Lymphocyte subsets in conjunctival mucosa-associated-lymphoid-tissue after exposure to retinal-S-antigen

Purpose: To evaluate the immune cell subsets in conjunctival mucosa-associated-lymphoid-tissue (C-MALT) following challenge with antigen. Methods: Ten adult female Lewis rats were studied. Five rats received one drop (5 µL) of retinal S-antigen (500 µg/mL in phosphate buffered saline, PBS) instilled into the lower fornix twice daily for 10 consecutive days. Five rats received PBS only and served as controls for the experiment. Two days after the last instillation the animals were sacrificed and the orbital contents prepared for immunohistological staining. A panel of monoclonal antibodies was used: CD5, CD4, CD8, CD25, and CD45RA. The number of positive cells were counted in sections of epibulbar, forniceal, and tarsal conjunctiva. Results: There was a significant increase in the number of CD8 T lymphocytes in the conjunctiva of animals receiving retinal S-antigen when compared to control animals. Conclusion: Conjunctival instillation of retinal S-antigen causes an immune response in the C-MALT with a significant increase in the CD8 T lymphocyte subset in this tissue. This response may be involved in the induction of tolerance to the encountered antigen.

Association of retinal arteriolar dilatation with lower verbal memory::The Edinburgh Type 2 Diabetes Study

Abstract


AIMS/HYPOTHESIS:

Retinal vascular calibre changes may reflect early subclinical microvascular disease in diabetes. Because of the considerable homology between retinal and cerebral microcirculation, we examined whether retinal vascular calibre, as a proxy of cerebral microvascular disease, was associated with cognitive function in older people with type 2 diabetes.

METHODS:

A cross-sectional analysis of 954 people aged 60-75 years with type 2 diabetes from the population-based Edinburgh Type 2 Diabetes Study was performed. Participants underwent standard seven-field binocular digital retinal photography and a battery of seven cognitive function tests. The Mill Hill Vocabulary Scale was used to estimate pre-morbid cognitive ability. Retinal vascular calibre was measured from an image field with the optic disc in the centre using a validated computer-based program.

RESULTS:

After age and sex adjustment, larger retinal arteriolar and venular calibres were significantly associated with lower scores for the Wechsler Logical Memory test, with standardised regression coefficients -0.119 and -0.084, respectively (p?<?0.01), but not with other cognitive tests. There was a significant interaction between sex and retinal vascular calibre for logical memory. In male participants, the association of increased retinal arteriolar calibre with logical memory persisted (p?<?0.05) when further adjusted for vocabulary, venular calibre, depression, cardiovascular risk factors and macrovascular disease. In female participants, this association was weaker and not significant.

CONCLUSIONS/INTERPRETATION:

Retinal arteriolar dilatation was associated with poorer memory, independent of estimated prior cognitive ability in older men with type 2 diabetes. The sex interaction with stronger findings in men requires confirmation. Nevertheless, these data suggest that impaired cerebral arteriolar autoregulation in smooth muscle cells, leading to arteriolar dilatation, may be a possible pathogenic mechanism in verbal declarative memory decrements in people with diabetes.

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.

Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?

Aims/hypothesis: Blood–retina barrier leakage in diabetes results in extravasation of plasma lipoproteins. Intra-retinal modified LDLs have been implicated in diabetic retinopathy (DR), but their effects on retinal pigment epithelial (RPE) cells and the added effects of extravasated modified HDLs are unknown.

Methods: In human retinas from individuals with and without diabetes and DR, immunohistochemistry was used to detect ApoB, ApoA1 and endoplasmic reticulum (ER) stress markers. In cell culture, human RPE cells were treated with native LDL (N-LDL) or heavily-oxidised glycated LDL (HOG-LDL) with or without pretreatment with native HDL (N-HDL) or heavilyoxidised glycated HDL (HOG-HDL). Cell viability, oxidative stress, ER stress, apoptosis and autophagy were assessed by Cell Counting Kit-8 assay, dichlorofluorescein assay, western blotting, immunofluorescence and TUNEL assay. In separate
experiments, RPE cells were treated with lipid oxidation products, 7-ketocholesterol (7-KC, 5–40 µmol/l) or 4-hydroxynonenal (4-HNE, 5–80 µmol/l), with or without pretreatment with N-HDL or HOG-HDL.

Results: ApoB, ApoA1 staining and RPE ER stress were increased in the presence of DR. HOG-LDL but not N-LDL significantly decreased RPE cell viability and increased reactive oxygen species generation, ER stress, apoptosis and autophagy. Similarly, 4-HNE and 7-KC decreased viability and induced ER stress. Pretreatment with N-HDL mitigated these effects, whereas HOG-HDL was less effective by most, but not all, measures.

Conclusions/interpretation: In DR, extravascular modified LDL may promote RPE injury through oxidative stress, ER stress, autophagy and apoptosis. N-HDL has protective effects, but HOG-HDL is less effective. Extravasation and modification of HDL may modulate the injurious effects of extravasated modified LDL on the retinal pigment epithelium.

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.

Oxidative and endoplasmic reticulum stresses mediate apoptosis induced by modified LDL in human retinal Müller cells

We previously showed that extravasated, modified LDL is implicated in pericyte loss in diabetic retinopathy (DR). Here, we investigate whether modified LDL induces apoptosis in retinal Müller glial cells.

Tyrosine nitration of prostacyclin synthase is associated with enhanced retinal cell apoptosis in diabetes

The risk of diabetic retinopathy is associated with the presence of both oxidative stress and toxic eicosanoids. Whether oxidative stress actually causes diabetic retinopathy via the generation of toxic eicosanoids, however, remains unknown. The aim of the present study was to determine whether tyrosine nitration of prostacyclin synthase (PGIS) contributes to retinal cell death in vitro and in vivo. Exposure of human retinal pericytes to heavily oxidized and glycated LDL (HOG-LDL), but not native forms of LDL (N-LDL), for 24 hours significantly increased pericyte apoptosis, accompanied by increased tyrosine nitration of PGIS and decreased PGIS activity. Inhibition of the thromboxane receptor or cyclooxygenase-2 dramatically attenuated HOG-LDL-induced apoptosis without restoring PGIS activity. Administration of superoxide dismutase (to scavenge superoxide anions) or L-N(G)-nitroarginine methyl ester (L-NAME, a nonselective nitric oxide synthase inhibitor) restored PGIS activity and attenuated pericyte apoptosis. In Akita mouse retinas, diabetes increased intraretinal levels of oxidized LDL and glycated LDL, induced PGIS nitration, enhanced apoptotic cell death, and impaired blood-retinal barrier function. Chronic administration of tempol, a superoxide scavenger, reduced intraretinal oxidized LDL and glycated LDL levels, PGIS nitration, and retina cell apoptosis, thereby preserving the integrity of blood-retinal barriers. In conclusion, oxidized LDL-mediated PGIS nitration and associated thromboxane receptor stimulation might be important in the initiation and progression of diabetic retinopathy.

Apoptosis induction by oxidized glycated LDL in human retinal capillary pericytes is independent of activation of MAPK signaling pathways

Pericyte loss is a cardinal feature of early diabetic retinopathy. We previously reported that highly oxidized-glycated low density lipoprotein (HOG-LDL) induces pericyte apoptosis in vitro. In this study, we investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathways in HOG-LDL-induced apoptosis in human pericytes.

Pigment epithelium-derived factor mitigates inflammation and oxidative stress in retinal pericytes exposed to oxidized low-density lipoprotein

Oxidized and/or glycated low-density lipoprotein (LDL) may mediate capillary injury in diabetic retinopathy. The mechanisms may involve pro-inflammatory and pro-oxidant effects on retinal capillary pericytes. In this study, these effects, and the protective effects of pigment epithelium-derived factor (PEDF), were defined in a primary human pericyte model. Human retinal pericytes were exposed to 100 microg/ml native LDL (N-LDL) or heavily oxidized glycated LDL (HOG-LDL) with or without PEDF at 10-160 nM for 24 h. To assess pro-inflammatory effects, monocyte chemoattractant protein-1 (MCP-1) secretion was measured by ELISA, and nuclear factor-kappaB (NF-kappaB) activation was detected by immunocytochemistry. Oxidative stress was determined by measuring intracellular reactive oxygen species (ROS), peroxynitrite (ONOO(-)) formation, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) production. The results showed that MCP-1 was significantly increased by HOG-LDL, and the effect was attenuated by PEDF in a dose-dependent manner. PEDF also attenuated the HOG-LDL-induced NF-kappaB activation, suggesting that the inhibitory effect of PEDF on MCP-1 was at least partially through the blockade of NF-kappaB activation. Further studies demonstrated that HOG-LDL, but not N-LDL, significantly increased ONOO(-) formation, NO production, and iNOS expression. These changes were also alleviated by PEDF. Moreover, PEDF significantly ameliorated HOG-LDL-induced ROS generation through up-regulation of superoxide dismutase 1 expression. Taken together, these results demonstrate pro-inflammatory and pro-oxidant effects of HOG-LDL on retinal pericytes, which were effectively ameliorated by PEDF. Suppressing MCP-1 production and thus inhibiting macrophage recruitment may represent a new mechanism for the salutary effect of PEDF in diabetic retinopathy and warrants more studies in future.

Intraretinal Leakage and Oxidation of LDL in Diabetic Retinopathy

Purpose: The pathogenesis of diabetic retinopathy (DR) is not fully understood. Clinical studies suggest that dyslipidemia is associated with the initiation and progression of DR. However, no direct evidence supports this theory.

Methods: Immunostaining of apolipoprotein B100 (ApoB100, a marker of low-density lipoprotein [LDL]), macrophages, and oxidized LDL was performed in retinal sections from four different groups of subjects: nondiabetic, type 2 diabetic without clinical retinopathy, diabetic with moderate nonproliferative diabetic retinopathy (NPDR), and diabetic with proliferative diabetic retinopathy (PDR). Apoptosis was characterized using the TUNEL assay. In addition, in cell culture studies using in vitro-modi?ed LDL, the induction of apoptosis by heavily oxidized-glycated LDL (HOG-LDL) in human retinal capillary
pericytes (HRCPs) was assessed.

Results: Intraretinal immuno?uorescence of ApoB100 increased with the severity of DR. Macrophages were prominent only in sections from diabetic patients with PDR. Merged images revealed that ApoB100 partially colocalized with macrophages. Intraretinal oxidized LDL was absent in nondiabetic subjects but present in all three diabetic groups, increasing with the severity of DR. TUNEL-positive cells were present in retinas from diabetic subjects but absent in those from nondiabetic subjects. In cell culture, HOG-LDL induced the activation of caspase, mitochondrial dysfunction, and apoptosis in
HRCPs.

Conclusions: These ?ndings suggest a potentially important role for extravasated, modi?ed LDL in promoting DR by promoting apoptotic pericyte loss.

Oxidised, glycated LDL selectively influences tissue inhibitor of metalloproteinase-3 gene expression and protein production in human retinal capillary pericytes

Aims/hypothesis: Matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitor of metalloproteinases (TIMPs), regulate important biological processes including the homeostasis of the extracellular matrix, proteolysis of cell surface proteins, proteinase zymogen activation, angiogenesis and inflammation. Studies have shown that their balance is altered in retinal microvascular tissues in diabetes. Since LDLs modified by oxidation/glycation are implicated in the pathogenesis of diabetic vascular complications, we examined the effects of modified LDL on the gene expression and protein production of MMPs and TIMPs in retinal pericytes. Methods: Quiescent human retinal pericytes were exposed to native LDL (N-LDL), glycated LDL (G-LDL) and heavily oxidised and glycated LDL (HOG-LDL) for 24 h. We studied the expression of the genes encoding MMPs and TIMPs mRNAs by analysis of microarray data and quantitative PCR, and protein levels by immunoblotting and ELISA. Results: Microarray analysis showed that MMP1, MMP2, MMP11, MMP14 and MMP25 and TIMP1, TIMP2, TIMP3 and TIMP4 were expressed in pericytes. Of these, only TIMP3 mRNA showed altered regulation, being expressed at significantly lower levels in response to HOG- vs N-LDL. Quantitative PCR and immunoblotting of cell/matrix proteins confirmed the reduction in TIMP3 mRNA and protein in response to HOG-LDL. In contrast to cellular TIMP3 protein, analysis of secreted TIMP1, TIMP2, MMP1 and collagenase activity indicated no changes in their production in response to modified LDL. Combined treatment with N- and HOG-LDL restored TIMP3 mRNA expression to a level comparable with that after N-LDL alone. Conclusions/interpretation: Among the genes encoding for MMPs and TIMPs expressed in retinal pericytes, TIMP3 is uniquely regulated by HOG-LDL. Reduced TIMP3 expression might contribute to microvascular abnormalities in diabetic retinopathy. © 2007 Springer-Verlag.

Effects of oxidized and glycated LDL on gene expression in human retinal capillary pericytes

Modified (oxidized and/or glycated) low-density lipoproteins (LDLs) have been implicated in retinal pericyte loss, one of the major pathologic features of early-stage diabetic retinopathy. To delineate underlying molecular mechanisms, the present study was designed to explore the global effects of modified LDL on pericyte gene expression.

Effects of modified low-density lipoproteins on human retinal pericyte survival

According to a current paradigm cardiovascular diseases can be initiated by exposure of vascular cells to qualitatively modified low-density lipoproteins (LDL). Capillary leakage, an early feature of diabetic retinopathy, results in the exposure of retinal pericytes to modified LDL, including glycated (G-LDL) and heavily oxidized glycated LDL (HOG-LDL). We demonstrate here that modified LDL inhibits the proliferation and survival of cultured human retinal pericytes. Modified LDL also induced DNA fragmentation in bovine retinal pericytes. Overall, HOG-LDL produced a significantly higher extent of cytotoxicity and apoptosis in retinal pericytes. These results indicate that exposure of pericytes to HOG-LDL could be implicated in the development of diabetic retinopathy.

Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells

Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL.

Pre-enrichment of modified low density lipoproteins with alpha-tocopherol mitigates adverse effects on cultured retinal capillary cells

We determined whether pre-enrichment of low density lipoproteins (LDL) with alpha-tocopherol mitigates their adverse effects, following in vitro glycation, oxidation or glycoxidation, towards cultured bovine retinal capillary endothelial cells (RCEC) and pericytes.