Protective Effects of Liraglutide and Linagliptin in C. elegans as a New Model for Glucose-Induced Neurodegeneration.

Liraglutide and linagliptin are novel drugs for the treatment of diabetes. Antioxidative and neuroprotective effects have been described for both compounds. However, it is not yet known, whether these mechanisms are also protective against diabetic retinal neurodegeneration. We assessed the antioxidative and neuroprotective capabilities of liraglutide and linagliptin as well as the signaling pathways involved, by using C. elegans as a model for glucose-induced neurodegeneration. C. elegans were cultivated under conditions, which mimic clinical hyperglycemia, and treated with 160 µmol/l liraglutide or 13 µmol/l linagliptin. Oxidative stress was reduced by 29 or 78% and methylglyoxal-derived advanced glycation endproducts (AGEs) by 33 or 22%, respectively. This resulted in an improved neuronal function by 42 or 60% and an extended mean lifespan by 9 or 11%, respectively. Antioxidative and AGE reducing effects of liraglutide and linagliptin were not dependent on v-akt murine thymoma viral oncogene homologue 1/forkhead box O1 (AKT1/FOXO). Neuroprotection by liraglutide was AKT1/FOXO dependent, yet AKT1/FOXO independent upon linagliptin treatment. Both liraglutide and linagliptin exert neuroprotective effects in an experimental model for glucose-induced neurodegeneration, however, the signaling pathways differ in the present study. Further pharmacological intervention with these pathways may help to delay the clinical onset of diabetic retinopathy by preserving neuronal integrity.

Diabetic retinopathy in type 1 diabetes-a contemporary analysis of 8,784 patients.

AIMS/HYPOTHESIS: The aim of this study was to analyse the risk profile for diabetic retinopathy under real-life conditions in a large cohort of patients with type 1 diabetes. METHODS: Patients (n = 18,891) with childhood, adolescent or adult onset of type 1 diabetes from the prospective German Diabetes Documentation System survey were analysed. A total of 8,784 patients fulfilled the inclusion criterion, which was availability of retinopathy status. Retinopathy grading (any retinopathy, advanced retinopathy), treatment regimens and risk factors were prospectively recorded and tested as covariates by Kaplan-Meier analysis and logistic regression. RESULTS: Any retinopathy was present in 27.4% and advanced retinopathy (severe non-proliferative or proliferative diabetic retinopathy) in 8.0% of the cohort. After 40 years of diabetes, the cumulative proportion of patients with any retinopathy and advanced retinopathy was 84.1% and 50.2%, respectively. In multiple regression analysis, risk factors for any retinopathy were diabetes duration (OR 1.167 per year), HbA(1c) >7.0% (53 mmol/mol) (OR 2.225), smoking (OR 1.295) and male sex (OR 1.187) (p < 0.0001 for all). Young age at onset (5 vs 15 years at disease onset) was protective (0.410, p < 0.0001). No glycaemic threshold was detected for retinopathy protection. Risk factors for advanced retinopathy were duration (1.124 per year, p < 0.0001), male sex (1.323, p = 0.0020), HbA(1c) >7.0% (53 mmol/mol) (1.499, p < 0.0001), triacylglycerol >1.7 mmol/l (1.398, p = 0.0013) and blood pressure >140/90 mmHg (1.911, p < 0.0001). CONCLUSIONS/INTERPRETATION: The prevalence of retinopathy remains significant in type 1 diabetes. Any improvement of metabolic control and non-smoking is protective, while hypertension affects progression to severe levels under real-life conditions. These data reinforce the validity of multifactorial concepts for morbidity protection in type 1 diabetes.

Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization.

Retinal neovascularization is a major cause of blindness in such disorders as retinopathy of prematurity, proliferative diabetic retinopathy and senile macular degeneration. Because ligation of vitronectin receptor-type integrins appears to be required for the survival and maturation of newly formed but not quiescent blood vessels in several vascular beds including the retina, blockade of this downstream adhesion receptor system was investigated. In a mouse model of hypoxia-induced retinal neovascularization twice daily administration of 1 to 20 mg cyclic alpha v-integrin antagonist peptide per kilogram of body weight reduced capillary proliferation in a dose-dependent fashion--maximum 76%--without obvious side effects. A cyclic control peptide displayed no inhibitory effect on neovascularization. These findings indicate that systemic application of vitronectin receptor antagonists appears to be clinically feasible and is efficient in preventing retinal neovascularization and superior to cytokine-blocking strategies.

[Diabetic retinopathy and maculopathy]. / Diabetische Retinopathie und Makulopathie.

Diabetic retinopathy is the most frequent microvascular complication in diabetes. Its early development indicates an increased cardiovascular risk. Since the early stages lack symptoms, yearly screening intervals are mandatory. Each retinopathy level determines evidence-based treatment with mutual interactive contributions from diabetology and ophthalmology. Intravitreal injection of inhibitors of the vascular endothelial growth factor may improve the so far only modestly successful therapy of diabetic macular edema.

Low-dose erythropoietin inhibits oxidative stress and early vascular changes in the experimental diabetic retina.

AIMS/HYPOTHESIS: Diabetic retinopathy is the result of increased oxidative and nitrosative stress induced by chronic hyperglycaemia, and affects the vasculature and the neuroglia. Erythropoietin is a neuroprotective and an endothelial survival factor. We assessed the effect of suberythropoietic epoetin delta doses on variables of oxidative stress in target tissues of diabetic complications and on pericyte loss in the diabetic retina. METHODS: We administered epoetin delta to streptozotocin-induced diabetic Wistar rats at doses of 384 IU/kg body weight once weekly or 128 IU/kg body weight three times a week. The treatment lasted for 3 months. Oxidative stress and formation of AGEs were assessed by immunoblotting, expression of Ang-2 (also known as Angpt2) by RT-PCR, activation of protein kinase B (AKT) and heat shock protein (HSP)-27 levels by immunofluorescence, and incipient retinal vascular changes by quantitative morphometry of retinal digest preparations. RESULTS: Diabetes increased variables of oxidative stress and nitrosative stress (N(epsilon)-carboxymethyl-lysine, nitrotyrosine and methylglyoxal-type AGEs) in retina, kidney and heart of diabetic rats. Epoetin delta reduced oxidative and nitrosative stress in all tissues, and AGEs in the retina. It also reduced increased retinal Ang-2 expression and pericyte loss, and ameliorated p-AKT and HSP-27 levels. CONCLUSIONS/INTERPRETATION: Epoetin delta has antioxidative properties in organs affected by diabetes and may prevent incipient microvascular damage in the diabetic retina.

Microvascular retinal changes in patients presenting with acute coronary syndromes.

BACKGROUND: Retinal microvascular changes predict cardiovascular morbidity and mortality independent of classical risk factors. However, it is unclear which retinal changes characterize patients with established coronary artery disease (CAD), and in particular, with acute coronary syndromes (ACS). The aim of the present preliminary study was to assess retinopathy in these patients. METHODS: 43 consecutive patients with ACS and 19 consecutive patients with stable CAD were investigated. Among the patient group with ACS, 20 patients presented with ST-Elevation Myocardial Infarction (STEMI) and 23 patients presented with Non-STEMI (NSTEMI). Standardized protocols were used and retinal fundus photography was taken within 48 h post-coronary angiography to assess retinopathy and general arteriolar narrowing as arterio-venous ratio (AVR). Clinical and laboratory cardiovascular risk factors were recorded. RESULTS: Despite comparable age and comparable frequency of diabetes and hypertension, patients with ACS had a much higher likelihood for retinal microaneurysms and dot bleedings than patients with stable CAD (17 (40%) vs. 1 (5%) patients, OR 11.77; 95%CI 1.43-96.59; p=0.006). Performing multivariate analysis, this association remains significant (OR 20.5, 95%CI 1.6-255, p=0.019). CAD patients presented more often with focal signs of arteriovenous nicking / focal vasoconstriction (10 (53%) vs. 9 (21%) patients, OR 4.2; 95%CI 1.31-13.4; p=0.018), however after multivariate analysis this association lost significance. The AVR was comparably low in both groups. CONCLUSION: Patients with ACS present more often with dot bleedings and microaneurysms. These findings provide preliminary evidence that retinal fundus examination may be useful to contribute to the risk profile of patients, enabling a more intensive survey and care.

Evaluation of urolithiasis: a link between stone formation and diabetes mellitus?

INTRODUCTION: The pathogenesis of calcium oxalate stone formation is not completely understood. Recently, an influence of vascular phenomena like arteriosclerosis on the crystallization process was hypothesized. Thus, stone formation should be more common in patients with diabetes mellitus (DM) who are at risk of developing angiopathy. The aim of the study was to determine the prevalence of urolithiasis (UL) in patients with DM and to identify specific risk factors. MATERIAL AND METHODS: 350 patients with DM were evaluated with respect to DM-related history, and a total of 179 patients was included (83 female, 96 male; age 23-84 years). All patients were interviewed to assess the history of stone formation. These data were compared to epidemiological data in Germany. RESULTS: The overall prevalence of UL in the diabetic group was 7.82% (vs. 4.73% in Germany, p = 0.0485; binominal test). The prevalence was significantly higher in patients with coronary heart disease (25%; p < 0.0001; Fisher's exact test). We could not demonstrate an increased prevalence of UL for patients with occlusive arterial disease or arterial hypertension as diabetic nephropathy was not a risk factor for developing urinary lithiasis (p = 0.7184, p = 1.000, p = 0.6266, respectively; Fisher's exact test). Thiazide medication lowered the prevalence of stone formation (p = 0.0399; Fisher's test). Calcium or magnesium supplementation did not influence stone formation significantly (p = 0.5279; p = 1.000; respectively; Fisher's test). CONCLUSIONS: In Germany, patients with DM are at higher risk of UL compared with patients without diabetes. We demonstrated a significantly higher prevalence of urinary stones in patients with coronary heart disease. These findings are consistent with the hypothesis that urinary stone formation has a vascular pathogenesis in part.

Identification of CML-modified proteins in hemofiltrate of diabetic patients by proteome analysis.

The posttranslational modification of extra- and intracellular proteins by non-enzymatic glycation results in the formation of advanced glycation end products (AGEs) in physiological systems and is associated with the loss of protein structure and function. Modification by N (epsilon)-carboxymethyl lysine (CML) correlates with the risk for retinopathy in diabetes mellitus and has been discussed as a marker for the prediction of mortality in hemodialysis patients. AGEing of proteins is particularly increased under hyperglycemia associated with different late complications of diabetes mellitus. Modification of proteins to form AGE residues is significantly more enhanced in patients suffering from chronic renal disease than in hyperglycemia and is associated with increased risk for cardiovascular complications and inflammation in patients with chronic renal insuffiency. In order to identify and define the protein "substrates" for non-enzymatic glycation we used a proteome approach combining two-dimensional gel electrophoresis and immunoblotting with Edman protein sequencing to identify specific CML-modified proteins in human hemofiltrate, which essentially resembles plasma with respect to protein composition. Albumin, Ig kappa chain, prostaglandin D2 synthase, lysozyme C, plasma retinol binding protein and beta-2-microglobulin were identified as the major CML-modified proteins. CML-modified fragments of these proteins were also found in hemofiltrate. All identified proteins have in common that they appeared in hemofiltrate predominantly in their CML-modified form(s). Further studies of the functional roles of proteins identified by this new experimental approach could lead to the development of diagnostic tools to follow the progression of diabetes and contribute to the understanding of the pathogenesis of AGE-related diseases.

Clinical benefit of a short term dietary oatmeal intervention in patients with type 2 diabetes and severe insulin resistance: a pilot study.

AIMS/HYPOTHESIS: To evaluate the potential effectiveness of 'carbohydrate days' as a dietary intervention to overcome insulin resistance in type 2 diabetes. MATERIALS AND METHODS: Patients (n=14) with uncontrolled type 2 diabetes and insulin resistance as defined by a dosage of more than 1 IU/day (*)kg BW were consecutively enrolled in this prospective study. Primary outcomes were daily insulin requirement and mean blood glucose levels which were evaluated before, after, and 4 weeks after the intervention. RESULTS: All patients had a metabolic syndrome, 75% had microvascular and 57.1% macrovascular complications. Hospital setting and diabetes adapted diet alone led to improved glycemic control with a mean blood glucose 158+/-47 mg/dl. Intervention with two days of oatmeal diet further decreased mean blood glucose to 118+/-37 mg/dl (p<0.05). This was associated with a significant reduction of insulin dosage by 42.5% (before: 145+/-68.9 U/d, after 83+/-34.2 U/d, p<0.001) as well as a significant reduction (-26.4%, p<0.01) of serum leptin levels. After the four weeks outpatient period, insulin dosage remained significantly decreased (83+/-20.2 U/kg (*)d, p<0.01). Glycemic control was comparable (mean blood glucose141+/-20.78 mg/dl) to glucose levels within the hospital setting. Adiponectin levels increased significantly by 53.8% (p<0.05). CONCLUSIONS: In this uncontrolled pilot study, hospital admission and diabetes adapted diet followed by oatmeal intervention achieved a approximately 40% reduction of insulin dosage required to achieve controlled glucose levels. This effect was conserved after a 4 week outpatient phase with normal diet.

Long-term treatment of central Cushing's syndrome with rosiglitazone.

CONTEXT: Central Cushing's syndrome is not always curable by surgery or radiation of the pituitary. Medical treatment is often not possible or effective. Some studies revealed beneficial effects of the PPARgamma (Peroxisome-Proliferator-Activator- Receptor-gamma)-agonist rosiglitazone (RG) in in vitro studies, animal models and short term clinical studies. OBJECTIVE: of this study was to observe the long-term effects of RG-treatment on cortisol- and ACTH -secretion, clinical outcomes and morphological changes of the pituitary in patients with persistent ACTH-overproduction despite previous operation and radiation. DESIGN, SETTING AND PATIENTS: 14 patients with persistent central ACTH -production were included and monitored over a period up to 12 months. RG was administered daily and increased to a maximum dosage of 24 mg daily, according to the response of ACTH and cortisol secretion. ACTH and cortisol were measured at least every 4 weeks during RG treatment. RESULTS: Patients were treated between 4 and 12 months with RG (mean 6.8 months). Compared to baseline, ACTH- and cortisol levels dropped significantly (p<0.01) after 12, 16, 20, 24 and 28 weeks but thereafter rose again during the study period, despite continuous RG- treatment and dose increase up to the maximum dosage. This was paralleled by reocurrence of clinical symptoms. MRI-scans were performed in 6 patients because of persisting visible adenoma, but showed no morphological changes. CONCLUSION: RG seems not to be a long-term treatment option for patients with persistent central ACTH-evcess. Though, in order to reduce perioperative complications, short term treatment of patients could be an alternative.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity.

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand-receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte-free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia-induced vascular remodeling using the pericyte reporter X-LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up-regulation of angiopoietin-1 and PDGF-B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie-2 function either by soluble Tie-2 or by a sulindac analog, an inhibitor of Tie-2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie-2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

Genetics of diabetic retinopathy.

Familial aggregation as well as racial and ethnic differences in incidence suggest that genetic components play a role in the development of diabetic retinopathy (DR), several approaches have been used to identify genes contributing to the development of retinopathy. We searched the literature database using the keywords [diabetes], [gene], for publications dealing with retinopathy. 88 original publications reporting data on genetics of retinopathy were found. For the purpose of this review, a simple scoring system was applied, that results in a score for each considered gene to indicate its potential relevance in genetic control of retinopathy. Based on published studies, the most intriguing genes for further genetic studies are aldose receptor, advanced glycation end products receptor, vascular endothelial growth factor, intercellular adhesion molecule 1, beta3-adrenergic receptor gene, hemochromatosis, and alpha2beta1 integrin. Pathways involving these gene products may represent a fruitful area for further studies aimed at investigating the genetics and pathophysiology of DR. Meta-analyses of candidate gene studies may provide further useful insights into their role. In addition, our paper addresses several issues challenging genetic studies of retinopathy such as replication of associations, patient ascertainment schemes, or accurately defined phenotypes.

[National guidelines for treatment of diabetic retinopathy : Second edition of the national guidelines for treatment of diabetic retinopathy]. / Nationale Versorgungsleitlinie (NVL) Diabetische Retinopathie : 2. Auflage der NVL zur Therapie der diabetischen Retinopathie.

The updated German clinical practice guidelines (second edition) describe the consensus recommendations for prevention and treatment of retinal complications secondary to diabetes. According to the updated numbers on epidemiology a further increase of persons affected is expected. The prevalence of diabetic retinopathy is estimated to be 9-16 % in type 2 diabetes and 24-27 % in type 1 diabetes. A prolongation of the screening interval from 1 to 2 years is recommended for those patients with a lower risk of progression, when retinopathy has not already occurred and no increased systemic risk factors are present. Standardized documentation forms are the foundation for improved communication between the disciplines. If diabetic retinopathy is present, control examinations follow the stipulations of the ophthalmologist. The guidelines define scenarios when the use of optical coherence tomography (OCT) is necessary, e. g. diagnosis and follow-up of macular edema. Besides focal and panretinal laser therapy, the efficacy and risks of intravitreal operative pharmacotherapy are discussed. Focal laser coagulation is recommended for therapy of macular edema without foveal involvement and for macular edema with foveal involvement patients should be informed about the effective alternative forms of treatment. Panretinal laser coagulation is recommended for first line treatment of proliferative diabetic retinopathy and is optional for severe non-proliferative retinopathy.

Dicarbonyl Stress Mimics Diabetic Neurovascular Damage in the Retina.

The net effect of euglycemic treatment is grossly overestimated in diabetes mellitus and retinopathy, similar to what is observed in diabetic individuals, is found in the absence of chronic hyperglycemia. Explanations of this clinical paradox include the excess generation of reactive intermediates of metabolism. Excess formation or impaired detoxification of reactive intermediates can also result in multiple posttranslational modifications with a wide range of cellular dysfunctions. The multicellular neurovascular unit represents the response element of the retina which is crucial for the development of diabetic retinopathy. Current evidence suggests that increased reactive intermediates in the retina induce (micro-)glial activation, neurodegeneration and vasoregression similar to alterations found in the diabetic retina. Reactive metabolites can be lowered by metabolic signal blockade, by an activation of detoxification pathways and by quenching. The translation of these novel findings into treatment of patients with complications is important to reduce individual suffering and financial burden for societies.Quick Summary:Increased levels of reactive intermediates, independent of blood glucose levels, are linked to damage of the neurovascular unit of the diabetic retina.

Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental background diabetic retinopathy of the rat.

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.

Fish oil-enriched diet and reduction of low-dose streptozocin-induced hyperglycemia. Inhibition of macrophage activation.

Repeated low doses of streptozocin (STZ; 40 mg/kg, 5 injections/day) induce hyperglycemia in certain strains of mice after a latency of 1 wk. Omega-3 polyunsaturated fatty acids (omega 3FA) have been reported to suppress immune processes by blockade of the cyclooxygenase pathway of arachidonic acid metabolism. We investigated the effects of diets high in omega 3FA on the development of diabetes in the low-dose STZ-induced diabetes (LDSTZ-D) model. Male C57BL/6J mice were on a fish oil diet (FOD) as a source of omega 3FA 8 wk before STZ injection. Controls received laboratory chow only or a coconut oil diet (COD). Blood glucose levels in FOD mice were reduced (12.5 vs. 28 mM for COD mice, P less than .001) 60 days after STZ injection with a diet in which 20% of the calories were from fish oil. In FOD mice, immunohistology showed reduced numbers of class II antigen-expressing cells in pancreatic islets followed by a decreased extent of insulitis. FOD significantly decreased the number of Fc receptor-negative dendritic cells in cytospin preparations of islets isolated from diabetic mice. Interleukin 1-like activity of peritoneal exudate cell supernatants isolated from mice on FOD was reduced. FOD did not improve insulin secretion of isolated islets from LDSTZ-D mice. These data indicate a beneficial effect of FOD on the immune component of the mouse LDSTZ-D model.

Loss of the antiangiogenic pigment epithelium-derived factor in patients with angiogenic eye disease.

Retinal neovascularization characterizes proliferative diabetic retinopathy (PDR). Pigment epithelium-derived factor (PEDF) has been shown to be a major antiangiogenic growth factor in the mammalian eye. PEDF expression is suppressed by hypoxia, and changes in PEDF have been correlated to the development of retinal neovascularization in animal models of hypoxic eye disease. However, whether this concept of a reduced angiogenesis inhibitor holds true in humans is as yet unclear. In this study, we analyzed the in vivo regulation of PEDF in patients with and without hypoxic eye disease. We used immunoblots to measure PEDF in ocular fluids obtained from 64 nondiabetic and diabetic patients. In addition, immunohistochemistry of PEDF was carried out in specimens of normal human retinas and retinas with various degrees of diabetic retinopathy. The PEDF concentrations in patients with PDR (P < 0.001) or extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion (P < 0.001) were lower than in control patients. Levels of PEDF were replenished in PDR patients with previous retinal scatter photocoagulation compared with PDR patients without previous photocoagulation (P = 0.01). Immunohistochemistry revealed an interstitial staining pattern as expected for a secreted protein, with an intense staining in retinas of patients without proliferative eye disease. However, in patients with PDR, little or no staining was detectable. Our data strongly support the concept that retinal angiogenesis is induced by loss of the major angiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor. Our findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.

Angiogenic capacity of endothelial cells in islets of Langerhans.

Transplantation of pancreatic islets reconstitutes glucose homeostasis in diabetes mellitus. Before transplantation, islets are disrupted from the surrounding blood vessels by the isolation procedure, with the grafted tissue being subject to ischemic damage. The survival of transplanted islets is assumed to depend on effective revascularization. Perfusion studies suggest that newly formed microvessels supplying the graft with nutrients are exclusively rebuilt by the host. It is generally not known whether isolated islets contain endothelial cells (EC), which potentially participate in the revascularization process. Therefore, we tried to detect immature EC in isolated islets by transformation with polyoma middle T antigen. Endothelioma cells were generated, implicating the presence of de-differentiated EC within isolated islets. When embedded in a fibrin gel, the islets developed cellular cords consisting of EC, whereas FGF-2 and VEGF stimulated the formation of cord-like structures. Furthermore, we studied the presence of donor EC in islet grafts by using transgenic mice with an EC lineage-specific promotor-LacZ reporter construct (Tie-2LacZ). Following islet transplantation, Tie-2LacZ-positive EC of both donor and recipient were identified in the vicinity of or within the graft up to 3 wk after transplantation. In conclusion, EC and/or their progenitors with angiogenic capacity reside within isolated islets of different species, and their proliferative potential can be stimulated by various inducers. These graft-related endothelia persist after islet transplantation and are integrated within newly formed microvessels.

Effect of triamcinolone acetonide on proliferation of retinal endothelial cells in vitro and in vivo.

AIM: To assess the effect of crystalline triamcinolone acetonide on retinal endothelial cell proliferation in vivo and in vitro. METHODS: For in vitro analysis, a sprouting assay was employed. Bovine retinal endothelial cells were stimulated with basic fibroblast growth factor (bFGF) and incubated with different concentrations of triamcinolone acetonide (0.05 mg/ml to 8 mg/ml). For in vivo analysis, a retinopathy of prematurity (ROP) model was used. 16 C57BL/J6 mice were exposed to 75% oxygen from postnatal day 7 to day 12. On day 12, triamcinolone acetonide was intravitreally injected into one eye ("study eye") and isotonic saline into the contralateral eye ("control eye"). On day 17, the mice were sacrificed and the eyes removed for quantitative analysis of preretinal neovascularisation. Four non-exposed mice served as negative control. RESULTS: The sprouting assay demonstrated a dose dependent inhibition of bovine retinal endothelial cell proliferation from 0.05 mg triamcinolone acetonide/ml (no inhibition) to 3 mg triamcinolone acetonide/ml (complete inhibition). Dosages of more than 2 mg/ml resulted in cytotoxic changes of endothelial cells. The ROP model demonstrated a significantly lower neovascular cell count of 58% in the study group compared to the control group (6.35 (SD 2.1) cells per histological section versus 14.9 (SD 5.3) cells; p<0.005). CONCLUSIONS: Triamcinolone acetonide inhibits bFGF induced proliferation of retinal endothelial cells in vivo and in vitro. These findings contribute to understanding the mode of action and effects of triamcinolone acetonide on retinal neovascularisation.

Early loss of arteriolar smooth muscle cells: more than just a pericyte loss in diabetic retinopathy.

Incipient diabetic retinopathy is characterized by increased capillary permeability and progressive capillary occlusion. The earliest structural change is the loss of pericytes (PC) from the retinal capillaries. With the availability of the XLacZ mouse, which expresses the LacZ reporter in a PC/vascular smooth muscle cell (vSMC) specific fashion, we quantitatively assessed the temporal dynamics of smooth muscle cells in arterioles under hyperglycemic conditions. We induced stable hyperglycemia in XLacZ mice. After 4, 8, and 12 weeks of diabetes retinae were isolated and beta-galactosidase/lectin stained. The numbers of smooth muscle cells were counted in retinal whole mounts, and diameters of retinal radial and branching arterioles and venules were analyzed at different distances apart from the center of the retina. After eight weeks of diabetes, the numbers of vSMCs were significantly reduced in radial arterioles 1000 microm distant from the optic disc. At proximal sites of branching arterioles (400 microm distant from the center), and at distal sites (1000 microm), vSMC were significantly reduced already after 4 weeks (to a maximum of 31 %). These changes were not associated with any measurable variation in vessel diameters. These data indicate quantitatively that hyperglycemia not only causes pericyte loss, but also loss of vSMCs in the retinal vasculature. Our data suggest that arteriolar vSMC in the eye underlie similar regulations which induce early pericyte loss in the diabetic retina.