Retinal Endothelium Research Articles

Pigment epithelium-derived factor (PEDF) prevents diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, suggesting that PEDF may protect against proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. Leukocyte adhesion to retinal capillary endothelium (leukostasis) is a critical event in early diabetic retinopathy, whose process is mainly mediated by intercellular adhesion molecule-1 (ICAM-1). We investigated here whether PEDF could prevent diabetes- or advanced glycation end products (AGE)-elicited retinal leukostasis by suppressing ICAM-1 expression. Immunohistochemistry of 8-hydroxydeoxyguanosine (8-OHdG), an oxidative stress marker, showed intense staining in the nuclei of cells in the inner and outer plexiform layers of streptozotocin-induced diabetic rat retinas. Administration of PEDF or pyridoxal phosphate, an AGE inhibitor, decreased retinal levels of 8-OHdG and subsequently suppressed ICAM-1 gene expression and retinal leukostasis in diabetic rats. Further, intravenous administration of AGE to normal rats increased ICAM-1 gene expression and retinal leukostasis, which were blocked by PEDF. PEDF also inhibited the AGE-induced T cell adhesion to microvascular endothelial cells by suppressing ICAM-1 expression. These results demonstrated that PEDF inhibited diabetes- or AGE-elicited retinal leukostasis by suppressing ICAM-1. Our present study suggests that PEDF may play a protective role against early diabetic retinopathy by attenuating the deleterious effect of AGE.

Choroidal neovascularization in transgenic mice expressing prokineticin 1: an animal model for age-related macular degeneration

The prognosis of choroidal neovascularization (CNV) in age-related macular degeneration (AMD) is poor and existing treatments are limited in retarding the progression of disease. The development of an animal model for AMD will be beneficial for finding potential treatments, including gene therapy. Recently prokineticin 1 (hPK1) was identified as a mitogen of fenestrated endothelium. We hypothesized that hPK1 could induce CNV, a hallmark of the exudative or wet form of AMD, since the endothelium of the choriocapillaris, but not retinal endothelium, has fenestration. We generated transgenic mice expressing hPK1 in the retina using the rhodopsin promoter. In these transgenic mice, an enlarged vascular bed of choroid resembling CNV was observed without any morphological changes in the retinal vasculature. In addition, the major fluorophore of lipofuscin, N-retinylidene-N-retinylethanolamine, which has several potential cytotoxic effects on the RPE, was accumulated approximately twice as much in the transgenic mouse eyes compared to controls. hPK1 could be one of the causative factors of AMD and the transgenic mouse exhibiting CNV may be useful to establish treatments for the wet form of AMD.

Thrombospondin‐1, PECAM‐1, and Regulation of Angiogenesis

Negative regulators of angiogenesis play a central role in keeping angiogenesis in check. Expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis, is down-regulated during the progression of many tumors and diabetes favoring a proangiogenic state. We have shown TSP1 expression in the endothelial cells (EC) promotes a quiescent, differentiated phenotype through regulation of a number of genes with important roles in angiogenesis. One such gene is PECAM-1, a proangiogenic molecule whose expression is down-regulated upon TSP1 expression in EC. We also showed down-regulation of PECAM-1 expression in EC results in increased expression of TSP1. Therefore, TSP1 and PECAM-1 are constituents of a regulatory switch whose reciprocal regulation, perhaps through the MAPK/ERKs pathway, promotes or inhibits angiogenesis. Our recent in vivo studies indicate TSP1 expression is essential for normal development of retinal vasculature and regression of embryonic and newly formed vessels during oxygen-induced ischemic retinopathy. Most recently, we have shown retinal endothelium continues to express proangiogenic isoforms of PECAM-1 at higher frequencies in the absence of TSP1. We have also found PECAM-1 is essential for normal retinal vascularization and neovascularization during oxygen-induced ischemic retinopathy. These studies demonstrate the important physiological roles of TSP1 and PECAM-1 in vascular development and neovascularization. Furthermore, TSP1 or PECAM-1 deficient EC maintain many of their respective in vivo characteristics in culture. Utilization of these EC and study of their adhesive and migratory properties upon restoration of TSP1 or a specific PECAM-1 isoform will provide additional insight into the cellular mechanisms utilized by these molecules to regulate EC phenotype.

Involvement of CCR5 in the passage of Th1-type cells across the blood-retina barrier in experimental autoimmune uveitis

Although the recruitment of T helper cell type 1 (Th1)/Th2 cells into peripheral tissues is essential for inflammation and the host response to infection, the traffic signals that enable the distinct positioning of Th1/Th2 cells are unclear. We have determined the role of CC chemokine receptor 5 (CCR5) in this using experimental autoimmune uveitis (EAU) as a model system. In EAU, Th1-like cells are preferentially recruited into the retina across the blood-retina barrier, partly as a result of expression of the adhesion molecules P-selectin glycoprotein ligand 1 and lymphocyte function-associated antigen-1 on these cells. CD3+ T cells, infiltrating the retina, also expressed the chemokine receptor CCR5, and CCR5 ligands, macrophage-inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated on activation, normal T expressed and secreted (RANTES), were strongly expressed in the retina at peak EAU. Th1-like cells, polarized in vitro, expressed high levels of CCR5. The trafficking of these CCR5+ cells was examined by tracking them after adoptive transfer in real time in vivo at an early disease stage using scanning laser ophthalmoscopy. Treatment of the cells with antibody against CCR5 prior to transfer resulted in a reduction in their infiltration into the retina. However, rolling velocity, rolling efficiency, and adherence of the cells to retinal endothelium were not reduced. CCR5 is clearly important for Th1 cell recruitment, and this study demonstrates for the first time in vivo that CCR5 may act at the level of transendothelial migration rather than at the earlier stage of rolling on the endothelium.

Cadherin-10 is a novel blood–brain barrier adhesion molecule in human and mouse

Maintenance of the specialised environment of the central nervous system requires barriers provided by the endothelium of brain microvessels (the blood–brain barrier (BBB)) or the epithelium lining the ventricles (CSF–brain barrier) or the choroid plexus (blood–CSF barrier). Inter-endothelial junctions are more extensive in the BBB than in other tissues, with elaborate tight junctions. However, few differences in the molecular composition of these junctions have been described. Here, we show, in both human and mouse brain, that the type II classical cadherin, cadherin-10, is expressed in BBB and retinal endothelia, but not in the leaky microvessels of brain circumventricular organs (CVO), or in those of non-CNS tissues. This expression pattern is distinct from, and reciprocal to, VE-cadherin, which is reduced or absent in tight cortical microvessels, but present in leaky CVO vessels. In CVO, the barrier function is switched from the microvasculature to the adjacent ventricular epithelium, which we also find to express cadherin-10. In the vessels of gliobastoma multiforme tumours, where BBB is lost, cadherin-10 is not detected. This demonstration of a distinctive expression pattern of cadherin-10 suggests that it has a pivotal role in the development and maintenance of brain barriers.

Conditional knockdown of tubedown-1 in endothelial cells leads to neovascular retinopathy.

Identification of novel proteins involved in retinal neovascularization may facilitate new and more effective molecular-based treatments for proliferative retinopathy. Tubedown-1 (Tbdn-1) is a novel protein that shows homology to the yeast acetyltransferase subunit NAT1 and copurifies with an acetyltransferase activity. Tbdn-1 is expressed in normal retinal endothelium but is specifically suppressed in retinal endothelial cells from patients with proliferative diabetic retinopathy. The purpose of this study was to investigate the importance of Tbdn-1 expression in retinal blood vessels in vivo. A bitransgenic mouse model that enables conditional knockdown of Tbdn-1 specifically in endothelial cells was produced and studied using molecular, histologic, and immunohistochemical techniques and morphometric analysis. Tbdn-1-suppressed mice exhibited retinal and choroidal neovascularization with intra- and preretinal fibrovascular lesions similar to human proliferative retinopathies. Retinal lesions observed in Tbdn-1-suppressed mice increased in severity with prolonged suppression of Tbdn-1. In comparison to normal retina, the retinal lesions displayed alterations in the basement membrane of blood vessels and in the distribution of glial and myofibroblastic cells. Moreover, the pathologic consequences of Tbdn-1 knockdown in endothelium were restricted to the retina and the choroid. These results indicate that the maintenance of Tbdn-1 expression is important for retinal blood vessel homeostasis and for controlling retinal neovascularization in adults. Restoration of Tbdn-1 protein expression and/or activity may provide a novel approach for treating proliferative retinopathies.

Simvastatin Inhibits Leukocyte Accumulation and Vascular Permeability in the Retinas of Rats with Streptozotocin-Induced Diabetes

Leukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common beta-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy.

The role of advanced glycation end products in retinal microvascular leukostasis.

A critical event in the pathogenesis of diabetic retinopathy is the inappropriate adherence of leukocytes to the retinal capillaries. Advanced glycation end-products (AGEs) are known to play a role in chronic inflammatory processes, and the authors postulated that these adducts may play a role in promoting pathogenic increases in proinflammatory pathways within the retinal microvasculature. Retinal microvascular endothelial cells (RMECs) were treated with glycoaldehyde-modified albumin (AGE-Alb) or unmodified albumin (Alb). NFkappaB DNA binding was measured by electromobility shift assay (EMSA) and quantified with an ELISA: In addition, the effect of AGEs on leukocyte adhesion to endothelial cell monolayers was investigated. Further studies were performed in an attempt to confirm that this was AGE-induced adhesion by co-incubation of AGE-treated cells with soluble receptor for AGE (sRAGE). Parallel in vivo studies of nondiabetic mice assessed the effect of intraperitoneal delivery of AGE-Alb on ICAM-1 mRNA expression, NFkappaB DNA-binding activity, leukostasis, and blood-retinal barrier breakdown. Treatment with AGE-Alb significantly enhanced the DNA-binding activity of NFkappaB (P = 0.0045) in retinal endothelial cells (RMECs) and increased the adhesion of leukocytes to RMEC monolayers (P = 0.04). The latter was significantly reduced by co-incubation with sRAGE (P < 0.01). Mice infused with AGE-Alb demonstrated a 1.8-fold increase in ICAM-1 mRNA when compared with control animals (P < 0.001, n = 20) as early as 48 hours, and this response remained for 7 days of treatment. Quantification of retinal NFkappaB demonstrated a threefold increase with AGE-Alb infusion in comparison to control levels (AGE Alb versus Alb, 0.23 vs. 0.076, P < 0.001, n = 10 mice). AGE-Alb treatment of mice also caused a significant increase in leukostasis in the retina (AGE-Alb versus Alb, 6.89 vs. 2.53, n = 12, P < 0.05) and a statistically significant increase in breakdown of the blood-retinal barrier (AGE Alb versus Alb, 8.2 vs. 1.6 n = 10, P < 0.001). AGEs caused upregulation of NFkappaB in the retinal microvascular endothelium and an AGE-specific increase in leukocyte adhesion in vitro was also observed. In addition, increased leukocyte adherence in vivo was demonstrated that was accompanied by blood-retinal barrier dysfunction. These findings add further evidence to the thinking that AGEs may play an important role in the pathogenesis of diabetic retinopathy.

Functional role of Egr-1 mediating VEGF-induced tissue factor expression in the retinal capillary endothelium.

To investigate the causal relationship between VEGF and tissue factor (TF) expression, and its intracellular signaling in the retinal capillary endothelium both in vitro and in vivo. TF mRNA and protein expression in cultured bovine retinal capillary endothelial cells (BRECs) were detected by RT-PCR and western blotting. The expression and subcellular localization of Egr-1 were analyzed by immunocytochemistry and western blotting. Involvement of p44/p42 MAPK pathway in this signaling was assessed using PD98059. Electrophoretic mobility shift assay (EMSA) was performed using human TF Egr-1/Sp-1 overlapping promoter region (-85 to -70). Decoy oligonucleotide was transfected into BRECs to clarify the critical transcription factor mediating VEGF-induced TF gene expression. To evaluate the importance of GC rich region in VEGF-induced TF protein expression in rat retinas, Mithramycin was intraperitoneally administered. VEGF stimulated TF mRNA and protein expression in cultured BRECs, reaching maximal effect after 4 h and 10 h, respectively. VEGF activated transcription factor Egr-1 within 60 min. Inactivation of Egr-1 by PD98059 resulted in the prohibition of VEGF-induced TF gene expression. EMSA revealed the increment of Egr-1 binding with TF promoter region by displacing Sp1 after treatment with VEGF. Transfection of the Egr-1/Sp-1 overlapping decoy into BRECs inhibited VEGF-dependent TF gene expression. Mithramycin almost completely suppressed VEGF-induced TF protein expression in retinal capillary system in vivo (80%, p<0.01). Transcription factor Egr-1, which lies downstream of p44/p42 MAPK, critically mediates VEGF-dependent TF expression in the retinal capillary endothelium.

Ischemia-Reperfusion Injury of Retinal Endothelium by Cyclooxygenase- and Xanthine Oxidase-Derived Superoxide

The formation of reactive oxygen species (ROS) may be important in the pathogenesis of microvascular dysfunction and injury in ischemic retinopathies. The authors hypothesized that retinal endothelial cells can generate injurious levels of superoxide radical in response to ischemia/reperfusion, that endothelial xanthine oxidase and cyclooxygenase are important enzymatic sources of superoxide radical under these conditions, and that superoxide scavengers and inhibitors of these enzymes can protect endothelium from ischemic injury. The authors used confluent cultures of mouse retinal endothelial cells (MREC) subjected to exogenously generated superoxide or simulated ischemia-reperfusion to test these hypotheses. Cell injury was assessed biochemically by lactate dehydrogenase release into the culture medium. MREC were injured in a duration-dependent fashion by exposure to the superoxide-generating mix of hypoxanthine and xanthine oxidase. Increasing periods of oxygen and glucose deprivation (OGD) for 5–9hr followed by replenishment of substrates for 2hr led to progressive increases in endothelial cell injury; a significant proportion of the injury occurred during the period of substrate replenishment. Significant MREC protection was achieved by the superoxide scavengers SOD (1000Uml−1) and a carboxylic acid derivative of carboxyfullerene (10μM), the xanthine oxidase inhibitors oxypurinol (100μM) and diphenyleneiodonium (DPI) (100n M), and the cyclooxygenase inhibitors indomethacin (300μM) and ibuprofen (300μM). It is concluded that MREC are vulnerable to auto-oxidative injury by superoxide radical generated following a period of OGD. Both xanthine oxidase- and cyclooxygenase-dependent pathways are important enzymatic sources of superoxide formation in this setting. These enzymes and the ROS produced from their activity may be viable therapeutic targets to reduce microvascular dysfunction and injury in ischemic retinopathies.

Increased numbers of caveolae in retinal endothelium and pericytes in hypertensive diabetic rats

Long-term clinical studies have now shown that tight control of blood pressure in type 2 diabetes reduces the risk of diabetes-related death and common diabetic complications, including diabetic retinopathy. However, the mechanisms by which hypertension enhances diabetic microvascular disease, especially diabetic retinopathy, are poorly understood. We developed an experimental model of hypertension in diabetic rats and studied the early ultrastructural changes in retinal capillaries under these conditions. Hypertension was induced in diabetic BioBreeding (BB) rats by unilateral nephrectomy, weekly subcutaneous mineralocorticoid and 0.9% oral saline. Serial blood pressures and ultrastructural features of retinal capillaries were recorded in four groups: normotensive Wistar rats, normotensive diabetic rats, hypertensive Wistar rats and hypertensive diabetic rats. A significant and sustained increase in systolic blood pressure occurred in both groups of nephrectomised rats. There was a significant increase in the number of caveolae (i) in both pericytes and endothelial cells in animals with hypertension and diabetes together compared with all other groups and (ii) in pericytes in animals with diabetes alone. The number of direct contacts between pericytes and endothelial cells was reduced in diabetic and hypertensive diabetic animals. Hypertension and diabetes had an interactive effect in producing retinal capillary basement membrane thickening. In the BB rat hypertension and diabetes have an interactive effect in increasing the number of caveolae in both endothelial cells and pericytes. We speculate that this may be a reflection of changes in calcium and nitric oxide metabolism in these animals.

Control of chemokine production at the blood-retina barrier.

Chemokine production at the blood-retina barrier probably plays a critical role in determining the influx of tissue-damaging cells from the circulation into the retina during inflammation. The blood-retina barrier comprises the retinal microvascular endothelium and the retinal pigment epithelium. Chemokine expression and production by human retinal microvascular endothelial cells (REC) have never been reported previously, so we examined the in vitro expression and production of monocyte chemoattractant protein-1 (MCP-1), regulated on activation of normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin (IL)-8, epithelial cell-derived neutrophil activating protein-78 (ENA-78) and growth related oncogene alpha (GROalpha) in these cells, both unstimulated and stimulated by cytokines likely to be present during the evolution of an inflammatory response. We compared this to expression and production of these chemokines in vitro in human retinal pigment epithelial cells (RPE). MCP-1 was expressed and produced constitutively by REC but all the chemokines were produced in greater amounts upon stimulation with the proinflammatory cytokines IL-1beta and tumour necrosis factor-alpha (TNF-alpha). MCP-1 and IL-8 were produced at much higher levels than the other chemokines tested. MIP-1alpha and MIP-1beta were present only at low levels, even after stimulation with IL-1beta and TNF-alpha. Cytokines with greater anti-inflammatory activity, such as IL-4, IL-10, IL-13, transforming growth factor-beta (TGF-beta) and IL-6, had little effect on chemokine production either by REC alone or after stimulation with IL-1beta and TNF-alpha. RPE, although a very different cell type, showed a similar pattern of expression and production of chemokines, indicating the site-specific nature of chemokine production. Chemokine production by REC and RPE is probably significant in selective leucocyte recruitment during the development of inflammation in the retina.

Advanced glycation end-product receptor interactions on microvascular cells occur within caveolin-rich membrane domains.

Advanced glycation end products (AGEs) have an important role in diabetic complications, with many responses mediated through AGE-receptors. The current study has investigated the binding and uptake of AGEs by retinal microvascular endothelium in an attempt to understand the nature of AGE-interaction with receptors on the cell surface. There has been special emphasis placed on the R1, R2, and R3 components of AGE-receptor complex (AGE-RC) and their localization to caveolin-rich membrane domains. Retinal microvascular endothelial cells (RMECs) were exposed to either AGE-modified BSA (AGE-BSA) or native BSA conjugated to colloidal gold (gAGE, gBSA) for various time periods, fixed, and processed for transmission electron microscopy (TEM). Localization of AGE-RC components in caveolae was investigated using confocal microscopy and ultrastructural immunogold labeling. Caveolae were extracted from RMECs using differential Triton X-100 solubility, and Western analysis was conducted to test for caveolae enrichment and the presence of AGE-RC complex components. Ligand blots determined 125I-AGE-BSA binding to caveolae-enriched extracts. Colloidal gold conjugates of AGE-BSA bound to caveolae and were internalized to be trafficked to lysosomal-like compartments. AGE-receptor complex components were significantly enriched within caveolae. The data suggest that AGEs interact with their receptors within caveolae. It is significant that the AGE-R complex localizes to these organelles, because this may have implications for AGE binding, internalization, signal transduction, and the modulation of AGE-receptor-mediated vascular cell dysfunction.

Distribution of monocarboxylate transporters MCT1 and MCT2 in rat retina

Transport of lactic acid and other monocarboxylates such as pyruvate and the ketone bodies through cellular membranes is facilitated by specific transport proteins. We used chicken polyclonal antibodies to the monocarboxylate transporters-1 and -2 to determine their cellular and subcellular distributions in rat retina, and we compared these distributions to those of the glucose transporters-1 and -3. Monocarboxylate transporter-1 was most highly expressed by the apical processes of retinal pigment epithelium that surround the outer segments of the photoreceptor cells. In contrast to glucose transporter-1, monocarboxylate transporter-1 was not detected on the basal membranes of pigment epithelium. The luminal and abluminal endothelial plasma membranes in retina also exhibited heavy labeling by antibody to monocarboxylate transporter-1. In addition, this transporter was associated with the Müller cell microvilli, the plasma membranes of the rod inner segments, and all retinal layers between the inner and external limiting membranes. Monocarboxylate transporter-2 was found to be abundantly expressed on the inner (basal) plasma membrane of Müller cells and by glial cell processes surrounding retinal microvessels. This transporter was also present in the plexiform and nuclear layers but was not detected beyond the external limiting membrane. Recent studies have shown that lactic acid transport is of particular importance at endothelial and epithelial barriers where membranes of adjoining cells are linked by tight junctions. Our results suggest that monocarboxylate transporter-1 functions to transport lactate between the retina and the blood, both at the retinal endothelium and the pigment epithelium. The location of monocarboxylate transporter-2 on glial foot processes surrounding retinal vessels suggests that this transporter is also important in blood–retinal lactate exchange. In addition, the abundance of these transporters in Müller cells and synaptic (plexiform) layers suggests that they function in lactate exchange between neurons and glia, supporting the notion that lactate plays a key role in neural metabolism.

Leukocyte trafficking in experimental autoimmune uveitis in vivo.

Leukocyte trafficking from blood into tissue is a fundamental process in immune surveillance and the immune response to stimuli. Experimental autoimmune uveitis (EAU) is an animal model for posterior uveitis and is mediated by T lymphocytes and macrophages that infiltrate the posterior segment of the eye. To analyze leukocyte migration into retinal tissue during the course of EAU, labeled cells were identified in vivo by scanning laser ophthalmoscopy and in retinal flatmounts by confocal microscopy. Adhesion of blood leukocytes to retinal endothelial cells in vivo was significantly raised 48 h before the appearance of clinical disease, and this correlated with the increased expression of CD54 on retinal vessels. Mitogen-activated spleen cells and CD4+ T cells only entered into retinal tissue in animals with clinical disease and not naive recipients. The disease status of the donor animal had no effect on leukocyte trafficking. These results, which identify leukocyte-endothelial cell interactions in vivo, suggest that the activation of the retinal endothelium is a prerequisite to leukocyte adhesion and extravasation into ocular tissue during EAU.

Polyunsaturated fatty acid metabolism in retinal and cerebral microvascular endothelial cells.

Docosahexaenoic acid (22:6n-3), an n-3 essential fatty acid derived from elongation and desaturation of linolenic acid (18:3n-3), is found in abundant proportion in the brain and the retina. It is generally assumed that the liver is the major source of 22:6n-3 for these organs, although some retinal and cerebral cells, such as retinal pigment epithelium (Wang and Anderson, 1993. Biochemistry. 32:13703-13709) and brain astrocytes (Moore et al. 1991. J. Neurochem. 56:518-524) have the ability to produce 22:6n-3. The aim of the present study was to determine whether retinal and cerebral microvascular endothelium could synthesize 22:6n-3. After incubation of both cultured bovine retinal and rat cerebral endothelial cells with [3-14C] 22:5n-3 in presence of serum, radioactivity was primarily recovered in 20:5n-3, indicating active retroconversion reactions in both tissues. However, 22:6n-3, 24:5n-3, and 24:6n-3 were also labeled. All of these metabolites were released in the medium as free fatty acids. Retinal endothelial cells preferentially released labeled 24-carbon metabolites, whereas cerebral endothelial cells released relatively more 20:5n-3 and 22:6n-3. With heat-inactivated serum or no serum, both endothelial cell preparations showed relatively higher retroconversion levels. However, in serum-deprived cells, the elongation/desaturation pattern was affected in retinal cells only, with an accumulation of 24:5n-3 relative to a decrease of 24:6n-3 and 22:6n-3. Fatty acid composition analyses revealed a decrease in long-chain polyunsaturated n-6 and n-3 fatty acids in retinal cells maintained in inactivated serum compared to normal serum, while no change was found in cerebral cells. Taken together, these results suggest that 1) the synthesis of 22:6n-3 by both retinal and cerebral endothelial cells is independent of a delta4-desaturase; 2) retinal and cerebral endothelia could be a source of 22:6n-3 for the retina and the brain, respectively; and 3) retinal endothelial delta6-desaturase, which converts 24:5n-3 to 24:6n-3, could be stimulated by serum components.

SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Lymphocyte Migration across the Anterior and Posterior Blood–Retinal Barrierin Vitro

The migration of lymphocytes through monolayers of rat retinal pigment epithelium (RPE) and retinal vascular endothelium, which form the posterior and anterior blood–retinal barrier (BRB) respectively, was investigatedin vitro.After a 4-hr assay the migration of untreated peripheral lymph node (PLN) cells through RPE monolayers was negligible (<1%) with only a small increase found after activation of the PLN cells with concanavalin A or by cross-linking CD3. Activation of the RPE with IFN-γ augmented migration with maximal PLN cell migration being achieved with a combination of CD3 cross-linking and IFN-γ activation (17% migration). The highest level of lymphocyte migration was observed with three CD4+antigen-specific T cell lines specific for purified protein derivative (PPD; 33% migration), ovalbumin (OA; 31%), and S-antigen (S-Ag; 57%). Migration of both untreated and Con A-activated PLN cells through retinal endothelial cells (EC) from PVG rats was negligible, whereas the migration of the antigen-specific T cell lines was 23, 29, and 23% for PPD, OA, and S-Ag lines, respectively. Migration of these cell lines through retinal endothelium derived from Lewis rats was significantly greater (44% for PPD, 39% for OA, and 39% for S-Ag) which corresponded with a greater expression of ICAM-1 on the EC.

Interferon-α 2b Enhances Barrier Function of Bovine Retinal Microvascular Endothelium in Vitro

Increased permeability of the vascular endothelium is a critical part of pathological processes such as inflammation and wound healing. While several cytokines have been shown to increase endothelial permeability, none has been shown to reduce it. We studied the effect of interferon-α 2b on the barrier function of bovine retinal capillary endothelial cells in vitro and found that it increased the electrical resistance of the monolayers (treated cells 211 ± 6 ohm.cm 2, untreated 109 ± 14 ohm.cm 2), decreased their permeability to inulin ( P c inulin of untreated cells 0.73 ± 0.21 × 10 -6 cm/sec, treated 0.31 ± 0.23 × 10 -6 cm/sec, P = 0.015), and enhanced their morphological differentiation. Interferon-γ had no effect. No effect of interferon-α treatment on the permeability of bovine aortic endothelial cells was found. We conclude that interferon-α 2b can enhance the barrier function of retinal microvascular endothelium in vitro. This is consistent with the hypothesis that interferon-α is an effector of a mechanism which actively promotes tissue homeostasis and suggests that it might have therapeutic potential in diseases characterized by leakage of the vascular endothelium.

Characterization of a rat retinal endothelial cell culture and the expression of P-glycoprotein in brain and retinal endothelium in vitro

Retinal vascular endothelia form one aspect of the blood-retinal barrier and, like the blood-brain barrier, control the passage of molecules and cells into the parenchyma. To facilitate comparative in vitro studies, rat retinal endothelial cells have been cultured and characterised. Using immunocytochemical techniques, retinal endothelium was positive for von Willebrand's factor, tight junction-associated polypeptide (ZO-1) and the transferrin receptor. The cells also expressed high-affinity uptake of acetylated low-density lipoprotein. Using the monoclonal antibodies JSB-1 and C219, the product of the multidrug resistancy gene, P-glycoprotein, was found to be expressed on primary cultures of both brain and retinal endothelium.