Exploring the Impact of Saccharin on Neovascular Age-Related Macular Degeneration: A Comprehensive Study in Patients and Mice.

Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.

Islet Co-Expression of CD133 and ABCB5 in Human Retinoblastoma Specimens.

BACKGROUND: The role of CD133 und ABCB5 is discussed in treatment resistance in several types of cancer. The objective of this study was to evaluate whether CD133+/ABCB5+ colocalization differs in untreated, in beam radiation treated, and in chemotherapy treated retinoblastoma specimens. Additionally, CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 gene expression was analyzed in WERI-RB1 (WERI RB1) and etoposide-resistant WERI RB1 subclones (WERI ETOR). METHODS: Active human untreated retinoblastoma specimens (n = 12), active human retinoblastoma specimens pretreated with beam radiation before enucleation (n = 8), and active human retinoblastoma specimens pretreated with chemotherapy before enucleation (n = 7) were investigated for localization and expression of CD133 and ABCB5 by immunohistochemistry. Only specimens with IIRC D, but not E, were included in this study. Furthermore, WERI RB1 and WERI ETOR cell lines were analyzed for CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 by the real-time polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical analysis revealed the same amount of CD133+/ABCB5+ colocalization islets in untreated and treated human retinoblastoma specimens. Quantitative RT-PCR analysis showed a statistically significant upregulation of CD133 in WERI ETOR (p = 0.002). No ABCB5 expression was detected in WERI RB1 and WERI ETOR. On the other hand, SPHK1 (p = 0.0027) and SPHK2 (p = 0.017) showed significant downregulation in WERI ETOR compared to WERI RB1. CONCLUSIONS: CD133+/ABCB5+ co-localization islets were noted in untreated and treated human retinoblastoma specimens. Therefore, we assume that CD133+/ABCB5+ islets might play a role in retinoblastoma genesis, but not in retinoblastoma treatment resistance.

Effects of TNFα receptor TNF-Rp55- or TNF-Rp75- deficiency on corneal neovascularization and lymphangiogenesis in the mouse.

Tumor necrosis factor (TNF)α is an inflammatory cytokine likely to be involved in the process of corneal inflammation and neovascularization. In the present study we evaluate the role of the two receptors, TNF-receptor (TNF-R)p55 and TNF-Rp75, in the mouse model of suture-induced corneal neovascularization and lymphangiogenesis. Corneal neovascularization and lymphangiogenesis were induced by three 11-0 intrastromal corneal sutures in wild-type (WT) C57BL/6J mice and TNF-Rp55-deficient (TNF-Rp55d) and TNF-Rp75-deficient (TNF-Rp75d) mice. The mRNA expression of VEGF-A, VEGF-C, Lyve-1 and TNFα and its receptors was quantified by qPCR. The area covered with blood- or lymphatic vessels, respectively, was analyzed by immunohistochemistry of corneal flatmounts. Expression and localization of TNFα and its receptors was assessed by immunohistochemistry of sagittal sections and Western Blot. Both receptors are expressed in the murine cornea and are not differentially regulated by the genetic alteration. Both TNF-Rp55d and TNF-Rp75d mice showed a decrease in vascularized area compared to wild-type mice 14 days after suture treatment. After 21 days there were no differences detectable between the groups. The number of VEGF-A-expressing macrophages did not differ when comparing WT to TNF-Rp55d and TNF-Rp75d. The mRNA expression of lymphangiogenic markers VEGF-C or LYVE-1 does not increase after suture in all 3 groups and lymphangiogenesis showed a delayed effect only for TNF-Rp75d. TNFα mRNA and protein expression increased after suture treatment but showed no difference between the three groups. In the suture-induced mouse model, TNFα and its ligands TNF-Rp55 and TNF-Rp75 do not play a significant role in the pathogenesis of neovascularisation and lymphangiogenesis.

FLASH proton irradiation setup with a modulator wheel for a single mouse eye.

PURPOSE: Recent studies indicate that FLASH irradiation, which involves ultra-high dose rates in a short time window (usually >40 Gy/s in <500 ms), might be equally efficient against tumors but less harmful to healthy tissues, compared to conventional irradiation with the same total dose. Aiming to verify the latter claim for ocular proton radiotherapy, in vivo experiments with mice are being carried out by Charité - Universitätsmedizin Berlin. This work presents the implemented setup for delivering FLASH proton radiation to a single eye of mice at the Helmholtz-Zentrum Berlin für Materialien und Energie (HZB). MATERIALS AND METHODS: The HZB cyclotron is tuned to provide a high-intensity 68 MeV focused proton beam. Outside the vacuum beamline, the protons hit a single scatterer, which also serves as range shifter, and a rotating modulator wheel, which produces a flat depth-dose distribution. Two transmission ionization chambers in between, read out by fast electronics, are used as dose monitors for triggering an in-vacuum beam shutter, which blocks the beam once the desired dose has been delivered. A collimating aperture shapes the radiation field at the isocenter, which is measured by a radioluminescent screen and a CCD camera. At the same position, a parallel-plate ionization chamber of type Advanced Markus® is used for absolute dosimetry and characterization of the spread-out Bragg peak inside a water phantom. A thin-foil mirror of adjustable tilt in the beam path assists the correct alignment of the target through side illumination. Radiochromic films of type EBT3 are used to supplement the dosimetry and assist the alignment. RESULTS: A dose rate of 75 Gy/s has been measured, delivering within 200 ms 15 Gy (RBE) with a reproducibility better than ±1%. A depth-dose curve with a range of 5.2 mm in water, 0.9 mm distal fall-off (90%-10%), and ±2.5% ripple has been demonstrated, with a PTV of 6.3 mm diameter, 1.7 mm lateral penumbra (90%-10%), 8% uniformity, and 3% symmetry. CONCLUSIONS: The implemented setup is able to accommodate ocular irradiation of narcotized mice with protons, targeting selectively the left or the right eye, under conventional and FLASH conditions. Switching between these two modes can be done within half an hour, including the calibration of the dose monitors and the verification of the dose delivery. Further upgrades are planned after the completion of the on-going experiment.

Local partial depletion of CD11b+ cells and their influence on choroidal neovascularization using the CD11b-HSVTK mouse model.

PURPOSE: To assess the influence of retinal macrophages and microglia on the formation of choroidal neovascularization (CNV). Therefore, we used a transgenic mouse (CD11b-HSVTK) in which the application of ganciclovir (GCV) results in a depletion of CD11b+ cells. METHODS: We first investigated if a local depletion of CD11b+ macrophages and microglia in the retina is feasible. In a second step, the influence of CD11b+ cell depletion on CNV formation was analysed. One eye of each CD11b-HSVTK mouse was injected with GCV, and the fellow eye received sodium chloride solution (NaCl). Cell counting was performed at day 3 and 7 (one injection) or at day 14 and 21 (two injections). Choroidal neovascularization (CNV) was induced by argon laser and analysed at day 14. RESULTS: The most effective CD11b+ cell depletion was achieved 7 days after a single injection and 14 days after two injections of GCV. After two injections of GCV, we found a significant reduction of CD11b+ cells in central (52 ± 23.9 cells/mm2 ) and peripheral retina (53 ± 20.6 cells/mm2 ); compared to eyes received NaCl (216 ± 49.0 and 210 ± 50.5 cells/mm2 , p < 0.001, respectively). Regarding CNV areas, no statistical significance was found between the groups. CONCLUSION: The CD11b-HSVTK mouse is a feasible model for a local depletion of CD11b+ cells in the retina. Nevertheless, only a partial depletion of CD11b+ cells could be achieved compared to baseline data without any intravitreal injections. Our results did not reveal a significant reduction in CNV areas. In the light of previous knowledge, the potential influence of systemic immune cells on CNV formation might be more relevant than expected.

Myeloid cells contribute indirectly to VEGF expression upon hypoxia via activation of Müller cells.

Anti-VEGF-directed therapies have been a milestone for treating retinal vascular diseases. Depletion of monocyte lineage cells suppresses pathological neovascularization in the oxygen-induced retinopathy mouse model. However, the question whether myeloid-derived VEGF-A expression is responsible for the pathogenesis in oxygen-induced retinopathy remained unknown. We analyzed LysMCre-driven myeloid cell-specific VEGF-A knockout mice as well as mice with complete depletion of circulating macrophages through clodronate-liposome treatment in the oxygen-induced retinopathy model by immunohistochemistry, qPCR, and flow cytometry. Furthermore, we analyzed VEGF-A mRNA expression in MIO-M1 cells alone and in co-culture with BV-2 cells in vitro. The myeloid cell-specific VEGF-A knockout did not change relative retinal VEGF-A mRNA levels, the relative avascular area or macrophage/granulocyte numbers in oxygen-induced retinopathy and under normoxic conditions. We observed an insignificantly attenuated pathology in systemically clodronate-liposome treated knockouts but evident VEGF-A expression in activated Müller cells on immunohistochemically stained sections. MIO-M1 cells had significantly higher expression levels of VEGF-A in co-culture with BV-2 cells compared to cultivating MIO-M1 cells alone. Our data show that myeloid-derived cells contribute to pathological neovascularization in oxygen-induced retinopathy through activation of VEGF-A expression in Müller cells.

Spatial distribution of CD115+ and CD11b+ cells and their temporal activation during oxygen-induced retinopathy in mice.

PURPOSE: The model of oxygen-induced retinopathy (OIR) is widely used to analyze pathomechanisms in retinal neovascularization. Previous studies have shown that macrophages (MP) play a key role in vessel formation in OIR, the influence of microglia (MG) having been discussed. The aim of our study was to analyze the spatial and temporal distribution and activation of MP/MG expressing CD115 and CD11b during the process of neovascularization in OIR. METHODS: We used MacGreen mice expressing the green fluorescence protein (GFP) under the promoter for CD115. CD115+ cells were investigated in vivo by scanning laser ophthalmoscopy at postnatal days (P) 17 and 21 in MacGreen mice with OIR (75% oxygen from P7 to P12), and were compared to MacGreen room-air controls. In addition MP/MG were examined ex vivo using immunohistochemistry for CD11b+ detection on retinal flatmounts at P14, P17, and P21 of wild type mice with OIR. RESULTS: In-vivo imaging revealed the highest density of activated MP/MG in tuft areas at P17 of MacGreen mice with OIR. Tufts and regions with a high density of CD115+ cells were detected close to veins, rather to arteries. In peripheral, fully vascularized areas, the distribution of CD115+ cells in MacGreen mice with OIR was similar to MacGreen room-air controls. Correspondingly, immunohistochemical analyses of retinal flatmounts from wild type mice with OIR induction revealed that the number of CD11b+ cells significantly varies between vascular, avascular, and tuft areas as well as between the retinal layers. Activated CD11b+ cells were almost exclusively found in avascular areas and tufts of wild type mice with OIR induction; here, the proportion of activated cells related to the total number of CD11b+ cells remained stable over the course of time. CONCLUSIONS: Using two different approaches to monitor MP/MG cells, our findings demonstrated that MP/MG concentrate within pathologically vascularized areas during OIR. We were able to clarify that reactive changes of CD11b+ cell distribution to OIR primarily occur in the deep retinal layers. Furthermore, we found the highest proportion of activated CD11b+ cells in regions with pathologic neovascularization processes. Our findings support previous reports about activated MP/MG guiding revascularization in avascular areas and playing a key role in the formation and regression of neovascular tufts.

Inhibition of Placenta Growth Factor Reduces Subretinal Mononuclear Phagocyte Accumulation in Choroidal Neovascularization.

Purpose: The cellular immune response driven by mononuclear phagocytes (MPs) is crucial for choroidal neovascularization (CNV) progression. Case reports show that a switch from pure anti-vascular endothelial growth factor-A (VEGF-A) intravitreal treatment to aflibercept, a drug with combined anti-VEGF-A and anti-placenta growth factor (PlGF) activity, can be beneficial for patients who do not respond to anti-VEGF-A alone. Since MPs harbor VEGFR1, we hypothesize that the interplay of P1GF/vascular endothelial growth factor receptor 1 (VEGFR1) in immune cells plays a pivotal role for CNV. Methods: CNV was induced with laser, and immune cells and neovascularization were analyzed in vivo and ex vivo. Immunohistochemistry was employed for protein detection. Differential expression of angiogenic factors and macrophage polarization markers were assessed by quantitative PCR (qPCR). One day after laser, intravitreal injection of aflibercept or anti-PlGF was performed. Results: In the early inflammatory phase after laser, Plgf but not Vegfa was significantly upregulated. VEGF-A upregulation is limited to the scar, whereas PlGF shows a wider distribution. M1 (proinflammatory) macrophage markers were upregulated in the early phase of CNV. However, M2 (proangiogenic) markers showed more inconsistent dynamics. We demonstrated that both aflibercept and anti-PlGF treatments decrease the overall amount of activated subretinal MPs, and especially of those expressing PlGF. These data correlated with a reduction in leakage associated to CNV. Aflibercept showed a stronger reduction in both parameters. Conclusions: The results hint at an interplay between PlGF/VEGFR1 and MPs that is important in the early phase of CNV. A combined inhibition of VEGF-A and PlGF is superior to a specific anti-PlGF treatment in terms of subretinal MP recruitment.

Vascular-Associated Muc4/Vwf Co-Localization in Human Conjunctival Malignant Melanoma Specimens-Tumor Metastasis by Migration?

PURPOSE: To investigate whether vascular differentiation marker von Willebrand factor (vWf) and proliferation marker KI67 expression correlate with MUC4 localization around stromal tumor vascularization in human conjunctival malignant melanoma (CMM). MATERIALS AND METHODS: For the purposes of this study, we analyzed samples from human CMMs (n = 4), conjunctival compound nevi (n = 7), and samples from healthy conjunctiva (n = 7) for MUC1, 4, and 16 by immunohistochemistry. To test CMM vessel association of MUC4, we investigated the co-localization of MUC4 with vWf or KI67 in human CMM specimens (n = 10) by immunohistochemistry. Also, we investigated the MUC4 localization around vessels of healthy conjunctiva (n = 10). RESULTS: The immunohistochemical analysis demonstrated membrane-associated mucin expression in epithelia of CMM, nevi and healthy conjunctiva, whereas only MUC4 was localized perivascular in CMM tissue in this preliminary analysis. Co-staining analysis with vWf and KI67 demonstrated MUC4 localization around stromal vessels in human CMM specimens. In contrast, no MUC4 localization has been seen around healthy conjunctiva stroma vessels. CONCLUSIONS: MUC4 was detected around vWf/KI67-positive CMM stromal vascular tissue, but not around healthy conjunctival stroma vessels. Therefore, we assume that MUC4 might play a role in tumor cell migration toward vessels inducing metastasis.

Netrin-4 Mediates Corneal Hemangiogenesis but Not Lymphangiogenesis in the Mouse-Model of Suture-Induced Neovascularization.

Purpose: Netrin-4, a secreted protein, is found in the basement membrane of blood vessels and acts as a key regulator of angiogenesis. Here we investigated the role of Netrin-4 in the mouse-model of suture-induced corneal hem- and lymphangiogenesis. Methods: Corneal hem- and lymphangiogenesis were induced in Netrin-4-deficient (Ntn4-/-) and wild-type (WT) mice by placing three 11-0 nylon sutures intrastromally. Fourteen days after suturing, the vascularized area was analyzed via corneal flat mount immunohistochemistry. Messenger RNA levels for VEGF-A, VEGF-C, Lyve-1, Netrin-4, Unc5H2, "deleted in colon cancer" receptor, and Neogenin in treated and nontreated mouse corneas, cultured human corneal keratocytes (HCK) and epithelial cells (HCEC+HCET) were analyzed by quantitative PCR. Results: In wild-type mice, Netrin-4 mRNA expression in the cornea decreased in growing corneal neovascularization after suturing. Correspondingly, Ntn4-/- mice showed an increased vascularized area compared to that in WT mice. Expression of VEGF-A mRNA was higher in Ntn4-/- versus WT mice. There was no Netrin-4 expression in lymphatic vessels and the area of lymphatic vascularization did not differ between Ntn4-/- and WT mice, nor did expression of VEGF-C and Lyve-1 mRNA. Human corneal epithelial cells showed mainly Netrin-4 mRNA expression, which increased after stimulation, while HCK demonstrated Unc5H2 mRNA expression. Expression of VEGF-A, Netrin-4, Unc5H2, and Neogenin mRNA in HCEC and HCK did not differ significantly between the serum-free condition and VEGF-A or Netrin-4 stimulation. Conclusions: Absence of Netrin-4 increased corneal hemangiogenesis but not lymphangiogenesis in the mouse-model of suture-induced neovascularization. Netrin-4 acted as an antiangiogenic factor in the cornea, with which the healthy cornea is enriched via its expression by corneal epithelial cells.

Lack of netrin-4 modulates pathologic neovascularization in the eye.

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4(-/-) mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4(-/-) mice. In OIR, Ntn-4(-/-) mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4(-/-) mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4(-/-) mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization.

B-Raf inhibition in conjunctival melanoma cell lines with PLX 4720.

PURPOSE: Mutations in the gene coding for the kinase B-Raf are associated with tumour growth in conjunctival melanoma. The purpose of this study is to explore effects of pharmacological B-Raf inhibition in conjunctival melanoma cell lines. METHODS: The B-Raf genotypes were assessed by PCR and subsequent sequencing. Cytotoxicity, cell viability, proliferation, apoptosis rate and phosphorylation rate of ERK and Akt were analysed in three different conjunctival melanoma cell lines under the influence of the B-Raf inhibitor PLX 4720 at various concentrations. RESULTS: The cell lines CRMM-1 and CM2005.1 showed the B-Raf V600E mutation, whereas CRMM-2 expressed a B-Raf wild type. CM2005.1 was highly sensitive to PLX 4720, showing a complete cytotoxic effect for >1 µM, as well as a significant concentration-dependent reduction of the proliferation rate and viability rate. Even though CRMM-1 also carries the B-Raf V600E mutation, it did not react as sensitive to PLX 4720 inhibition as CM2005.1, but showed a significant concentration-dependent reduction regarding proliferation and viability. PLX 4720 had only slight impact on CRMM-2 in high concentrations (10 µM) regarding cytotoxicity, proliferation and viability. Fluorescence-activated cell sorting analysis revealed that PLX 4720 acted predominantly antiproliferative and not via an induction of apoptosis. The phosphorylation rate of ERK was significantly reduced in CRMM-1 and CM2005.1, while it remained unchanged in CRMM-2. The phosphorylation rate of Akt was significantly elevated in CRMM-2. CONCLUSIONS: Proliferation inhibition of conjunctival melanoma cells by PLX 4720 depends on their B-Raf genotype. Therefore, therapeutic application of B-Raf inhibitors should take into account the specific B-Raf genotype.

Aldose reductase deficiency reduced vascular changes in neonatal mouse retina in oxygen-induced retinopathy.

PURPOSE: Retinal neovascularization is the major pathologic process in many ocular diseases and is associated with oxidative stress. Deficiency of aldose reductase (AR), the first enzyme in the polyol pathway for glucose metabolism, has been shown to reduce oxidative stress and blood vessel leakage. The present study aimed to investigate the effect of AR deficiency on retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model. METHODS: Seven-day-old wild-type (WT) and AR-deficient (AR(-/-)) mice were exposed to 75% oxygen for 5 days and then returned to room air. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. Immunohistochemistry for AR, nitrotyrosine (NT), poly(ADP-ribose) (PAR), glial fibrillary acidic protein (GFAP), and Iba-1, as well as Western blots for vascular endothelial growth factor (VEGF), phospho-Erk (p-Erk), phospho-Akt (p-Akt), and phospho-IκB (p-IκB) were performed. RESULTS: Compared with WT OIR retinae, AR(-/-) OIR retinae displayed significantly smaller central retinal vaso-obliterated area, less neovascularization, and reduced blood vessel leakage. Significantly reduced oxidative stress and glial responses were also observed in AR(-/-) OIR retinae. Moreover, reduced microglial response in the avascular area but increased microglial responses in the neovascular area were found with AR deficiency. Furthermore, expression levels of VEGF, p-Erk, p-Akt, and p-IκB were significantly reduced in AR(-/-) OIR retinae. CONCLUSIONS: Our observations indicated that AR deficiency reduced retinal vascular changes in the mouse model of OIR, indicating that AR can be a potential therapeutic target in ischemia-induced retinopathy.

Altered calcium regulation by thermosensitive transient receptor potential channels in etoposide-resistant WERI-Rb1 retinoblastoma cells.

Differences in transient receptor potential (TRP) and cannabinoid receptor type 1 (CB1) expression levels can serve as prognostic factors for retinoblastoma (RB) tumor progression. We hypothesized in RB tissue that such differences are also indicators of whether or not they are sensitive to etoposide. Accordingly, we compared in malignant etoposide-sensitive and etoposide-resistant WERI-Rb1 cells TRPV1, TRPM8 and TRPA1 subtype and CB1 gene expression pattern levels and accompanying functional activity using quantitative real-time RT-PCR, immunohistochemistry, immunofluorescence microscopy, calcium imaging as well as patch-clamp technology. Gene expression patterns were evaluated in enucleated human RB tissues (n = 4). Both etoposide-resistant and etoposide-sensitive WERI-Rb1 cells expressed all of the aforementioned channels based on responses to known activators and thermal challenges. However, TRPA1 was absent in the etoposide-resistant counterpart. Even though both types of RB cells express TRPV1 as well as TRPM8 and CB1, the capsaicin (50 µM) (CAP)-induced Ca(2+) rise caused by TRPV1 activation was prompt and transient only in etoposide-resistant RB cells (n = 8). In this cell type, the inability of CB1 activation (10 µM WIN) to suppress Ca(2+) responses to CAP (50 µM; n = 4) may be attributable to the absence of TRPA1 gene expression. Therefore, using genetic approaches to upregulate TRPA1 expression could provide a means to induce etoposide sensitivity and suppress RB cell tumorigenesis.

Recruitment of blood-derived inflammatory cells mediated via tumor necrosis factor-α receptor 1b exacerbates choroidal neovascularization.

Purpose. Tumor necrosis factor (TNF)-α contributes to inflammation-associated angiogenesis, and TNF-α receptor 1b is selectively expressed on immuno-competent and endothelial cells. This study investigated the role of TNF-α receptor 1b in the recruitment of circulating inflammatory cells and the development of choroidal neovascularization (CNV). Methods. Lethally irradiated Tnfrsf1b(-/-) mice and their wild-type (WT) controls were transplanted with whole adult bone marrow (BM) cells, competent for both TNF-α receptors 1a and 1b (gfp(+) labeled), as well as with BM cells deficient for TNF-α receptor 1b. One month after transplantation CNV was induced by laser damage of Bruch's membrane. Pathologic angiogenesis was estimated qualitatively and quantitatively by histology on choroidal flatmounts and paraffin cross sections. Macrophage invasion was investigated by immunochemistry. Results. One month after transplantation the reconstitution rate measured by FACS analysis was >80% in gfp(+)-chimeric mice. Two weeks after laser injury reduced gfp(+)-cell invasion to the laser scars and decreased pathologic angiogenesis were observed in Tnfrsf1b(-/-) versus WT recipients. Approximately 70% of the invaded gfp(+) cells were labeled with macrophage marker F4/80. Transplantation of TNF-α receptor 1b-deficient BM cells in WT recipients reduced the CNV lesion compared with WT and Tnfrsf1b(-/-) recipients that received TNF-α receptor-competent BM cells. Transplantation of receptor 1b-deficient cells to Tnfrsf1b(-/-) recipients further reduced the degree of CNV formation. Conclusions. Signals through TNF-α receptor 1b expressed on BM -derived inflammatory cells mediate an increased inflammatory cell invasion and enhanced angiogenic response after laser-induced rupture of Bruch's membrane.

Conditions of retinal glial and inflammatory cell activation after irradiation in a GFP-chimeric mouse model.

PURPOSE: Microglia cells have been associated with immunologic defense and repair. The course of retinal disease after lethal irradiation for bone marrow depletion and substitution was evaluated with respect to macrophage and microglial involvement. METHODS: Lethal irradiation in C57BL/6 mice was conducted with a low-voltage radiation unit. The animals were randomized to shielded or unshielded radiation and subsequently received transplants of GFP+ bone marrow cells (beta-actin promoter). The GFP transformation rate was analyzed by flow cytometry. GFP+ cells in the retina were examined for co-localization with macrophage and dendritic cell markers at various time points between 1 and 7 months after irradiation. Clodronate liposomes were used to investigate the fate of migrated and residential microglia cells. Pathologic angiogenesis was investigated in laser-induced choroidal neovascularization (CNV) after unshielded and shielded irradiation. RESULTS: Flow cytometry revealed average transformation rates of 78.2% in unshielded and 64.1% in shielded group. Four weeks after transplantation, perfused flat mounts were virtually free of extravasal GFP+ cells in both groups, whereas 4 months after irradiation, cluster cell infiltrations, preferentially in the peripheral retina, became apparent exclusively in the unshielded group. Cell morphology ranged from oval, to a few extensions, to dendritiform with long-branched extensions. Clodronate treatment resulted in a reduction of GFP+ cells in the retinal tissue when applied 3 months after unshielded irradiation. Although GFP+ cells accumulated in the choroidal scar after laser treatment, in both the shielded and unshielded groups, GFP+ cells in the overlying retina were restricted to the unshielded group. CONCLUSIONS: Approximately 3 months after lethal full-body irradiation including the eye, bone marrow-derived leukocytes exhibit a wound-healing reaction, and unlike physiological turnover, infiltrate the retina and form microglial cells.

Differential role of tumor necrosis factor (TNF)-alpha receptors in the development of choroidal neovascularization.

PURPOSE: Tumor necrosis factor alpha (TNF)-alpha contributes to inflammation-associated angiogenesis. This study investigates the role of TNF-alpha receptors 1a and 1b in the development of choroidal neovascularization (CNV). METHODS: CNV was induced in Tnfrsf1a(-/-) and Tnfrsf1b(-/-) mice with C57Bl6/J background and their wild-type (WT) (C57Bl/6J) controls by laser damage to the Bruch's membrane. TNF-alpha expression in RPE/choroid was determined by Western blot analysis. Pathologic angiogenesis was estimated qualitatively and quantitatively by fluorescein angiography and histology on choroidal flat mounts and paraffin cross-sections. Inflammatory cell invasion was investigated by clodronic acid depletion of circulating macrophages and immunochemistry, and the apoptotic activity was investigated by TUNEL assay and by caspase-3 and caspase-8 expression. Receptor 1b-specific Bmx/Etk kinase was detected by immunochemistry. RESULTS: TNF-alpha levels were elevated after laser treatment. Severe CNV lesions and increased macrophage invasion were observed in Tnfrsf1a(-/-) compared with WT and Tnfrsf1b(-/-) mice. Increased immunoreactivity for Bmx/Etk kinase corresponded to the severity of CNV formation. Reduced pathologic angiogenesis and macrophage invasion in Tnfrsf1b(-/-) mice (vs. WT and Tnfrsf1a(-/-)) was accompanied by enhanced endothelial cell apoptosis and by caspase-3 and caspase-8 activation. CONCLUSIONS: Receptor 1b promotes the recruitment of inflammatory cells to the site of injury and exacerbated pathologic angiogenesis probably by way of the Bmx/Etk-kinase-dependent pathway in the absence of receptor 1a. On the other hand, receptor 1a-dependent apoptosis in the absence of receptor 1b leads to reduced inflammatory response and CNV lesions after laser treatment. This demonstrates the potential for specific targeting of TNF-alpha receptors for future therapies of inflammation-associated choroidal neovascularization.

TNF-alpha mediated apoptosis plays an important role in the development of early diabetic retinopathy and long-term histopathological alterations.

PURPOSE: The pathophysiology of diabetic retinopathy involves leukocyte adhesion to retinal vasculature, early blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell death. We investigated the involvement of tumor necrosis factor alpha (TNF-alpha) in diabetes-related histopathological changes in two relevant rodent models. METHODS: In short-term studies, Long-Evans rats with streptozotocin-induced diabetes were treated with or without the TNF-alpha inhibitor, etanercept. For long-term studies, tumor necrosis factor receptor I (TNF-RI)-deficient mice and TNF-RII-deficient mice, as well as C57/Bl6 wild-type mice, were fed 30% galactose for up to 20 months. The retinal histopathological alterations of hypergalactosemia were analyzed in trypsin digest preparations. Endothelial cell injury and apoptosis in rat retinas were evaluated by propidium iodide, TUNEL, CytoDeath staining, and DNA fragmentation ELISA. Caspase 3 and 8 activity was evaluated by immunoblotting and quantitative enzymatic activity assay. RESULTS: Etanercept suppressed caspase activation, retinal cell injury, and apoptosis in short-term diabetic rats. Pericyte and endothelial cell loss were also reduced in long-term hypergalactosemic mice. Long-term studies demonstrated that pericyte loss and endothelial cell loss were reduced in comparison to wild-type diabetic controls. CONCLUSIONS: Our study identifies an important role for TNF-alpha in the pathogenesis of signature diabetic retinopathy pathologies and demonstrates that etanercept can inhibit retinal cell death and long-term complication of diabetes. Taken together, our results suggest that etanercept could prove beneficial in preventing both early and late vascular diabetic complications.