Biomedical subjects

S J Ryan

Publications and source records attributed to S J Ryan.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cellular response in subretinal neovascularization induced by bFGF-impregnated microspheres.

PURPOSE: To determine the sequence of cellular changes associated with a new rabbit model of subretinal neovascularization (SRN) induced by subretinal injection of basic fibroblast growth factor (bFGF)-impregnated microspheres. METHODS: bFGF-impregnated gelatin microspheres, prepared by forming a polyion complex between gelatin and bFGF, were subretinally implanted into rabbit eyes. The eyes were studied by immunochemistry at 3 days to 8 weeks after implantation. Antibodies to CD4, CD8, cytokeratin, CD31, glial fibrillary acidic protein (GFAP), and RAM11 were used. RESULTS: Cytokeratin-positive retinal pigment epithelial (RPE) cells appeared on day 3 and continued to increase in number in the subretinal space throughout the growth of the SRN membrane, becoming the predominant cell type. Macrophages (RAM11-positive) appeared early, but most disappeared within 7 days. GFAP-positive Müller cells were evident early in the retina but migrated into the subretinal space after 7 days; the gliotic adhesion they formed between the retina and the SRN membrane was prominent at 8 weeks. CD31-positive endothelial cells were first evident at 14 days and formed neovascular channels that were still present for up to 8 weeks. CD4- and CD8-positive lymphocytes appeared in the early stages but were few in number. CONCLUSIONS: SRN membranes are primarily composed of RPE cells and vascular endothelial cells. The membrane adheres to the retina by a gliotic band. The cellular components involved in the membrane of this model resemble those found in SRN membranes removed from patients with age-related macular degeneration.

Animals↗

PubMed1998

Retinal pigment epithelial cells secrete and respond to hepatocyte growth factor.

Hepatocyte growth factor (HGF) is normally expressed by mesenchymal cells while its receptor, c-Met, is expressed in epithelial cells. Since HGF is critically involved in epithelial-mesenchyme interactions and the retinal pigment epithelium (RPE) is present at the interface between the retina and choroid, this study was initiated to determine whether the RPE expresses or responds to HGF in vitro. Cultured adult and fetal human RPE expressed mRNA for HGF and c-Met by RT-PCR. ELISA assay demonstrated the secretion of HGF into RPE culture supernatants. Tyrosine phosphorylation of c-Met was constitutively found in 72 hour RPE cultures and could be rapidly induced in serum-starved cells by concentrated RPE supernatants. HGF was mitogenic for cultured RPE (100 ng/ml.) and stimulated their chemotaxis (maximal response at 50 ng/ml). RPE are one of only a very limited number of epithelia that express both HGF and its receptor, suggesting the possibility of an autocrine action for this growth factor.

Adult↗

PubMed1998

Mitogen-activated protein kinase activation mediates PDGF-directed migration of RPE cells.

Growth factor-directed migration is a critical component of the wound healing response although little is known about the signaling pathways involved. We examined the effect of inhibiting the mitogen-activated protein kinase (MAPK) pathway on platelet-derived growth factor (PDGF) and fibronectin-induced cell migration of human retinal pigment epithelial (RPE) cells. Using transwell cell-culture chambers, the effect of PDGF-BB (10-50 ng/ml) and fibronectin on components of migration was measured with or without the MAPK pathway inhibitor PD98059 (10-30 microM) MAPK activation of serum-starved cells by PDGF-BB was demonstrated by an immunoprecipitation/kinase assay and by immunohistochemistry using antibody specific for phosphorylated MAPK. PDGF-BB (10 ng/ml) stimulated MAPK activity in RPE (10 min) and its nuclear localization (1 h). PD98059 inhibited the activation of MAPK by PDGF-BB or serum. PDGF-BB stimulated RPE chemokinesis, chemotaxis, and haptotaxis; chemokinesis was additively increased and chemotaxis synergistically increased by the presence of a fibronectin substratum. PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis but inhibited chemokinesis only at higher PDGF-BB (50 ng/ml) concentrations in the presence of fibronectin substratum. These results demonstrate that MAPK is critically involved in multiple components of RPE migration in vitro and suggest the potential of targeting MAPK to inhibit RPE migration in vivo.

Becaplermin↗

PubMed1998

Advanced glycation end products in age-related macular degeneration.

OBJECTIVE: To investigate the localization of N epsilon-(carboxymethyl)lysine (CML), a component and major immunologic epitope of advanced glycation end products, in aged eyes and choroidal neovascular membranes (CNVMs) surgically excised from eyes with age-related macular degeneration. METHODS: Immunohistochemistry for CML was performed using 8 snap-frozen, surgically excised CNVMs. Twelve eyes from patients aged 69 to 82 years and 2 donor eyes, 1 each from a 23-week-old fetus and 21-year-old patient, without age-related macular degeneration or diabetic retinopathy were also examined. To determine if retinal pigment epithelial cells in CNVMs accumulate advanced glycation end products, cytokeratin and CML were stained in paired serial sections. RESULTS: Soft, macular drusen and/or basal laminar and basal linear deposits were observed in 8 of 12 aged eyes. Each case showed CML accumulation, while overlying retinal pigment epithelial cells showed no accumulation in all 12 eyes. In CNVMs, however, retinal pigment epithelial cells showed CML accumulation in their cytoplasm. CONCLUSION: The additional accumulation of advanced glycation end products in soft, macular drusen and/or retinal pigment epithelial cells may play a role in the pathogenesis of CNVM formation in age-related macular degeneration. CLINICAL RELEVANCE: Recently, advanced glycation end products have been found to play a role both in aging changes and neovascularization. Localization of advanced glycation end products in the above-mentioned tissue may lead to a better understanding of the pathogenesis of age-related macular degeneration.

Adult↗

PubMed1998

Verapamil inhibits proliferation, migration and protein kinase C activity in human retinal pigment epithelial cells.

The effects of three calcium channel blockers, verapamil, diltiazem and nifedipine, were examined on in vitro proliferation and migration of human retinal pigment epithelial cells. Human retinal pigment epithelial cells were seeded in Dulbecco's modified essential medium with 10% fetal bovine serum and different concentrations of the three calcium channel blockers. After 3 days of treatment, cell proliferation was determined by cell counting and by [3H]-thymidine uptake. Cell viability was determined with trypan blue exclusion. For determination of cell migration, retinal pigment epithelial cells were grown to confluence and then growth-inhibited with mitomycin C. After a 3 mm zone was denuded, the cells were treated with different concentrations of the calcium channel antagonists. After 24 hr, the cells that had migrated over the wound edge were counted. To determine the involvement of protein kinase C in the verapamil effect, its activity was measured in both verapamil-treated and untreated cells. Verapamil dose dependently inhibited serum-induced proliferation of retinal pigment epithelial cells, when measured by cell number (IC50 14.6 microM) or [3H]-thymidine incorporation (IC50 11.3 microM). At concentrations of 15 microM and below, there was no effect on cell viability, as determined by morphology and trypan blue exclusion. Diltiazem inhibited cell proliferation at a concentration of 100 microM; however, 100 microM nifedipine had no effect. Verapamil showed a significant inhibition of serum-induced migration in the range of 10 microM to 0.1 microM. The IC50 of the inhibition of retinal pigment epithelial cell proliferation and migration by verapamil is significantly higher than that seen for effects on calcium channel blockage. Eight micromolar verapamil reversibly inhibited total protein kinase-C activity in retinal pigment epithelial cells suggesting the possibility that the drug may act by inhibiting the protein kinase-C pathway. These data suggest the potential of the calcium channel blocker verapamil as a pharmacological modulator of disorders such as proliferative vitreoretinopathy in which there is increased retinal pigment epithelial cell proliferation and migration.

Calcium Channel Blockers↗

PubMed1998

Retrovirus-mediated gene transfer targeted to retinal photocoagulation sites.

Diabetic retinopathy is a major cause of acquired blindness due to the development of retinal neovascularization and associated traction retinal detachment. It is commonly treated with retinal photocoagulation therapy; however, progression to blindness remains a significant problem. To determine the feasibility of adjunctive anti-angiogenic gene therapy, we evaluated the capability of retroviral vectors, which transfer exogenous genes only into dividing cells, to transfer and express a beta-galactosidase gene selectively into photocoagulation sites. Thirty-five rabbits received 30 retinal photocoagulation burns in the right eye followed 2 days later by beta-galactosidase (G1nBgSvNa) or control (G1XSvNa) vector injection into the subretinal space. Beta-galactosidase expression was observed in the photocoagulation sites from 5 days after vector administration (31.7+/-7.0%) to 12 weeks (6.7+/-3.4%). Immunohistochemical studies of the treated retinas using antibody Ber-MAC3 and anti-cytokeratin antibodies revealed that transduced cells were macrophages and retinal pigment epithelial cells. To determine feasibility in a primate, two monkeys received 10 laser burns in the macula superior to the fovea followed 2 days later by G1nBgSvNa vector. beta-galactosidase expression was found in photocoagulation sites and foveal retina was well preserved. We conclude that gene transfer to retinal photocoagulation sites provides stable expression of the transduced gene with relatively high efficiency. This feasibility study suggests the possibility of transferring genes encoding for anti-angiogenic factors into photocoagulation sites to improve the efficacy of laser photocoagulation therapy.

Angiography↗

PubMed1998

Ability of retroviral transduction to modify the angiogenic characteristics of RPE cells.

BACKGROUND: Retinal pigment epithelial (RPE) cells play an important role in the modulation of ocular angiogenesis. Transduction of RPE cells with retroviral vectors bearing modulating genes can result in long-term transgene expression and may alter the angiogenic characteristics of RPE cells. This study was designed to determine whether changes in angiogenic characteristics of RPE cells result from transduction with retroviral vectors bearing modulating genes, using in vitro angiogenic assays, including analysis of endothelial proliferation and wound healing. METHODS: Human RPE cells were transduced with retroviral vectors bearing either a urokinase-type plasminogen activator (u-PA) or a tissue-type plasminogen activator (t-PA) cDNA. Ten weeks after gene transfer, RPE cells transduced with the u-PA (u-PA-RPE cells) or the t-PA cDNA (t-PA-RPE cells), or untransduced (control) RPE cells, were cocultured with human umbilical vein endothelial cells (HUVECs) by contacting and non-contacting coculture methods. The effects of these cells on proliferation and in vitro "wound healing" of HUVECs were evaluated. RESULTS: Over 18 weeks, u-PA-RPE cells released large amounts of biologically active u-PA (total amount, 50.2 +/- 9.7 ng/10(6) cells/24 h), while t-PA-RPE cells released large amounts of functional t-PA (15.4 +/- 3.2 ng/10(6) cells/24 h). Control RPE cells did not release any detectable t-PA or u-PA. In the proliferation assay, u-PA-RPE cells stimulated HUVEC proliferation in contacting cell cultures, but not in non-contacting cell cultures. In contrast, t-PA-RPE cells, normal RPE cells or exogenous u-PA had no effect on HUVEC proliferation. In the wound healing assay, u-PA-RPE cells in contacting coculture and exogenous u-PA stimulated wound healing of HUVECs, while non-contacting u-PA-RPE cells, t-PA-RPE cells and normal RPE cells had no effect on HUVEC wound healing. RPE cells transduced with u-PA secreted large amounts of u-PA for as long as 18 weeks, and these cells stimulate HUVEC proliferation and in vitro wound healing. As a result, the angiogenic characteristics of RPE cells can undergo long-term changes. CONCLUSIONS: These results suggest that genetically modified RPE cells can be used to modulate ocular angiogenesis and may have potential for gene therapy of ocular diseases.

Cell Division↗

PubMed1998

Rapid isolation of choriocapillary endothelial cells by Lycopersicon esculentum-coated Dynabeads.

In vitro studies of choroidal endothelial cells may be critical for understanding the pathogenesis of neovascularization in age-related macular degeneration, since endothelial cells from different sites are highly heterogeneous in their morphology and behavior. Isolation of choroidal endothelial cells is complicated and labor intensive because of the small size of the choroid and the difficulty of excluding contaminating cells. We describe a rapid, simplified method for the isolation of bovine choroidal endothelial cells using microdissection followed by the use of superparamagnetic beads (Dynabeads) coated with the endothelial cell-specific lectin Lycopersicon esculentum, which selectively binds to fucose residues on the endothelial cell surface. Cells bound to beads are isolated using a magnetic particle concentrator. Isolated cells grew to confluence in a monolayer with a cobblestone morphology and were shown to be endothelial cells by their greater than 95% immunoreactivity to von Willebrand factor and phagocytosis of dil-acetylated LDL. Isolated cells grew as tubes in three-dimensional cultures. This method markedly reduces the time needed for pure culture of cells and makes the in vitro study of choroidal endothelial cells practical and reproducible.

Animals↗

PubMed1998

Long-term studies on allotransplantation of rabbit retinal pigment epithelial cells double-labelled with 5-bromodeoxyuridine and natural pigment.

OBJECTIVE: To facilitate the identification of transplanted retinal pigment epithelial (RPE) cells, we sought to double-label the cells with 5-bromodeoxyuridine (BrdU) and with natural pigment. The BrdU is not lost during cell division but does require immunohistochemical methods for visualization; the pigment on the other hand, allows immediate, obvious identification, but is gradually lost with cell division. Together they provide a convenient, long-term double label. METHODS: Non-confluent RPE cells at the second to the fifth passages were labelled with 5-BrdU and pigment. The double-labelled RPE cells were transplanted onto Bruch's membrane of 72 eyes of New Zealand albino rabbits. The labelled cells were localized by anti-BrdU antibody and the avidin biotin-alkaline phosphatase complex (ABC-AP) method, and by visible inclusions of pigment. RESULTS: The transplanted RPE cells had distinct basal and apical morphology, and were in close contact with the photoreceptor outer segments of the host. The BrdU label was restricted to the nuclei of the RPE cells, which were stained blue. The pigment was located in the cytoplasm of the apical portion of these RPE cells. No evidence of severe rejection was seen. CONCLUSIONS: Using this double-label method, transplanted RPE cells could be readily and reliably identified till one year after transplantation. The transplanted RPE cells revealed normal morphology with some function: they had distinct basal and apical morphology as seen in close contact with the outer segment of photoreceptor cells. The junctional complexes were well formed with neighboring RPE cells. The transplanted RPE cells phagocytose shed outer segments of the host. No evidence of rejection was observed, suggesting that the subretinal space of the rabbit may possess some degree of immunologic privilege. This experiment provides reliable evidence for the clinical research of allotransplantation of RPE cells.

Affinity Labels↗

PubMed1998

Retrovirus-mediated gene transfer to photocoagulation-induced choroidal neovascular membranes.

PURPOSE: To determine the feasibility of experimental gene transfer to laser-induced choroidal neovascular membrane (CNVM) in rats, with a retroviral vector containing the reporter construct beta-galactosidase (beta-gal). METHODS: Laser photocoagulation was used to induce CNVM in rats. To ascertain the duration of beta-gal expression in the CNVM, 23 rats received 10 burns (75 microm, 100 mW, 0.1 seconds) in their right eyes, and beta-gal expression was examined from day 3 to 4 months. In addition, 14 pigmented rats were treated with 3 photocoagulation burns in their right eyes. beta-gal vector was injected into the vitreous or subretinal space 2 days later. On day 14, fluorescein angiography was performed to detect choroidal neovascularization. Then, beta-gal expression in each photocoagulation-induced CNVM was examined by observing the exposed fundus of the eyes stained with the beta-gal substrate X-Gal. RESULTS: beta-gal expression was identified in the CNVM induced by photocoagulation from day 5 (16.2% +/- 6.8% of the lesions) to 4 months (3.7% +/- 2.4%). Histopathologic examination revealed beta-gal-transduced macrophages and spindle-shaped cells, which amounted to 1.12% +/- 0.58% (at 2 weeks) of the total cells in the CNVM. beta-gal expression was restricted to the CNVM, and there was no beta-gal transduction in surrounding normal retinochoroidal tissue. There was no correlation between choroidal neovascularization formation and beta-gal expression. CONCLUSIONS: The feasibility of gene transduction targeted to the photocoagulation-induced CNVM was demonstrated using retroviral vectors. By transducing functional genes, this model could be useful for investigating the possibility of gene therapy to inhibit formation of the CNVM in age-related macular degeneration.

Animals↗

PubMed1997

Vitrectomy with silicone oil or long-acting gas in eyes with severe proliferative vitreoretinopathy: results of additional and long-term follow-up. Silicone Study report 11.

BACKGROUND: The Silicone Study evaluated the outcomes of vitreoretinal surgery for retinal detachment with proliferative vitreoretinopathy (PVR). OBJECTIVE: To evaluate short-term (up to 36 months) outcomes in eyes randomized to silicone oil or perfluoropropane gas and long-term (up to 72 months) outcomes in eyes with attached maculas at 36 months. DESIGN: Prospective, randomized, multicentered surgical trial. SETTING: Community- and university-based vitreoretinal practices. PATIENTS: Two-hundred sixty-five eyes with PVR randomized to perfluoropropane gas and silicone oil with follow-up through 3 years (cohort 1) and 249 eyes with attached maculas at 36 months (121 eyes randomized to long-acting gas [either sulfur hexafluoride or perfluoropropane] and 128 eyes randomized to silicone oil) with follow-up up to 6 years (cohort 2). Both cohorts consisted of eyes that had and had not undergone vitrectomy for PVR (groups 1 and 2, respectively) before randomization. Of the 265 eyes in cohort 1, 24-month follow-up data were available for 218 eyes (82%) and 36-month follow-up data were available for 196 eyes (74%). Of 208 eyes in cohort 2, 48-month follow-up data were available for 146 eyes (70%), 60-month follow-up data for 119 eyes (57%), and 72-month follow-up data for 73 eyes (35%). INTERVENTIONS: Vitrectomy surgery for PVR with a long-acting gas or silicone oil as the intraocular tamponade. MAIN OUTCOME MEASURES: Changes in visual acuity, recurrent retinal detachment, and incidence of complications. RESULTS: In group 1 of cohort 1, compared with oil-treated eyes, gas-treated eyes had a higher rate of complete retinal reattachment from 18 to 36 months (P < .05). No other differences were found. In group 2 of cohort 1, no notable differences were found between treatment arms. In cohort 2, during 6 years of follow-up, attachment of the macula was maintained for all eyes. No notable differences in the rates of complete retinal attachment, visual acuity of 5/200 or better, or glaucoma were found between treatment groups. In contrast, gas-treated eyes had more hypotony (P < .001). Silicone oil-treated eyes that underwent subsequent surgery were more likely to have the oil retained (P = .02). Compared with oil-retained eyes, oil-removed eyes had higher rates of complete posterior attachment (P = .01) and of a visual acuity of 5/200 or better (P < .001) and less keratopathy (P < .05). Compared with oil-removed eyes, gas-treated eyes had a worse visual acuity outcome (P < .05) and more hypotony (P < .01). CONCLUSION: The Silicone Study showed that silicone oil and perfluoropropane gas were equal in most respects for the management of retinal detachments with PVR. Success in the first surgery for PVR is paramount for obtaining better visual results. Overall, surgery for PVR had a high likelihood of retinal reattachment, and if anatomically and visually successful at 3 years, there is an excellent chance that the results will be maintained over the long-term.

Adult↗

PubMed1997

Post-traumatic proliferative vitreoretinopathy. The epidemiologic profile, onset, risk factors, and visual outcome.

PURPOSE: The purpose of the study was to characterize the clinical development of proliferative vitreoretinopathy (PVR) after trauma in the human eye. METHODS: A chart review was performed on the records of 1564 patients with ocular trauma seen at a large metropolitan hospital. The frequency, type of ocular trauma, time to onset, potential risk factors, and visual outcome for PVR were evaluated. RESULTS: Proliferative vitreoretinopathy occurred in 71 (4%) of 1654 injured eyes. Of these 71 injured eyes, 30 (42%) resulted from rupture, 15 (21%) from penetration, 13 (18%) from perforation, and 7 (10%) from confusion. Six (9%) were associated with an intraocular foreign body (IOFB). The frequency of PVR following perforation, rupture, penetration, IOFB, and contusion was 43%, 21%, 15%, 11%, and 1%, respectively. Overall, those eyes that developed PVR had a poorer visual outcome, with PVR being the primary reason for visual loss. The time from injury to onset of PVR was shortest after perforation (median, 1.3 months), followed by rupture (2.1 months), IOFB (3.1 months), penetration (3.2 months), and contusion (5.7 months). Vitreous hemorrhage was the strongest independent predictive factor for the development of PVR. A long, posteriorly located wound and persistent intraocular inflammation were also important risk factors for PVR. CONCLUSIONS: These results suggest that PVR is a common complication following a variety of ocular injuries, and that it is associated with a poor visual outcome. Its frequency, onset, and outcome are strongly dependent on the nature of the trauma. Specific high-risk groups are identified as candidates for more aggressive therapy.

Adolescent↗

PubMed1997

Hypericin inhibits choroidal endothelial cell proliferation and cord formation in vitro.

PURPOSE: To evaluate the effect of hypericin on bovine choroidal endothelial cell proliferation and cord formation and on protein kinase C activity. METHODS: The effect of hypericin (0.1-5 microM) on bovine choroidal endothelial cell proliferation was determined by cell number counting and a 3H-thymidine uptake assay in media containing 1, 5 or 10% serum. For the cord formation assay, bovine choroidal endothelial cells were seeded on basement membrane matrix, and the lengths of the capillary-like structures (cords) formed were quantified by image analysis. The effect of hypericin on cord formation was evaluated in the presence of serum or vascular endothelial growth factor. The effect of hypericin on protein kinase C activity was also measured in the presence or absence of light. RESULTS: Hypericin inhibited bovine choroidal endothelial cell proliferation in a dose-dependent manner in the presence of light but not in the dark. Serum dose-dependently masked the inhibition of DNA synthesis by hypericin. Cord formation by bovine choroidal endothelial cells was stimulated by serum or vascular endothelial growth factor and inhibited by hypericin in the presence of light. Protein kinase C activity was completely inhibited by hypericin in the presence of light but only mildly inhibited in the absence of light. CONCLUSIONS: Hypericin inhibits bovine choroidal endothelial cell proliferation and cord formation and choroidal endothelial cell protein kinase C activity. These results suggest that hypericin should be further investigated in animal models for its potential to inhibit subretinal neovascularization.

Animals↗

PubMed1997

Retrovirus-mediated transfer of the suicide gene into retinal pigment epithelial cells in vitro.

PURPOSE: Human retinal pigment epithelial (HRPE) cells are a major cell component in proliferative vitreoretinopathy (PVR) membranes. We investigated the feasibility of killing HRPE cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, also known as the suicide gene, into HRPE cells followed by ganciclovir treatment, and to study the so-called bystander effect. Such a treatment plan might serve as a possible therapy for PVR. METHODS: Transduction efficiency was determined using retroviral vectors encoding the beta-galactosidase reporter gene. To evaluate the efficacy of suicide gene therapy. HRPE cells were transduced with retroviral vectors encoding the HSV-tk gene (G1TkSvNa), with empty vectors or without vectors, and were treated with 5 micrograms/ml ganciclovir. Sensitivity of HSV-tk positive HRPE cells to various concentrations of ganciclovir was evaluated. To demonstrate the bystander effect, HSV-tk positive cells were cultured with HSV-tk negative cells at varying proportions. RESULTS: Transduction efficiency in vitro was 15.1 +/- 4.8%. Cell growth was significantly inhibited after transduction with G1TkSvNa followed by ganciclovir treatment (P < 0.01). Ganciclovir showed dose- and time-dependent cytotoxicity only on HSV-tk positive cells. The concentration that resulted in 50% inhibited was 0.1 micrograms/ml. In terms of the bystander effect, after ganciclovir treatment the viability of co-cultured cells decreased with increasing populations of HSV-tk positive cells. CONCLUSIONS: HRPE cells were successfully transduced with the HSV-tk gene via retroviral vectors and displayed a strong bystander effect after treatment with ganciclovir. These results suggest that retrovirus-mediated suicide gene therapy might be a feasible treatment strategy for PVR.

Antiviral Agents↗

PubMed1997

Retinal pigment epithelium cells promote the maturation of monocytes to macrophages in vitro.

Proliferative vitreoretinopathy is characterized by excessive cell proliferation within the eye; retinal pigment epithelial (RPE) cells form the majority of proliferating cells and interact with infiltrating leukocytes including monocytes. The purpose of this study was to determine the effect of RPE cells on the maturation of monocytes to macrophages. The enriched monocyte fraction of peripheral blood mononuclear cells was either cultured with or without RPE cells. The expression of the maturation-associated antigen CD16 on monocytes was assessed by flow cytometry, and the concentration of bioactive transforming growth factor-beta (TGF-beta) in the culture supernatant measured by mink lung epithelial cell (Mv1Lu) bioassay. The cellular density of CD16 in terms of mean fluorescence intensity was significantly higher on monocytes in coculture with RPE cells (p = 0.0153) than on monocytes in monoculture. The CD16 expression was significantly (p = 0.0093) reduced when antibodies to TGF-beta were added to the culture medium. RPE cells did not express CD16. Supernatants from cocultures also contained active TGF-beta (76.7 +/- 23.8 pg/ml), while in those of cell monocultures TGF-beta was close to the detection limit. We conclude that RPE cells stimulate and modulate the differentiation of monocytes to macrophages. Bioactive TGF-beta generated in the coculture was in part responsible for this effect. It seems likely that RPE cells or interactions between RPE cells and monocytes could be an important factor in inflammatory/immune processes and wound healing in the eye, which are probably involved in proliferative vitreoretinopathy.

Cell Differentiation↗

PubMed1997

Monocyte-macrophage differentiation induced by coculture of retinal pigment epithelium cells with monocytes.

Macrophages play an important role in proliferative vitreoretinopathy (PVR). Since macrophage maturation may be modulated by the local microenvironment, we determined the effect of retinal pigment epithelium (RPE) cells interacting with monocytes on macrophage maturation. The enriched monocyte fraction of peripheral blood mononuclear cells was cocultured with RPE cells. Cell-free supernatants conditioned during the culture periods 0-4 days (growing RPE cells) and 4-8 days (confluent RPE cells) were tested for their capacity to induce monocyte/macrophage differentiation of the promyelocytic cell line HL-60, which was measured by the expression of CD11c and CD14 and flow cytometry. RPE cells released factors that increased the CD14 expression on HL-60 cells in terms of percentage of positive cells (22.7% vs. control 10.4%). RPE-cell-conditioned supernatants had no effect on the CD11c expression. Monocytes secreted substances that increased the expression of CD11c (20.5% vs. control 9.1%; p = 0.003) and CD14 (31.6% vs. control 10.4%; p < 0.0001). Supernatants from cocultures increased the CD11c (19.8%) and CD14 expression (40.8%) to values that were similar to the sum of those of cell monocultures. Supernatants conditioned during the later culture had no effect on CD14 and CD11c expression. We conclude that invading monocytes and RPE cells could create an intraocular microenvironment that supports macrophage maturation during the initial stage of PVR.

CD11 Antigens↗

PubMed1997

Ocular gene therapy: experimental studies and clinical possibilities.

The Human Genome Project will identify, map and sequence all 50,000-100,000 human genes and will provide the tools to determine the genetic basis of both common and rare diseases. Understanding the genetic basis of human disease will allow for the development of highly specific drugs and for replacement of the altered gene through gene therapy. Gene therapy may also be used to introduce a new function into cells with resulting therapeutic benefit. Genes may be delivered into cells in vitro or in vivo utilizing viral or nonviral vectors. Viral vectors which have been used include retroviruses, adenoviruses, adeno-associated viruses and herpes viruses. Ocular disorders with the greatest potential for benefit of gene therapy at the current time include hereditary ocular diseases, including retinitis pigmentosa, tumors such as retinoblastoma or melanoma, and acquired proliferative and neovascular retinal disorders. We have demonstrated the feasibility of ocular gene therapy in a rabbit model of proliferative vitreoretinopathy, using retroviral vectors containing the herpes simplex virus thymidine kinase 'suicide' gene. Although in vivo transduction efficiency is low, the strong bystander effect results in prominent killing of proliferating cells in this model leading to inhibition of disease. In the future, gene therapy has the potential for the replacement of defective gene products or introduction of new gene products into ocular cells. The selection of appropriate target genes and cells will be critical, as will the development of a methodology for safe, targeted gene transfer.

Animals↗

PubMed1997

Interleukin 1 in experimental proliferative vitreoretinopathy.

Since proliferative vitreoretinopathy (PVR) is associated with an inflammatory reaction and interleukin-1 (IL-1) initiates a cascade of inflammatory mediators, we determined the intravitreal levels of IL-1 in experimental PVR. In the blood-trauma model of PVR in rabbits, we measured the intravitreal levels of IL-1 alpha and IL-1 beta using a radioimmunoassay and performed immunohistochemical staining of cells from the vitreous. We found that on day 1 polymorphonuclear and from day 2 on mononuclear inflammatory cells predominated in the vitreous. IL-1 alpha- and IL-1 beta-positive cells with a large cytoplasm were observed on days 4 and 8. However, the IL-1 alpha and IL-1 beta concentrations in the vitreous did not exceed the radioimmunoassay sensitivity limit 8 h, 1, 2, 4 and 8 days after trauma, while in 2 control rabbits injected with additional endotoxins we found 5.6 and 8 ng/ml IL-1 beta. We conclude that despite IL-1-positive cells, presumably macrophages, PVR is not associated with high levels of IL-1. No conclusions concerning the low levels of IL-1 alpha and IL-beta can be drawn due to the performance of the RIA.

Animals↗