studies so that the pathogenesis of diabetic retinopathy can be understood more clearly.

Effect of shear stress on the hydraulic conductivity of cultured bovine retinal microvascular endothelial cell monolayers.

The shear stress of flowing blood on endothelial cells increases water transport (hydraulic conductivity, Lp) in several vascular beds in vivo and has been hypothesized to play a role in elevating vascular transport in ocular diseases such as diabetic retinopathy. The purpose of this study is to determine the response of Lp to varying levels of shear stress using an in vitro model of the blood-retinal barrier: bovine retinal endothelial cells (BRECs) grown on polycarbonate filters. The study also addresses the role of nitric oxide (NO) and other downstream effectors in mediating shear-induced changes in water transport. A step change in shear stress of 10 dyn/cm(2) did not produce a significant change in Lp over 3 hours, whereas a 20 dyn/cm(2) step change elevated Lp by 14.6-fold relative to stationary controls at the end of 3h of shear exposure. 20 dyn/cm( 2) of shear stress stimulated the endothelial monolayers to release nitric oxide in a biphasic manner and incubation of the BRECs with a nitric oxide synthase (NOS) inhibitor, L-NMMA, significantly attenuated the shear-induced Lp response. These experiments demonstrate that NO is a key signaling molecule in the pathway linking shear stress and Lp in BRECs. A widely studied pathway downstream of NO involves the activation of guanylate cyclase (GC), guanosine 3', 5' -- cyclic monophosphate (cGMP) and protein kinase G (PKG). It was observed that incubation of BRECs with the GC inhibitor, LY83583 (10 microM) or the PKG inhibitor, KT5823 (1 microM) did not significantly alter the shear-induced Lp response. Also the cGMP analogue, 8-br-cGMP (1mM), did not affect the baseline Lp over 4h. These results demonstrate that shear stress elevates hydraulic conductivity in BRECs through a signaling mechanism that involves NO but not the GC/cGMP/PKG pathway.

New insights into the pathophysiology of diabetic retinopathy: potential cell-specific therapeutic targets.

Diabetic retinopathy, a leading cause of vision impairment, is classically defined by its vascular lesions. This review examines how diabetes affects vascular cells, as well as neurons, macroglia, and microglia. The cellular and clinical elements of diabetic retinopathy have many features of chronic inflammation. Understanding the individual cell-specific and global inflammatory changes in the retina may lead to novel therapeutic approaches to prevent vision loss.

Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors.

Vascular endothelial growth factor (VEGF) may have a physiologic role in regulating vessel permeability and contributes to the pathophysiology of diabetic retinopathy as well as tumor development. We set out to ascertain the mechanism by which VEGF regulates paracellular permeability in rats. Intra-ocular injection of VEGF caused a post-translational modification of occludin as determined by a gel shift from 60 to 62 kDa. This event began by 15 min post-injection and was maximal by 45 min. Alkaline phosphatase treatment revealed this modification was caused by a change in occludin phosphorylation. In addition, the quantity of extracted occludin increased 2-fold in the same time frame. The phosphorylation and increased extraction of occludin was recapitulated in retinal endothelial cells in culture after VEGF stimulation. The data presented herein are the first demonstration of a change in the phosphorylation of this transmembrane protein under conditions of increased endothelial permeability. In addition, intra-ocular injection of VEGF also caused tyrosine phosphorylation of ZO-1 as early as 15 min and increased phosphorylation 4-fold after 90 min. In conclusion, VEGF rapidly increases occludin phosphorylation as well as the tyrosine phosphorylation of ZO-1. Phosphorylation of occludin and ZO-1 likely contribute to regulated endothelial paracellular permeability.

Molecular mechanisms of vascular permeability in diabetic retinopathy.

Diabetes leads to a wide array of complications in humans, including kidney failure, vascular disease, peripheral nerve degeneration, and vision loss. Diabetic retinopathy causes blindness in more working-age people in the United States than any other disease and contributes greatly to blindness in the young and old as well. The increasing rate of diabetes occurring in our society can only bring about a further decrease in the visual health of this country unless new modalities are discovered to prevent and cure diabetic retinopathy. Breakdown of the blood-retinal barrier and the resultant vascular permeability remains one of the first observable alterations in diabetic retinopathy and strongly correlates with vision loss. In this article, we examine the molecular components that form this blood-retinal barrier and explore how changes in the production of growth factors in the neural parenchyma cause an increase in vascular permeability and contribute to retinal degeneration.

Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group.

Blood-retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, but the molecular changes that cause this pathology are unclear. Occludin is a transmembrane component of interendothelial tight junctions that may regulate permeability at the BRB. In this study, we examined the effects of vascular endothelial growth factor (VEGF) and diabetes on vascular occludin content and barrier function. Sprague-Dawley rats were made diabetic by intravenous streptozotocin injection, and age-matched animals served as controls. After 3 months, BRB permeability was quantified by intravenous injection of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), Mr 66 kDa, and 10-kDa rhodamine-dextran (R-D), followed by digital image analysis of retinal sections. Retinal fluorescence intensity for FITC-BSA increased 62% (P < or = 0.05), but R-D fluorescence did not change significantly. Occludin localization at interendothelial junctions was confirmed by immunofluorescence, and relative protein content was determined by immunoblotting of retinal homogenates. Retinal occludin content decreased approximately 35% (P < or = 0.03) in the diabetic versus the control animals, whereas the glucose transporter GLUT1 content was unchanged in rat retinas. Additionally, treatment of bovine retinal endothelial cells in culture with 0.12 nmol/l or 12 nmol/l VEGF for 6 h reduced occludin content 46 and 54%, respectively. These data show that diabetes selectively reduces retinal occludin protein expression and increases BRB permeability. Our findings suggest that the elevated VEGF in the vitreous of patients with diabetic retinopathy increases vascular permeability by downregulating occludin content. Decreased tight junction protein expression may be an important means by which diabetes causes increased vascular permeability and contributes to macular edema.

Diabetic retinopathy.

Nonproliferative diabetic retinopathy may cause visual loss when associated with macular edema or macular ischemia (secondary to retinal capillary nonperfusion). Proliferative diabetic retinopathy may cause severe visual loss if complicated by vitreous hemorrhage or traction detachment of the macula. Patients with diabetes benefit from collaboration between the internist and ophthalmologist. Tighter control of blood glucose levels and lower blood pressure reduce the risk of progression of diabetic retinopathy. Regular dilated eye examinations and appropriate intervention with laser or vitrectomy surgery help to preserve vision in patients with established macular edema or proliferative diabetic retinopathy.