Microglia/macrophages migrate through retinal epithelium barrier by a transcellular route in diabetic retinopathy: role of PKCζ in the Goto Kakizaki rat model.

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Proliferative and Osteogenic Differentiation Potentials of Human Fetal Bone Cells

BACKGROUND. Human primary fetal bone cells (hFBC) are being characterized for use in bone tissue regeneration. Unlike human mesenchymal stem cells (hMSC), hFBC are partially differentiated with high expansion and regeneration potential. To date, proliferative and osteoblastic differentiation capacities of fetal bone cells remain poorly examined. The goal of this study was to define an environmental culture conditions for optimal proliferation and production of extracellular bone matrix leading to efficient bone repair. METHODS. Human primary FBC derived from our dedicated, consistent banks of bone cells comprising several fetal donors. For proliferation study, monolayer cultures of both cell types were expanded in DMEM or α- MEM media. Osteoblastic differentiation potentials of both hFBC and hMSC were evaluated through RT-PCR. Regulation of osteogenic differentiation by protein ligands Wnt3a and Wnt5a was studied by ALP enzymatic activity measurement. RESULTS. Evaluation of the proliferation rate demonstrated that hFBC proliferated more rapidly in α-MEM medium. Regarding growth factors that could stimulate cell proliferation rate, we observed that PDGF, FGF2 and Wnt3a had positive effects on proliferation of hFBC. Gene expression analysis demonstrated a higher expression of runx2 in hFBC cultured in basal conditions, which was was similar than that was observed in hMSC in osteoinductive culture conditions. Expression of sox9 was very low in hBFC and hMSC, compared to expression observed in fetal cartilage cells. Looking at osteogenic differentiation capacity, ALP activity was positively regulated byWnt5awhen hFBCwere cultured inα-MEM, but not in DMEM. Conversely, Wnt3a was shown to block the effect of osteogenic inductors on differentiation of both cell types. CONCLUSION. Data presented in this study indicate that the proliferation and differentiation of fetal and mesenchymal stem cells is optimal in α- MEM. Evidence for a pre-differentiated state of hBFC was given by extracellular matrix spontaneous mineralization as well as by higher ALP activity levels observed for these cells in baseline culture conditions, in comparison with hMSC. As we showed that, in vitro, hFBC express a higher capacity to differentiate in osteoblasts, they represent an attractive and promising prospect for fundamental research, and specifically for a new generation of skeletal tissue engineering.

Integrating quantitative proteomics and metabolomics in a cellular model of diabetic retinopathy

Diabetic retinopathy is the leading cause of visual loss in individuals under the age of 55. Most investigations into the pathogenesis of diabetic retinopathy have been concentrated on the neural retina since this is where clinical lesions are manifested. Recently, however, various abnormalities in the structural and secretory functions of retinal pigment epithelium that are essential for neuroretina survival, have been found in diabetic retinopathy. In this context, here we study the effect of hyperglycemic and hypoxic conditions on the metabolism of a human retinal pigment epithelial cell line (ARPE-19) by integrating quantitative proteomics using tandem mass tagging (TMT), untargeted metabolomics using MS and NMR, and 13C-glucose isotopic labeling for metabolic tracking. We observed a remarkable metabolic diversification under our simulated in vitro hyperglycemic conditions of diabetes, characterized increased flux through polyol pathways and inhibition of the Krebs cycle and oxidative phosphorylation. Importantly, under low oxygen supply RPE cells seem to consume rapidly glycogen storages and stimulate anaerobic glycolysis. Our results therefore pave the way to future scenarios involving new therapeutic strategies addressed to modulating RPE metabolic impairment, with the aim of regulating structural and secretory alterations of RPE. Finally, this study shows the importance of tackling biomedical problems by integrating metabolomic and proteomics results.

Eye Fundus Image Analysis for Automatic Detection of Diabetic Retinopathy

Diabetes is a rapidly increasing worldwide problem which is characterised by defective metabolism of glucose that causes long-term dysfunction and failure of various organs. The most common complication of diabetes is diabetic retinopathy (DR), which is one of the primary causes of blindness and visual impairment in adults. The rapid increase of diabetes pushes the limits of the current DR screening capabilities for which the digital imaging of the eye fundus (retinal imaging), and automatic or semi-automatic image analysis algorithms provide a potential solution. In this work, the use of colour in the detection of diabetic retinopathy is statistically studied using a supervised algorithm based on one-class classification and Gaussian mixture model estimation. The presented algorithm distinguishes a certain diabetic lesion type from all other possible objects in eye fundus images by only estimating the probability density function of that certain lesion type. For the training and ground truth estimation, the algorithm combines manual annotations of several experts for which the best practices were experimentally selected. By assessing the algorithm’s performance while conducting experiments with the colour space selection, both illuminance and colour correction, and background class information, the use of colour in the detection of diabetic retinopathy was quantitatively evaluated. Another contribution of this work is the benchmarking framework for eye fundus image analysis algorithms needed for the development of the automatic DR detection algorithms. The benchmarking framework provides guidelines on how to construct a benchmarking database that comprises true patient images, ground truth, and an evaluation protocol. The evaluation is based on the standard receiver operating characteristics analysis and it follows the medical practice in the decision making providing protocols for image- and pixel-based evaluations. During the work, two public medical image databases with ground truth were published: DIARETDB0 and DIARETDB1. The framework, DR databases and the final algorithm, are made public in the web to set the baseline results for automatic detection of diabetic retinopathy. Although deviating from the general context of the thesis, a simple and effective optic disc localisation method is presented. The optic disc localisation is discussed, since normal eye fundus structures are fundamental in the characterisation of DR.

Proliferative enteropathy (Lawsonia intracellularis) outbreak in rabbits in Brazil

An outbreak of Lawsonia intracellularis infection in rabbits, which occurred in 1988 in Rio de Janeiro state, Brazil, is reported. The disease had an acute course (24-48 hours) with clinical signs characterized by brownish or green diarrhea and dehydration. Occasionally, the animals died one day after the onset of diarrhea, without showing any other clinical signs. At necropsy, the ileum was prominent, firm and had a thickened wall; it was dilated in the caudal direction and had a somewhat reticulated appearance, perceptible through the serosa. The thickened mucous membrane had finely corrugated aspect and a shiny surface. The ileocecal valve and surrounding areas were slightly edematous and irregular. The Peyer's patches were sometimes more evident. There was moderate enlargement of the mesenteric lymph nodes. The histological examination revealed different degrees of hyperplasia of the epithelial cells of intestinal crypts consisting of poorly differentiated, hyperchromatic cells with high mitotic index, arranged in a pseudostratified layer which, in some cases, reached the apical portions of the villi. The inflammatory infiltrate between the hyperplastic epithelial cells was composed of lymphocytes, plasma cells, macrophages, some eosinophils and globular leukocytes. Silver impregnation revealed large numbers of bacteria with morphology of the genus Lawsonia in the apical pole of cryptal enterocytes. These bacteria reacted positively to a Lawsonia intracellularis polyclonal antibody by the avidin-biotin immunohistochemistry method.

Equine proliferative enteropathy on a Brazilian farm

Abstract:Lawsonia intracellularis infection on a horse farm in the Midwest region of Brazil is described. Thirty-nine foals a few days to months old from a herd with 300 horses, experienced diarrhea with variable characteristics and intensities, weight loss, hyperemic mucous membranes and dehydration. In foals 3 to 6 months of age, hypoproteinemia associated with submandibular edema were also common. Intestinal fragments of a 7-month-old foal were sent to an animal disease laboratory for diagnosis. The observed macroscopic lesions were hyperemic serosa, thickening of the intestinal wall with a corrugation, thickening of the mucosa folds and reduction of intestinal lumen. Histological analysis of the small and large intestine revealed enterocyte hyperplasia of the crypts associated with diffuse marked decrease in the number of goblet cells and positive L. intracellularis antigen labeling by immunohistochemistry. Three out of 11 animals of the same property were seropositive for L. intracellularis, demonstrating the circulation of the agent throughout the farm, but none were PCR positive in fecal samples. Based on clinical signs and pathological findings, the diagnosis of equine proliferative enteropathy was confirmed.

Feeding manipulation elicits different proliferative responses in the gastrointestinal tract of suckling and weanling rats

Food deprivation has been found to stimulate cell proliferation in the gastric mucosa of suckling rats, whereas the weanling period has been reported to be unresponsive in terms of proliferative activity. In the present study we analyze regional differences in the effect of milk or food deprivation on cell proliferation of the epithelia of the esophagus and of five segments of small intestine in suckling, weanling and newly weaned Wistar rats of both sexes. DNA synthesis was determined using tritiated thymidine to obtain labeling indices (LI); crypt depth and villus height were also determined. Milk deprivation decreased LI by 50% in the esophagus (from 15 to 8.35%) and small intestine (from 40 to 20%) of 14-day-old rats. In 18-day-old rats, milk and food deprivation decreased LI in the esophagus (from 13 to 5%) and in the distal segments of the small intestine (from 36-40 to 24-32%). In contrast, the LI of the epithelia of the esophagus (5%) and of all small intestine segments (around 30%) of 22-day-old rats were not modified by food deprivation. Crypt depth did not change after treatment (80 to 120 µm in 14- and 22-day-old rats, respectively). Villus height decreased in some small intestine segments of unfed 14- (from 400 to 300 µm) and 18-day-old rats (from 480 to 360 µm). The results show that, contrary to the stomach response, milk deprivation inhibited cell proliferation in the esophagus and small intestine of suckling rats, demonstrating the regional variability of each segment of the gastrointestinal tract in suckling rats. In newly weaned rats, food deprivation did not alter the proliferation of these epithelia, similarly to the stomach, indicating that weanling is a period marked by the insensitivity of gastrointestinal epithelia to dietary alterations

Analysis of p53 expression and proliferative assessment using PCNA in localized prostate carcinoma

The surgical specimens from 51 men submitted to radical prostatectomy for localized prostate cancer were examined by immunohistochemistry using proliferation cell nuclear antigen (PCNA) monoclonal antibody to evaluate the proliferative index (PI). The relationship between PI, biological variables and p53 protein expression was evaluated by immunohistochemistry. PI was low in invasive localized prostate carcinoma (mean, 12.4%) and the incidence of PCNA-positive cells was significantly higher in tumors with p53 expression (P = 0.0226). There was no statistical difference in PCNA values when biological parameters such as Gleason score, tumor volume, extraprostatic involvement, seminal vesicle infiltration or lymph node metastasis were considered. We conclude that proliferative activity is usually low in prostate carcinoma but is correlated with p53 immune staining, indicating that p53 is important in cell cycle control in this neoplasm.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 ± 1.9 mV) compared to NWR (-69 ± 1.4 mV), NWR 1K-1C (-60.8 ± 1.6 mV), WKY (-67.1 ± 3.2 mV) and WKY 1K-1C (-56.9 ± 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 ± 0.86) when compared to arteries of all the other groups (NWR 0.93 ± 0.65, NWR 1K-1C 1.24 ± 0.45, WKY 1.22 ± 0.32, WKY 1K-1C 1.15 ± 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

Proliferative signaling initiated in ACTH receptors

This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0->G1->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a) rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK) (2 to 10 min), b) transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min), c) induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d) onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.