Immunohistochemical and Immunocytochemical Analyses in Patients with Vitreoretinal Lymphoma.

Purpose: The aim of this study was to analyze immunohistochemical and immunocytological findings by examining enucleated eyes and vitreous cell block (CB) in patients with vitreoretinal lymphoma (VRL).Methods: Histological specimens were obtained from two enucleated eyes with VRL associated with neovascular glaucoma. CB specimens were prepared in 18 patients from diluted waste fluids containing shredded vitreous. Histological and cytological specimens were submitted for hematoxylin-eosin staining and immunopathological analyses.Results: Both specimens demonstrated massive infiltration of large lymphoma cells. The lymphoma cells were positive for CD20 and MUM-1 in enucleated eyes. Membranous immunoreactivity for CD20 was observed in lymphoma cells in CB with VRL. Bcl-6 and MUM-1 were marked in five and eight out of nine cases examined, respectivelyConclusions: Cytological findings in CB specimens indicated similar histopathological characteristics of enucleated eyes. CB specimens obtained from vitreous waste diluted fluids may serve as effective materials for cytological diagnosis of VRL.

Effect of geranylgeranylacetone on the protection of retinal ganglion cells in a mouse model of normal tension glaucoma.

Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies, and lowering intraocular pressure is associated with an attenuation of progressive optic nerve damage. Nevertheless, intraocular pressure (IOP) reduction alone was not enough to inhibit the progression of disease, which suggests the contribution of other factors to the glaucoma pathogenesis. In this study, we investigated the cytoprotective effect of geranylgeranylacetone (GGA) on RGCs degeneration using a normal tension glaucoma (NTG) mouse model, which lacks glutamate/aspartate transporter (GLAST) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure (IOP). Three-week-old GLAST+/- mice were given oral administration of GGA at 100, 300, or 600 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. At 5 weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. In addition, retrograde labeling technique was also used to quantify the number of RGC. Expression and localization of heat shock protein 70 (HSP70) in retinas were evaluated by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Activities of caspase-9 and -3 in retinas were also assessed. The number of RGCs of GLAST+/- mice significantly decreased, as compared to that of control mice. RGC loss was significantly suppressed by administration of GGA at 600 mg/kg/day, compared with vehicle alone. Following GGA administration, HSP70 was significantly upregulated together with reduction in the activities of caspase-9 and -3. Our studies highlight HSP70 induction in the retina is available to suppress RGC degeneration, and thus GGA may be applicable for NTG as a promising therapy.

Phosphorylation of alphaB-crystallin in epiretinal membrane of human proliferative diabetic retinopathy.

AIM: To examine phosphorylation of alphaB-crystallin (p-αBC), a vascular endothelial growth factor (VEGF) chaperone, and immunohistochemically investigate relationship between p-αBC, VEGF and phosphorylated p38-mitogen-activated protein kinase (p-p38 MAPK) in the epiretinal membrane of human proliferative diabetic retinopathy (PDR). METHODS: Eleven epiretinal membranes of PDR surgically excised were included in this study. Two normal retinas were also collected from enucleation tissues due to choroidal melanoma. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with anti-p-αBC, VEGF, CD31, and p-p38 MAPK antibodies. RESULTS: Immunoreactivity for p-αBC was observed in all of the epiretinal membranes examined, where phosphorylation on serine (Ser) 59 showed strongest immunoreactivity in over 70% of the membranes. The immunolocalization of p-αBC was detected in the CD31-positive endothelial cells, and co-localized with VEGF and p-p38 MAPK in PDR membranes. Immunoreactivity for p-αBC, however, was undetectable in endothelial cells of the normal retinas, where p-p38 MAPK immunoreactivity was less marked than PDR membranes. CONCLUSION: Phosphorylation of αBC, in particular, phosphorylation on Ser59 by p-p38 MAPK may play a potential role as a molecular chaperon for VEGF in the pathogenesis of epiretinal membranes in PDR.

Regulation of vascular endothelial growth factor-C by tumor necrosis factor-α in the conjunctiva and pterygium.

Vascular endothelial growth factor C (VEGF-C) plays an important role in the development of a pterygium through lymphangiogenesis. We examined the association between VEGF-C and tumor necrosis factor-α (TNF-α) in the pathogenesis of pterygia. Cultured conjunctival epithelial cells were treated with TNF-α, and the gene expression levels of VEGFC were evaluated by quantitative polymerase chain reaction (qPCR) and VEGF-C protein expression levels were measured using an enzyme-linked immunosorbent assay (ELISA). In addition, using ELISA, we evaluated the VEGF-C protein expression in the supernatants of cultured conjunctival epithelial cells, in which we neutralized TNF-α using anti­TNF-α antibody. The gene expression of tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A), known as TNF receptor 1 (TNFR1), was confirmed using reverse transcription PCR in cultured conjunctival epithelial cells. Immunofluorescence microscopy was used to examine the localization of VEGF-C and TNFR1 in pterygium tissues and TNFR1 expression in cultured conjunctival epithelial cells. Immunohistochemistry was used to examine the localization of TNFR1 in pterygia and normal conjunctival tissues. VEGFC gene expression increased in cultured conjunctival epithelial cells 24 h after the addition of TNF-α. The secretion of VEGF-C protein was significantly increased 48 h after the stimulation of cultured conjunctival epithelial cells with TNF-α. Increased VEGF-C protein secretion stimulated by TNF-α was significantly reduced by anti-TNF-α neutralizing antibody treatment. In cultured conjunctival epithelial cells, TNFRSF1A and TNFR1 were expressed. TNFR1 was immunolocalized in normal conjunctival tissues and in human pterygium tissues as well as in VEGF­C­positive epithelial cells from human pterygia. Our data demonstrate that TNF-α mediates VEGF-C expression, which plays a critical role in the pathogenesis of pterygia.

Identification of a Dual Inhibitor of SRPK1 and CK2 That Attenuates Pathological Angiogenesis of Macular Degeneration in Mice.

Excessive angiogenesis contributes to numerous diseases, including cancer and blinding retinopathy. Antibodies against vascular endothelial growth factor (VEGF) have been approved and are widely used in clinical treatment. Our previous studies using SRPIN340, a small molecule inhibitor of SRPK1 (serine-arginine protein kinase 1), demonstrated that SRPK1 is a potential target for the development of antiangiogenic drugs. In this study, we solved the structure of SRPK1 bound to SRPIN340 by X-ray crystallography. Using pharmacophore docking models followed by in vitro kinase assays, we screened a large-scale chemical library, and thus identified a new inhibitor of SRPK1. This inhibitor, SRPIN803, prevented VEGF production more effectively than SRPIN340 owing to the dual inhibition of SRPK1 and CK2 (casein kinase 2). In a mouse model of age-related macular degeneration, topical administration of eye ointment containing SRPIN803 significantly inhibited choroidal neovascularization, suggesting a clinical potential of SRPIN803 as a topical ointment for ocular neovascularization. Thus SRPIN803 merits further investigation as a novel inhibitor of VEGF.

Amelioration of experimental autoimmune uveoretinitis by inhibition of glyceraldehyde-derived advanced glycation end-product formation.

AGEs are permanently modified macromolecule derivatives that form through nonenzymatic glycation of amino groups of proteins. Glycer-AGEs are highly toxic and play an important role in the pathogenesis of chronic inflammatory diseases. However, the contribution of glycer-AGEs to the pathogenesis of uveitis is unclear. In this study, we measured serum levels of glycer-AGEs in 100 patients with endogenous uveitis (22 with HLA-B27-associated uveitis, 20 with VKH disease, 14 with Behçet's disease, and 44 with sarcoidosis) and 33 healthy volunteers. We then examined the effect of the AGE inhibitor in a mouse model of human endogenous uveitis (EAU) by continuous oral administration of pyridoxamine at 200 or 400 mg/kg/day. Regardless of the etiology, serum glycer-AGE levels were significantly higher in patients with uveitis than in healthy subjects. Treatment with 400 mg/kg pyridoxamine significantly reduced the clinical and histological severity of EAU and was accompanied by a significant decrease in serum and retinal glycer-AGE levels and suppression of translocation of NF-κB p65 into the nucleus of retinal cells. Serum glycer-AGE levels may therefore serve as a biomarker of human uveitis, as well as systemic inflammation, and may contribute to the progression of uveitis, including diabetic iritis, via the activation of NF-κB.

Genistein attenuates choroidal neovascularization.

Genistein is a dietary-derived flavonoid abundantly present in soybeans and known to possess various biological effects including anti-inflammation and anti-angiogenic activity. To investigate the effects of genistein on intraocular neovascularization, we used an animal model of laser-induced choroidal neovascularization (CNV). Male C57BL/6J mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CNV was induced by laser photocoagulation. The animals were fed a mixture diet containing 0.5% genistein or a control diet ad libitum for 7 days before laser photocoagulation and the treatment was continued until the end of the study. Seven days after laser injury, the size of CNV lesions was quantified. Retinal pigment epithelium (RPE)-choroid complex was also harvested 1 or 3 days after laser injury and the level of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and matrix metalloproteinase (MMP)-9 were measured by enzyme-linked immunosorbent assay. Expression levels of Ets-1 and F4/80 were examined by real-time PCR. A significant decrease in CNV size was observed in animals treated with genistein (15441.9±1511.8 µm(2)) compared to control mice (21074.0±1940.7µm(2), P<.05). Genistein significantly reduced the protein level of MCP-1, ICAM-1, and MMP-9 in the RPE-choroid complex (P<.05). In addition, genistein suppressed the expression levels of Ets-1 and F4/80 (P<.05). The current data indicate the anti-angiogenic property of genistein during CNV formation.

Expression of vascular endothelial growth factor in human ocular adnexal lymphoma.

PURPOSE: To examine the expression of VEGF in extranodal marginal zone B-cell lymphoma (EMZL) and reactive lymphoid hyperplasia (RLH) of human ocular adnexa, and analyze the correlation with the intratumoral microvessel density (MVD). METHODS: Twenty-two EMZL and 16 RLH tissues were examined in this study. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with antibodies against VEGF and CD20. Vascular endothelial growth factor expression was analyzed using the ELISA and RT-PCR in the EMZL tissues. Microvessel density was determined based on the immunoreactivity for anti-CD34 antibody. RESULTS: Vascular endothelial growth factor immunoreactivity was detected in the cytoplasm of lymphoid cells in EMZL and RLH. ELISA and RT-PCR confirmed VEGF protein and mRNA expressions in the EMZL tissue, respectively. Vascular endothelial growth factor-immunopositive rate in B-cells was significantly higher in 12 conjunctival EMZLs than four RLHs (P < 0.01) and 10 orbital EMZLs than 12 RLHs (P < 0.05). The MVD showed a significant positive correlation with the VEGF-immunopositive rate in conjunctival and orbital EMZLs. CONCLUSIONS: This study demonstrated increased VEGF expression in human conjunctival and orbital EMZL compared with that in RLH, suggesting that VEGF plays a significant role in the pathogenesis and tumor angiogenesis of ocular adnexal lymphoma.

Amelioration of endotoxin-induced uveitis treated with the sea urchin pigment echinochrome in rats.

PURPOSE: Echinochrome is a pigment present in the shells and spines of sea urchins. It has been reported to have several biologic protective effects, including in experimental models of myocardial ischemia/reperfusion injury, for which the proposed mechanisms are scavenging reactive oxygen species (ROS) and chelating iron. Endotoxin-induced uveitis (EIU) is an animal model of acute anterior segment intraocular inflammation that is induced by the injection of lipopolysaccharide (LPS). In this study, the therapeutic effect of echinochrome was examined in uveitis using the EIU model. METHODS: EIU was induced in Lewis rats via 200 µg subcutaneous injections of LPS from Escherichia coli. Echinochrome was administered intravenously in 10, 1, or 0.1 mg/kg doses suspended in PBS (controls were injected with PBS only). Twenty-four hours after LPS injection, the number of infiltrating cells and the protein concentration in aqueous humor were determined. Aqueous tumor necrosis factor α (TNF-α) concentration was quantified with enzyme-linked immunosorbent assay, eyes were stained with nuclear factor (NF) κB antibodies, and ROS production was determined by dihydroethidium staining in fresh frozen samples. RESULTS: The number of inflammatory aqueous cells and protein levels were lower in the groups treated with 10 and 1 mg/kg of echinochrome than in the untreated LPS group (p<0.01). Treatment with 10 and 1 mg/kg of echinochrome significantly reduced TNF-α concentrations in aqueous humor (p<0.01). The numbers of NFκB-positive cells and ROS signals were also reduced by echinochrome administration (p<0.05). CONCLUSIONS: Echinochrome ameliorated intraocular inflammation caused by EIU by reducing ROS production, thereby also decreasing the expression of NFκB and TNF-α. As a natural pigment, echinochrome may therefore be a promising candidate for the safe treatment of intraocular inflammation. The use of sea urchin shells and spines in health foods and medical products is thus both economically and environmentally meaningful.

Expression of αB-crystallin and vascular endothelial growth factor in conjunctival squamous cell carcinoma.

AIM: To examine the expression of αB-crystallin and vascular endothelial growth factor (VEGF) in conjunctival squamous cell carcinoma (CSCC). MATERIALS AND METHODS: Seven CSCCs and three normal conjunctivas that were surgically excised were studied. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with antibodies against αB-crystallin, its phosphorylated forms, and VEGF. In vitro experiments were conducted to investigate the effects of mitomycin C (MMC) treatment on the expression of αB-crystallin and VEGF secretion. RESULTS: αB-Crystallin and VEGF were strongly expressed in CSCCs compared to normal conjunctivas. αB-Crystallin immunoreactivity was co-localized with that for VEGF in CSCCs, whereas these signals were reduced in CSCC tissues treated with MMC before excision. MMC treatment suppressed the αB-crystallin expression and VEGF secretion in cultured conjunctival cells in a dose-dependent manner. CONCLUSION: This study demonstrated αB-crystallin and VEGF expressions in human CSCCs, which may play a role in the pathogenesis. αB-Crystallin expression, and VEGF secretion were reduced by MMC, indicating a novel therapeutic mechanism in MMC treatment for human CSCC.

Specific inhibition of serine/arginine-rich protein kinase attenuates choroidal neovascularization.

PURPOSE: To investigate the applicability of serine/arginine-rich protein kinase (SRPK)-specific inhibitor, SRPIN340, for attenuation of choroidal neovascularization (CNV) formation using a mouse model. METHODS: Laser photocoagulation was performed to induce CNV in C57BL/6J mice, followed by intravitreal injection of SRPIN340 or vehicle. Seven days after the treatment, the CNV size was evaluated using a flatmount technique. Protein levels of vascular endothelial growth factor (VEGF) and inflammation-associated molecules, such as monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, in the retinal pigment epithelium-choroid complex were measured with enzyme-linked immunosorbent assay. Expression levels of total Vegf, exon 8a-containing Vegf isoforms, and F4/80 (a specific marker for macrophage) were assessed using real-time PCR. RESULTS: SRPIN340 inhibited CNV formation in a dose-dependent manner. Compared with the vehicle, SRPIN340 significantly decreased the protein levels of VEGF, MCP-1, ICAM-1, and consequently inhibited macrophage infiltration. Furthermore, SRPIN340 suppressed the gene expression levels of total Vegf and exon 8a-containing Vegf isoforms. CONCLUSIONS: SRPIN340, a specific inhibitor of SRPK, suppressed Vegf expression and attenuated CNV formation. Our data suggest the possibility that SRPIN340 is applicable for neovascular age-related macular degeneration as a novel chemical therapeutics.

Expression of vascular endothelial growth factor C in human pterygium.

Vascular endothelial growth factor C (VEGF-C) and its receptor VEGFR-3 mediate lymphangiogenesis. In this study, we analyzed the expression of VEGF-C and VEGFR-3 as well as lymphatic vessels in the pterygium and normal conjunctiva of humans. Fifteen primary nasal pterygia and three normal bulbar conjunctivas, surgically removed, were examined in this study. The lymphatic vessel density (LVD) and blood vessel density were determined by the immunolabeling of D2-40 and CD31, markers for lymphatic and blood vessels, respectively. VEGF-C and VEGFR-3 expression in pterygial and conjunctival tissue proteins was detected by Western blotting and were evaluated using immunohistochemistry. The LVD was significantly higher in the pterygium than normal conjunctiva (p < 0.05). Western blot demonstrated high-level expression of VEGF-C and VEGFR-3 in the pterygium compared with normal conjunctiva. VEGF-C immunoreactivity was detected in the cytoplasm of pterygial and normal conjunctival epithelial cells. The number of VEGF-C-immunopositive cells in pterygial epithelial cells was significantly higher than in normal conjunctival cells (p < 0.05). VEGFR-3 immunoreactivity was localized in the D2-40-positive lymphatic endothelial cells. The present findings suggest the potential role of VEGF-C in the pathogenesis and development of a pterygium through lymphangiogenesis and the VEGF-C/VEGFR-3 pathway as a novel therapeutic target for the human pterygium.

Tissue kallikrein attenuates choroidal neovascularization via cleavage of vascular endothelial growth factor.

PURPOSE: To investigate the antiangiogenic properties of tissue kallikrein in a murine model of laser-induced choroidal neovascularization (CNV). METHODS: CNV was induced in male C57BL/6J mice by laser photocoagulation. The animals received daily subcutaneous injections of tissue kallikrein (50 µg/kg) or vehicle control for 2 days before the laser photocoagulation, and this treatment continued until sample collection. Seven days after laser injury, the CNV size was quantified. The levels of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 were assessed by enzyme-linked immunosorbent assay 3 days after laser injury. Cleavage of mouse VEGF with tissue kallikrein was assessed in vivo and in vitro. The protein levels of bradykinin were assessed in the RPE-choroid complexes and hearts. RESULTS: A significant decrease in CNV size was observed in animals treated with tissue kallikrein (27,168.3 ± 2432.2 µm(2)) compared with vehicle-treated controls (36,374.6 ± 3204.1 µm(2), P < 0.05). Tissue kallikrein treatment significantly reduced MCP-1, ICAM-1, and IL-6 levels in RPE-choroid complexes. Furthermore, immunoblotting showed the bands, presumably corresponding to the fragmented VEGF(164) protein, in the samples of both mouse VEGF preincubated with tissue kallikrein and RPE-choroid complexes obtained from animals treated with tissue kallikrein. In addition, bradykinin was unchanged in the RPE-choroid complexes of animals treated with tissue kallikrein, whereas the level of bradykinin was increased in the heart obtained from these experimental animals. CONCLUSIONS: The current data indicate that kallikrein exhibits antiangiogenic properties by cleaving VEGF(164) in a laser-induced CNV model.

Expression of vascular endothelial growth factor in eyes with Coats' disease.

PURPOSE: To examine the expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR)-2 in enucleated eyes with Coats' disease. METHODS: Formalin-fixed, paraffin-embedded tissue sections from nine globes with Coats' disease were submitted for hematoxylin and eosin staining and immunohistochemistry with anti-VEGF and VEGFR antibodies. RESULTS: Histologically, the enucleated eyes demonstrated the presence of macrophage infiltration and cholesterol clefts in the subretinal space. There were marked retinal vascular abnormalities, including dilated vessels with hyalinized vessel walls in six globes. Exudative retinal detachment was noted in all globes. VEGF immunoreactivity was observed in macrophages infiltrating the subretinal space, and in the detached retina including several blood vessels. VEGF-positivity in macrophages was significantly higher in cases containing retinal vessel abnormalities than those without the abnormalities (P < 0.01). VEGFR-2 immunoreactivity was detected in endothelial cells lining the abnormal retinal vessels, where VEGFR-1 or VEGFR-3 was not expressed. CONCLUSIONS: Immunoreactivity for VEGF and VEGFR-2 was detected in macrophages and endothelia of abnormal vessels in eyes with Coats' disease. These results suggest that anti-VEGF approach is a promising therapy for patients with Coats' disease.

Amelioration of endotoxin-induced uveitis treated with an IκB kinase ß inhibitor in rats.

PURPOSE: Endotoxin-induced uveitis (EIU) is an animal model for acute ocular inflammation. Several substances play major roles in the development of inflammatory changes in EIU, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. These inflammatory cytokines trigger the degradation of IκB by activating IκB kinases (IKKs). Released nuclear factor kappaB (NFκB) subsequently translocates to the nucleus, where NFκB expresses its proinflammatory function. IMD-0354, N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide, selectively inhibits IKKß, particularly when induced by proinflammatory cytokines, such as TNF-α and IL-1ß. In the present study, we examined whether IKKß inhibition has therapeutic effects on EIU by using IMD-0354 and its prodrug IMD-1041. METHODS: Six-week-old male Lewis rats were used. EIU was induced with subcutaneous injections of 200 µg of lipopolysaccharide (LPS) from Escherichia coli that had been diluted in 0.1 ml of phosphate-buffered saline. IMD-0354 was administered intraperitoneally at 30, 10, 3, or 0 mg/kg, suspended in 1.0 ml of 0.5% carboxymethyl cellulose sodium. The prodrug IMD-1041 (100 mg/kg) was also administered orally. The rats were euthanized 24 h after LPS injection, and EIU severity was evaluated histologically. The number of infiltrating cells and the protein, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) concentrations in the aqueous humor were determined. TNF-α and MCP-1 concentrations were quantified with enzyme-linked immunosorbent assay. Eye sections were also stained with anti-NFκB and phosphorylated I-κBα antibodies. RESULTS: The number of infiltrating cells in aqueous humor was 53.6±9.8×10(5), 72.5±17.0×10(5), 127.25±32.0×10(5), and 132.0±25.0×10(5) cells/ml in rats treated with 30, 10, 3, or 0 mg/kg of IMD-0354, respectively. The total protein concentrations of aqueous humor were 92.6±3.1 mg/ml, 101.5±6.8 mg/ml, 112.6±1.9 mg/ml, and 117.33±1.8 mg/ml in rats treated with 30, 10, 3, and 0 mg/kg of IMD-0354, respectively. Infiltrating cells and protein concentrations were significantly decreased by treatment with IMD-0354 (p<0.01). IMD-0354 treatment significantly reduced the concentration of TNF-α (p<0.05) and MCP-1 (p<0.01) in aqueous humor. The number of NFκB positive nuclei was reduced when treated with IMD-0354. Furthermore, IMD-0354-treated EIU rats showed only background levels of phosphorylated I-κBα; however, it was strongly expressed in the iris-ciliary body cell cytoplasm of the IMD-0354 untreated EIU rats. Oral administration of IMD-1041 also decreased the cell number (p<0.01) and protein concentration (p<0.05) of aqueous humor in EIU. CONCLUSIONS: Acute uveitis was ameliorated by inhibition of IKKß in rats. IMD-0354 and its prodrug IMD-1041 seem to be promising candidates for treating intraocular inflammation/uveitis.

Conjunctival lymphoma arising from reactive lymphoid hyperplasia.

Extra nodal marginal zone B-cell lymphoma (EMZL) of the conjunctiva typically arises in the marginal zone of mucosa-associated lymphoid tissue. The pathogenesis of conjunctival EMZL remains unknown. We describe an unusual case of EMZL arising from reactive lymphoid hyperplasia (RLH) of the conjunctiva. A 35-year-old woman had fleshy salmon-pink conjunctival tumors in both eyes, oculus uterque (OU). Specimens from conjunctival tumors in the right eye, oculus dexter (OD), revealed a collection of small lymphoid cells in the stroma. Immunohistochemically, immunoglobulin (Ig) light chain restriction was not detected. In contrast, diffuse atypical lymphoid cell infiltration was noted in the left eye, oculus sinister (OS), and positive for CD20, a marker for B cells OS. The tumors were histologically diagnosed as RLH OD, and EMZL OS. PCR analysis detected IgH gene rearrangement in the joining region (JH) region OU. After 11 months, a re-biopsy specimen demonstrated EMZL based on compatible pathological and genetic findings OD, arising from RLH. This case suggests that even if the diagnosis of the conjunctival lymphoproliferative lesions is histologically benign, confirmation of the B-cell clonality by checking IgH gene rearrangement should be useful to predict the incidence of malignancy.

Soluble vascular adhesion protein-1 accumulates in proliferative diabetic retinopathy.

PURPOSE: Vascular adhesion protein (VAP)-1, a multifunctional molecule with adhesive and enzymatic properties, is expressed at the surface of vascular endothelial cells of mammals. It also exists as a soluble form (sVAP-1), which is implicated in oxidative stress via its enzymatic activity. This study explores a link between increased level of sVAP-1 and oxidative stress in proliferative diabetic retinopathy (PDR) with a focus on mechanistic components to form sVAP-1 by shedding from retinal endothelial cells. METHODS: Protein levels of sVAP-1 and N epsilon-(hexanoyl)lysine (HEL), an oxidative stress marker, in the vitreous samples from patients with PDR or non-PDR were measured by ELISA. The mechanism of VAP-1 shedding under diabetic condition, exposure to high glucose and/or inflammatory cytokines, was explored using cultured retinal capillary endothelial cells. RESULTS: Protein level of sVAP-1 was increased and correlated with HEL in the vitreous fluid of patients with PDR. Retinal capillary endothelial cells released sVAP-1 when stimulated with high glucose or inflammatory cytokines, such as TNF-α and IL-1ß in vitro. Furthermore, matrix metalloproteinase-2 and -9, type IV collagenases, were the key molecules to mediate the protein cleavage of VAP-1 from retinal capillary endothelial cells. CONCLUSIONS: Our data for the first time provide evidence on the link between sVAP-1 and type IV collagenases in the pathogenesis of PDR.

Immunolocalization of vascular adhesion protein-1 in human conjunctival tumors.

OBJECTIVE: We analyzed the expression and immunolocalization of vascular adhesion protein (VAP)-1 in conjunctival tumors and normal conjunctival tissue of humans. METHODS: Nine conjunctival tumors, including pyogenic granuloma and extranodal marginal zone B-cell lymphoma (EMZL), and 2 normal conjunctivas were analyzed by immunohistochemistry for VAP-1 and CD31 expression. RESULTS: Immunoreactivity for VAP-1 was detected in the lumen of microvessels in pyogenic granuloma and in EMZLs. In contrast, normal bulbar conjunctival tissues demonstrated weak cytoplasmic immunoreactivity for VAP-1 in the blood vessels. CONCLUSIONS: The immunolocalization of VAP-1 varied in the histopathology of the conjunctiva, involving the pathology of inflammatory conjunctival disorders.

Alphab-crystallin expression in epiretinal membrane of human proliferative diabetic retinopathy.

PURPOSE: To examine the expression of alphaB-crystallin and its colocalization with vascular endothelial growth factor in the epiretinal membrane of human proliferative diabetic retinopathy. METHODS: Ten epiretinal membranes of proliferative diabetic retinopathy and three normal retinas surgically excised were included in this study. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with alphaB-crystallin, vascular endothelial growth factor, and CD31 antibodies. RESULTS: AlphaB-crystallin was expressed in all epiretinal membranes examined. The immunolocalization of alphaB-crystallin was detected in the cytoplasm of CD31-positive endothelial cells, but not in normal retinal blood vessels. Furthermore, alphaB-crystallin immunoreactivity was colocalized in vascular endothelial growth factor-positive endothelial cells in proliferative diabetic retinopathy membranes. CONCLUSION: AlphaB-crystallin was expressed in proliferative diabetic retinopathy membranes, and colocalized with vascular endothelial growth factor-positive neovessels. AlphaB-crystallin may play a potential role in the pathogenesis of epiretinal membranes in proliferative diabetic retinopathy, together with vascular endothelial growth factor.