1. Activation of MMP-2 by human GCT23 giant cell tumour cells induced by osteopontin, bone sialoprotein and GRGDSP peptides is RGD and cell shape change dependent
A Gulino, A Tiberio, A R Farina, I Villanova, A F Chambers, A Teti, G Sciortino, A Tacconelli, A R Mackay Int J Cancer . 1998 Jul 3;77(1):82-93. doi: 10.1002/(sici)1097-0215(19980703)77:13.0.co;2-b.
We show that osteopontin (OPN), bone sialoprotein (BSP) and GRGDSP peptides, in solution, induce activation of metalloproteinase-2 (MMP-2) secreted by human GCT23 giant cell tumour cells. Activation of MMP-2 is RGD sequence dependent, possibly involves anti-alphaVbeta3 integrins, is preceded by a change from spread to rounded cell morphology and is mimicked by the actin depolymerising agent cytochalasin B. Cells that had spread on OPN, BSP and GRGDSP substrata failed to activate MMP-2, but subsequent addition of soluble GRGDSP induced rounding and MMP-2 activation. Activation induced by GRGDSP and cytochalasin B was cell mediated, inhibited by EDTA, tissue inhibitor of metalloproteinase-2 (TIMP-2) and carboxyl terminal MMP-2 consistent with a role for membrane type (MT)-MMP but did not involve urokinase, plasmin or thrombin activity. Activation induced by GRGDSP and cytochalasin B, but not cell rounding, was inhibited by herbimycin A, cycloheximide and actinomycin D, suggesting a role for tyrosine kinases, protein and RNA synthesis, but was not associated with changes in mRNA for MT-MMP-1, MMP-1, MMP-2, TIMP-1 or TIMP-2. GRGDSP and cytochalasin B enhanced levels of membrane-associated pro- and active form MMP-1 and MMP-2 but not MT-MMP-1, stimulated cell surface MMP-1 staining and induced that of MT-MMP-1, MMP-2 and TIMP-2. This was consistent with the possible relocation of constitutive MT-MMP-1 to the cell surface as a prerequisite for subsequent cell surface MMP-2/TIMP-2/MT-MMP-1 complex formation and to the potential induction of conditions favourable for reciprocal cell surface MMP-1/MMP-2 activation. Our data provide a novel insight into interactions between RGD containing bone matrices, GCT cells and MMPs of potential relevance to GCT pathology.
2. Regulation of endothelial cell function by GRGDSP peptide grafted on interpenetrating polymers
Gregory M Harbers, Kevin E Healy, Jonathan Tsang, Shyam Patel, Song Li J Biomed Mater Res A . 2007 Nov;83(2):423-33. doi: 10.1002/jbm.a.31320.
Vascular endothelium plays an important role in preventing thrombogenesis. Bioactive molecules such as fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) can be used to modify the surface of cardiovascular implants such as vascular grafts to promote endothelialization. Here we conjugated GRGDSP peptide to the nonfouling surface of an interpenetrating polymer network (IPN), and investigated the effects of the immobilized GRGDSP molecules on EC functions under static and flow conditions at well-defined GRGDSP surface densities (approximately 0 to 3 pmol/cm2). EC adhesion and spreading increased with GRGDSP surface density, reached a plateau at 1.5 pmol/cm2, and increased further beyond 2.8 pmol/cm2. Cell adhesion and spreading on GRGDSP induced two waves of extracellular signal-regulated kinase (ERK) activation, and 0.2 pmol/cm2 density of GRGDSP was sufficient to activate ERK. EC proliferation rate was not sensitive to GRGDSP surface density, suggesting that cell spreading at low-density of GRGDSP is sufficient to maintain EC proliferation. EC migration on lower-density GRGDSP-IPN surfaces was faster under static condition. With the increase of GRGDSP density, the speed and persistence of EC migration dropped quickly (0.2-0.8 pmol/cm2) and reached a plateau, followed by a slower and gradual decrease (1.5-3.0 pmol/cm2). These data suggest that the changes of EC functions were more sensitive to the increase of GRGDSP density at lower range. Under flow condition with shear stress at 12 dyn/cm2, EC migration was inhibited on GRGDSP-IPN surfaces, which may be attributed to the assembly of large focal adhesions induced by shear stress, suggesting a catch-bond characteristic for RGD-integrin binding. This study provides a rational base for surface engineering of cardiovascular implants.
3. GRGDSP peptide-bound silicone membranes withstand mechanical flexing in vitro and display enhanced fibroblast adhesion
Thomas J Hartman, Samuel Boateng, Carrie A Crot, Syed S Lateef, Brenda Russell, Luke Hanley Biomaterials . 2002 Aug;23(15):3159-68. doi: 10.1016/s0142-9612(02)00062-5.
Mechanobiological studies of cardiac tissue require devices that allow forces to be exerted on cells in vitro. Silicone elastomer is often used in these devices because it is flexible and transparent, permitting optical imaging of the cells. However, native untreated silicone is hydrophobic and is unsuitable for cell culture. Peptides covalently bound to silicone surfaces are examined here for the enhancement of cellular adhesion during in vitro dynamic flexing. A procedure is described for the chemical modification of medical grade silicone membranes with covalently bound GRGDSP peptides. The conditions for mechanical studies of cardiac cell cultures are then duplicated and it is demonstrated that the peptide layers survive 48 h of mechanical flexing in vitro. Specifically, mechanical flexing in vitro of the 30 pmol/cm2 peptide-modified silicone membranes has no significant effect on the amount of peptides that remains bound to the surface. Cardiac fibroblasts display enhanced adhesion to these peptide-bound silicone membranes for at least 24 h of growth, compared with native silicone or tissue culture polystyrene. The effects of serum versus serum-free media on fibroblast growth are also examined.
