Insulin-like growth factor binding proteins modulate Müller cell responses to insulin-like growth factors

PURPOSE: Müller cells are consistently identified in diabetic fibrocontractive ocular tissues and, in response to insulin-like growth factor I, generate tractional forces of the type that cause retinal detachment. Recent studies suggest that diabetes-associated increases in vitreous insulin-like growth factor activity cannot be attributed to simple increases in concentration alone, suggesting that more complex biochemical changes in vitreous growth factor control mechanisms are involved. The goal of this study was to evaluate the contributions of vitreous insulin-like growth factor-binding proteins (IGFBPs) toward control of growth factor activity. METHODS: Native and recombinant IGFBPs effects were evaluated on IGF-I- and -II-stimulated Müller cells in tissue culture assays that involved cell incubation on three-dimensional collagen gels and that monitored progressive matrix condensation. IGFBP degradation by Müller cell-secreted proteases was assessed in Western ligand blots, and direct stimulatory effects were evaluated by incubating cells with IGFBPs alone. RESULTS: IGFBP direct stimulatory effects on Müller cells were significant, but relatively modest, and IGFBP modulation through Müller cell-secreted proteases was undetectable. In contrast, IGFBP inhibitory effects on IGF-I and -II were highly variable and, in some cases, profound. IGFBP-3 effectively inhibited IGF-I and -II stimulation with detectable effects at concentrations equimolar to the growth factor. IGFBP-1, -2, -4, and -5 were of intermediate effectiveness as inhibitors, 3- to 11-fold less active than IGFBP-3. IGFBP-6 had virtually no inhibitory effects on IGF-I, but was moderately effective against IGF-II. CONCLUSIONS: IGFBP effects on IGF-I- and -II-stimulated Müller cells are primarily inhibitory with only modest direct stimulatory effects of limited physiologic relevance. IGFBP-2 and -3, the major binding proteins identified in vitreous, most likely function as the vitreous growth factor sink and control ligand activity through sequestration.     

Müller cell production of insulin-like growth factor-binding proteins in vitro: modulation with phenotype and growth factor stimulation

PURPOSE: Müller cells are present in diabetic fibrocontractive ocular tissues and generate tractional forces in response to insulin-like growth factors. Recent studies indicate that diabetes-associated increases in vitreous insulin-like growth factor activity are, in part, attributable to changes in insulin-like growth factor binding proteins (IGFBPs). The objectives of this study were to evaluate Müller cells as a source of IGFBPs and characterize changes associated with cell phenotype and growth factor stimuli known to be present in diabetic vitreous. METHODS: Müller cells isolated from normal porcine retina were maintained in culture for 1 and 5 weeks, yielding phenotypes described as proliferative and myofibroblastic. RNA preparations from porcine liver, retina, and Müller cell cultures were evaluated by RT-PCR and Northern blot analysis. IGFBP production was verified by Western ligand and Western blot analysis of Müller-cell-conditioned media and detergent-extracted proteins. RESULTS: Molecular biological analyses of RNA from normal retina and from proliferative and myofibroblastic Müller cells did not detect message for IGFBP-1, but revealed progressive increases in message abundance for IGFBP-2, -3, -4, and -6. IGFBP-5 message was detected in all samples, but was least abundant in myofibroblastic Müller cells. Stimulation of myofibroblastic Müller cells by IGF-I and -II, but not PDGF, further increased message abundance and production of IGFBP-2, -4, -5, and -6, but not IGFBP-3. CONCLUSIONS: Müller cell production of IGFBPs changes with phenotype and, in most cases, is highest in the cells most likely to participate in fibrocontractive retinal disease. IGFBP production by these cells is further increased by IGF-I and -II, growth factors known to be present and active in proliferative vitreoretinal disorders, suggesting that Müller cells represent a potential source of vitreous IGFBPs in disorders involving this cell type.     

Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment epithelium.

Insulin-like growth factors (IGFs) I and II are mitogenic polypeptides structurally homologous to insulin, which are thought to mediate important neurobiologic actions in the CNS. The purpose of this study was to determine if cultured bovine retinal pigment epithelial cells (RPE) express IGF receptors and secrete soluble IGF binding proteins, and to characterize these receptors and binding proteins. We also characterized the soluble IGF binding proteins present in juvenile and adult rat vitreous and serum, as well as those in fetal bovine vitreous and serum, in order to facilitate identification of the RPE IGF binding protein, and to determine potential destinations for this protein once produced. Affinity labeling was used to characterize insulin, IGF-I and IGF-II receptors. Western radioligand blotting and immunoprecipitation were used to characterize IGF binding proteins. We found that RPE cells in culture express virtually no insulin receptors, and only modest amounts of IGF-I receptors. IGF-II receptors were abundantly expressed. Additionally, RPE cells secrete a soluble IGF binding protein which is immunologically related to IGFBP-2, the primary IGF binding protein produced in the central nervous system. Bovine vitreous was found to contain a mixture of IGF binding proteins (IGFBP). The most prominent IGFBP in this mixture is immunologically related to IGFBP-2. Likewise, juvenile and adult rat vitreous contained only one IGF binding protein that was shown to be immunologically related to IGFBP-2. Juvenile rat vitreous contained more binding activity corresponding to IGFBP-2 than did adult vitreous, suggesting developmental regulation. These data suggest that IGF's and their binding proteins may have important, and as yet undefined, roles in retinal neurophysiology.     

Involvement of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in corneal fibroblasts during corneal wound healing

PURPOSE: The involvement of downstream messengers of transforming growth factor (TGF)-beta in the differentiation of corneal fibroblasts into myofibroblasts was investigated. The effects of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 upregulated by TGF-beta were examined in human corneal fibroblasts, and the possible involvement of IGF axis components in corneal wound healing was assessed in a mouse model. METHODS: Human corneal fibroblasts were incubated with TGF-beta2 or IGF-I, to investigate IGF-I, IGF-II, IGFBP-3, type I collagen, and alpha-smooth muscle actin (alpha-SMA) mRNA, as well as IGFBP-3 protein expression, during myofibroblast differentiation. DNA synthesis was evaluated with a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. IGFBP-3 mRNA expression, protein expression, and immunolocalization were investigated in mouse corneas after photorefractive keratectomy (PRK). RESULTS: TGF-beta2 treatment induced expression of IGF-I and IGFBP-3 mRNA and of IGFBP-3 protein in human corneal fibroblasts. TGF-beta2 and IGF-I both stimulated expression of type I collagen. TGF-beta2 but not IGF-I potently stimulated alpha-SMA mRNA expression. IGF-I potently stimulated basal DNA synthesis, whereas IGFBP-3 inhibited it. IGF-I potently stimulated proliferation of TGF-beta2-activated myofibroblasts without reversing the activated fibrogenic phenotype, whereas IGFBP-3 suppressed IGF-I-induced proliferation of corneal fibroblasts. IGFBP-3 mRNA and protein increased in mouse corneas soon after PRK, when in vivo immunostaining of the corneas showed expression of IGFBP-3 in the deep layer of the corneal stroma. CONCLUSIONS: These results suggest that during corneal wound healing, TGF-beta stimulates IGF axis components, whereas IGFBP-3 may modulate IGF-I-induced myofibroblast proliferation to suppress corneal mesenchymal overgrowth.     

Expression of the IGF system in normal and diabetic transgenic (mRen-2)27 rat eye

PURPOSE: In the present study, a recently described model of diabetic eye disease was used to investigate the distribution of the insulin-like growth factor (IGF) system in the eyes of transgenic (mRen-2)27 rats (exhibiting hypertension and elevated serum and ocular renin levels) with streptozotocin-induced diabetes. METHODS: Female transgenic (mRen-2)27 rats were randomized to receive either streptozotocin (diabetic) or citrate buffer (control). After 10 months, the rats were killed and the eyes fixed and embedded in paraffin. In situ hybridization (ISH) was used to document the cellular distribution of mRNAs for components of the IGF system (IGF-I, IGF-I receptor [IGFIR] and IGF binding proteins [IGFBP]1 to -6) in the eyes. RESULTS: In nondiabetic rats, mRNA for IGFBP-1, -5, and -6; IGF-I; and IGFIR were detected in the retina. In addition, IGF-I mRNA was present in the cornea, IGFBP-1 mRNA was observed in the cornea and iris, and IGFBP-5 and -6 mRNAs were identified in the ciliary body, iris, and cornea. mRNAs for IGFBP-2, -3, and -4 were not found in the eyes. In diabetic rats, reduced levels of IGFBP-6 mRNA were detectable, whereas levels of IGFBP-5 mRNA were increased in the inner and outer retina, rods and cones, iris, cornea, and ciliary body. Other components of the IGF system in the eye were unchanged with diabetes. CONCLUSIONS: In the diabetic (mRen-2)27 rat, IGFBP-6 is downregulated and IGFBP-5 is upregulated by induction of diabetes. Because these IGFBPs may respectively have IGF-enhancing and IGF-inhibitory effects, these findings suggest a possible net IGF-enhancing effect induced by diabetes, providing further evidence for a role of the IGF system in the development of diabetic retinopathy.     

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres^®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.     

IGF-1-induced VEGF and IGFBP-3 secretion correlates with increased HIF-1 alpha expression and activity in retinal pigment epithelial cell line D407

PURPOSE: To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1 alpha and secretion of vascular endothelial growth factor (VEGF) and IGF binding protein (IGFBP)-3 in human retinal pigment epithelial (RPE) cell line D407. METHODS: D407 cells cultured in dishes or Transwell inserts were treated with cobalt chloride or varying doses of IGF-1. Whole cell lysates were assayed by immunoblot for HIF-1 alpha expression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1 alpha, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na(+)/K(+)-ATPase alpha-1 subunit and either the alpha or beta subunits of the IGF-1 receptor and visualized in Z-section using confocal microscopy. RESULTS: Immunoblot analysis of whole cell lysates from IGF-1-treated D407 cells revealed the upregulation of HIF-1 alpha protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1 alpha expression and nuclear localization, VEGF and IGFBP-3 synthesis and export, and IGF-1 action. Western and ligand blot analyses of RPE cell-conditioned medium indicated that IGF-1 induced increases in VEGF and IGFBP-3 secretion. Cells grown on Transwell inserts exhibited constitutive apical secretion of VEGF and IGFBP-3, which increased on apical or basolateral treatment with IGF-1. Confocal analysis of Transwell-cultured D407 cells confirmed the apical localization of the Na(+)/K(+)-ATPase alpha-1 subunit, characteristic of polarized RPE, with IGF-1 receptor alpha and beta subunits exhibiting a nonpolarized distribution. CONCLUSIONS: IGF-1 stimulates increased HIF-1 alpha expression as well as VEGF and IGFBP-3 secretion in D407 cells. Similar to their in vivo counterparts, D407 cells maintain reversed epithelial polarity. Apical secretion of VEGF and IGFBP-3 increases in response to either apical or basolateral IGF-1 stimulation consistent with the nonpolarized distribution of IGF-1 receptors.     

Insulin-like growth factor I stimulates proliferation, migration, and plasminogen activator release by human retinal pigment epithelial cells

The migration of retinal pigment epithelial (RPE) cells from their normal anatomic position to a new position in the vitreous cavity is a critical feature of proliferative vitreous retinopathy. To determine if insulin-like growth factor I (IGF I), which is present in the vitreous fluid of diabetics, stimulates RPE cells, we examined the effects of IGF I on the proliferation, chemotaxis, and release of plasminogen activator by these cells. At the concentrations of IGF I tested, significant proliferation of RPE cells is seen. Significant chemotaxis of the RPE cells also is seen at all the concentrations of IGF I tested. The mean number of migrating cells per high-powered field in control studies was 43 +/- 13 (x +/- SEM), and for IGF I at 2.5 ng and 50 ng/ml the mean numbers of migrating cells were 96 +/- 17 and 483 +/- 62, respectively (P less than 0.001 for each comparison). The IGF I response was noted to be dose-dependent. The chemotactic response noted at 50 ng/ml of IGF I was greater than the positive chemotactic control of 10% fetal calf serum. Addition of alpha IR-3, an IGF I receptor antibody, eliminated the IGF I chemotactic response. The effect of IGF I on the secretion of plasminogen activators was assessed using an immunological assay for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). Media conditioned by RPE cells have measureable levels of PAI and t-PA antigen. IGF I supplementation resulted in an increase of t-PA secretion and PAI secretion over basal levels. These studies support a role for IGF I in modulating RPE cell function.     

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF,and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.     

Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation

Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.     

Expression of IGFBP-3 by human retinal endothelial cell cultures: IGFBP-3 involvement in growth inhibition and apoptosis

PURPOSE: Growth hormone (GH), insulin-like growth factor (IGF), and somatostatin (SST) modulate each other's actions. SST analogues have been successfully used to treat proliferative diabetic retinopathy (PDR) that is unresponsive to laser therapy and to retard the progression of severe nonproliferative retinopathy to PDR. In this study, the endogenous expression of IGF-binding protein (IGFBP)-3 was examined in human retinal endothelial cells (HRECs), the direct effects of IGFBP-3 on HRECs were evaluated, and the possible involvement of IGFBP-3 in mediating the growth inhibitory effects of SST receptor (SSTR) agonists in HRECs was assessed. METHODS: The cellular localization of IGFBP-3 was examined with anti-IGFBP-3 and fluorescein-conjugated goat anti-rabbit IgG. HRECs were exposed to varying concentrations of human recombinant IGFBP-3, and growth inhibition was evaluated by thiazolyl blue (MTT) conversion. Apoptosis was examined using fluorochrome-annexin V staining. Conditioned media (CM) from SSTR2 agonist (L779976)-treated or SSTR3 agonist (L796778)-treated HRECs were analyzed by ELISA for changes in expression of IGFBP-3. RESULTS: HREC immunostaining showed cell surface and cytoplasmic IGFBP-3. Exogenous IGFBP-3 induced a dose-dependent inhibition of HREC proliferation and staining with fluorochrome-annexin V showed numerous apoptotic HRECs. HRECs exposed to the SSTR2 or SSTR3 agonists expressed IGFBP-3 in a concentration-dependent manner. CONCLUSIONS: Cultured HRECs expressed endogenous IGFBP-3. Exogenous administration of IGFBP-3-induced growth inhibition and apoptosis, supporting a regulatory role for IGFBP-3 in endothelial cells. SSTR agonists mediate their growth-inhibitory effect, in part, by increasing expression of IGFBP-3.     

Age and topographic variation of insulin-like growth factor-binding protein 2 in the human rpe

PURPOSE: Previous studies have shown that insulin-like growth factor-binding protein (IGFBP)-2 is markedly upregulated in senescent RPE cells in vitro, and might therefore be a marker of senescent cells in vivo. This study was conducted to determine whether IGFBP-2 expression in human RPE cells from the macula and periphery varies with age in vivo. METHODS: Paraformaldehyde (4%)-fixed and optimal cutting temperature (OCT) compound-embedded human eyes from 17 patients were cryosectioned and subjected to high-sensitivity digoxigenin (DIG)-labeled cRNA in situ hybridization to determine the expression of IGFBP-2. Complementary immunohistochemistry experiments using a polyclonal anti-IGFBP-2 antibody were performed to confirm IGFBP-2 protein expression. Specimens were examined by light microscopy, and images were captured with a digital camera. The total numbers of RPE cells and IGFBP-2 mRNA expression-positive RPE cells were counted for each section, and the ratio of labeled RPE cells to total RPE cells counted was calculated for both macular and peripheral regions of each donor. RESULTS: IGFBP-2 mRNA expression was detected in the ganglion cell layer, inner and outer nuclear layers, and inner segments of photoreceptor cells in all 17 eyes. In 16 of 17 eyes, IGFBP-2 mRNA expression was detected in the RPE. In 11, the ratio of labeled cells to total RPE cells counted per section in the macula was 1.2 times greater than the ratio in the periphery (P = 0.008). The ratio of labeled RPE cells in the macula decreased with age (P = 0.0064). Immunohistochemistry studies for IGFBP-2 confirmed the expression pattern found by in situ hybridization. CONCLUSIONS: There is a topographical and age-related change in IGFBP-2 expression in RPE cells from human donor eyes. This distribution is likely not to represent senescent RPE cells in vivo.     

生长因子对培养人眼视网膜色素上皮细胞增的调控作用

探讨生长因子对人眼视网膜色素上皮细胞增殖的调控作用。方法建立人眼ＲＰＥ细胞的培养体系，利用＾３Ｈ－ＴｄＲ掺入法和细胞计数观察表皮生长因子、碱性成纤维细胞生长因子胰岛素样生长因子对培养ＲＰＥ细胞的作用。     

Insulin-like growth factor-I and its receptor in neovascular age-related macular degeneration

PURPOSE: The insulin-like growth factor (IGF)-I protein is a growth-promoting polypeptide that can act as an angiogenic agent in the eye. The purpose of the current study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in the normal human eye and to examine the presence of expression in eyes with neovascular age-related macular degeneration (AMD). METHODS: Formalin-fixed, paraffin-embedded slides of 4 normal control eyes and 14 eyes with choroidal neovascularization (CNV) secondary to AMD were examined. Three eyes with proliferative diabetic retinopathy were studied as the positive control. IGF-I and IGF-IR mRNA was detected by in situ hybridization with digoxigenin-labeled RNA probes. IGF-IR protein was studied by immunohistochemistry. RESULTS: In the normal retina, IGF-I and IGF-IR mRNA expression was found throughout the neuroretinal layers, in the retinal pigment epithelium (RPE), and in some choriocapillary and retinal capillary endothelial cells. In eyes with CNV we found IGF and IGF-IR mRNA in capillary endothelial cells, some transdifferentiated RPE, and fibroblast-like cells. IGF-IR protein was found in normal eyes in all neuroretinal layers, in the RPE, and in the choroidal vessels. In eyes with CNV, IGF-IR protein was present in the RPE monolayer, in transdifferentiated RPE, and in newly formed vessels. CONCLUSIONS: The colocalization of protein and receptor indicates an autocrine function of IGF-I in the normal human retina. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in endothelial cells, RPE cells, and fibroblast-like cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD.     

Tractional force generation by human müller cells: growth factor responsiveness and integrin receptor involvement

PURPOSE: To assess the ability of human Müller cells to generate tractional forces and to determine the role of growth factors and collagen binding integrins in this process. METHODS: Müller cells were isolated from papain-DNase-digested human retina. Cell identity and changes in cell phenotype were confirmed by immunodetection of glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), vimentin, and alpha-smooth muscle actin (alpha-SMA). Generation of tractional force was assessed with a tissue culture assay involving incubation of cells on three-dimensional collagen gels. The effects of contraction-promoting growth factors were examined by adding these directly to the culture medium. Müller cell expression and the involvement of specific integrin receptors were assessed by immunodetection, RT-PCR, and subunit-specific blocking antibodies. RESULTS: During maintenance in culture, human Müller cells adopted a fibroblast-like phenotype capable of generating tractional forces. Matrix contraction was stimulated in a dose-dependent fashion by insulin-like growth factor I and platelet-derived growth factor. Modest responses were observed with high concentrations of transforming growth factor (TGF)-beta1 and -beta2. Müller cells express all four integrin subunits that comprise the collagen-binding receptors including alpha1, alpha2, alpha3, and beta1. Blocking antibodies against receptor subunits alpha2 and beta1 significantly reduced the overall rate of matrix contraction. Antibodies against the alpha1 subunit were modestly inhibitory, whereas anti-alpha3 was without effect. CONCLUSIONS: Human Müller cells acquire the capacity to generate tractional forces in vitro and the contraction-promoting growth factors insulin-like growth factor (IGF)-I and platelet-derived growth factor (PDGF) are potent stimuli. Generation of tractional force by Müller cells primarily involves integrin receptors containing alpha2 and beta1 subunits.     

Production of IGF-I and IGF binding proteins by retinal cells in vitro.

Insulin-like growth factor I (IGF-I) and its associated binding proteins (IGFBPs) have been identified in retinal tissues but their precise cellular origin remains unclear. The aim of this study was to examine the ability of three retinal cell types (microvascular endothelial cells, pericytes, and pigment epithelial cells) and a non-retinal cell type (Tenon's fibroblasts) to produce IGF-I and cell specific IGFBPs in vitro. Using a radioimmunoassay we demonstrated that all four cell types produce IGF-I and that this production continued over a 72 hour period. In addition, western ligand blotting revealed that each cell type produced at least one IGFBP and that each cell type produced a different IGFBP profile. Endothelial cells produced a 24 kDa band only, pericytes produced both a 24 kDa and a 34 kDa band, retinal pigment epithelial cells produced a 38-41 kDa band, while fibroblasts produced both a 24 kDa and a 30 kDa band. Laser scanning densitometry demonstrated that in the majority of experiments the IGFBPs accumulated in the culture medium over a 72 hour period. Neither IGF-I nor IGFBPs were observed in cell lysates indicating de novo synthesis and secretion in vitro. These results suggest an autocrine/paracrine function for IGF-I and its associated binding proteins which may play a significant role in both retinal physiology and disease.     

Ontogeny of VEGF, IGF-I, and GH in neonatal rat serum, vitreous fluid, and retina from birth to weaning

PURPOSE: Vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-I, and growth hormone (GH) are major regulators of physical growth, as well as normal and pathologic retinal development. Ocular tissues are protected by the blood-ocular barrier. This study was conducted to test the hypothesis that the ontogenic profiles of VEGF, IGF-I, and GH in the rat serum, vitreous fluid, and retina are compartment specific, and that the vitreous is a reservoir for retinal growth factors. METHODS: Sprague-Dawley rat pups were killed at birth (postnatal day [P]0) and at P7, P14, and P21. At death, serum, vitreous fluid, and retinal homogenates were analyzed for ontogeny of VEGF, IGF-I, and GH. RESULTS: VEGF levels were 10 times higher in the vitreous than in serum at all stages of development. Vitreous and serum VEGF levels progressively declined, with lowest concentrations at P21. Retinal VEGF levels increased with the highest concentration at P21. IGF-I levels in the vitreous decreased from P7 through P21. IGF-I levels in serum and retinal homogenates increased with advancing postnatal age. Although IGF-I levels were four times higher in the vitreous than in the retina at P0, equilibration was achieved at P21. GH levels in the vitreous were 10 times lower than serum levels, were decreased at P14 and P21, and remained unchanged from P0 through P21 in the retina. CONCLUSIONS: VEGF and IGF-I act in concert to promote retinal development with the vitreous fluid as a reservoir. The ontogenic profiles of VEGF, IGF-I and GH in the serum and ocular compartments are specific. These differences should be considered when therapies for ROP are proposed.     

Increased extraocular muscle strength with direct injection of insulin-like growth factor-I

PURPOSE: Previous work has demonstrated the effectiveness of insulin-like growth factor (IGF)-II in increasing force generation in extraocular muscle (EOM). Studies in the literature have suggested that IGF-I would be even more effective than IGF-II. This study was performed to assess the effects on muscle mass and force generation of IGF-I injection in adult rabbit superior rectus muscle. METHODS: Adult rabbits received a single injection of IGF-I at one of several doses into one superior rectus muscle. One week after treatment, the rabbits were euthanatized, and the superior rectus muscle from each orbit was removed. Force generation was measured using an in vitro apparatus, and injected muscles were compared with the contralateral control. A second group of animals were injected similarly, and the muscles were examined at 1 week for changes in cross-sectional area of individual myofibers. RESULTS: EOMs demonstrate significant numbers of cells expressing the IGF receptor. After the EOMs were injected with IGF-I, there were significant increases both in muscle force generation and cross-sectional area at all doses tested in this study. Doses of 10 and 25 microg IGF-I were most effective. CONCLUSIONS: Direct muscular injection of IGF-I effectively increases EOM force generation without the potential biomechanical hazards of surgery such as permanently altered muscle length or insertional position on the globe.     

RPE-mediated collagen gel contraction. Inhibition by colchicine and stimulation by TGF-beta

Retinal detachment secondary to proliferative vitreoretinopathy (PVR) is often precipitated by traction on the retina from preretinal membranes and traction on the vitreous base. Contraction and proliferation of retinal pigment epithelial (RPE) cells appear to play an important role in the pathogenesis of PVR. Contraction of collagen gels by human RPE cells was evaluated as a model of tractional forces on the vitreous gel. The area of collagen gels with admixed RPE cells in 15-mm wells was measured daily. The collagen gels contracted to less than 50% of original area in 89% of wells. Colchicine (0.01-1 microM) inhibited RPE-induced collagen gel contraction, whereas 1 ng/ml of transforming growth factor-beta enhanced it. Potential stimulators or inhibitors of RPE-mediated gel contraction can be tested using this model.