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Results for “Integrin alphaVbeta3”

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PubMed2006

Efficient gene transfer into differentiated human trophoblast cells with adenovirus vector containing RGD motif in the fiber protein.

Previously we reported fiber-modified adenovirus (Ad) vectors containing the Arg-Gly-Asp (RGD) motif on the HI loop of the fiber knob (Ad-RGD vectors) have high gene transfer efficacy into some human trophoblast cell lines. In the current study, we investigate transgene activity of Ad-RGD during differentiation of human cytotrophoblast BeWo cells into syncytiotrophoblast-like cells. Although cellular differentiation into syncytiotrophoblast cells was followed by a decrease in the coxsackievirus and adenovirus receptor levels on the cell membrane, the alphaVbeta3 and alphaVbeta5 integrin levels did not change. Conventional adenovirus vector had lower transduction activity in the differentiated cells than non-differentiated cells. In contrast, Ad-RGD vector had no influence on differentiation and had a ca. 2-5 fold higher transduction activity than that of the conventional Ad vector. Thus, Ad-RGD vector can be a powerful tool for gene transfer experiments in syncytiotrophoblast cells.

Adenoviridae↗

PubMed2002

CD36 or alphavbeta3 and alphavbeta5 integrins are not essential for MHC class I cross-presentation of cell-associated antigen by CD8 alpha+ murine dendritic cells.

Cross-presentation of cell-associated Ag is thought to involve receptor-mediated uptake of apoptotic cells by dendritic cells (DC), and studies with human DC strongly implicate the endocytic receptor CD36 and the integrins alpha(v)beta(3) and/or alpha(v)beta(5) in this process. In the mouse, cross-presentation was recently shown to be a function of CD8alpha(+) DC. Here we report that CD36 is expressed on CD8alpha(+), but not on CD8alpha(-), DC. To address the role of CD36 in cross-presentation we compared CD36(-/-) and CD36(+/+) H-2(b) DC for their ability to stimulate naive OT-1 T cells specific for OVA plus H-2K(b) in the presence of OVA-loaded MHC-mismatched splenocytes as a source of cell-associated Ag for cross-presentation. Surprisingly, no difference was seen between CD36(-/-) and CD36(+/+) CD8alpha(+) DC in their ability to cross-present cell-associated OVA or to capture OVA-bearing cells. Furthermore, the proliferation of CFSE-labeled OT-1 cells in response to OVA cross-presentation in vivo was normal in CD36(-/-) bone marrow chimeras, also arguing against a necessary role for CD36 in cross-presentation by DC or other APC. DC doubly deficient for beta(3) and beta(5) integrins were similarly unimpaired in their ability to cross-present OVA-bearing cells in vitro. These data demonstrate that in the mouse, receptors other than CD36 or beta(3) and beta(5) integrins can support the specialized cross-presenting function of CD8alpha(+) DC.

Animals↗

PubMed1999

Evidence for alphavbeta3 and alphavbeta5 integrin-like vitronectin (VN) receptors in Candida albicans and their involvement in yeast cell adhesion to VN.

The expression of integrin vitronectin (VN) receptors on Candida albicans yeasts and their involvement in the adhesion to VN were investigated. By immunofluorescence and cytofluorimetric analysis, several antibodies directed against human alphav, beta3, beta5, alphavbeta3, or alphavbeta5 integrin positively stained C. albicans yeasts. Biochemical analysis on yeast lysates with anti-human alphav, beta3, or beta5 antibody revealed molecular species of 130, 110, 100, and 84 kDa. The 130-kDa band was identified as alphav, whereas the doublet of 110/100 kDa and the 84-kDa band likely correspond to the beta3 and beta5 subunits, respectively. Some 48%-54% of Candida yeasts specifically adhered to VN, and this binding was strongly inhibited by anti-human alphav, beta3, alphavbeta3, and alphavbeta5 antibodies and by RGD- but not RGE-containing peptides. In addition, VN inhibited C. albicans adherence to a human endothelial cell line. Thus, C. albicans in the yeast phase expresses VN receptors antigenically related to the vertebrate alphavbeta3 and alphavbeta5 integrins, which mediate its adhesion to VN.

Candida albicans↗

PubMed2003

CAR is a cell-cell adhesion protein in human cancer cells and is expressionally modulated by dexamethasone, TNFalpha, and TGFbeta.

The coxsackie adenovirus receptor (CAR) has become of interest for gene therapy due to its crucial function in adenoviral cell entry. In clinical trials with adenoviral vectors, dexamethasone is applied to reduce side effects such as inflammatory reactions or emesis. By using a beta-galactosidase-expressing adenovirus (AdGal), we observed that dexamethasone treatment resulted in decreased adenoviral gene transfer into human cancer cells. Expression of CAR and integrin alpha5beta1 was transcriptionally downregulated by dexamethasone as shown for HeLa cervical cancer cells and U87MG glioblastoma cells. TNFalpha increased CAR expression in HeLa and ovarian cancer cells but decreased CAR expression in U87MG cells. In all tested cancer cell lines, TNFalpha induced a significant increase in the expression of adenovirus-binding integrins alpha5beta1, alphavbeta3 and alphavbeta5. Pretreatment with TNFalpha increased AdGal gene transfer into cancer cells and enhanced the cytotoxic effect of a p53-expressing adenovirus. In contrast, TGFbeta reduced CAR expression level and adenoviral gene transfer into OV-UL-2 ovarian cancer cells. Confocal immunofluorescence analysis revealed localization of CAR at cell-cell adhesions in several human cancer cell lines and disruption of cell-cell contacts increased adenoviral gene transfer into human cancer cells. In clinical cancer gene therapy, efficiency of adenoviral gene delivery could be altered by cell adhesion, TNFalpha, TGFbeta, and dexamethasone.

Adenoviridae↗

PubMed2004

Bone sialoprotein, matrix metalloproteinase 2, and alpha(v)beta3 integrin in osteotropic cancer cell invasion.

BACKGROUND: Bone sialoprotein (BSP) interacts separately with both matrix metalloproteinase 2 (MMP-2) and integrin alpha(v)beta3 and is overexpressed in many metastatic tumors. Its role in tumor biology, however, remains unclear. We investigated whether BSP enhances cancer cell invasiveness by forming a trimolecular complex with MMP-2 and cell-surface integrin alpha(v)beta3. METHODS: Invasiveness of breast, prostate, lung, and thyroid tumor cell lines was measured with a modified Boyden chamber assay. Binding and co-localization of BSP, MMP-2, and integrin alpha(v)beta3 were investigated with immunoprecipitation and in situ hybridization. All statistical tests were two-sided. RESULTS: Treatment with BSP increased invasiveness of many breast, prostate, lung, and thyroid cancer cells through Matrigel in a dose-dependent manner. BSP at 50 nM increased the invasiveness of SW-579 thyroid cancer cells (95.2 units, 95% confidence interval [CI] = 90.4 to 100 units) by approximately 10-fold compared with that of untreated control SW-579 cells (9.1 units, 95% CI = 5.7 to 12.5 units) (P<.001). Addition of an inactive mutated BSP, in which BSP's integrin-binding RGD tripeptide was altered, or addition of integrin alpha(v)beta3-blocking antibodies resulted in invasiveness equivalent to that of untreated cells. Inhibiting cellular MMP-2 activity with chemical inhibitors or a specific antibody also blocked BSP-enhanced invasiveness. Osteopontin and dentin matrix protein 1, proteins related to BSP that also bind integrin alpha(v)beta3 and form complexes with other MMPs (but not MMP-2), did not enhance invasiveness. Immunoprecipitation showed that a complex containing BSP, integrin alpha(v)beta3, and MMP-2 formed in vitro. Addition of BSP increased the amount of MMP-2 bound by cells in an integrin-dependent fashion. Co-expression of BSP, integrin alpha(v)beta3, and MMP-2 in papillary thyroid carcinoma cells was shown by in situ hybridization. CONCLUSION: Cancer cells appear to become more invasive when BSP forms a cell-surface trimolecular complex by linking MMP-2 to integrin alpha(v)beta3.

Bone Neoplasms↗

PubMed2002

Integrin alphavbeta1 is a receptor for foot-and-mouth disease virus.

Infection by field strains of Foot-and-mouth disease virus (FMDV) is initiated by binding to certain species of arginine-glycine-aspartic acid (RGD)-dependent integrin including alphavbeta3 and the epithelial integrin alphavbeta6. In this report we show that the integrin alphavbeta1, when expressed as a human/hamster heterodimer on transfected CHOB2 cells, is a receptor for FMDV. Virus binding and infection mediated by alphavbeta1 was inefficient in the presence of physiological concentrations of calcium and magnesium but were significantly enhanced by reagents that activate the integrin and promote ligand binding. The ability of chimeric alpha5/alphav integrin subunits, in association with the beta1 chain, to bind FMDV and mediate infection matched the ligand binding specificity of alphavbeta1, not alpha5beta1, thus providing further evidence for the receptor role of alphavbeta1. In addition, data are presented suggesting that amino acid residues near the RGD motif may be important for differentiating between the binding specificities of alphavbeta1 and alphavbeta6.

Animals↗

PubMed2006

Bone sialoprotein enhances migration of bone marrow stromal cells through matrices by bridging MMP-2 to alpha(v)beta3-integrin.

UNLABELLED: BMSCs migrate through matrix barriers and differentiate into osteoblasts. BSP enhances osteogenic cell migration through basement membrane and collagen matrices in vitro by localizing MMP-2 on the cell surface through alpha(v)beta(3)-integrin. INTRODUCTION: The specific mechanisms by which bone marrow stromal cells (BMSCs) leave their primary sites, move through matrices encountered during homing to their site of final differentiation, and remove preexisting matrices in preparation for bone matrix production are not well understood. MATERIALS AND METHODS: The enhanced migration of human osteoblast precursor cells through matrix barriers by bone sialoprotein (BSP) was studied by a modified Boyden-chamber assay. The bridging of normally soluble matrix metalloproteinase 2 (MMP-2) to the cell surface receptor, alpha(v)beta(3)-integrin, by BSP was analyzed by flow cytometry. RESULTS: BSP enhanced the in vitro passage of BMSCs and pre-osteoblasts through matrix barriers (Matrigel and denatured type I collagen) in a dose-dependent manner. An intact ArgGlyAsp (RGD) was required in the BSP for enhanced migration through the barriers but was not sufficient, as shown by the inactivity of two other SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family members, osteopontin and dentin matrix protein-1. The specificity of the BSP enhancement activity was apparently caused by this molecule's ability to bridge MMP-2 to the cell surfaces. CONCLUSIONS: Pre-osteoblasts and their BMSC precursors may use MMP-2/BSP/integrin complexes to disrupt matrix barriers during migration to their final destinations in vivo.

Bone Marrow Cells↗

PubMed2005

Monitoring of integrin-mediated adhesion of human ovarian cancer cells to model protein surfaces by quartz crystal resonators: evaluation in the impedance analysis mode.

The quartz crystal microbalance (QCM) was used to monitor specific, integrin-mediated adhesion of human ovarian cancer cells to distinct extracellular matrix (ECM) proteins immobilized on gold-coated quartz crystal resonators. The QCM was operated in the impedance analysis mode, where frequency shift as well as bandwidth are accessible on a broad range of overtones. The increase in bandwidth caused by covering the quartz resonator with cells was reproducible and largely independent of overtone order, whereas the frequency shift displayed some variability. Thus the bandwidth proved to be the more robust parameter for sensing cell adhesive events. The bandwidth increased in proportion to the number of seeded cells to the quartz crystal as long as the number was below 150,000 cells/ml. Comparing the resonance parameters on different harmonics, one finds that viscoelastic modeling with homogeneous layer systems cannot reproduce the results: lateral heterogeneity has to be taken into account. The differences in adhesive strength of human ovarian cancer cells towards selected ECM proteins monitored by QCM was in good agreement with data obtained by conventional cell adhesion assays. Strong cell adhesion was observed to the ECM proteins vitronectin (VN) and fibronectin (FN), while only weak attachment occurred on laminin. In order to prove specific, integrin alphavbeta3-mediated cell adhesion to its ligands FN and VN, the cyclic integrin alphavbeta3-directed peptide c(RGDfV) was used as competitor and significantly reversed cell adhesion. Since integrin interaction with ECM proteins is dependent on the presence of bivalent cations, cell detachment was also seen after treatment of cell monolayers with the chelator ethylene-dinitro-tetra-acetic acid (EDTA). The QCM technique is a reliable method to monitor cell adsorption to ECM-pretreated surfaces in real time. It may be an alternative tool for screening specific and selective antagonists of integrin/ECM interaction.

Biosensing Techniques↗

PubMed2000

Expression of integrin subunit beta1B in integrin beta1-deficient GD25 cells does not interfere with alphaVbeta3 functions.

We have expressed the beta1B integrin subunit in beta1-deficient GD25 cells to examine beta1B functions without the interference of endogenous beta1A expression. As previously reported [Retta et al., 1998, Mol. Biol. Cell 9, 715-731], the beta1B integrins did not mediate cell adhesion under normal culture conditions, while the presence of 0.3 mM Mn(2+) allowed beta1B integrins to support adhesion. Mn(2+), as well as the small soluble peptide GRGDS, induced a beta1B conformation, which was recognized by the mAb 9EG7, a marker for active or ligand-bound integrins. beta1B integrins were found to localize to a subset of focal contacts in a ligand-independent manner on fibronectin, but not on vitronectin. However, clustering of beta1B did not induce tyrosine phosphorylation of FAK, p130(Cas), or paxillin, as studied by beta1B-mediated adhesion, to fibronectin in the presence of Mn(2+) or to anti-beta1 antibody in DMEM. Induction of ligand-occupied conformation by the GRGDS peptide during the adhesion to anti-beta1 antibody also failed to trigger FAK phosphorylation. Stimulation of tyrosine phosphorylation on FAK, p130(Cas), and paxillin by adhesion via integrin alphaVbeta3 to fibronectin or vitronectin was not disturbed in GD25-beta1B cells compared to the untransfected GD25 cells, nor were any negative effects of beta1B observed on alphaVbeta3-mediated cell attachment, spreading, and actin organization, or on the cell proliferation rate. These results show that the reported negative effects of beta1B on adhesive events do not apply to alphaVbeta3-dependent interactions and suggest that they may specifically act on beta1 integrins.

Actins↗

PubMed2005

Grafting aminocyclopentane carboxylic acids onto the RGD tripeptide sequence generates low nanomolar alphaVbeta3/alphaVbeta5 integrin dual binders.

Eleven gamma-aminocyclopentane carboxylic acid (Acpca) platforms, including four dihydroxy representatives (19-22), three hydroxy analogues (34-36), and four deoxy derivatives (30-33), were prepared in a chiral nonracemic format. These simple units were then grafted onto an Arg-Gly-Asp (RGD) tripeptide framework by a mixed solid phase/solution protocol delivering an ensemble of 11 macrocyclic analogues of type cyclo-[-Arg-Gly-Asp-Acpca-], 1-11. The individual compounds were evaluated for their binding affinity toward the alphaVbeta3 and alphaVbeta5 integrin receptors. The analogue 10 exhibited a very interesting activity profile (IC50/alphaVbeta3= 1.5 nM; IC50/alphaVbeta5= 0.59 nM), comparable to that of reference compounds EMD121974 and ST1646. Closely related congeners 6, 8, and 9 also proved to be excellent dual binders with activity levels in the low nanomolar range. The three-dimensional (3D) NMR solution structures were determined, and docking studies to X-ray crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with the reference compound EMD121974 were performed on selected analogues to elucidate the interplay between structure and function in these systems and to evidence the subtle bases for receptorial recognition. The results prove that the principle of isosteric dipeptide replacement for peptidomimetics design and synthesis can be violated, without detriment to the development of highly effective integrin binders.

Binding, Competitive↗

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↗

PubMed2005

Cartilage oligomeric matrix protein/thrombospondin 5 supports chondrocyte attachment through interaction with integrins.

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix of the musculoskeletal system. Although COMP/TSP5 abnormalities are associated with several pathological conditions, its normal function remains unclear. This study was undertaken to delineate the function(s) of COMP/TSP5 in cartilage, especially regarding its interaction with chondrocytes. We show that COMP/TSP5 can support chondrocyte attachment and that the RGD sequence in COMP/TSP5 and the integrin receptors alpha5beta1 and alphaVbeta3 on the chondrocytes are involved in mediating this attachment. The interactions of COMP/TSP5 with the integrins are dependent on COMP/TSP5 conformation. Chondrocyte attachment to COMP/TSP5 in the calcium-replete conformation was inhibited by function-blocking integrin alpha5 and beta1 antibodies, suggesting the involvement of the alpha5beta1 integrin. Under this condition, a function-blocking antibody against alphaVbeta3 did not have any effect on cell attachment. On the other hand, chondrocyte attachment to reduced COMP/TSP5 was instead sensitive to alphaVbeta3 function-blocking antibodies, suggesting that COMP/TSP5 mediates attachment through chondrocyte alphaVbeta3 integrin under this condition. Cell attachment to reduced COMP/TSP5 was not inhibited by beta1 antibodies. These data indicate that COMP/TSP5 in different conformations can utilize different integrin receptors. These results are the first to demonstrate that COMP/TSP5 can mediate chondrocyte attachment through interactions with integrins. Through these interactions, COMP/TSP5 may be able to regulate cellular activities and respond to environment in the surrounding cartilage matrix.

Calcium↗

PubMed2002

Determination of molecules regulating gene delivery using adenoviral vectors in ovarian carcinomas.

Gene therapeutic approaches currently favor adenoviral vectors over alternatively available vector systems. Ovarian cancer represents an attractive model for an intraperitoneal adenovirus-based gene therapy, which is now under intensive clinical investigation. Adenovirus-mediated gene transfer depends on adequate virus uptake and thus on the presence of sufficient amounts of high-affinity coxsackie-adenovirus receptor (CAR) and alphavbeta3- and alphavbeta5 integrins on target cells. This fact has been ignored in most ongoing clinical trials. This investigation, therefore, determined expression of CAR by immunohistochemistry in 37 ovarian carcinomas and compared it with that of alphavbeta3 and alphavbeta5 integrins. In all samples, except one undifferentiated carcinoma, CAR was immunohistochemically demonstrable. Grade 1 tumors exhibited stronger CAR immunostaining as compared with higher-grade cancers (P < 0.03). Integrins alphavbeta3 and alphavbeta5 were detectable in 62% and 65% of carcinomas, respectively, and staining for both classes correlated positively (P < 0.005). Cancers classified as undifferentiated completely lacked alphavbeta3 expression. Furthermore, in undifferentiated and grade 3 carcinomas the three molecules studied exhibited marked distributional heterogeneity with regard to focal positivity and negativity within the same tumor. Either the absence of CAR, alphavbeta3 and alphavbeta5 or the pronounced heterogeneity in their expression might seriously compromise the efficiency of adenovirus-based gene therapy in ovarian cancer.

Adenoviridae↗

PubMed2005

alphavbeta3 and macrophage colony-stimulating factor: partners in osteoclast biology.

Osteoclasts, the sole bone-resorbing cells, arise by fusion and differentiation of monocyte/macrophage precursors. Matrix degradation requires adhesion of the osteoclast to bone, an integrin alphavbeta3-mediated event that also stimulates signals which polarize the cell and secrete resorptive molecules such as hydrochloric acid and acidic proteases. Two cytokines are necessary and sufficient for osteoclastogenesis, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), both produced by mesenchymal cells in the bone marrow environment. M-CSF promotes survival and proliferation of osteoclast precursors. It also contributes to their differentiation and regulates the cytoskeletal changes that accompany bone resorption. Binding of M-CSF to c-Fms, its receptor, recruits adapter proteins and cytosolic kinases, thereby activating a variety of intracellular signals. We herein review how alphavbeta3 and M-CSF, alone and in concert, impact production, survival, and function of the osteoclast, thereby controlling skeletal mass. Signals from alphavbeta3 and/or c-Fms activate Syk and Vav3, originally defined by their function in lymphoid cells. Genetic depletion of either protein generates a strong bone phenotype, underscoring the promise of osteoimmunobiology.

Animals↗

PubMed2006

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation.

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.

Alkaline Phosphatase↗

PubMed2003

Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis.

The integrin receptor alphavbeta3 has been shown to play a critical role in several distinct processes, such as angiogenesis, osteoclast-mediated bone resorption and tumor metastasis. Its expression is upregulated in newly synthesized blood vessels produced in response to a variety of tumors and purified angiogenic factors. Studies show that alphavbeta3 is a critical target downstream from perhaps all angiogcnic factors. Proof-of-principle that alphavbeta3 antagonists such as monoclonal antibodies and small molecules block angiogenesis and tumor growth has been obtained in several animal models. Many endogenous inhibitors of angiogenesis such as angiostatin, endostatin and tumstatin seem to work through the alphavbeta3 receptor further emphasizing the critical role of this receptor in angiogenesis. In addition, the alphavbeta3 receptor has been clearly implicated in several pathological processes such as rheumatoid arthritis, osteoporosis, and metastasis of prostate cancer to bone. Thus alphavbeta3 may prove to be an important target for pharmacological intervention in more than one clinical setting.

Angiogenesis Inhibitors↗

PubMed2004

Cholesterol-independent interactions with CD47 enhance alphavbeta3 avidity.

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced alpha(v)beta(3) activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of "activable" integrin molecules. Expression of either CD47 species also increased alpha(v)beta(3)-mediated adhesion to vitronectin, and to surfaces coated with the anti-beta(3) antibody AP3, because of enhanced clustering of alpha(v)beta(3) as confirmed by chemical cross-linking. Cholesterol depletion with methyl-beta-cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit alpha(v)beta(3) and its associated signaling molecules to these domains. Thus CD47-alpha(v)beta(3) complexes in cholesterol-rich raft domains appear to engage in G(i)-dependent signaling whereas CD47-alpha(v)beta(3) interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.

Antigens, CD↗

PubMed2003

Matrix-fibrinogen enhances wound closure by increasing both cell proliferation and migration.

Fibrinogen (FBG) assembles into matrix fibrils of fibroblasts, lung and mammary epithelial cells, but not endothelial cells. Furthermore, cryptic beta15-21 residues are exposed in FBG fibrils with no evidence of thrombin or plasmin proteolysis. Herein, the effects of FBG on migration and proliferation of wounded dermal fibroblasts were investigated. FBG preassembled into matrix prior to scrape-wounding induced 3H-thymidine incorporation 8-fold and shortened the time to wound closure 1.6-fold +/- 0.1-fold. FBG added immediately after wounding did not enhance either response. Fibroblast growth factor-2/platelet-derived growth factor (FGF-2/PDGF) stimulated cell proliferation 2.2-fold for FGF-2 and 3.2-fold for PDGF and wound closure 1.5-fold +/- 0.1-fold in the absence of matrix-FBG. Surprisingly, exogenous growth factors had negligible effect on wound closure and cell proliferation already enhanced by matrix-FBG. Matrix-FBG-enhanced wound closure required active assembly of an FBG-fibronectin matrix, engagement of alphavbeta3, and FBG Aalpha-RGDS572-575 integrin recognition sites; Aalpha-RGDF95-98 sites were not sufficient for matrix-FBG assembly, enhanced wound closure, or cell proliferation. Although Bbeta1-42 was not necessary for matrix assembly, it was required for matrix-FBG-enhanced cell migration. These data indicate that FBG serves as an important matrix constituent in the absence of fibrin formation to enhance wound repair and implicate Bbeta1-42 as a physiologic inducer of signal transduction to promote an intermediate state of cell adhesion and a migratory cell phenotype.

Amino Acid Sequence↗