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PubMed1997

[Distribution of cell adhesion glycoproteins in the human retrobulbar optic nerve].

We examined the immunohistochemical distribution of six types of cell adhesion glycoproteins in the human retrobulbar optic nerve. The pial septa had laminin and entactin on the surface at the border with the optic nerve fibers, and fibronectin and vitronectin were distributed in the stroma of the septa. Some tenastin was also detectable both on the surface and in the stroma of the septa. Thrombospondin was associated with capillaries within the stroma. The surface of the arachnoid membrane, the pia mater, and the dura mater was covered by laminin and entactin. Diffuse staining with antifibronectin antibody was seen in all these meningeal tissues, but vitronectin showed a fine fibrillar appearance in these tissues. No significant staining could be found for tenastin. Thrombospondin showed only faint staining on the capillaries of the pia mater and the dura mater. These findings can provide clues for the discussion of how the extracellular matrix works and what roles it plays in disorders involving the retrobulbar optic nerve, such as optic neuritis and glaucoma.

Cell Adhesion↗
PubMed1997

Protective effect of nafamostat mesilate in early wound healing.

BACKGROUND: Tensile strength in rat ileal anastomosis is diminished after injection with superoxide dismutase. We have studied the immunohistochemistry of extracellular matrix to investigate changes associated with this loss of tensile strength. A serine proteinase inhibitor, Nafamostat mesilate, was used to evaluate its potential for maintaining tensile strength. STUDY DESIGN: Four groups of rats underwent ileal anastomosis, Groups 1 and 2 were controls. Groups 3 and 4 were given superoxide dismutase and Nafamostat mesilate, respectively, after anastomosis. RESULTS: In controls, groups 1 and 2, tensile strength decreased to 58 percent and 57 percent, respectively, of the initial value measured immediately after anastomosis 1 day postoperatively and both dropped to 33 percent 3 days postoperatively. After 5 days, tensile strength recovered to 83 percent of initial value. In comparison with controls, administration of superoxide dismutase significantly attenuated the loss of tensile strength on day 1 (94 percent of initial value, p < 0.01) and on days 3, 5 and 7 (p < 0.05). Similar results were found after administration of Nafamostat mesilate on days 3 and 5 (p < 0.05). In group 1, degradation of the collagen layer in the anastomosis was observed, with disappearance of immunostaining for fibronectin and vitronectin on day 3. In both groups 3 and 4, these changes were significantly attenuated with intense immunostaining for plasminogen activator inhibitor-1; fibronectin and vitronectin were seen particularly among collagen fibers on both sides of the anastomosis. CONCLUSIONS: These findings suggest that degradation of extracellular matrix causes loss of tensile strength early after anastomosis. Nafamostat mesilate may be clinically useful to prevent breakdown of intestinal anastomoses.

Anastomosis, Surgical↗
PubMed1998

Combinatorial chemistry techniques applied to nonpeptide integrin antagonists.

The integrins are cell surface receptors that recognize extracellular matrix adhesive proteins such as fibrinogen, fibronectin, vitronectin, and VCAM-1 (vascular cell adhesion molecule-1). Nonpeptide integrin antagonists designed after the adhesion recognition sequence RGD (Arg-Gly-Asp) not only have displayed efficacy as antithrombotic agents, but also have promise for the treatment of cancer and osteoporosis. Combinatorial organic syntheses of chemical mini-libraries have facilitated nonpeptide lead optimization of integrin antagonists with marked success. Although these accomplishments have been realized primarily for the discovery of orally active GPIIb/IIIa antagonist antithrombotics, vitronectin receptor (avb3) antagonist research has also benefited from such rapid synthesis. The purpose of this review is to report progress in combinatorial synthesis lead optimization by highlighting the drug design strategies and synthetic tactics that have led to improved integrin antagonists.

Animals↗
PubMed1998

Orientation of ECM protein deposition, fibroblast cytoskeleton, and attachment complex components on silicone microgrooved surfaces.

The microfilaments and vinculin-containing attachment complexes of rat dermal fibroblasts (RDF) incubated on microtextured surfaces were investigated with confocal laser scanning microscopy (CLSM) and digital image analysis (DIA). In addition, depositions of bovine and endogenous fibronectin and vitronectin were studied. Smooth and microtextured silicone substrata were produced that possessed parallel surface grooves with a groove and ridge width of 2.0, 5.0, and 10.0 microns. The groove depth was approximately 0.5 micron. CLSM and DIA make it possible to visualize and analyze intracellular and extracellular proteins and the underlying surface simultaneously. It was observed that the microfilaments and vinculin aggregates of the RDFs on the 2.0 microns grooved substrata were oriented along the surface grooves after 1, 3, 5, and 7 days of incubation while these proteins were significantly less oriented on the 5.0 and 10.0 microns grooved surfaces. Vinculin was located mainly on the surface ridges on all textured surfaces. In contrast, bovine and endogenous fibronectin and vitronectin were oriented along the surface grooves on all textured surfaces. These proteins did not seem to be hindered by the surface grooves since many groove-spanning filaments were found on all the microgrooved surfaces. In conclusion, it can be said that microtextured surfaces influence the orientation of intracellular and extracellular proteins. Although results corroborate three earlier published hypotheses, they do not justify a specific choice of any one of these hypotheses.

Animals↗
PubMed1996

Extracellular matrix regulates the amount of the beta-amyloid precursor protein and its amyloidogenic fragments.

We have studied the influence of the extracellular matrix (ECM) on the amount of beta-amyloid precursor protein (APP) and C-terminal amyloid-bearing fragments in 313 fibroblasts. After incubation with ECM components, the cellular APP content of 3T3 cells changed. Besides, different substrata including collagen, fibronectin, laminin, vitronectin, and heparin, determined changes in the amount of a C-terminal 22 kDa-fragment. The regulation of amyloidogenic fragments by the ECM was transient; in fact, when 3T3 cells were plated on tissue culture dishes coated with collagen or vitronectin, maximal levels of the 22 kDa fragment were observed 12 h after plating; in the presence of fibronectin, the maximum level of the amyloidogenic fragment was obtained 36 h after plating. These results indicate that the ECM modulates in a transient way the generation of APP-derived polypeptides containing the amyloid-beta-peptide (A beta). The ECM does not have a generalized effect on 3T3 fibroblasts, because no significant differences in cell attachment, growth rate, whole-cell polypeptide pattern beta 1 integrin and alpha-tubulin levels were observed on cells grown on various matrix proteins. Laminin, collagen, and heparin also influence the level of an amyloidogenic fragment of 35 kDa in Neuro 2A neuronal cells, without a significant change in the neuronal marker acetylcholinesterase. In this case, however, a long-lasting response to ECM molecules was observed. These observations provide evidence that ECM molecules influence APP biogenesis, including the generation of amyloidogenic fragments containing the A beta peptide. Our studies might prove significant to understand the localized increment of beta-amyloid deposition in selected areas of the brain of Alzheimer's patients.

3T3 Cells↗
PubMed1993

Thrombin stimulates melanoma tumor-cell binding to endothelial cells and subendothelial matrix.

Thrombin has been shown to activate tumor-cell adhesion to platelets, fibronectin and von Willebrand factor 2- to 3-fold in vitro, and enhance metastasis 10- to 156-fold in vivo. We therefore elected to determine whether thrombin binds to tumor cells and whether thrombin-treated tumor cells enhance their adhesion to endothelial cells, the first barrier to tumor invasion and metastasis. Thrombin-treated human and hamster melanoma cells (SK-Mel-28 and HM-29) enhanced their adhesion to bovine aortic endothelial cells 2.1- to 2.3-fold, respectively. Similar results were obtained with bovine capillary endothelial cells. Thrombin activation of tumor cells was rapid, reaching its peak 15 min after thrombin activation; and transient, declining to baseline levels by 60 min. 125I-thrombin bound to both SK-Mel-28 and HM-29 cells in a saturation-dependent manner, was inhibitable by unlabelled thrombin, and could be 90% washed away with buffer following 30 min of incubation. Electron microscopy of tumor cells bound to fibronectin-coated millipore filters revealed adhesion of naive as well as thrombin-treated tumor cells to endothelial cells and subendothelial matrix between endothelial cells. Neither mode of adhesion was preferentially enhanced by thrombin-treated tumor cells. Both naive and thrombin-treated SK-Mel-28 cells had the adhesive ligand integrin receptors: alpha 3 beta l (fibronectin, laminin, collagen); alpha 5 beta l (fibronectin); alpha v beta x (vitronectin). Receptors for the beta 2 integrin family (LFA-I and Mac-I) were not found, nor were receptors of the beta 3 integrin family, GPIIIa. The receptor ligands fibronectin and vitronectin were present. None of the above receptors or ligands increased their density or appeared de novo after thrombin stimulation. Thus, 2 melanoma cell lines have thrombin receptors which, when occupied, lead to enhanced adhesion of tumor cells to endothelium and subendothelial matrix.

Animals↗
PubMed1993

12(S)-HETE promotes tumor-cell adhesion by increasing surface expression of alpha V beta 3 integrins on endothelial cells.

The present work was undertaken to investigate the regulatory role of 12(S)-HETE, a lipoxygenase metabolite of arachidonic acid, in the surface expression of alpha v beta 3 integrin receptors in endothelial cells (rat aortic endothelial cells, or RAEC). Several monoclonal and polyclonal antibodies localized alpha v beta 3 in focal adhesions in both subconfluent and post-confluent RAEC. RAEC alpha v beta 3 integrins were further characterized by immunoblotting and immunoprecipitation. 12(S)-HETE, but not 12(R)-HETE or other lipoxygenase-derived hydroxy fatty acids, induced a dose-dependent increase in alpha v beta 3 surface expression in RAEC, which was antagonized by prostacyclin or its analog iloprost as well as by 13-HODE, a 15-lipoxygenase product of linoleic acid. 12(S)-HETE promoted RAEC adhesion to vitronectin, an effect inhibited by antibodies against alpha v beta 3. 12(S)-HETE also promoted tumor-cell (W256 carcinosarcoma) adhesion to vitronectin, which was inhibited by various antibodies against alpha IIb beta 3 but not by an antibody against alpha v. W256 adhesion to 12(S)-HETE-treated RAEC demonstrated a significant increase, which was inhibited by anti-alpha v, -beta 3, or -alpha v beta 3 antibodies and by 13-HODE. Western blotting, immunoprecipitation and reverse transcription-polymerase chain reaction indicated that W256 carcinosarcoma cells expressed alpha IIb beta 3 integrins but not alpha v beta 3. The results suggest that the lipoxygenase metabolites [i.e., 12(S)-HETE and 13-HODE] play a significant role in modulating tumor-cell interactions with endothelium by enhancing endothelial cell integrin (e.g., alpha v beta 3) expression.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1992

Rat osteoclasts adhere to a wide range of RGD (Arg-Gly-Asp) peptide-containing proteins, including the bone sialoproteins and fibronectin, via a beta 3 integrin.

The ligand binding ability of rat osteoclast adhesion receptors was investigated in an attachment assay using osteoclasts disaggregated from bone. Osteoclasts adhered well to the Arg-Gly-Asp (RGD)-containing proteins osteopontin (bone sialoprotein I) and BSP (bone sialoprotein II), vitronectin, fibrinogen, von Willebrand factor, and fibronectin. Osteoclasts also adhered, but less strongly, to type I collagen. No attachment of osteoclasts was observed to thrombospondin, tenascin, laminin, or a range of non-RGD-containing bone proteins and proteins from other sources. The attachment of osteoclasts to all ligands was abolished in the presence of GRGDSP peptide, indicating the involvement of the RGD cell binding sequence in ligand binding. Attachment of osteoclasts to all substrates, with the exception of type I collagen, was also strongly inhibited by the addition of monoclonal antibody F11 to the beta 3 integrin subunit, indicating that a beta 3 integrin, probably the vitronectin receptor, was involved. Attachment to type I collagen was blocked by EDTA chelation of divalent cations and was not significantly affected by anti-beta 3 or anti-beta 1 antibodies; when taken with the inhibition by RGD peptide, this suggests the involvement of various receptors, possibly including nonintegrin collagen receptors, in the binding of osteoclasts to this protein. These results define the wide range of ligands for extracellular matrix receptors in osteoclasts in vitro. It remains to be established which of these proteins are important in osteoclast adhesion and osteoclastic bone resorption in vivo.

Amino Acid Sequence↗
PubMed1994

Integrins and osteoclastic resorption in three bone organ cultures: differential sensitivity to synthetic Arg-Gly-Asp peptides during osteoclast formation.

We investigated possible inhibitory effects of five synthetic Arg-Gly-Asp (RGD)-containing peptides on osteoclastic resorption in three distinct in vitro resorption assays (17-day-old fetal mouse bone organ cultures) that differ in stages of osteoclast differentiation. RGD peptides, which can bind the adhesion receptors called integrins, inhibited osteoclastic resorption (45Ca release) in fetal mouse bone explants in which osteoclast precursors have yet to adhere to the mineralized matrix and develop into mature osteoclasts (metacarpals and coculture system). Treatment of metacarpals with RGD peptides inhibited the formation of multinucleated TRAP+ osteoclasts in the mineralized matrix because their mononuclear TRAP+ osteoclast precursors remained localized in the periosteum. In particular, echistatin, a viper venom protein with known affinity for alpha v beta 3 integrin, and GdRGDSP inhibited osteoclastic resorption dose dependently in these systems (ED50 10(-9) and 10(-4) M, respectively) but did not alter the activity of mature resorbing osteoclasts in radii. In addition, 45Ca release was significantly inhibited by the cyclic peptide GPenGRGDSPCA, which has a relatively higher affinity for the vitronectin than fibronectin receptor(s). In contrast, GRDGdSP, which has a much higher affinity for the fibronectin receptor (than the vitronectin receptors), had no effect on resorption at similar concentrations in any resorption system used. In summary, the data presented in this paper show that peptides with RGD motifs are capable of inhibiting osteoclastic resorption in bone organ cultures. Our studies not only support the hypothesis concerning the importance of alpha v beta 3 in osteoclastic resorption but also suggest an important role of integrin(s) in events preceding the actual resorption of calcified matrix by osteoclasts.

Amino Acid Sequence↗
PubMed1992

Identification of integrin cell-substratum adhesion receptors on cultured rat bone cells.

The interactions of bone cells with their extracellular matrix is of major importance in bone development, repair, and disease. We examined the ability of rat calvarial bone cells to adhere to various matrix proteins and to define the role of integrin cell-substrate adhesion receptors in these interactions. Isolated newborn rat calvarial bone cells prelabeled with 3H-thymidine and plated on plastic wells that had been precoated with serial dilutions of various substrates showed typical dose-response adherence curves to fibronectin, fibrinogen, laminin, vitronectin, and collagen I and IV. Cell adherence to poly-D-lysine, a nonspecific cell adherent, was high at all substrate concentrations > 0.0001 micrograms/ml. A polyclonal anti-rat integrin antibody blocked cell adhesion to all substrates tested except poly-D-lysine. Isolated rat calvarial bone cells were surface labeled with 125I, extracted, and immunoprecipitated with polyclonal antibodies made against the rat integrin complex and peptides derived from the cytoplasmic domains of the alpha 2, alpha 3, and alpha 5 subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (nonreduced) identified four bands representing a mixture of integrins including the alpha 1 beta 1 laminin/collagen receptor, the alpha 5 beta 1 fibronectin receptor, and the alpha V beta 3 (or possibly alpha V beta 5) vitronectin receptor. These experiments show that bone cells adhere to a wide variety of extracellular matrix proteins via specific integrins. Increased knowledge about the regulation of these receptors and the mechanisms by which they transmit information to the cell will be important for a more complete understanding of bone physiology and pathophysiology.

Animals↗
PubMed1996

Osteoblast adhesion to orthopaedic implant alloys: effects of cell adhesion molecules and diamond-like carbon coating.

In total joint arthroplasty, long-term outcomes depend in part on the biocompatibility of implant alloys. This study analyzed effects of surface finish and diamond-like carbon coating on osteoblast cell adhesion to polished titanium-aluminum-vanadium and polished or grit-blasted cobalt-chromium-molybdenum alloys. Osteoblast binding was tested in the presence and absence of the cell adhesion proteins fibronectin, laminin, fibrinogen, and vitronectin and was quantified by measurement of DNA content. Although adherence occurred in serum-free medium, maximal osteoblast binding required serum and was similar for titanium and cobalt alloys at 2 and 12 hours. With the grit-blasted cobalt alloy, cell binding was reduced 48% (p < 0.05) by 24 hours. Coating the alloys with diamond-like carbon did not alter osteoblast adhesion, whereas fibronectin pretreatment increased cell binding 2.6-fold (p < 0.05). In contrast, fibrinogen, vitronectin, and laminin did not enhance cell adhesion. These results support the hypothesis that cell adhesion proteins can modify cell binding to orthopaedic alloys. Although osteoblast binding was not affected by the presence of diamond-like carbon, this coating substance may influence other longer term processes, such as bone formation, and deserves further study.

Alloys↗
PubMed1999

Macrophage colony-stimulating factor and interleukin-6 release by periprosthetic cells stimulates osteoclast formation and bone resorption.

Periprosthetic bone loss is an important contributory factor for aseptic loosening of total joint replacements. It has recently been shown that osteoclast precursor cells are present in the wear particle-associated macrophage infiltrate found in the membrane surrounding loose implants and that these cells are capable of differentiating into osteoclastic bone-resorbing cells. Long-term co-culture of arthroplasty-derived macrophages and the rat osteoblast-like cell line, UMR-106, in the presence of 1,25(OH)2D3 results in the formation of numerous multinucleated cells that are positive for tartrate-resistant acid phosphatase and vitronectin receptor and capable of extensive lacunar bone resorption. The aim of this study was to determine the effect of cytokines/growth factors, known to be present in the arthroplasty membrane, on this process of osteoclast differentiation. During osteoclast formation, increased levels of macrophage colony-stimulating factor, interleukin-6, and to a lesser extent, interleukin-1beta, but not tumour necrosis factor alpha, were detected in the co-culture supernatants. Addition of neutralising antibodies to human interleukin-1beta or tumour necrosis factor alpha to the co-culture system did not inhibit osteoclast formation. In contrast, co-cultures to which neutralising antibodies to human macrophage colony-stimulating factor or interleukin-6 were added contained fewer cells positive for tartrate-resistant acid phosphatase and vitronectin receptor and formed significantly fewer resorption pits. Time-course studies showed that macrophage colony-stimulating factor and interleukin-6 increase osteoclast formation mainly in the early stages of osteoclast differentiation. These results indicate that the release of macrophage colony-stimulating factor and interleukin-6 by activated cells in the arthroplasty membrane is likely to contribute to pathological bone resorption associated with aseptic loosening by stimulating differentiation of mononuclear phagocyte osteoclast precursors into mature bone-resorbing cells.

Acetabulum↗
PubMed2006

Synthetic RGD-containing alpha-helical coiled coil peptides promote integrin-dependent cell adhesion.

Integrin receptors are the main mediators of cell adhesion to the extracellular matrix. They bind to their ligands by interacting with short amino acid sequences, such as the RGD sequence. Soluble, small RGD-based peptides have been used to block integrin-binding to ligands, thereby interfering with cell adhesion, migration and survival, while substrate-immobilized RGD sequences have been used to enhance cell binding to artificial surfaces. This approach has several important medical applications, e.g. in suppression of tumor angiogenesis or stimulation of bone formation around implants. However, the relatively weak affinity of short RGD-containing peptides often results in incomplete integrin inhibition or ineffective ligation. In this work, we designed and synthesized several new multivalent RGD-containing molecules and tested their ability to inhibit or to promote integrin-dependent cell adhesion when used in solution or immobilized on substrates, respectively. These molecules consist of an oligomeric structure formed by alpha-helical coiled coil peptides fused at their amino-terminal ends with an RGD-containing fragment. When immobilized on a substrate, these peptides specifically promoted integrin alphaVbeta3-dependent cell adhesion, but when used in solution, they blocked alphaVbeta3-dependent cell adhesion to the natural substrates fibronectin and vitronectin. One of the peptides was nearly 10-fold more efficient than fibronectin or vitronectin in promoting cell adhesion, and almost 100-fold more efficient than a linear RGD tripeptide in blocking adhesion. These results indicate that alpha-helical coiled coil peptides carrying an amino-terminal RGD motif can be used as soluble antagonists or surface-immobilized agonists to efficiently inhibit or promote integrin alphaVbeta3-mediated cell adhesion, respectively.

Cell Adhesion↗
PubMedDate not supplied

Binding of extracellular matrix proteins to the surface of Bacteroides spp.

The binding of fibronectin an vitronectin to 207 Bacteroides strains and the binding of collagen and sialoproteins to 55 Bacteroides strains were investigated by means of latex agglutination tests. The binding of fibronectin, collagen and lactoferrin to the same 55 strains was also tested by using 125I-labelled proteins. The 207 strains, belonging to ten Bacteroides species, were isolated from different infections (51%) and from faeces of healthy subjects (49%). Most of the strains displaying fibronectin binding belonged in the species B. fragilis or B. vulgatus. The binding could be inhibited by preincubation of the cells with an excess amount of fibronectin. No inhibition of the binding was observed with carbohydrates. The vitronectin binding of the strains was less common, but was always observed to accompany fibronectin binding. None of the examined 55 strains exhibited any binding to fetuin or asialofetuin. The radiolabelling method indicated a low binding to 125I-fibronectin. The binding of 125I-collagen-I and 125I-lactoferrin in the Bacteroides strains tested was higher than that of 125I-fibronectin.

Journal Article↗
PubMed1996

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate.

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

Alendronate↗
PubMed1997

RGD-containing peptides trigger apoptosis in glomerular mesangial cells of adult human kidneys.

Substantial evidence was given that different Arg-Gly-Asp (RGD)-containing peptides, linear Arg-Gly-Asp-Ser (RGDS) and cyclic Arg-Gly-Asp-Phe (RGDF), which are sequences present in fibronectin and vitronectin and which can bind alpha5beta1 and alpha(v)beta3 integrins, respectively, both induced apoptosis and expression of interleukin-1beta-converting enzyme (ICE) in cultured glomerular mesangial cells from adult human kidneys, with consistent apoptosis features appearing as nuclei condensation and fragmentation, internucleosomal DNA fragmentation, and DNA content decrease. These results indicated that both fibronectin and vitronectin may be important in the survival of human glomerular mesangial cells and that stable cyclic RGD(D)FV peptide may be a candidate to be used for regulating apoptosis in vivo.

Adult↗
PubMed1997

Vascular permeability factor/vascular endothelial growth factor inhibits anchorage-disruption-induced apoptosis in microvessel endothelial cells by inducing scaffold formation.

Survival and proliferation of endothelial cells requires both growth factors and an appropriate extracellular matrix to which cells can attach. In the absence of either, endothelial cells rapidly undergo apoptosis. Thus, when human microvascular endothelial cells (HDMEC) are plated on a hydrophobic surface such as untreated polystyrene, they rapidly undergo apoptosis and die. The present study demonstrates that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-selective cytokine, inhibits apoptosis of HDMEC cultured on untreated polystyrene and induces these cells to adhere, spread, and proliferate. VPF/VEGF-induced HDMEC adhesion was time-dependent, required de novo protein synthesis, and was inhibited by a soluble RGD peptide but not by an inhibitor of collagen synthesis. Under the conditions of these experiments, VPF/VEGF downregulated expression of collagen IV and fibronectin but did not change collagen I mRNA levels. VPF/VEGF-induced HDMEC adhesion was inhibited by antibodies to alpha(v)beta5 and vitronectin but not by antibodies to alpha(v)beta3. Other endothelial growth factors and cytokines such as bFGF, HGF, and TGFbeta did not reproduce the VPF/VEGF effect. We suggest that VPF/VEGF induces endothelial cells to deposit a scaffolding (likely involving vitronectin) that allows them to attach to and proliferate on an otherwise nonsupportive surface (hydrophobic polystyrene) and in this manner serves as both a survival factor and a growth factor.

Actin Cytoskeleton↗
PubMed1998

Modulation of apoptotic cell death by extracellular matrix proteins and a fibronectin-derived antiadhesive peptide.

Cell adhesion to the extracellular matrix (ECM) has been implicated in apoptosis in anchorage-dependent cell types. We recently found that a peptide derived from fibronectin (termed III14-2) inhibits the integrin-mediated cell adhesion to ECM. Using this antiadhesive peptide and a variety of ECM proteins, we show here a critical role of the integrin-ECM protein interaction in apoptotic regulation of human umbilical vein endothelial cells (HUVEC). HUVEC in suspension underwent apoptosis under the serum-free conditions, as judged by nuclear and DNA fragmentations. This apoptosis was suppressed to varying degrees when alpha 5 beta 1, alpha v beta 3, and alpha 2 beta 1 integrins were occupied with either soluble or immobilized ECM proteins such as fibronectin, vitronectin, and type I collagen, respectively. Peptide III14-2, which had no effect by itself on the HUVEC apoptosis, disrupted the ligation of alpha 5 beta 1 and alpha v beta 3 but no alpha 2 beta 1 and ultimately led the cells to apoptosis, indicating that this antiadhesive peptide indirectly induces apoptosis by blocking cell survival signal delivered from alpha 5 beta 1 and alpha v beta 3 integrins. Genistein, a protein tyrosine kinase inhibitor, slightly reduced the rescuing effect of fibronectin, whereas sodium orthovanadate and bombesin, which increase in the level of protein tyrosine phosphorylation, made HUVEC less susceptible to apoptosis and blocked the effect of peptide III14-2. HUVEC adhesion to fibronectin substrate raised the tyrosine phosphorylation level of focal adhesion kinase and the expression of cytoprotective Bcl-2 protein, both of which were reversed by the antiadhesive effect of peptide III14-2. Thus, the opposing effects of ECM proteins, including fibronectin and vitronectin, and peptide III14-2 on HUVEC apoptosis appear to be due to the opposing effects of these factors on the signaling pathway which includes tyrosine phosphorylation of FAK and Bcl-2 expression.

Amino Acid Sequence↗