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.

Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin.

This study determined whether retinal degeneration during diabetes includes retinal neural cell apoptosis. Image analysis of retinal sections from streptozotocin (STZ) diabetic rats after 7.5 months of STZ diabetes identified 22% and 14% reductions in the thickness of the inner plexiform and inner nuclear layers, respectively (P < 0. 001). The number of surviving ganglion cells was also reduced by 10% compared to controls (P < 0.001). In situ end labeling of DNA terminal dUTP nick end labeling (TUNEL) identified a 10-fold increase in the frequency of retinal apoptosis in whole-mounted rat retinas after 1, 3, 6, and 12 months of diabetes (P < 0.001, P < 0. 001, P < 0.01, and P < 0.01, respectively). Most TUNEL-positive cells were not associated with blood vessels and did not colocalize with the endothelial cell-specific antigen, von Willebrand factor. Insulin implants significantly reduced the number of TUNEL-positive cells (P < 0.05). The number of TUNEL-positive cells was also increased in retinas from humans with diabetes. These data indicate that retinal neural cell death occurs early in diabetes. This is the first quantitative report of an increase in neural cell apoptosis in the retina during diabetes, and indicates that neurodegeneration is an important component of diabetic retinopathy.

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.

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.

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.

Digoxin does not accelerate progression of diabetic retinopathy.

OBJECTIVE: To test the hypothesis that digoxin, an inhibitor of Na(+)-K(+)-ATPase activity, accelerates the progression of diabetic retinopathy. RESEARCH DESIGN AND METHODS: We compared the incidence and risk of retinopathy in 120 digoxin-taking vs. 867 non-digoxin-taking diabetic participants in the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) and in 117 digoxin-taking vs. 1,883 non-digoxin-taking diabetic subjects in the Early Treatment Diabetic Retinopathy Study (ETDRS). In both studies, retinopathy was detected by grading stereoscopic color photographs using the modified Airlie House classification scheme, and a two-step difference in baseline retinopathy grade was considered significant. RESULTS: After controlling for other risk factors, we found no statistically significant association with either 4-year incidence of retinopathy (WESDR) or progression of retinopathy (WESDR and ETDRS) in patients taking digoxin at baseline compared with those not taking digoxin. CONCLUSIONS: These data suggest that digoxin therapy does not adversely affect the course of diabetic retinopathy.

Retinal histamine synthesis is increased in experimental diabetes.

We examined retinal de novo histamine synthesis mediated by retinal histidine decarboxylase in normal and streptozotocin-diabetic male, Sprague Dawley rats that were diabetic for 21 days. We also examined effects of insulin and alpha-hydrazinohistidine (alpha HH) treatments on retinal histamine synthesis in this diabetic model. alpha HH is a specific inhibitor of histidine decarboxylase. Results indicate that the retina contains an active histidine decarboxylase enzyme system, and that in streptozotocin diabetes retinal histamine synthesis is increased 197%. Both insulin and alpha HH independently reverse and normalize retinal histamine synthesis. These data thus indicate that the retinal inducible histamine pool is increased in experimental diabetes, and that insulin is an important modulator of retinal histamine metabolism. This newly described retinal metabolic alteration may be one factor responsible for increased retinal vascular permeability in diabetic retinopathy.

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 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.

Astrocytes increase barrier properties and ZO-1 expression in retinal vascular endothelial cells.

PURPOSE: Diabetic retinopathy and other diseases associated with retinal edema are characterized by increased microvascular leakage. Astrocytes have been proposed to maintain endothelial function in the brain, suggesting that glial impairment may underlie the development of retinal edema. The purpose of this study was to test the effects of astrocytes on barrier properties in retinal microvascular endothelial cells. METHODS: Bovine retinal microvascular endothelial cells were exposed to conditioned media from rat brain astrocytes. Transendothelial electrical resistance (TER) was determined on 24-mm Transwell (Cambridge, MA) polycarbonate filters with the End-Ohm device (World Precision Instruments, Sarasota, FL). ZO-1 protein content was quantified by microtiter enzyme-linked immunosorbent assay. RESULTS: Astrocyte-conditioned medium (ACM) significantly increased TER (P < 0.0001) and ZO-1 content (P < 0.01). Both serum-containing and serum-free N1B defined ACM increased ZO-1 expression, but heating abolished the effect. Serum-free ACM decreased cell proliferation by 16%. CONCLUSIONS: Astrocytes release soluble, heat-labile factors that increase barrier properties and tight junction protein content. These results suggest that astrocytes enhance blood-retinal barrier properties, at least in part by increasing tight junction protein expression. Our findings suggest that glial malfunction plays an important role in the pathogenesis of vasogenic retinal edema.

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.

Current patterns of intraocular gas use in North America.

A 1987 survey of 365 members of the Retina and Vitreous societies revealed that use of long-acting intraocular gases was both widespread and frequent. However, despite the expanded use of intravitreal gases and the stated intention of the Food and Drug Administration to approve premarket applications for use of sulfur hexafluoride and perfluoropropane, there are still limitations in the ready availability of these substances. Therefore, we conducted another survey to determine current patterns of intraocular gas use. The results indicate that more surgeons are using long-acting gases. The data also demonstrate the selective use of pneumatic retinopexy with geographic pockets of increased retinopexy use in California and Florida. Finally, 100% of respondents now indicate that they consider the use of sulfur hexafluoride and perfluoropropane to be a standard of care.

Mucinous adenocarcinoma of the eyelid. A case report.

A 52-year-old man had a rapidly growing cystic-appearing lesion on his lower eyelid. Excisional biopsy disclosed a mucinous sweat gland adenocarcinoma and the mucinous material (presumably sialomucin) accounted for the transillumination property of the tumor. These carcinomas should be considered in the differential diagnosis of cystic eyelid lesions.

Histamine H1 receptors mediate increased blood-retinal barrier permeability in experimental diabetes.

To test the hypothesis that histamine receptors mediate increased blood-retinal barrier permeability in experimental diabetes, 51 rats were made diabetic by streptozocin injection (65 mg/kg; jugular vein) and were held for four weeks. The seven animal groups were as follows: untreated controls; untreated diabetic rats; diabetic rats receiving diphenhydramine hydrochloride (Benadryl); diabetic rats receiving cimetidine hydrochloride (Tagamet); diabetic rats receiving diphenhydramine and cimetidine; diabetic rats receiving purified pork insulin (Iletin II); and diabetic rats receiving insulin and diphenhydramine. All treatments were given during the last week. Blood-retinal barrier permeability was assessed through measurement of the vitreous content of fluorescein isothiocyanate conjugated to bovine serum albumin (FITCBSA) after 20 minutes of FITCBSA circulation. Vitreous FITCBSA content of the diabetic group was 64% greater than control content. Diabetic rats treated with either diphenhydramine or diphenhydramine and insulin had respective decreases of 43% and 40% in vitreous FITCBSA content. The vitreous content of the diabetic group receiving insulin was lowered 37% below untreated diabetic values, while the vitreous FITCBSA content of the diabetic group receiving both insulin and diphenhydramine was reduced 63%. These data indicate that retinal histamine H1-receptor activation may be partially responsible for initial blood-retinal barrier leakage of macromolecules into the vitreous and that this abnormal leakage can be prevented both by diphenhydramine and by insulin. Histamine H1 receptors may play an important role in mediating increased blood-retinal barrier permeability in experimental diabetes.

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.

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.

Astemizole reduces blood-retinal barrier leakage in experimental diabetes.

We examined the potential of astemizole, a histamine H1-receptor antagonist that does not cross the blood-brain barrier, to reverse blood-retinal barrier leakage to albumin in streptozotocin diabetic rats. Four groups of nondiabetic and four groups of diabetic rats received vehicle or astemizole at dosages of 5, 10, or 20 mg/kg body weight for days 22-28 of a 28-day holding period. There were no significant differences in nondiabetic plasma-vitreous albumin ratios between animals receiving vehicle or any of the three astemizole dosages. Only diabetic rats receiving vehicle showed a significant (p < 0.05) 100% increase in the plasma-vitreous albumin ratio over their nondiabetic counterparts. Diabetic rats receiving either 5, 10, or 20 mg/kg astemizole exhibited total normalization of vitreous albumin accumulation, despite persistence of diabetes. These data indicate that astemizole, an H1-receptor antagonist that does not cross the blood-retinal barrier, is effective in reversing blood-retinal barrier leakage of albumin in experimental diabetes.

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.

Physiological transport properties of cultured retinal microvascular endothelial cell monolayers.

PURPOSE: To characterize baseline transport properties: hydraulic conductivity (Lp), albumin permeability (Pe), and transendothelial electrical resistance (TER) of bovine retinal microvascular endothelial cells (RMEC) in the development of an in vitro model of the blood-retinal barrier (BRB). METHODS: RMEC were grown on porous, polycarbonate filters for determination of the number of days required to achieve minimal transport rates. Lp, Pe, and TER were measured by utilizing a bubble tracking spectrophotometer, by quantifying the diffusional movement of fluorescein isothiocyanate-labeled albumin, and by utilizing a Millipore electrical resistance meter, respectively. RESULTS: Lp decreased significantly from 7.82 +/- 0.85 x 10(-7) (mean +/- SEM) cm/sec/cm H2O at post-plating Day 5 to 1.44 +/- 0.26 x 10(-7) cm/sec/cm H2O at Day 9. Pe of the monolayer also decreased progressively with days post-plating from 3.44 +/- 0.53 x 10(-6) cm/sec at Day 7 to a minimum of 1.95 +/- 0.29 x 10(-6) cm/sec at Day II. Peak TER fluctuated until Day 7, when it began to steadily increase from 17.14 ohm-cm2 to a peak value of 25.42 ohm-cm2 at Day 10, decreasing from then on to 22.24 ohm.cm2 on Day 12. Known disrupters of the BRB, NECA and VEGF, elicited significant increase in RMEC Lp showing the sensitivity of this model to pharmacological alterations. CONCLUSIONS: Our data indicate that RMEC grown on polycarbonate filters form a restrictive monolayer of cells, which exhibit dynamic alterations in response to pharmacological agents, thus demonstrating an in vitro model of the BRB. Future studies with the model may offer insights into the pathogenesis of retinal vascular diseases and allow convenient testing of pharmacological interventions.

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.