Showing papers by "Michael D. Pierschbacher published in 2006"
01 Jan 2006
TL;DR: In this article, a series of RGD peptides were designed, synthesized and tested for binding to integrin receptors, and a peptide that binds potently to the αν β3, ανβ5 and α5β1 was selected for further investigations with regards to its effect on glioma cells.
Abstract: Summary A panel of human glioma cell explants was screened for integrin expression by flow cytometry using ανβ-specific antibodies. A lower percentage of the glioma cells were positive for the ανβ3 (mean % positive = 20.8%) integrin, whereas higher percentages were positive for the ανβ5 (mean % positive = 72.7%), VLA5α (mean % positive = 87%) and VLAβ1 (mean % positive = 41.7%) integrins. A series of RGD peptides was designed, synthesized and tested for binding to integrin receptors. Based on the results of the binding to the isolated integrin receptors and the expression of integrins on glioma cell lines, a peptide that binds potently to the ανβ3, ανβ5 and α5β1 was selected for further investigations with regards to its effect on glioma cells. The peptide, Ac-c[(Pen)-Tyr(Me)-Ala-ArgGly-Asp-Asn-Tic-Cys]NH2 (RGD peptide), exhibited high potential for use in clinical intracranial administration since it had good stability in rat brain cell homogenates placed into artificial cerebrospinal fluid. Using an HPLC method for quantification of peptides in rat brain cell homogenates, we could demonstrate the half-life of the RGD peptide approximated 20 hr. Relative to a scrambled peptide control (non-RGD sequence, same amino acids), the experimental RGD peptide significantly decreased glioma cell proliferation of the entire panel of rat and human glioma cells tested. Adhesion of recently passaged glioma cells to glioma-derived extracellular matrix protein-coated plates was inhibited significantly by the RGD peptide. The peptide also reversed attachment of plated glioma cells. The RGD peptide caused some, but not substantial, glioma cell injury, as evidenced by a quantitative in vitro nuclear DNA morphologic assay and by a flow cytometric assay employing 7-amino actinomycin D (7AAD). We histologically monitored for toxicity caused by various doses of the RGD peptide infused repeatedly into normal cannulated rat brain. At safe doses, the experimental RGD peptide-treated brains did not show significant differences from those infused with scrambled peptide or buffer-treated controls. In tumor-bearing brains, slightly smaller tumor areas were measured with a higher necrotic-to-tumor index in the RGD peptide treated relative to the scrambled peptide-treated controls. This was obtained with intracranial peptide administrations or combined intracranial and intraperitoneal injections. From this in vitro work, we conclude that the anti-glioma effects of the RGD peptide tested resulted from lowered glioma proliferation and adhesion/mobility, rather than from significant glioma cell injury in the timeframe analyzed. Although other mechanisms not discerned from our limited histopathological observations may be operational, from our in vivo work, we conclude that repeated administration of RGD peptide into brain is safe but that better delivery of the peptides to infiltrating tumor cells is necessary.
35 citations
TL;DR: The observation that cell attachment can be directed to surfaces modified with small synthetic RGD-containing peptides has provided the incentive to modify and fabricate the sequence into biomaterials.
Abstract: The development of materials for use in the fabrication of implantable medical devices was initially based on the concept of using materials that were refractile to cell interaction. It has become increasingly clear, however, that the relatively inert nature of materials seen in vitro does not translate to devices implanted for long periods of time. In addition, material surfaces can elicit varying types of cell responses, some resulting in adverse resctions. This has prompted an effort to create or modify materials in ways that promote controlled cell response at the material surface.' One such strategy for creating a cell interactive surface of a biomaterial involves the use of adsorbed extracellular matrix proteins (ECM) to provide specific signals for cell attachment.2 This method has been refined by the use of short synthetic peptides containing the cell-binding domains of ECM proteins6 One sequence that functions well as a cell-binding site is the tripeptide ArgGly-Asp (RGD), originally isolated as the cell-binding domain of fibronectin.' The RGD sequence has been identified as a cell-binding domain in a number of other ECM proteins, each of which is recognized by distinct cell-surface receptors, termed in tegr in~ .~ ' The observation that cell attachment can be directed to surfaces modified with small synthetic RGD-containing peptides has provided the incentive to modify and fabricate the sequence into biomaterials. A number of investigators have created cell attachment-competent surfaces by covalently coupling RGD peptides to materials, such as glycophase glass,s polyacrylamide? ethylene-acrylic acid copolymer,I0 polyethylene terephthalate," polytetrafluoroethylene,'2 and polyvinyl alcohol. The RGD sequence has also been used to modify the base monomer of polyurethane and thus form a material having sites for cell attachment throughout the material.I4 The use of covalent methods to immobilize the bioactive species is somewhat limited due to the constraints imposed by the material surface chemistry as well as the difficulties associated with modifying complex prefabricated medical devices. An alternative approach to modify the surface of a material involves the use of adsorptive interactions to bind molecules to the surface. This has proved successful for the immobilization of block copolymers that act to inhibit protein adsorption to blood-contacting biomaterial~.'~ We used a similar strategy to create RGD-containing peptides having a stretch of hydrophobic amino acids that mediate the interaction
31 citations