Rgd receptor



Keywords: rgd receptor
Description: Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal

Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors αv β3 and αv β5. was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins αv β3 and αv β5 on RPE cells was examined.

The effect of a cyclic integrin antagonist and a control peptide (0.01 μg/ml to 300 μg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H 3 -thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors αv β3 and αv β5 was evaluated by flow cytometry.

A selective inhibition of the integrin receptors αv β3 and αv β5 through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease.

Integrins are a family of heterodimeric, non-covalently bound cell surface receptors, which mediate cell-cell and cell to extracellular matrix (ECM) adhesion. They are transmembrane glycoproteins consisting of a larger α and a smaller β subunit. In mammals, 18 α- and 8 β integrin genes encode polypeptides that combine to form 24 αβ heterodimeric receptors [1 ]. By mediating cell-cell and cell-matrix contact, the integrins are involved in a spectrum of physiologic and pathologic processes. Integrin expression is modulated by several cytokines, growth factors and the extracellular matrix in different cell types [2 ].

The integrins αv β3 and αv β5 have been shown to be of particular significance for the progression of neovascularization. Both integrin receptors contain RGD sequences containing the amino acid sequence Arg-Gly-Asp. Vitronectin receptor integrins of the αv – chain subfamily interact with their target proteins via the tripeptide sequence RGD (NH2-arginine-glycine-aspartic acid-COOH) [3 ,4 ]. The blockage of these RGD receptor sequences induces endothelial cell apoptosis by inhibiting their binding to the ECM [5 ,6 ]. In addition fibroblast attachment onto fibrinogen, a substep involved in wound healing, is inhibited by blockage of the αv β3 integrin receptor [7 ]. Therefore, the integrin αv β3 plays a critical role during wound repair as an adhesion receptor and as a signaling receptor [8 ]. As an adhesion receptor, it has the exceptional ability to bind vitronectin, fibronectin and fibrinogen, the three major proteins present in the wound provisional matrix [9 ].

In proliferative vitreoretinopathy (PVR), a disease of the posterior eye characteristically induced by trauma or failed retinal reattachment surgery, integrins are also of importance for the progression of the disease [10 ,11 ]. PVR, an exaggerated wound healing process, is characterized by the proliferation, migration and contraction of retinal pigment epithelial cells (RPE), fibroblasts, glial cells and macrophages. PVR membranes are formed on, or under the sensory retina; their contraction may lead to retinal detachment and blindness. RPE cells play a dominant role in the pathobiology of the disease [12 ]. Growth factors including basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF), and the ECM molecule fibronectin, regulate proliferation, migration and invasion of the RPE cell and are expressed in PVR membranes [12 ,13 ]. Fibronectin is an adhesive substrate for both the integrin receptors αv β3 and αv β5 [14 ]. Several studies have identified integrins αv β3. αv β5. integrin alpha subunits 2,3,4,5,6, V, and integrin beta subunits 1, 2, 3 on RPE cells and cells within PVR membranes [15 -17 ]. The ECM-cell interactions are thought to be mediated largely through this family of cell surface receptors. Because cell-ECM interaction is important in the process of wound healing, one could predict that integrins would be actively involved in the pathogenesis of PVR. For PVR, it was shown in vivo. that tractional retinal detachment could be inhibited [11 ] by blockage of integrin-ECM binding by an RGD containing disintegrin.

Peptide antagonists specific for individual integrins have been developed, including cyclic RGD peptides selective for the αv integrin [18 ], the αv β3 integrin [19 ], and the αv β5 integrin [18 -21 ]. Sequence alterations in the backbone of the RGD-containing cyclic peptides that result in conformational differences in the interatomic distance between the Cβ atoms of Arg and Asp, have been shown to correlate with selective peptide recognition [18 ,22 ,23 ].

In this study we investigated the modulatory effect of the cytokines PDGF-BB and bFGF on the expression of the RGD containing integrin receptors αv β3 and αv β5 on human fetal RPE cells. Furthermore, the effect of a specific cyclic integrin antagonist that blocks the integrin receptors αv β3 and αv β5. was investigated for its effects on RPE attachment, proliferation, migration and invasion. A specific cyclic integrin antagonist was chosen for its higher affinity and stronger binding capacity to the αv β3 and αv β5 integrins compared to a non-cyclic integrin antagonist [24 ,18 ].

All cell culture solutions, media and ECM molecules (fibronectin, laminin and vitronectin) were purchased from Sigma (Sigma, St. Louis, MO).

Human RPE cells were isolated from fetal donor eyes (gestation time over 22 weeks) which were obtained from the Anatomic Gift foundation (Woodbine, GA) as has been described [25 ]. RPE cells were seeded onto laminin-coated 6 well plates (Fisher Scientific, Tustin, CA) using DMEM with 10 % fetal bovine serum, 1 % penicillin/streptomycin and glutamine. Cells from passage 3 to 5 were used in all experiments.

The cyclic integrin antagonist RGDfV, specific for the inhibition of integrins αv β3 and αv β5 [18 -21 ] and the control peptide RADfV were synthesized as described [6 ] at the USC/ Norris Cancer Peptide synthesis facility. The peptides were dissolved in Hank's Balanced Salt Solution (HBSS), and stored at -80°C. HPLC analysis revealed a purity > 99% of the synthesized peptide.

RPE cells (30,000 cells/well) were treated for 5 days with the cytokines bFGF and PDGF-BB at a concentration of 10 ng/ml in 6 well plates. Cells were detached, fixed with 4 % paraformaldehyde, blocked with 5 % goat serum for 30 minutes, centrifuged at 4°C, and incubated with a monoclonal mouse antibody against αv β3 or αv β5 (5 μg/ml) (Chemicon, CA) for one hour. After washing, a fluorescein conjugated secondary antibody (Chemicon, CA) directed against the mouse antibodies was added in a dilution of 1:100 for one hour. The expression of the integrin receptors αv β3 or αv β5 was measured using a fluorescent-activated cell sorter (FACStar plus, Becton Dickinson, Mountain View, CA). Forward and side light scatter was used to gate the desired scattered events (RPE cells) from dead cells and debris. The fluorescent index was determined by multiplying the percentage of cells by their mean fluorescence. At least 10,000 cells were evaluated/experiment and all experiments were performed in triplicate.

All experiments were repeated in triplicate. Standard deviations and averages were calculated. The data obtained were analyzed for significance using one way analysis of variance (ANOVA). Data with a p-value of less then 0.05 were considered to be statistically significant.

The RPE cells were stimulated to incorporate thymidine by either 10% serum or by 10 ng/ml bFGF or 10 ng/ml PDGF-BB (p < 0.01). Neither the integrin antagonist, nor control peptide showed any significant effect on serum, PDGF-BB or bFGF stimulated DNA synthesis. (data not shown). In addition, cell counting showed no effects of the integrin antagonist or the control peptide on serum or cytokine stimulated proliferation. Furthermore, a lack of toxicity for the integrin antagonist and the control peptide in the concentration range tested (0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, 30 μg/ml and 300 μg/ml) was determined by use of the trypan blue exclusion assay (data not shown).






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