Complement expression in retinal pigment epithelial cells is modulated by activated macrophages

Complement activation is involved in a variety of retinal diseases. We have shown previously that a number of complement components and regulators can be produced locally in the eye, and that retinal pigment epithelial (RPE) cells are the major source of complement expression at the retina-choroidal interface. The expression of complement components by RPE cells is regulated by inflammatory cytokines. Under aging or inflammatory conditions, microglia and macrophages accumulate in the subretinal space, where they are in close contact with RPE cells. In this study, we investigated the effect of activated macrophages on complement expression by RPE cells. Mouse RPE cells were treated with the supernatants from un-activated bone marrow-derived macrophages (BM-DMs), the classically activated BM-DMs (M1) and different types of the alternatively activated BM-DMs (M2a by IL-4, M2b by immune complex and lipopolysaccharide (LPS), M2c by IL-10). The expression of inflammatory cytokines and complement genes by RPE cells were determined by real-time RT-PCR. The protein expression of CFB, C3, C1INH, and C1r was examined by Western blot. Our results show that un-stimulated RPE cells express a variety of complement-related genes, and that the expression levels of complement regulators, including C1r, factor H (CFH), DAF1, CD59, C1INH, Crry, and C4BP genes are significantly higher than those of complement component genes (C2, C4, CFB, C3, and C5). Macrophage supernatants increased inflammatory cytokine (IL-1ß, IL-6, iNOS), chemokine (CCL2) and complement expression in RPE cells. The supernatants from M0, M2a and M2c macrophages mildly up-regulated (2~3.5-fold) CFB, CFH and C3 gene expression in RPE cells, whereas the supernatants from M1 and M2b macrophages massively increased (10~30-fold) CFB and C3 gene expression in RPE cells. The expression of other genes, including C1r, C2, C4, CFH, Masp1, C1INH, and C4BP in RPE cells was also increased by the supernatants of M1 and M2b macrophages; however, the increment levels were significantly lower than CFB and C3 genes. M1 and M2b macrophage supernatants enhanced CFB (Bb fragment) protein expression and C3 secretion by RPE cells. M1 macrophages may affect complement expression in RPE cells through the STAT1 pathway. Our results suggest that under inflammatory conditions, activated macrophages could promote the alternative pathway of complement activation in the retina via induction of RPE cell CFB and C3 expression.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression

PURPOSE. Vascular endothelial growth factor (VEGF)-A and placental growth factor (PIGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PIGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS. C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PIGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS. Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PIGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PIGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PIGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS. Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Mechanisms of modified LDL-induced pericyte loss and retinal injury in diabetic retinopathy

Aims/hypothesis: In previous studies we have shown that extravasated, modified LDL is associated with pericyte loss, an early feature of diabetic retinopathy (DR). Here we sought to determine detailed mechanisms of this LDLinduced pericyte loss.

Methods: Human retinal capillary pericytes (HRCP) were exposed to ‘highly-oxidised glycated’ LDL (HOG-LDL) (a model of extravasated and modified LDL) and to 4-hydroxynonenal or 7-ketocholesterol (components of oxidised LDL), or to native LDL for 1 to 24 h with or without 1 h of pretreatment with inhibitors of the following: (1) the scavenger receptor (polyinosinic acid); (2) oxidative stress (N-acetyl cysteine); (3) endoplasmic reticulum (ER) stress (4-phenyl butyric acid); and (4) mitochondrial dysfunction (cyclosporin A). Oxidative stress, ER stress, mitochondrial dysfunction, apoptosis and autophagy were assessed using techniques including western blotting, immunofluorescence, RT-PCR, flow cytometry and TUNEL assay. To assess the relevance of the results in vivo, immunohistochemistry was used to detect the ER stress chaperon, 78 kDa glucose-regulated protein, and the ER sensor, activating transcription factor 6, in retinas from a mouse model of DR that mimics exposure of the retina to elevated glucose and elevated LDL levels, and in retinas from human participants with and without diabetes and DR.

Results: Compared with native LDL, HOG-LDL activated oxidative and ER stress in HRCP, resulting in mitochondrial dysfunction, apoptosis and autophagy. In a mouse model of diabetes and hyperlipidaemia (vs mouse models of either condition alone), retinal ER stress was enhanced. ER stress was also enhanced in diabetic human retina and correlated with the severity of DR.

Conclusions/interpretation: Cell culture, animal, and human data suggest that oxidative stress and ER stress are induced by modified LDL, and are implicated in pericyte loss in DR.

Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

Extremely Complex Populations of Small RNAs in the Mouse Retina and RPE/Choroid

Purpose: MicroRNAs (miRNAs) are small non-coding RNAs of ~18-22 nucleotides in length that regulate gene expression. They are widely expressed in the retina, being both required for its normal development and perturbed in disease. The aim of this study was to apply new high-throughput sequencing techniques to more fully characterise the microRNAs and other small RNAs expressed in the retina and retinal pigment epithelium (RPE)/choroid of the mouse.

Methods: Retina and RPE/choroid were dissected from eyes of 3 month-old C57BL/6J mice. Small RNA libraries were prepared and deep sequencing performed on a Genome Analyzer (Illumina). Reads were annotated by alignment to miRBase, other non-coding RNA databases and the mouse genome.

Results: Annotation of 9 million reads to 320 microRNAs in retina and 340 in RPE/choroid provides the most comprehensive profiling of microRNAs to date. Two novel microRNAs were identified in retina. Members of the sensory organ specific miR-183,-182,-96 cluster were amongst the most highly expressed, retina-enriched microRNAs. Remarkably, microRNA 'isomiRs', which vary slightly in length and are differentially detected by Taqman RT-PCR assays, existed for all the microRNAs identified in both tissues. More variation occurred at the 3' ends, including non-templated additions of T and A. Drosha-independent mirtron microRNAs and other small RNAs derived from snoRNAs were also detected.

Conclusions: Deep sequencing has revealed the complexity of small RNA expression in the mouse retina and RPE/choroid. This knowledge will improve the design and interpretation of future functional studies of the role of microRNAs and other small RNAs in retinal disease.

STAT3 activation in circulating immune cells is related to neovascular age-related macular degeneration

Purpose:The Signal Transducer and Activator of Transcription 3 (STAT3) pathway is known to play an important role in inflammation and angiogenesis. STAT3 can be activated by IL-6 family cytokines through the receptor IL-6R/gp130. Increased IL-6 has been detected in the plasma and vitreous in neovascular age-related macular degeneration (nAMD) patients. The aim of this study was to investigate the role of the STAT3 pathway in the pathogenesis of nAMD.

Methods:Blood cells from nAMD patients (n = 11) and age-, gender-matched healthy controls (n = 13) were stimulated with IL-6 for 20 minutes. The expression of the activated form of STAT3 (p-STAT3) was examined by flow cytometry. The mRNA levels of gp130, IL-6R and the suppressor of cytokine signalling 3 (SOCS3, a negative regulator of p-STAT3) were evaluated by real-time RT-PCR. Laser-induced choroidal neovascularisation (CNV) was performed in WT C57BL/6J mice as well as in the myeloid cell specific SOCS3 deficiency mice i.e., the LysMCre-SOCS3fl/fl mice. STAT3 activation in CNV lesions was examined by western blot. The size of CNV at different times after laser treatment was measured by confocal microscopy of RPE/choroidal flatmounts.

Results:The expression of p-STAT3 in CD11b+ monocytes was significantly increased in nAMD patients compared to healthy controls, although mRNA expression of gp130, IL-6R and SOCS3 did not differ between patients and controls. The expression of p-STAT3 in the retinal and RPE/choroidal tissues was increased at 1 and 3 days after laser treatment. The administration of a STAT3 inhibitor LLL12 significantly suppressed CNV. CD11b+ monocytes from LysMCre-SOCS3fl/fl mice expressed higher levels of p-STAT3 compared to the cells from WT mice. Laser induced CNV developed earlier and were larger in LysMCre-SOCS3fl/fl mice compared to WT C57BL/6J mice.

Conclusions:Our results suggest that STAT3 activation in circulating monocytes may contribute to the development of choroidal neovascularisation in AMD, and targeting the STAT3 pathway may have therapeutic potential in nAMD.

Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.

Acute respiratory distress syndrome caused by Mycoplasma pneumoniae diagnosed by polymerase chain reaction

Mycoplasma pneumoniae (M. pneumoniae) is a common pathogen in cases of atypical pneumonia. Most individuals with Mycoplasma pneumonia run a benign course, with non-specific symptoms of malaise, fever and non-productive cough that usually resolve with no long-term sequelae. Acute lung injury is not commonly seen in Mycoplasma pneumonia. We report a case of acute respiratory distress syndrome cause by M. pneumoniae diagnosed by quantitative real-time polymerase chain reaction (RT-PCR).

KIF2A silencing inhibits the proliferation and migration of breast cancer cells and correlates with unfavorable prognosis in breast cancer

Background: Kinesin family member 2a (KIF2A), a type of motor protein found in eukaryotic cells, is associated with development and progression of various human cancers. The role of KIF2A during breast cancer tumorigenesis and progression was studied.

Methods: Immunohistochemical staining, real time RT-PCR and western blot were used to examine the expression of KIF2A in cancer tissues and adjacent normal tissues from breast cancer patients. Patients' survival in relation to KIF2A expression was estimated using the Kaplan-Meier survival and multivariate analysis. Breast cancer cell line, MDA-MB-231 was used to study the proliferation, migration and invasion of cells following KIF2A-siRNA transfection.

Results: The expression of KIF2A in cancer tissues was higher than that in normal adjacent tissues from the same patient (P <0.05). KIF2A expression in cancer tissue with lymph node metastasis and HER2 positive cancer were higher than that in cancer tissue without (P <0.05). A negative correlation was found between KIF2A expression levels in breast cancer and the survival time of breast cancer patients (P <0.05). In addition, multivariate analysis indicated that KIF2A was an independent prognostic for outcome in breast cancer (OR: 16.55, 95% CI: 2.216-123.631, P = 0.006). The proliferation, migration and invasion of cancer cells in vitro were suppressed by KIF2A gene silencing (P <0.05).

Conclusions: KIF2A may play an important role in breast cancer progression and is potentially a novel predictive and prognostic marker for breast cancer.

Gene expression in normal Urothelium depends on location within the bladder:A possible link to bladder carcinogenesis

Objectives: Clinical studies have shown that more than 70% of primary bladder tumours arise in the area around the ureteric orifice. In this study a genomic approach was taken to explore the molecular mechanisms that may influence this phenomenon.

Methods: RNA was isolated from each individual normal ureteric orifice and the dome biopsy from 33 male patients. Equal amounts of the pooled ureteric orifice and dome mRNAs were labelled with Cy3 and Cy5, respectively before hybridising to the gene chip (UniGEM 2.0, Incyte Genomics Inc., Wilmington, Delaware, USA). Results: Significant changes (more than a twofold difference) in gene expression were observed in 3.1% (312) of the 10,176 gene array: 211 genes upregulated and 101 downregulated. Analysis of Cdc25B, TK1, PKM, and PDGFra with RT-PCR supported the reliability of the microarray result. Seladin-1 was the most upregulated gene in the ureteric orifice: 8.3-fold on the microarray and 11.4-fold by real time PCR.

Conclusions: Overall, this study suggests significant altered gene expression between these two anatomically distinct areas of the normal human bladder. Of particular note is Seladin-1, whose significance in cancer is yet to be clarified. Further studies of the genes discovered by this work will help clarify which of these differences influence primary bladder carcinogenesis. (c) 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Epidermal growth factor receptor (EGFR), HER2 and insulin-like growth factor-1 receptor (IGF-1R) status in ovarian adult granulosa cell tumours

AIMS: Adult granulosa cell tumours (AGCTs) are uncommon ovarian sex cord-stromal tumours which recur following surgical removal in up to 50% of patients. Treatment options for recurrent and advanced stage AGCTs are limited, with poor response to chemotherapy and radiotherapy. We aimed to assess epidermal growth factor receptor (EGFR), HER2 and insulin-like growth factor-1 receptor (IGF-1R) status in AGCTs with a view to investigating whether or not these receptors might be potential therapeutic targets in these neoplasms.

METHODS AND RESULTS: Immunohistochemical staining for EGFR, HER2 and IGF-1R was undertaken in 31 AGCTs. Tumour DNA was also analysed for mutations in the tyrosine kinase domain of EGFR (exons 18-21) by Cobas mutation RT-PCR. Twenty-three of 31 (74%) AGCTs showed some degree of EGFR expression, generally with cytoplasmic or mixed membranous and cytoplasmic staining of variable intensity. Eleven of 27 (41%) cases exhibited strong membranous and cytoplasmic expression of IGF-1R. HER2 expression was not seen. No mutations were found in exons 18-21 of the EGFR gene in hot-spots of therapeutic relevance.

CONCLUSIONS: This study raises the possibility that anti-EGFR and/or anti-IGF-1R therapies may be of potential benefit in ovarian AGCTs, and this requires further study. Lack of known mutations within the tyrosine kinase domain of EGFR suggests that EGFR-related tyrosine kinase inhibitors may not be useful therapeutically.

Seroepidemiology of norovirus-associated travelers' diarrhea

Background: Noroviruses (NoVs) are the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and were recently identified as a leading cause of travelers' diarrhea (TD) in US and European travelers to Mexico, Guatemala, and India.

Methods: Serum and diarrheic stool samples were collected from 75 US student travelers to Cuernavaca, Mexico, who developed TD. NoV RNA was detected in acute diarrheic stool samples using reverse transcription-polymerase chain reaction (RT-PCR). Serology assays were performed using GI.1 Norwalk virus (NV) and GII.4 Houston virus (HOV) virus-like particles (VLPs) to measure serum levels of immunoglobulin A (IgA) and IgG by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA); serum IgM was measured by capture enzyme-linked immunosorbent assay (ELISA), and the 50% antibody-blocking titer (BT50 ) was determined by a carbohydrate-blocking assay.

Results: NoV infection was identified in 12 (16%; 9 GI-NoV and 3 GII-NoV) of 75 travelers by either RT-PCR or fourfold or more rise in antibody titer. Significantly more individuals had detectable preexisting IgA antibodies against HOV (62/75, 83%) than against NV (49/75, 65%) (p = 0.025) VLPs. A significant difference was observed between NV- and HOV-specific preexisting IgA antibody levels (p = 0.0037), IgG (p = 0.003), and BT50 (p = <0.0001). None of the NoV-infected TD travelers had BT50  > 200, a level that has been described previously as a possible correlate of protection.

Conclusion: We found that GI-NoVs are commonly associated with TD cases identified in US adults traveling to Mexico, and seroprevalence rates and geometric mean antibody levels to a GI-NoV were lower than to a GII-NoV strain.

The role of SOCS3/pSTAT3 pathway in the development of diabetic retinopathy

Purpose: Suppressor of cytokine signalling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 critically controls STAT3 activation, cytokine signalling and inflammatory gene expression in macrophages and microglia. In this study, we investigated the role of SOCS3/STAT3 in myeloid cells in the initiation and progression of diabetic retinopathy (DR). 
Methods: Mice with a conditional deletion of SOCS3 in myeloid cells (LysMCre-SOCS3 fl/fl) and C57BL/6J (as control) were rendered diabetic by a low-dose multiple intraperitoneal injections of Stroptozocine. Diabetes related retinal changes, including leukostasis, acellular capilliaries, and microglial activation were assessed at different stages of disease. Bone marrow derived macrophages (BMDMs) from LysMCreSOCS3 fl/fl and C57BL/6J mice were cultured in high glucose (HG) medium, and cell activation was evaluated by real-time RT-PCR.
Results: In C57BL/6J diabetic mice the expression of phosphorylated STAT3 (pSTAT3) was increased and SOCS3 was decreased in the retina. Interleukin 6 (IL-6), the main cytokine that stimulates STAT3 activation, was increased in the plamsa in diabetic mice. Although blood glucose levels and Hbac-1 were comparable between LysMCre-SOCS3fl/fl and WT mice after STZ injection, the LysMCreSOCS3 fl/fl diabetic mice developed severe retinal vasculopathy, including increased leukostasis and microglial activation at one month and enhanced acellular capillary formation at 6 months after diabetes induction. 
Conclusions: our study suggests that the JAK/STAT3 pathway is involved in the initiation and progression of DR, and uncontrolled STAT3 activation results in accelerated DR progression. Targeting the STAT3 pathway may be a novel approach for the management of DR.

In Helicobacter pylori auto-inducer-2, but not LuxS/MccAB catalysed reverse transsulphuration, regulates motility through modulation of flagellar gene transcription

BACKGROUND: LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2); hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB), synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only.

RESULTS: We report that disruption of luxS renders H. pylori non-motile in soft agar and by microscopy, whereas disruption of mccAHp or mccBHp (other genes in the cysteine provision pathway) does not, implying that the lost phenotype is not due to disrupted cysteine provision. The motility defect of the DeltaluxSHp mutant was complemented genetically by luxSHp and also by addition of in vitro synthesised AI-2 or 4, 5-dihydroxy-2, 3-pentanedione (DPD, the precursor of AI-2). In contrast, exogenously added cysteine could not restore motility to the DeltaluxSHp mutant, confirming that AI-2 synthesis, but not the metabolic effect of LuxS was important. Microscopy showed reduced number and length of flagella in the DeltaluxSHp mutant. Immunoblotting identified decreased levels of FlaA and FlgE but not FlaB in the DeltaluxSHp mutant, and RT-PCR showed that the expression of flaA, flgE, motA, motB, flhA and fliI but not flaB was reduced. Addition of DPD but not cysteine to the DeltaluxSHp mutant restored flagellar gene transcription, and the number and length of flagella.

CONCLUSIONS: Our data show that as well as being a metabolic enzyme, H. pylori LuxS has an alternative role in regulation of motility by modulating flagellar transcripts and flagellar biosynthesis through production of the signalling molecule AI-2.

Evidence supporting a role for N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine accumulation in Muller glia dysfunction and death in diabetic retinopathy

Purpose: Recent evidence suggests that neuroglial dysfunction and degeneration contributes to the etiology and progression of diabetic retinopathy. Advanced lipoxidation end products (ALEs) have been implicated in the pathology of various diseases, including diabetes and several neurodegenerative disorders. The purpose of the present study was to investigate the possible link between the accumulation of ALEs and neuroretinal changes in diabetic retinopathy.

Methods: Retinal sections obtained from diabetic rats and age-matched controls were processed for immunohistochemistry using antibodies against several well defined ALEs. In vitro experiments were also performed using a human Muller (Moorfields/Institute of Ophthalmology-Muller 1 [ MIO-M1]) glia cell line. Western blot analysis was used to measure the accumulation of the acrolein-derived ALE adduct N epsilon-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) in Muller cells preincubated with FDP-lysine-modified human serum albumin (FDP-lysine-HSA). Responses of Muller cells to FDP-lysine accumulation were investigated by analyzing changes in the protein expression of heme oxygenase-1 (HO-1), glial fibrillary acidic protein (GFAP), and the inwardly rectifying potassium channel Kir4.1. In addition, mRNA expression levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha) were determined by reverse transcriptase PCR (RT-PCR). Apoptotic cell death was evaluated by fluorescence-activated cell sorting (FACS) analysis after staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide.

Results: No significant differences in the levels of malondialdehyde-, 4-hydroxy-2-nonenal-, and 4-hydroxyhexenal-derived ALEs were evident between control and diabetic retinas after 4 months of diabetes. By contrast, FDP-lysine immunoreactivity was markedly increased in the Muller glia of diabetic rats. Time-course studies revealed that FDP-lysine initially accumulated within Muller glial end feet after only a few months of diabetes and thereafter spread distally throughout their inner radial processes. Exposure of human Muller glia to FDP-lysine-HSA led to a concentration-dependent accumulation of FDP-lysine-modified proteins across a broad molecular mass range. FDP-lysine accumulation was associated with the induction of HO-1, no change in GFAP, a decrease in protein levels of the potassium channel subunit Kir4.1, and upregulation of transcripts for VEGF, IL-6, and TNF-alpha. Incubation of Muller glia with FDP-lysine-HSA also caused apoptosis at high concentrations.

Conclusions: Collectively, these data strongly suggest that FDP-lysine accumulation could be a major factor contributing to the Muller glial abnormalities occurring in the early stages of diabetic retinopathy.