Ultrastructural and proliferative features of the ventral lobe of the prostate in non-obese diabetic mice (NOD) following androgen and estrogen replacement associated to insulin therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The absence of angiopoietin-2 leads to abnormal vascular maturation and persistent proliferative retinopathy

Angiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice, Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF. Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF) has been implicated in the proliferation of new blood vessels. We have investigated the use of antisense phosphorothioate oligodeoxynucleotides against murine VEGF to inhibit retinal neovascularization and VEGF synthesis in a murine model of proliferative retinopathy. Intravitreal injections of two different antisense phosphorothioate oligodeoxynucleotides prior to the onset of proliferative retinopathy reduced new blood vessel growth a mean of 25 and 31% compared with controls. This inhibition was dependent on the concentration of antisense phosphorothioate oligodeoxynucleotides and resulted in a 40-66% reduction in the level of VEGF protein, as determined by Western blot analysis. Control (sense, nonspecific) phosphorothioate oligodeoxynucleotides did not cause a significant reduction in retinal neovascularization or VEGF protein levels. These data further establish a fundamental role for VEGF expression in ischemia-induced proliferative retinopathies and a potential therapeutic use for antisense phosphorothioate oligodeoxynucleotides.

Influence of insulin treatment on the lacrimal gland and ocular surface of diabetic rats

Previous studies have observed changes in the lacrimal gland and ocular surface related to diabetes mellitus and related it to insulin resistance or insufficiency and oxidative damage. The aim of this study was to evaluate whether insulin treatment inhibits those changes. Diabetes was induced in male Wistar rats with a single intravenous injection of streptozotocin and a subgroup was treated with insulin. After 5 and 10 weeks, the three groups (n = 5-10/group/experimental procedure) were compared for biochemical, functional, and histological parameters. After 5 weeks, changes in morphology and increased numbers of lipofucsin-like inclusions were observed in lacrimal glands of diabetic but not insulin-treated rats. After 5 weeks, malonaldehyde and total peroxidase activity were significantly higher in diabetic rats, but similar to control in insulin-treated diabetic rats (P = 0.03, P = 0.02, respectively). Our data indicate that diabetes induces histological alterations in lacrimal gland and suggests that hyperglycemia-related oxidative stress may participate in diabetic dry eye syndrome. Prevention by insulin replacement suggests direct hormone action and/or benefit by early sub optimal metabolic control.

Thirty-Day Morbidity and Mortality of the Laparoscopic Ileal Interposition Associated with Sleeve Gastrectomy for the Treatment of Type 2 Diabetic Patients with BMI < 35: An Analysis of 454 Consecutive Patients

Background The objective of this study was to evaluate the early results of the laparoscopic interposition of a segment of ileum associated with a sleeve gastrectomy (LII-SG) in order to treat patients with type 2 diabetes mellitus (T2DM) and BMI <35. Data regarding morbidly obese diabetic patients subjected to surgery has consistently been validated. To date, there is scarce information about morbidity and mortality related to the surgical treatment of a ""true"" typical diabetic population with BMI <35. Methods The procedures were performed in 454 patients (322 male, 132 female). Mean age was 53.6 +/- 8 years (range = 27-75). Mean BMI was 29.7 +/- 3.6 kg/m(2) (range = 19-34.8). All patients had the diagnosis of T2DM for at least 3 years. Insulin therapy was used by 45.6% of patients. Mean duration of T2DM was 10.8 +/- 5.9 years (range = 3-35). Mean hemoglobin A(1c) was 8.8 +/- 1.9%. Dyslipidemia was observed in 78.4%, hypertension in 64.8%, nephropathy in 28.6%, retinopathy in 32.6%, neuropathy in 34.6%, and coronary heart disease in 13%. Results There was no conversion to open surgery. All patients were evaluated postoperatively. Mortality was 0.4%. There were 29 major complications (6.4%) in 22 patients (4.8%) and 51 minor complications (11.2%). Reoperations were performed on 8 patients (1.7%). Twenty patients (4.4%) were readmitted to the hospital. Mean postoperative BMI was 25.8 +/- 3.5 kg/m(2). Mean fasting plasma glucose decreased from 198 +/- 69 to 128 +/- 67 mg/dl and mean postprandial plasma glucose decreased from 262 +/- 101 to 136 +/- 43 mg/dl. Conclusions The laparoscopic ileal interposition associated with a sleeve gastrectomy was considered a safe operation with low rates of morbidity and mortality in a diabetic population with BMI < 35. An early control of postprandial glycemia was observed.

The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity

Recent work has demonstrated that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron-transport chain triggers several pathways of injury [(protein kinase C (PKC), hexosamine and polyol pathway fluxes, advanced glycation end product formation (AGE)] involved in the pathogenesis of diabetic complications by inhibiting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity. Increased oxidative and nitrosative stress activates the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP). PARP activation, on one hand, depletes its substrate, NAD+, slowing the rate of glycolysis, electron transport and ATP formation. On the other hand, PARP activation results in inhibition of GAPDH by poly-ADP-ribosylation. These processes result in acute endothelial dysfunction in diabetic blood vessels, which importantly contributes to the development of various diabetic complications. Accordingly, hyperglycemia-induced activation of PKC and AGE formation are prevented by inhibition of PARP activity. Furthermore, inhibition of PARP protects against diabetic cardiovascular dysfunction in rodent models of cardiomyopathy, nephropathy, neuropathy, and retinopathy. PARP activation is also present in microvasculature of human diabetic subjects. The present review focuses on the role of PARP in diabetic complications and emphasizes the therapeutic potential of PARP inhibition in the prevention or reversal of diabetic complications.

Multimodal Highlighting of Structural Abnormalities in Diabetic Rat and Human Corneas.

PURPOSE: This study aimed to highlight structural corneal changes in a model of type 2 diabetes, using in vivo corneal confocal microscopy (CCM). The abnormalities were also characterized by transmission electron microscopy (TEM) and second harmonic generation (SHG) microscopy in rat and human corneas. METHODS: Goto-Kakizaki (GK) rats were observed at age 12 weeks (n = 3) and 1 year (n = 6), and compared to age-matched controls. After in vivo CCM examination, TEM and SHG microscopy were used to characterize the ultrastructure and the three-dimensional organization of the abnormalities. Human corneas from diabetic (n = 3) and nondiabetic (n = 3) patients were also included in the study. RESULTS: In the basal epithelium of GK rats, CCM revealed focal hyper-reflective areas, and histology showed proliferative cells with irregular basement membrane. In the anterior stroma, extracellular matrix modifications were detected by CCM and confirmed in histology. In the Descemet's membrane periphery of all the diabetic corneas, hyper-reflective deposits were highlighted using CCM and characterized as long-spacing collagen fibrils by TEM. SHG microscopy revealed these deposits with high contrast, allowing specific detection in diabetic human and rat corneas without preparation and characterization of their three-dimensional organization. CONCLUSION: Pathologic findings were observed early in the development of diabetes in GK rats. Similar abnormalities have been found in corneas from diabetic patients. TRANSLATIONAL RELEVANCE: This multidisciplinary study highlights diabetes-induced corneal abnormalities in an animal model, but also in diabetic donors. This could constitute a potential early marker for diagnosis of hyperglycemia-induced tissue changes.

The wound watch: an objective staging system for wounds in the diabetic (db/db) mouse model.

As in cancer biology, in wound healing there is a need for objective staging systems to decide for the best treatment and predictors of outcome. We developed in the diabetic (db/db) wound healing model, a staging system, the "wound watch," based on the quantification of angiogenesis and cell proliferation in open wounds. In chronic wounds, there is often a lack of cellular proliferation and angiogenesis that leads to impaired healing. The wound watch addresses this by quantifying the proliferative phase of wound healing in two dimensions (cellular division and angiogenesis). The results are plotted in a two-dimensional graph to monitor the course of healing and compare the response to different treatments.

Épidémiologie et physiopathologie de la rétinopathie du prématuré [Epidemiology and pathophysiology of retinopathy of prematurity].

Retinopathy of prematurity (ROP) is a major cause of visual impairment in premature infants. It is characterized by an arrest in normal retinal vascular development associated with microvascular degeneration, followed by an abnormal hypoxiainduced neovascularization. Recent studies point out that ROP is a multifactorial disease, implicating both oxygen-dependent and oxygen-independent mechanisms. Oxygen-dependent factors leading to microvascular degeneration include generation of reactive oxygen species and suppression of specific oxygen-regulated vascular survival factors, such as vascular endothelial growth factor (VEGF) and erythropoietin. The other major mechanism for the initial capillary loss is oxygen-independent and implicates a deficit in growth factor IGF-1/IGFBP3. The proliferative, second phase of ROP is triggered by increases in vascular growth factors concentrations, in an attempt to compensate for the hypoxic retina. Novel signaling pathways for vascular repair, implicating both metabolite signaling and inflammatory lipids signaling, represent new therapeutic avenues for ROP.

Myocardial perfusion scintigraphy in the detection of silent ischemia in asymptomatic diabetic patients

OBJECTIVE: This study was aimed to evaluate myocardial perfusion in asymptomatic patients with type 1 (DM1) and type 2 diabetes mellitus (DM2) without previous diagnoses of coronary artery disease (CAD) or cerebral infarction. MATERIALS AND METHODS: Fifty-nine consecutive asymptomatic patients (16 DM1, 43 DM2) underwent myocardial perfusion scintigraphy with 99mTc-sestamibi (MPS). They were evaluated for body mass index, metabolic control of DM, type of therapy, systemic arterial hypertension, dyslipidemia, nephropathy, retinopathy, peripheral neuropathy, smoking, and familial history of CAD. RESULTS: MPS was abnormal in 15 patients (25.4%): 12 (20.3%) with perfusion abnormalities, and 3 with isolated left ventricular dysfunction. The strongest predictors for abnormal myocardial perfusion were: age 60 years and above (p = 0.017; odds ratio [OR] = 6.0), peripheral neuropathy (p = 0.028; OR = 6.1), nephropathy (p = 0.031; OR = 5.6), and stress ECG positive for ischemia (p = 0.049; OR = 4.08). CONCLUSION: Silent myocardial ischemia occurs in more than one in five asymptomatic diabetic patients. The strongest predictors of ischemia in this study were: patient age, peripheral neuropathy, nephropathy, retinopathy and a stress ECG positive for ischemia.

Residual ß-cell function and microvascular complications in type 1 diabetic patients

To determine the influence of residual ß-cell function on retinopathy and microalbuminuria we measured basal C-peptide in 50 type 1 diabetic outpatients aged 24.96 ± 7.14 years, with a duration of diabetes of 9.1 ± 6.2 years. Forty-three patients (86%) with low C-peptide (<0.74 ng/ml) had longer duration of diabetes than 7 patients (14%) with high C-peptide (³0.74 ng/ml) (9 (2-34) vs 3 (1-10) years, P = 0.01) and a tendency to high glycated hemoglobin (HBA1) (8.8 (6-17.9) vs 7.7 (6.9-8.7)%, P = 0.08). Nine patients (18%) had microalbuminuria (two out of three overnight urine samples with an albumin excretion rate (AER) ³20 and <200 µg/min) and 13 (26%) had background retinopathy. No association was found between low C-peptide, microalbuminuria and retinopathy and no difference in basal C-peptide was observed between microalbuminuric and normoalbuminuric patients (0.4 ± 0.5 vs 0.19 ± 0.22 ng/ml, P = 0.61) and between patients with or without retinopathy (0.4 ± 0.6 vs 0.2 ± 0.3 ng/ml, P = 0.43). Multiple regression analysis showed that duration of diabetes (r = 0.30, r2 = 0.09, P = 0.031) followed by HBA1 (r = 0.41, r2 = 0.17, P = 0.01) influenced basal C-peptide, and this duration of diabetes was the only variable affecting AER (r = 0.40, r2 = 0.16, P = 0.004). In our sample of type 1 diabetic patients residual ß-cell function was not associated with microalbuminuria or retinopathy.

Dopamine Receptor Gene Expression In Streptozotocin Induced Diabetic Rats And Its Role In Insulin Secretion

Diabetes Mellitus is a metabolic disorder associated with insulin deficiency, which not.only affects the carbohydrate metabolism but also is associated with various central and peripheral complications. Chronic hyperglycemia during diabetes mellitus is a major initiator of diabetic microvascular complications like retinopathy, neuropathy, The central nervous system (CNS) neurotransmitters play an important role in the regulation of glucose homeostasis. These neurotransmitters mediate rapid intracellular communications not only within the central nervous system but also in the peripheral tissues. They exert their function through receptors present in both neuronal and non neuronal cell surface that trigger second messenger signaling pathways. Dopamine is a neurotransmitter that has been implicated in various central neuronal degenerative disorders like Parkinson's disease and behavioral diseases like Schizophrenia. Dopamine is synthesised from tyrosine, stored in vesicles in axon terminals and released when the neuron is depolarised. Dopamine interacts with specific membrane receptors to produce its effect. Dopamine plays an important role both centrally and peripherally. The recent identification of five dopamine receptor subtypes provides a basis for understanding dopamine's central and peripheral actions . Dopamine receptors are classified into two major groups : DA D1 like and DA D2 like. Dopamine D1 like receptors consists of DA D1 and DA D5 receptors . Dopamine D2 like receptors consists of DA D2, DA D3 and DA D4 receptors. Stimulation of the DA D1 receptor gives rise to increased production of cAMP. Dopamine D2 receptors inhibit cAMP production, but activate the inositol phosphate second messenger system . Impairment of central dopamine neurotransmission causes muscle rigidity, hormonal regulation , thought disorder and cocaine addiction. Peripheral dopamine receptors mediate changes in blood flow, glomerular filtration rate, sodium excretion and catecholamine release. The dopamine D2 receptors increased in the corpus striatum and cerebral cortex but decreased in the hypothalamus and brain stem indicating their involvement in regulating insulin secretion. Dopamine D2 receptor which has a stimulatory effecton insulin secretion decreased in the pancreatic islets during diabetes. Our in vitro studies confirmed the stimulatory role of dopamine D2 receptors in stimulation of glucose induced insulin secretion. A detailed study at the molecular level on the mechanisms involved in the role of dopamine in insulin secretion, its functional modification could lead to therapeutic interventions that will have immense clinical importance.

Temporal relationship between successful pancreas transplantation and control of ocular complications in alloxan-induced diabetic rats

Purpose. The impact of pancreas transplantation (PT) on the progression of eye disease is still controversial. This study evaluated the course of retinopathy in transplanted rats in two different diabetic stages.Methods. Sixty inbred male Lewis rats were assigned to four experimental groups: NC-15 nondiabetic control rats; DC-15 untreated diabetic control rats; PT1-15 diabetic rats that received syngeneic pancreas transplants 2 weeks after alloxan diabetes induction; PT2-15 diabetic rats that received pancreas transplants 12 weeks after diabetes onset. Clinical and laboratory parameters and tens opacity were examined in all rats prior to treatment and at 1-, 6-, and 12-months follow-up. Nucleated eyes from five rats in each group processed for ultrastructural study of the retinal at 6 and 12 months after PT or at follow-up.Results. Cataracts were observed in 20%, 60%, and 100% of DC rats at 1-, 6-, and 12-months follow-up, respectively. Early PT (2 weeks) significantly reduced the prevalence of this complication but not late (12 weeks) PT. PT1 rats also showed improved ultrastructure of the superficial and deep capillary plexuses of the retina, and of Muller cells, compared with DC and PT2. In the last group, retinopathy continued to evolve despite successful PT.Conclusion. Our results suggested that prevention of diabetic ocular lesions by PT was closely dependent on earlier performance of the procedure.

Malignant lesions in the ventral prostate of alloxan-induced diabetic rats

The aim of this study was to evaluate the changes caused by chronic diabetes in the rat ventral prostate and to establish a correlation between diabetes and the development of prostatic lesions. Male rats received alloxan (42 mg/kg b.w.) to induce diabetes. Ninety days after diabetes diagnosis, animals were sacrificed and the ventral prostate was removed and prepared for general and immunohistochemical analyses. The total area showing different types of lesions was estimated. Diabetes led to a decrease in the body and prostatic weights, as well as in testosterone levels. The prostate morphology and stereology showed high variation in the diabetic group. Some animals had light changes; the great majority had an intense epithelial atrophy; and other rats showed premalignant and malignant lesions in the prostate. Such epithelial atrophy was, in some samples, combined with chronic inflammation, similar to proliferative inflammatory atrophy (PIA). The diabetic group also presented high incidence of prostatitis, adenocarcinoma and prostatic intra-epithelial neoplasia (PIN). Samples with adenocarcinoma had poorly differentiated acini with high levels of cellular proliferation and nuclear atypia. These lesions exhibited an invasive feature showing Bcl-2-positive cells and interruptions in the basement membrane. An association of PIA, PIN and adenocarcinoma was detected in one sample. Reduced androgen levels have a synergic effect to insulin dysfunction promoting negative effects in the rat prostate. Diabetic individuals had a high incidence of prostatitis, and this inflammation could stimulate the incidence of other forms of prostatic pathology.