Search

Progressive retinal ganglion cell (RGC) loss underlies a number of retinal neurodegenerative disorders, which may lead to permanent vision loss. However, secreted neuroprotective factors, such as PEDF, VEGF and IL-6, which are produced by Muller cells, have been shown to promote RGC survival. Assuming that the communication of RGCs with Muller cells involves a release of glioactive substances we sought to determine whether retinal neurons are able to modulate expression levels of Muller cell-derived PEDF, VEGF and IL-6. We demonstrate elevated mRNA levels of these factors in Muller cells in co-cultures with RGCs or R28 cells when compared to homotypic Muller cell cultures. Furthermore, R28 cells were more protected from apoptosis when co-cultured with Muller cells. IL-6 and VEGF were upregulated in Muller cells under hypoxia. Both cytokines, as well as PEDF, induced an altered neuronal expression of members of the Bcl-2 family, which are central molecules in the regulation of apoptosis. These results suggest that in retinal ischemia, via own secreted mediators, RGCs can resist a potential demise by stimulating Muller cells to increase production of neuroprotective factors, which counteract RGC apoptosis.

Zusammenfassung Einleitung Die Müllerʼschen Gliazellen übernehmen in der Netzhaut vielfältige Aufgaben und gewährleisten damit deren regelrechte Funktion. Wie stark die neuroprotektive Wirkung von Müller-Zellen auf retinale Ganglienzellen (RGC) ist, soll untersucht werden. Material und Methoden RGC wurden für 24 Stunden alleine oder in Kokultur mit Müllerʼschen Gliazellen unter normoxischen (20% O2, 5% CO2) und hypoxischen (0,2% O2, 5% CO2, 94,8% N2) Bedingungen kultiviert. Die Anzahl lebender RGC und die Länge der neu ausgebildeten Neuriten dienten zur Beurteilung der Zellvitalität. Ergebnisse Unter normoxischen Kulturbedingungen war die Anzahl vitaler RGC in Kokultur (62,85 ± 2,06%) signifikant höher (p Schlussfolgerung Müllerʼsche Gliazellen unterstützen das Überleben von RGC unter normoxischen und hypoxischen Bedingungen. Ein weiterer Parameter zur Bewertung der Zellvitalität neben der Anzahl lebender RGC ist die Länge der neu ausgebildeten Neuriten.

Background/ Aims: This study was performed to reveal signaling pathways exploited by pigment epithelium-derived factor (PEDF) derived from retinal (glial) Müller cells to protect retinal ganglion cells (RGCs) from cell death. Methods: The survival of RGCs was determined in the presence of conditioned culture media (MCM) from or in co-cultures with Müller cells. The significance of PEDF-induced STAT3 activation was evaluated in viability assays and using Western blotting analyses and siRNA-transfected cells. Results: Secreted mediators of Müller cells increased survival of RGCs under normoxia or hypoxia to a similar degree as of PEDF- or IL-6-exposed cells. PEDF and MCM induced an increased STAT3 activation in RGCs and R28 cells, and neutralization of PEDF in MCM attenuated STAT3 activation. Inhibition of STAT3 reduced PEDF-promoted survival of RGCs. Similar to IL-6, PEDF induced STAT3 activation, acting in a dose-dependent manner via the PEDF receptor (PEDF-R) encoded by the PNPLA2 gene. Ablation of PEDF-R attenuated MCM-induced STAT3 activation and compromised the viability of PEDF-exposed R28 cells. Conclusions: Müller cells are an important source of PEDF, which promotes RGC survival through STAT3 activation and, at least in part, via PEDF-R. Enhancing the secretory function of Müller cells may be useful to promote RGC survival in retinal neurodegenerative diseases.

Age-related macular degeneration (AMD) is a leading cause of legal blindness in people over 50 years of age in many developed countries. Drusen are yellowish extracellular deposits beneath retinal pigment epithelium (RPE) found in aging eyes and considered as a biomarker of AMD. However, the biogenesis of drusen has not been elucidated. We reported previously that multicellular spheroids comprised of human RPE cells constructed a well-differentiated monolayer of RPE with Bruch's membrane. We determined that RPE spheroids exhibited drusen formation between RPE and Bruch's membrane with expression of many drusen-associated proteins such as amyloid ß and complement components, the expression of which was altered by a challenge with oxidative stress. Artificial lipofuscin-loaded RPE spheroids yielded drusen more frequently. In the current study, we showed that drusen originates from the RPE. This culture system is an attractive tool for use as an in vitro drusen model, which might help elucidate the biogenesis of drusen and the pathogenesis of related diseases such as AMD.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Müller glial cells carry out different tasks to warrant normal retinal functions. The aim of this study was to investigate if Müller cells also support retinal ganglion cells (RGC).RGC were cultured for 24 hours in the presence or absence of Müller glial cells under normoxic (20% OUnder normoxic conditions, RGC vitality was significantly higher (p 0.01) when cultured with Müller cells (62.85 ± 2.06%) than without (47.29 ± 2.83%). Under hypoxia, RGC vitality was significantly higher (p 0.01) in co-cultures (41.07 ± 2.28%) than in homotypic RGC cultures (28.49 ± 2.16%). The maximum length of the newly developed neurites was found in the normoxic co-culture (90.7 ± 7.4 µm), but showed only a minor difference (p = 0.04) when compared to the normoxic homotypic RGC culture.Müller glial cells support RGC under normoxic and hypoxic culture conditions. Length of newly developed neurites and number of surviving RGC are both parameters to evaluate cell vitality.Die Müllerʼschen Gliazellen übernehmen in der Netzhaut vielfältige Aufgaben und gewährleisten damit deren regelrechte Funktion. Wie stark die neuroprotektive Wirkung von Müller-Zellen auf retinale Ganglienzellen (RGC) ist, soll untersucht werden.RGC wurden für 24 Stunden alleine oder in Kokultur mit Müllerʼschen Gliazellen unter normoxischen (20% OUnter normoxischen Kulturbedingungen war die Anzahl vitaler RGC in Kokultur (62,85 ± 2,06%) signifikant höher (p 0,01) als in der homotypischen RGC-Kultur (47,29 ± 2,83%). Unter Hypoxie war die Anzahl vitaler RGC in der Kokultur (41,07 ± 2,28%) ebenfalls signifikant höher (p 0,01) als in der homotypischen RGC-Kultur (28,49 ± 2,16%). Die Länge der neu gebildeten Neuriten war in der normoxischen Kokultur am größten (90,7 ± 7,4 µm), zeigte aber nur im Vergleich zwischen normoxischer Kokultur und homotypischer RGC-Kultur einen signifikanten Unterschied (p = 0,04).Müllerʼsche Gliazellen unterstützen das Überleben von RGC unter normoxischen und hypoxischen Bedingungen. Ein weiterer Parameter zur Bewertung der Zellvitalität neben der Anzahl lebender RGC ist die Länge der neu ausgebildeten Neuriten.

Neovascularization is a sight-threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)-β, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Muller) cells produce TGF-β2 under physiological and post-ischemic conditions. To characterize glial cell-derived mediators of angiogenesis regulation in glial-endothelial interactions in the retina, we co-cultured primary Muller cells and bovine microvascular retinal endothelial cells (BRECs). Muller cell-derived TGF-β2 was bound by the BRECs, which were found to express serine/threonine kinase TGF-β receptors, and stimulated TGF-β-dependent anti-proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF-β2 as well as by the presence of Muller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti-angiogenic action of Muller cell-derived TGF-β2: (i) binding of TGF-β2 to BRECs is mediated by the type-II TGF-β receptor, leading to (ii) activation and phosphorylation of receptor-activated Smads; (iii) Muller cell-derived TGF-β2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal-regulated kinase (ERK)-1/-2. Neutralizing TGF-β or TGF-β type-II receptor or blocking the activation of Smads partially abrogated the effect of Muller cell-conditioned media on BRECs. Together, our data suggest that Muller cells release TGF-β2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context-dependent action of TGF-β2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti-angiogenic role of TGF-β therein.

Background/Aims: By the release of antiangiogenic factors, Müller glial cells provide an angiostatic environment in the normal and ischemic retina. We determined whether Müller cells produce thrombospondin-1 (TSP-1), a known inhibitor of angiogenesis. Methods: Secretion of TSP-1 by cultured Müller cells was determined with ELISA. Slices of rat retinas and surgically excised retinal membranes of human subjects were immunostained against TSP-1 and the glial marker vimentin. The effects of TSP-1 on the growth of bovine retinal endothelial cells (BRECs) and activation of ERK1/2 were determined with DNA synthesis and migration assays, and Western blotting, respectively. Results: Cultured Müller cells secrete TSP-1 under normoxic and hypoxic (0.2% O2) conditions. Secretion of TSP-1 was increased in hypoxia compared to normoxia. In rat retinal slices, glial, retinal ganglion, and possibly horizontal cells were stained for TSP-1. Retinal glial cells in preretinal membranes from human subjects with nonhypoxic epiretinal gliosis (macular pucker) and proliferative diabetic retinopathy, respectively, were immunopositive for TSP-1. Exogenous TSP-1 reduced the VEGF-induced proliferation and migration of BRECs and decreased the phosphorylation level of ERK1/2 in BRECs. Conclusion: The data suggest that Müller cells are one major source of TSP-1 in the normal and ischemic retina. Glia-derived TSP1 may inhibit angiogenic responses in the ischemic retina.

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Muller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Muller cell-RGC-co-cultures we observed that activity of Muller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Muller cells towards RGC.

Abnormal fundus autofluorescence (FAF) is associated with the incidence or progression of dry and wet age-related macular degeneration (AMD). We previously developed a rabbit AMD model with drusen and type-1 choroidal neovascularization (CNV) that mimics the accumulation of lipofuscin using artificial glycoxidized particles. The objective of the current study was to investigate in vitro effects of glycoxidized particles on retinal pigment epithelial (RPE) cells, and the FAF and fate of injected particles in this model.Glycoxidized particles were prepared by a 4-day incubation of water-in-oil emulsions of serum albumin and glycolaldehyde to allow glycoxidation and consequent cross-linking. After particles were added in the culture medium of confluent human RPE cells, cell viability, adhesion activity, and proliferation activity were assessed by cell counting. In anesthetized rabbits, 250 microg of glycoxidized particles were injected into the subretinal space to induce experimental AMD. FAF measurement and angiography with sodium fluorescein and indocyanine green were performed periodically using the Heidelberg Retina Angiograph 2 (HRA2). The eyes enucleated, and the lung and the spleen, excised at week 4 or 12, were histologically evaluated by light and fluorescence microscopy.Glycoxidized particles phagocytosed did not impair the cell viability, adhesion, and proliferation of RPE cells, as compared with RPE cells in controls. HRA2 showed different patterns of abnormal FAF in the area with the implanted glycoxidized particles, similar to pathological FAF patterns in aging human eyes with or without AMD. Histologic examination showed accumulated glycoxidized particles and large lipofuscin granules with green autofluorescence in and under the RPE and at the margins of or beneath drusen, possibly associated with abnormal FAF. In addition, some particles were detected in the lung and the spleen.Glycoxidized particles phagocytosed might stay in RPE cells without any acute biological reaction. Our rabbit model of AMD simulated abnormal FAF patterns observed in aging human eyes with or without AMD. Glycoxidized particles phagocytosed by RPE cells could be deposited on Bruch's membrane in rabbits, possibly excreted in part into choroidal circulation. This model may be useful for understanding various patterns of abnormal FAF histologically, and for elucidating the pathogenesis of AMD.

Mounting evidence suggests that oxidative stress caused by reactive oxygen intermediates is a significant mechanism in the pathogenesis of age-related macular degeneration (AMD). Although vascular endothelial growth factor (VEGF) and other cytokines are involved in choroidal neovascularization (CNV) it is largely unknown whether oxidative stress may predispose the eye to increased levels of proangiogenic factors. In an in vitro study we have determined viability and proliferation of both human retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (CECs) and assessed the release of basic fibroblast growth factor (bFGF) and VEGF from RPE cells after exposing them to oxidative stress. Permanent presence of tert -butyl-hydroperoxide ( t BH), a pro-oxidative stressor, in the cell cultures resulted in decreasing viability and proliferation of RPE cells and CECs. Loss of RPE cell viability was associated with activation of apoptosis by t BH in a dose-dependent manner. The antioxidant, N -acetyl- l -cysteine (NAC), and secreted soluble mediators of RPE cells were appropriate to attenuate the effects of t BH-mediated oxidative stress. RPE cells exposed to t BH were found to release increasing amounts of bFGF but not VEGF after 24 h of culture, thereby supporting proliferation of CECs. These findings suggest that oxidative stress compromises the viability of RPE cells and CECs. However, increased bFGF levels concomitantly released from RPE cells may attenuate the CEC-directed effect, protect CECs from oxidative insults, and are likely to promote CNV.

Pigment epithelium-derived factor (PEDF), a glycoprotein with pleiotropic functions, is naturally occuring in the eye and considered as crucial to prevent pathological angiogenesis. Since retinal glial (Müller) cells produce PEDF, the authors have studied its impact on glial-endothelial cellular interactions. Bovine retinal endothelial cells were cultured in the presence of culture media originating from primary Müller cells, and endothelial proliferation as well as phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinases (ERK)-1/-2 were investigated. The concerted activity of Müller-cell derived soluble mediators attenuated endothelial proliferation and ERK-1/-2 activation, regardless of whether the Müller cells were preincubated under normoxia or hypoxia, and even though the endothelial cells were stimulated by vascular endothelial growth factor-A (VEGF). This inhibitory activity was no longer demonstrable if high levels of basic fibroblast growth factor or VEGF were supplied, suggesting that in cases of pathological neovascularization, overproduction of proangiogenic mediators overrides the "antiangiogenic background" provided by Müller cells. However, neutralizing the activity of PEDF partially restored endothelial cell proliferation and resulted in increased ERK-1/-2 activation, which is in concordance with findings demonstrating that exogenously applied PEDF is able to suppress VEGF-induced ERK-1/-2 phosphorylation. PEDF production by Müller cells is not only regulated by retinal oxygen but also by the activity of soluble factors released from retinal endothelial cells. For instance, PEDF levels were significantly elevated in glial (Müller)-endothelial cell cocultures as compared with bovine retinal endothelial cell-free Müller cell cultures. These results have implications for the pathogenesis of retinal neovascularization since the Müller cell may be regarded as a central control element which modulates retinal PEDF levels and, thus, is of critical importance for adjusting the balance between proangiogenic and antiangiogenic mediators.

Neovascularization is a common and potentially visually threatening complication of eye diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). An antiangiogenic therapy is aimed at inhibiting the growth of new blood vessels and should prevent onset or progression of neovascularization. Accumulated evidence indicates that growth factors, endothelial cell surface receptors, and extracellular matrix (ECM) proteins are major mediators of neovascularization and appealing targets for pharmacotherapeutical intervention. Vascular endothelial growth factor (VEGF) plays a critical role in the pathogenesis of retinal neovascularization (in linking tissue ischemia to angiogenesis), and is likely to contribute also significantly to choroidal neovascularization (CNV). Several antineovascular agents antagonize the function of VEGF, by blocking its proangiogenic activity. Indeed, VEGF targeting or disruption of VEGF signalling is the most effective strategy known so far in the pharmacological treatment of ocular neovascularization. Other compounds such as pigment epithelium-derived factor (PEDF) either aim at balancing the levels of pro-angiogenic and angiostatic molecules, target inflammation (cyclooxygenase inhibitors, steroids) or comprise modifiers of the ECM such as inhibitors of matrix metalloproteinases (MMPs) and agents that block the action of integrins. Vascular targeting agents (combretastatin) promote removal of newly formed vessels. This review provides an update on recent investigations directed at the pharmacotherapeutical management of ocular neovascular diseases, placing special emphasis on the underlying target molecules and relevant intracellular signalling pathways.

To investigate the potential role of endostatin, an endogenous angiogenesis inhibitor, in the prevention of choroidal angiogenesis-related disorders.Bovine choroidal endothelial cells (CEC) were cultured and treated with basic fibroblast growth factor (bFGF) alone or combined with endostatin at concentrations ranging from 0.1 to 10 microg/ml. The proliferation and migration of CECs were evaluated by using 3, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and modified Boyden chamber assay, respectively. For evaluating expression and secretion of matrix metalloproteinase-2 (MMP-2), CEC-conditioned media were subjected to zymography and/or Western blot analysis, and the cells were used for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis.Endostatin did not inhibit bFGF-induced or nonstimulated CEC proliferation (p0.05). The bFGF-induced migration was significantly inhibited by endostatin at concentrations of 1 and 10 microg/ml (p0.05). The bFGF-upregulated expression of mRNA in CECs and the secretion of MMP-2 protein of CECs were both suppressed by endostatin.Inhibitory effect of endostatin on expression and secretion of MMP-2 and cell migration, but not on proliferation of CECs, could respond to its therapeutic action for choroidal neovascularization-dependent disorders.

CD97, a cell surface molecule on immune cells with potential adhesive function, is a member of the epidermal growth factor seven-span transmembrane (EGF-TM7) family. We have cloned and characterized bovine CD97 and determined its expression in various cells and tissues. Based on sequence alignment with human genomic DNA, as well as human cDNA sequences encoding various CD97 isoforms, we predict that bovine CD97 mRNA occurs in four splice variants. The encoded CD97 isoforms (800, 751, 756, and 707 amino acids in length) contain different numbers of EGF-like modules, a stalk region, and a TM7 domain with a cytoplasmic tail. RT-PCR demonstrated expression of CD97 mRNA in leukocytes and several other tissues. In each cell type, CD97 mRNA encoding the shortest isoform, CD97 (EGF 1,2,5), was detected at the highest level, which was consistent with the cell surface expression of a 90-kDa polypeptide precipitated from lysates of peripheral blood mononuclear cells.

Background. Advanced glycation end products (AGEs) are considered to be important modulators of angiogenesis and accumulate in choroidal neovascularization (CNV). Their effects regarding cells involved in proliferation of CNV [retinal pigment epithelial (RPE) cells, Muller cells and choroidal endothelial cells (CECs)] were investigated. Furthermore, the effects of AGEs on expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) by CECs were explored. Methods. RPE cells, CECs and Muller cells were exposed to AGEs (10 µg/ml, 50 µg/ml and 100 µg/ml) for a time course of three days in their desired medium and proliferation was estimated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MMP-2 expression of AGE-stimulated CECs was determined by zymography and reverse-transcription polymerase chain reaction (RT-PCR) after 36 h of exposure. Furthermore, VEGF expression of AGE-stimulated CECs (50 µg/ml and 100 µg/ml) was determined by RT-PCR after an exposure time of 36 h. Results. AGEs in a concentration of 50 µg/ml and 100 µg/ml increased the proliferation of CECs (41% vs 46.1%; P

International audience; The death of retinal ganglion cells (RGC) leads to visual impairment and blindness in ocular neurodegenerative diseases, primarily in glaucoma and diabetic retinopathy; hence, mechanisms that contribute to protecting RGC from ischemia/hypoxia are of great interest. We here address the role of retinal glial (Muller) cells and of pigment-epithelium-derived factor (PEDF), one of the main neuroprotectants released from the glial cells. We show that the hypoxia-induced loss in the viability of cultured purified RGC is due to apoptosis, but that the number of viable RGC increases when co-cultured with Muller glial cells suggesting that glial soluble mediators attenuate the death of RGC. When PEDF was ablated from Muller cells a significantly lower number of RGC survived in RGC-Muller cell co-cultures indicating that PEDF is a major survival factor allowing RGC to escape cell death. We further found that RGC express a PEDF receptor known as patatin-like phospholipase domain-containing protein 2 (PNPLA2) and that PEDF exposure, as well as the presence of Muller cells, leads to an activation of nuclear factor (NF)-kappa B in RGC. Furthermore, adding an NF-kappa B inhibitor (SN50) to PEDF-treated RGC cultures reduced the survival of RGC. These findings strongly suggest that NF-kappa B activation in RGC is critically involved in the pro-survival action of Muller-cell derived PEDF and plays an important role in maintaining neuronal survival.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Neovascularization is a sight-threatening complication of ischemic proliferative retinopathies. Transforming growth factor (TGF)-β, a cytokine with multiple functions in the retina, participates in the control of pathological angiogenesis and neovascularization. Retinal glial (Müller) cells produce TGF-β2 under physiological and post-ischemic conditions. To characterize glial cell-derived mediators of angiogenesis regulation in glial-endothelial interactions in the retina, we co-cultured primary Müller cells and bovine microvascular retinal endothelial cells (BRECs). Müller cell-derived TGF-β2 was bound by the BRECs, which were found to express serine/threonine kinase TGF-β receptors, and stimulated TGF-β-dependent anti-proliferative signaling pathways. The proliferation of BRECs was attenuated by exogenous TGF-β2 as well as by the presence of Müller cell culture media. The following intracellular signaling mechanisms were found to be involved in the anti-angiogenic action of Müller cell-derived TGF-β2: (i) binding of TGF-β2 to BRECs is mediated by the type-II TGF-β receptor, leading to (ii) activation and phosphorylation of receptor-activated Smads; (iii) Müller cell-derived TGF-β2 activates Smad2 and Smad3 to (iv) attenuate the phosphorylation state of the MAP kinases, extracellular signal-regulated kinase (ERK)-1/-2. Neutralizing TGF-β or TGF-β type-II receptor or blocking the activation of Smads partially abrogated the effect of Müller cell-conditioned media on BRECs. Together, our data suggest that Müller cells release TGF-β2, inhibiting the proliferation of retinal endothelial cells via activation of Smad2/Smad3 and attenuation of ERK signaling. Given the context-dependent action of TGF-β2 on angiogenesis, our results may have implications for understanding the pathogenesis of retinal angiopathies, such as diabetic retinopathy, and the anti-angiogenic role of TGF-β therein.

The human NK gene complex encodes for the leucocyte C-type lectins, CD69, AICL (activation-induced C-type lectin), LLT1 (lectin-like transcript), CD161/NKR-P1A, CD94, and for NKG-2 molecules. These gene products have been implicated in the regulation of the function of natural killer (NK) cells and other lymphocytes. In this study the expression of C-type lectins during the early activation of PMA-stimulated peripheral blood lymphocytes was examined. To investigate the influence of de novo protein synthesis on activation-dependent expression of C-type lectins, cells were cultured in presence of cycloheximide (CHX) and mRNA levels were analyzed by semi-quantitative reverse transcription-polymerase chain reaction. Upregulated levels of CD69, AICL, and LLT1, but less pronounced changes of CD161/NKR-P1A and CD94 mRNA were found at early time points of cellular activation. CD69 was superinduced by CHX at the nuclear precursor transcript and the mRNA level suggesting that regulation of transcriptional activity and mRNA stability contribute to extent of CD69 mRNA accumulation. CHX treatment resulted also in an overexpression of AICL, LLT1, and CD161/NKR-P1A mRNAs. Conversely, CHX blocked CD94 mRNA expression in PMA-stimulated cells, demonstrating that this process is dependent on new protein synthesis. Expression kinetics in context with susceptibility to CHX indicate that the mechanisms responsible for upregulated CD69, AICL, and LLT1 expression are distinct from those which control CD161/NKR-P1A or CD94 expression. J. Cell. Biochem. Suppl. 36: 201-208, 2001.

Isolated retinae or isolated Muller cells were cultured in vitro, and vascular endothelial growth factor (VEGF) was assayed as protein (by ELISA) and as mRNA (by semi-quantitative RT- PCR). In both types of cultures, hypoxia (5% O2) resulted in an upregulated VEGF release. While the unstimulated VEGF secretion was virtually independent of glucose (0.125-25 mM), elevated glucose concentrations (10-25 mM) blocked most of the stimulatory effect of hypoxia on VEGF mRNA synthesis (determined in Muller cell cultures) as well as on VEGF release (in both retina and Muller cell cultures). It is concluded that in retinal glial (Muller) cells, being responsible for retinal VEGF synthesis (and, thus, for undesirable neovascularization), the metabolic effects of hypoxia can be compensated by a surplus of glucose. NeuroReport 11:3533-3537 & 2000 Lippincott Williams & Wilkins.

Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine with pro-angiogenic and neurotrophic effects. The angioregulatory role of this molecule may become especially significant in retinal neovascularization, which is a hallmark of a number of ischemic eye diseases. This study was undertaken to reveal expression characteristics of bFGF, produced by retinal glial (Müller) cells, and to determine conditions under which glial bFGF may stimulate the proliferation of retinal microvascular endothelial cells. Immunofluorescence labeling detected bFGF in Müller cells of the rat retina and in acutely isolated Müller cells with bFGF levels, which increased after ischemia-reperfusion in postischemic retinas. In patients with proliferative diabetic retinopathy or myopia, the immunoreactivity of bFGF co-localized to glial fibrillary acidic protein (GFAP)-positive cells in surgically excised retinal tissues. RT-PCR and ELISA analyses indicated that cultured Müller cells produce bFGF, which is elevated under hypoxia or oxidative stress, as well as under stimulation with various growth factors and cytokines, including pro-inflammatory factors. When retinal endothelial cells were cultured in the presence of media from hypoxia (0.2%)-conditioned Müller cells, a distinct picture of endothelial cell proliferation emerged. Media from 24-h cultured Müller cells inhibited proliferation, whereas 72-h conditioned media elicited a stimulatory effect. BFGF-neutralizing antibodies suppressed the enhanced endothelial cell proliferation to a similar extent as anti-VEGF antibodies. Furthermore, phosphorylation of extracellular signal-regulated kinases (ERK-1/-2) in retinal endothelial cells was increased when the cells were cultured in 72-h conditioned media, while neutralizing bFGF attenuated the activation of this signaling pathway. These data provide evidence that retinal (glial) Müller cells are major sources of bFGF in the ischemic retina. Müller cells under physiological conditions or transient hypoxia seem to provide an anti-angiogenic environment, but long-lasting hypoxia causes the release of bFGF, which might significantly co-stimulate neovascularization in the retina.

Purpose Aging changes in the RPE involve lipid accumulation and membranous basal deposits onto the underlying Bruch's membrane, which may be related to AMD. Conventional in vitro cell culture is limited in its ability to observe the epithelial functions on the basal side. The purpose of this study was to develop a three-dimensional culture system to observe basolateral functions of the RPE. Methods Isolated human RPE cells were cultured in a viscous medium on a rounded-bottom culture dish, resulting in spheroid formation. The appearance and size of the spheroids were assessed by light microscopy. Spheroids were fixed in 4% paraformaldehyde for immunohistochemistry or sampled for Western blotting. For transmission electron microscopy (TEM) and scanning electron microscopy (SEM), spheroids were postfixed in 1% osmium tetroxide. Results The spheroids had a differentiated RPE monolayer with a thin elastic layer, a main layer of Bruch's membrane, on their surface and showed outward deposition of lipoproteins with apoB-100. TEM revealed widely spaced collagen, which was identified as condensation of collagen fibrils by SEM. SEM showed deposition of membranous debris and lipid particles, which have been observed in human Bruch's membrane. Western blotting showed expression of RPE differentiation markers and components of Bruch's membrane and RPE lipoproteins. Conclusions This model provides direct views of epithelialization processes involving elastogenesis and functions at the basolateral side such as lipoprotein deposition and may elucidate not only unknown epithelial behaviors but also the pathogenesis of RPE-related diseases.

Retinal glial (Muller) cells are involved in a wide range of developmental mechanisms, including axon guidance and angiogenesis. This study was undertaken to explore whether Netrin-4, an axonal guidance molecule, is expressed by Muller cells and promotes angiogenesis-related activities. Netrin-4 was found through all retinal layers, and its expression was demonstrated in Muller cells, retinal pigment epithelium cells and bovine retinal endothelial cells (BRECs). Co-localization of Netrin-4 with Muller cell-specific molecules [cellular retinaldehyde-binding protein (cRALBP), vimentin] was observed in the ganglion cell layer, nerve fiber layer, and at the outer limiting membrane. Under hypoxic conditions, the release of Netrin-4 from Muller cells was increased, with mRNA levels upregulated in a hypoxia-inducible factor-1-dependent manner and dependent on the concomitantly induced release of vascular endothelial growth factor. These findings were consistent with an intensified immunofluorescence of Netrin-4 labeling in the postischemic retinas after ischemia-reperfusion. Netrin-4 stimulated BRECs to increase phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK)-1/-2, and p38, in a dose-dependent manner. Synthetic inhibitors of the MAP kinases were able to suppress Netrin-4-induced migration and proliferation of BRECs suggesting that both MAP kinases are differentially involved in Netrin-4-induced angiogenesis. Two receptors for Netrins, i.e., deleted in colorectal cancer (DCC) and uncoordinated-5-homolog 1 (Unc5H1), were detected in BRECs. DCC is at least partially required for Netrin-4-induced activation of ERK-1/-2. These data suggest that Muller glial cells contribute to, and may modulate, retinal Netrin-4 levels. This may be a novel pathway of Muller cell-mediated control of retinal angiogenesis, particularly under hypoxic/ischemic conditions when the cells upregulate Netrin-4 expression.

Neovascularization in the eye is a major cause of irreversible vision loss. The present study was undertaken to determine mechanisms through which pazopanib, a drug that targets multiple receptor tyrosine kinases such as VEGF receptors, inhibits angiogenesis and experimental choroidal neovascularization (CNV). Pazopanib inhibited VEGF expression by retinal pigment epithelium (RPE) cells and choroidal endothelial cells (CEC), decreased VEGF-induced cellular migration in a dose-dependent manner and suppressed extracellular signal-regulated kinase (ERK)-1/-2 phosphorylation. To assess the impact of pazopanib in vivo, CNV was induced in rats by rupturing the Bruch's membrane by laser coagulation. These experiments demonstrated that twice-daily topical eye drop treatment significantly (P

Neuronal degeneration and aberrant neovascularization are common problems of ischemic retinopathies. Pigment epithelium-derived factor (PEDF), a neuroprotective protein and an inhibitor of angiogenesis, is produced by retinal glial (Muller) cells and can counterbalance elevated levels of vascular endothelial growth factor (VEGF), the expression of which is regulated primarily by hypoxia-inducible factor (HIF)-1. In an approach to mimic transient ischemia in vitro, primary Muller cells were cultured under transient and strong hypoxia (0.2% O(2) ), followed by reoxygenation at 2.5% O(2) , and molecular mechanisms that might contribute to changes in the intraretinal PEDF level were determined. Hypoxic conditions caused an increasing expression of HIF-1α and led to upregulation of both PEDF and VEGF. Treatment of the cells with synthetic HIF-1α blockers or neutralization of VEGF binding to VEGF receptors (VEGFR-1 and-2) suppressed hypoxia-induced PEDF upregulation. Furthermore, the presence of CoCl(2) (a hypoxia mimetic) induced an accumulation of elevated HIF-1α protein in the nucleus and an upregulation of PEDF expression in Muller cells. Increasing PEDF expression was attenuated when HIF-1α levels were suppressed using HIF-1α small interfering RNA (siRNA). On the other hand, siRNA-mediated depletion of PEDF facilitated HIF-1α upregulation caused by CoCl(2) and resulted in increasing VEGF mRNA and protein levels. These results demonstrate that VEGF and PEDF may be unidirectionally regulated in hypoxia through HIF-1α activation, with upregulation of PEDF, which may occur in a VEGF-dependent manner. However, endogenously produced PEDF seems to be an inherent control element of HIF-1α expression in Muller cells, indicating an important feedback mechanism for limiting upregulation of VEGF.

PURPOSE. To assess the regulation of pigment epithelium- derived factor (PEDF) production by retinal Miiller glial cells, especially under ischemic or hypoxic conditions. METHODS. PEDF was determined in surgically excised retinal tissue originating from patients with ischemic diabetic retinopathy and in primary guinea pig Miiller cell cultures exposed to the protein synthesis inhibitor cycloheximide (CHX) and to hypoxia. PEDF production and secretion were studied by immunohistochemistry, quantitative RT-PCR, ELISA, fluorescence-activated cell sorter analysis, and Western blotting. RESULTS. Gliotic Miiller cells displayed decreased PEDF immunoreactivity in fibrovascular tissue from patients with diabetes compared with tissue from subjects with pathologic myopia. In Muller cell cultures, CHX treatment resulted in an increase, whereas mild hypoxia (2.5%-10% O 2 ) induced a decrease, of PEDF mRNA and protein levels. However, strong hypoxia (0.2% O 2 ) induced an upregulation of PEDF mRNA expression and resulted in only slightly reduced PEDF levels after 24 hours, detected as either a released, soluble, or cell surface-linked protein. CONCLUSIONS. These results suggest that under certain conditions including mild hypoxia, Miiller cells synthesize a protein factor that downregulates PEDF expression or its turnover. Generally, the cells appear to generate a biphasic response to hypoxia. In moderate hypoxia, PEDF is downregulated such that the VEGF-to-PEDF ratio increases (and angiogenesis is facilitated). During severe (or chronic) oxygen deficiency, however, the PEDF decline is arrested or even reversed; thus, the neurotrophic effects of PEDF remain available.

Purpose: Alkylphosphocholines (APCs) are investigated for their effect on Muller glial cell proliferation and F-actin stress fiber distribution in vitro. Materials and Methods: Muller cells were incubated with APCs (C18:1-PC and C22:1-PC) ± fetal calf serum. Proliferation was assessed by BrdU labeling and with the tetrazolium dye-reduction assay. Toxicity was measured using the trypan blue exclusion assay. The distribution of F-actin stress fibers was determined using FITC-phalloidin staining. Results: APCs are effective inhibitors of human and rat Muller glial cell proliferation and hypoxia-induced up-regulation of F-actin stress fibers in vitro in non-toxic concentrations. Conclusions: APCs might prevent intraretinal changes as a result of serum stimulation and hypoxia following retinal detachment.

The biogenesis of drusen, a hallmark of age-related macular degeneration (AMD), is still unclear. Lipofuscin, which extensively accumulates with age in RPE cells, is hardly soluble, derived in part from oxidation byproducts of the photoreceptor outer segments. The purpose of the current study is to develop a new AMD model in rabbits using glycoxidized particles as imitation lipofuscin, and determine whether accumulation of lipofuscin as insoluble material may play a role in drusen biogenesis and other pathogenesis of AMD.To mimic the accumulation of insoluble lipofuscin, glycoxidized microspheres (glycox-MS) were made through a glycoxidation process with albumin and glycolaldehyde, alpha-hydroxy aldehyde. As a control, microspheres made with glutaraldehyde (cMS) and soluble glycoxidized (glycox-) albumin were prepared. Each material was implanted into the subretinal space in rabbits. The implanted area was assessed by funduscopy, fluorescein angiography, histology, and transmission electron microscopy (TEM).Compared with control microspheres, glycox-MS stagnated for a prolonged period in the cytoplasm of RPE cells. Eyes implanted with glycox-MS produced drusen-like deposits at a significantly higher frequency, when compared with the controls. Glycox-MS were observed at the margin of or beneath the drusen-like deposits in all cases. In some eyes with glycox-MS, late-onset sub-RPE choroidal neovascularization was observed, while control groups did not have these findings.These results suggest that the accumulation of indigestible granules such as lipofuscin in RPE or subsequent depositions toward Bruch's membrane may play a role in drusen biogenesis as a trigger of inflammation or via other mechanisms. This model of AMD may be useful to elucidate drusen biogenesis and pathogenesis of AMD.

The structure of cell surface carbohydrates expressed on human leukocytes is dependent on the cell's developmental stage, differentiation, and activation. Although modification of oligosaccharide side chains by sialylation is quite common, antigenic determinants on lymphocytes associated with the presence of sialoglycans are still incompletely defined. In the study presented here, monoclonal antibodies (mAbs) were used to characterize two novel but related cell surface carbohydrate antigens. One antigen, denominated as B8, is largely masked by sialyl residues on most lymphocytes, while it is detectable on the majority of B cells. Treatment with sialidase resulted in the exposure of B8 on the surface of blood cells including lymphocytes. Although the second carbohydrate antigen, C1, was sialidase-sensitive, its molecular properties and cellular distribution place it in close vicinity to B8. B8(+) as well as C1(+) lymphocytes were found predominantly in the mantle zone of secondary follicles of tonsillar tissue. These findings raised the possibility that B8 and C1 are closely related to a category of carbohydrate antigens previously classified as CDw76 (recently assigned to CD75s). MAbs directed against B8 or C1 precipitated 34, 37, 43, and 200kDa glycoproteins from tonsillar lymphocytes, indicating that identical cell surface proteins are associated with both antigens. In contrast to B8, however, the expression of C1 was increased on lymphocytes upon activation. Together the results suggest that CD75-related epitopes are distinct molecular entities which may be exposed on glycoproteins and are differently expressed on lymphocytes.

Purpose: Transplantation of pigment epithelial cells is a promising treatment modality to repair retinal damage in age-related macular degeneration. For this purpose, it is necessary to establish cell culture techniques that allow acquisition of proper functional and morphological characteristics by the cells to be transplanted. Methods: Primary retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells grown to confluence on collagen membranes were examined for morphology, adhesion, proliferation, apoptosis, as well as viability after ex vivo transplantation. Results: Pigment epithelial cells adhere, proliferate, form monolayers, acquire differentiated properties, and remain viable during transplantation to the subretinal space. Conclusions: Pigment epithelial cells cultured on collagen membranes acquire differentiated characteristics and are amenable to be transplanted as cell monolayers.

CD97 is a member of a new subgroup of seven-span transmembrane (7-TM) molecules which belong to the secretin receptor superfamily. Different from other members of the secretin receptor family, these recently characterized molecules have extended extracellular regions comprising several EGF domains near the NH2 terminus. We recently demonstrated that the extracellular part of CD97 is involved in intercellular adhesion since it specifically binds to CD55 (decay accelerating factor), a regulatory protein of the complement cascade. To our knowledge this is the first demonstration of a cellular ligand for a 7-TM molecule.

To explore the impact of retinal glial (Müller) cells on survival and neovascularization-related activities of cultured retinal endothelial cells under normoxic and hypoxic conditions.Bovine retinal endothelial cells (BRECs) were cultured under normoxia or hypoxia (0.5% O2) either alone, together with the human Müller cell line MIO-M1, or in normoxia- or hypoxia-conditioned media of MIO-M1 cells. Cell number, proliferation, apoptotic cell death, and migration of BRECs were determined.Exposure of BRECs to hypoxia for 24 h decreased the number of adherent cells and the proliferation rate, but increased apoptosis and cell migration. Increased apoptosis and decreased proliferation of the BRECs occurred also in the presence of conditioned media of MIO-M1 cells. Under normoxic conditions, co-culture with MIO-M1 cells resulted in increased proliferation, but decreased apoptosis and migration rates of BRECs. Under hypoxic conditions, the Müller cells released elevated amounts of VEGF but their presence decreased proliferation, apoptosis and the migration rates of BRECs.Hypoxia inhibits the proliferation of retinal endothelial cells. Müller cells release soluble mediators that enhance this hypoxia-mediated effect but, under certain conditions (i.e., in co-culture), may protect retinal endothelial cells from apoptosis, thus supporting their survival. Altogether the findings indicate that the key signal necessary to trigger retinal endothelial proliferation under hypoxia remains to be determined.

Integrin alpha(v)beta3 is predominantly expressed on endothelial cells in choroidal neovascularization (CNV). We evaluated the efficacy of cyclic RGD (Arg-Gly-Asp) peptide, an alpha(v)-integrin antagonist, in a rat model of laser-induced CNV METHODS: We evaluated the effect of cyclic RGD on the adhesion and cell viability of bovine choroidal endothelial cells (BCECs) by cell counting and the trypan blue dye exclusion test. CNV was induced by laser photocoagulation in female Long Evans rats (day 0), followed by intravitreal administration of one dose of cyclic RGD of 200 (n = 9), 100 (n = 10), 50 (n = 4), or 0 microg (n = 9) on days 9 and 11. We assessed the area of CNV by fluorescein angiography and the thickness microscopically on histologic sections. Neovascular vessels were detected by an antibody against factor VIII.Cyclic RGD (0.02 to 200 microg/ml) inhibited adhesion of BCECs in a dose-dependent manner without changing the cell viability (p0.01). In eyes treated with two injections of 200 or 100 microg of cyclic RGD peptide, the development of CNV was significantly (p0.01) inhibited in the area of leakage on fluorescein angiography. Histologically, the CNV membrane was observed beneath the retina and the factor VIII-positive cells and red blood cells were involved. The thickness of the lesions was significantly (p0.01) reduced in eyes that received 200 or 100 microg of RGD.Cyclic RGD effectively inhibited CNV progression in a rat model of laser-induced CNV, suggesting that this alpha(v)-integrin antagonist may be beneficial in the treatment of CNV.

Retinal glial (Muller) cells may play a major role in vascular eye diseases as they secrete vascular endothelial growth factor (VEGF), a hypoxia-induced angiogenic cytokine. They also release significant amounts of the anti-angiogenic factors, transforming growth factor (TGF)-beta2, pigment epithelium derived factor (PEDF), and thrombospondin-1 (TSP-1). Exposure of human (MIO-M1) and guinea-pig Muller cells to hypoxia resulted in a decreased release of TGF-beta2 and PEDF but in an elevated secretion of TSP-1. When retinal endothelial cells were exposed to VEGF/anti-angiogenic factor ratios mimicking those found in culture media of Muller cells under normoxia or hypoxia, their proliferation was significantly inhibited by TGF-beta2, PEDF or TSP-1. Thus Muller cells may provide a permanent anti-proliferative condition for retinal endothelial cells.

A precise balance between stimulators and inhibitors of angiogenesis, such as vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), respectively, is essential for angiogenic homeostasis in ocular tissues. Retinal hypoxia is accompanied by some pathological conditions that may promote intraocular neovascularization. Here we demonstrate that retinal glial (Muller) cells express and release pigment epithelium-derived factor (PEDF). Decreasing oxygen concentrations cause strong attenuation of PEDF release resulting in enhanced VEGF/PEDF ratios. Exposure of Muller cells to VEGF suppressed PEDF release in a dose-dependent manner. This may represent a novel mechanism of ocular angiogenic homeostasis sufficient in the control of PEDF levels during normoxia or mild hypoxia but supplemented by other (hitherto unknown) mechanisms in cases of strong hypoxia. In spite of the enhanced VEGF/PEDF ratios resulting from hypoxia, conditioned media of Muller cells failed to stimulate additional proliferation of retinal endothelial cells. These findings suggest that in the ischemic retina, Muller cells generate a permissive condition for angiogenesis by secreting more VEGF and less PEDF, but the onset of retinal endothelial cell proliferation requires another triggering signal that remains to be identified.

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated.Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers.MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor.Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.

Background: Angiostatic drugs might provide desirable modulation of choroidal angiogenesis-related diseases, including histoplasmosis and the exudative form of age-related macular degeneration. However, the precise effects of this class of compounds in the choroidal neovascularization are still unclear. In the present study, we investigated the effects of triamcinolone acetonide (TA), an angiostatic steroid, on choroidal angiogenesis in vitro. Methods: Bovine choroidal endothelial cells (CEC), which are the critical cellular component of choroidal angiogenesis in vivo, were isolated with Lycopersicon esculentum agglutinin-coated Dynabeads and cultured in EGM medium. CEC were treated with basic fibroblast growth factor (bFGF) and TA at various concentrations ranging from 50 to 300 mg/l. The capacities for CEC migration and tube formation were evaluated with the modified Boyden chamber and the Vitrogen collagen assay, respectively. The activities of matrix metalloproteinases (MMP)-2 and -9 were examined using gelatin zymography. Results: The stimulation of CEC with 50 ng/ml bFGF resulted in an increase of about 100% in migration activity (P

Vascular endothelial growth factor (VEGF) and nitric oxide (NO) seem to be involved in the process of angiogenesis, but their interactions are not clearly understood. The aim of this study was to investigate the influence of VEGF on NO production of choroidal endothelial cells (CEC) and its importance in angiogenesis. Experiments were performed using cultured bovine CEC. Basal NO release of unstimulated CEC was measured and compared to NO release of VEGF-stimulated CEC (1, 10, and 100 ng/ml). Further, cells were pretreated with Nω-nitro- l -arginine methyl ester ( l -NAME, 1 and 2 mM) and incubated with and without VEGF (10 ng/ml) to investigate the effect of blocking NO synthase. NO release into the medium was assessed by an amperometric NO sensor. To show the importance of NO in angiogenesis, proliferation and migration of CEC were measured after VEGF stimulation and in the presence or absence of l -NAME (1 and 2 mM). Unstimulated CEC continuously produced low levels of NO. Stimulation of the cells with VEGF resulted in a dose-dependent increase in NO release. The time course after stimulation with 10 ng/ml VEGF was characterized by a prompt initial rise up to 140% of unstimulated levels and a subsequent sustained increase over 120 min. Pretreatment with l -NAME attenuated the VEGF-induced response. l -NAME incubation alone led to a reduction in basal NO release. l -NAME also significantly diminished the VEGF-enhanced CEC proliferation and migration.The results demonstrate that VEGF enhances the formation of NO in cultured CEC. The blockade of NO production reduces CEC proliferation and migration, an effect which may be important for controlling angiogenesis, especially in reducing neovascularization in age-related macular degeneration in the eye.

Different adhesive capacity in interactions with CD55 has been ascribed to the isoforms of the leukocyte CD97 antigen, CD97 (EGF 1,2,5), CD97 (EGF 1,2,3,5), and CD97 (EGF 1,2,3,4,5). In the study, coexpression of the three CD97 isoforms and predominance of CD97 (EGF 1,2,5) transcripts in leukocytes are demonstrated. The contribution of CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) to total CD97 levels varied among most cell types only slightly, although relatively higher mRNA levels of both isoforms were detected in U 937 cells and monocytes. In peripheral blood lymphocytes, CD97 isoforms did not show clear variation after PMA stimulation and were down-regulated equally after CD97 cross-linking. Moreover, the CD97 isoform pattern was not altered in monocytes after interferon-γ stimulation and in synovial T cells from patients with rheumatoid arthritis. CD97 mRNA levels did not necessarily correspond to CD97 surface density. The findings suggest that adhesive activity of CD97 toward CD55 is unlikely to be regulated by differential CD97 isoform expression.

CD97 is an activation-induced antigen on leukocytes which belongs to a new group of seven-span transmembrane (7-TM) molecules, designated EGF-TM7 family. Family members, including EMR1 and F4/80, are characterized by an extended extracellular region with several N-terminal epidermal growth factor-like (EGF) domains. Alternative splicing of CD97 results in isoforms possessing either three (EGF1, 2, 5), four (EGF1, 2, 3, 5) or five EGF domains (EGF1, 2, 3, 4, 5). We recently identified decay accelerating factor (DAF, CD55), a regulatory protein of the complement cascade, as a cellular ligand of the smallest isoform. Employing mutants of CD97(EGF1, 2, 5) in which the EGF domains have been systematically deleted, we here demonstrate the necessity of at least three tandemly linked EGF domains for the interaction with CD55. Consistent with the involvement of different EGF domains, monoclonal antibodies directed against the first EGF domain as well as the removal of Ca2+, for which binding sites exist in the second and fifth EGF domain, blocked binding to CD55. Compared to CD97(EGF1, 2 ,5) the larger isoforms CD97(EGF1, 2, 3, 5) and CD97(EGF1, 2, 3, 4, 5) have a significantly lower affinity for CD55. Thus, alternative splicing may regulate the ligand specificity of CD97 and probably other members of the EGF-TM7 family.

The CD45 protein tyrosine phosphatase is expressed in different isoforms that result from alternative splicing of three exons (A, B, and C) encoding regions near the N-terminus of the extracellular part of the molecule. We describe here a novel epitope on the N-terminal end of CD45 that is recognized by the MAb BL-TSub/2. Crossblocking studies showed that BL-TSub/2 and UCHL1 (CD45RO) binding sites are partially overlapping. However, in marked contrast to the CD45RO epitope, protease treatment of cells strongly diminished BL-TSub/2 binding. Similar to the UCHL1 epitope, the BL-TSub/2 binding site involves carbohydrate moieties, since neuraminidase treatment abrogated the reactivity of the MAb. Markedly, preincubation of cells with both CD45 common and CD45RA MAb induced a pronounced increase of BL-TSub/2 binding. This latter finding suggests that crosslinking of the CD45 molecule leads to conformational changes that could influence association of the molecule with putative ligands.

To improve our knowledge on the pathophysiology of rheumatoid arthritis (RA), we investigated gene expression patterns in synovial tissue from RA and osteoarthritis (OA) patients. DNA oligonucleotide microarray analysis was employed to identify differentially expressed genes in synovial tissue from pathologically classified tissue samples from RA (n = 20) and OA patients (n = 10). From 7131 gene sets displayed on the microarray chip, 101 genes were found to be upregulated and 300 genes to be downregulated in RA as compared with OA. Semiquantitative reverse-transcription polymerase chain reaction, Western blotting and immunohistochemistry were used to validate microarray expression levels. These experiments revealed that Cys–X–Cys receptor (CXCR)1, CXCR2 and CXCR3 mRNAs, as well as Cys–X–Cys ligand (CXCL)9 (monokine induced by IFN-γ) and CXCL10 (IFN-γ inducible protein 10) mRNAs, were significantly upregulated in RA as compared with OA disease. Elevated protein levels in RA synovial tissue were detected for CXCR1 and CXCR3 by Western blotting. Using immunohistochemistry, CXCR3 protein was found to be preferentially expressed on mast cells within synovial tissue from RA patients. These findings suggest that substantial expression of CXCR3 protein on mast cells within synovial tissue from RA patients plays a significant role in the pathophysiology of RA, accompanied by elevated levels of the chemokines CXCL9 and CXCL10. Mature mast cells are likely to contribute to and sustain the inflamed state in arthritic lesions (e.g. by production of inflammatory mediators such as histamine, proteinases, arachidonic acid metabolites and cytokines). Thus, the mast cell could become a potential target in therapeutic intervention.