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PubMed2004

Distribution of the extracellular matrix glycoproteins in ependymomas--an immunohistochemical study with follow-up analysis.

The extracellular matrix (ECM) plays a critical role in influencing the biological behavior of brain tumors and the diagnostic detection of ECM components in ependymomas might be of prognostic value. In the present study we evaluated immunohistochemically the expression of a spectrum of ECM glycoproteins (tenascin, vitronectin, fibronectin, laminin, collagen types II, IV and VI) in a series of 36 pediatric intracranial ependymomas. The distribution of the ECM glycoproteins was evaluated both within the tumor tissue and at the tumor invasion front, and the prognostic value of the results was tested in a survival analysis. The expression of most of the ECM glycoproteins was associated only with blood vessels. Tenascin and vitronectin were found in a more diffuse pattern around the tumor cells and at the tumor invasion fronts of several cases. The progression-free survival was significantly decreased for patients with tenascin positive tumors (in any of the studied compartments) and for the tumors with vitronectin accumulation at their invasion fronts. In one ependymoma containing foci of cartilage with metaplastic ossification we demonstrated that collagen types II and VI and tenascin were present in ECM of both the cartilage and the ependymoma, and were accompanied by areas of necrosis and dystrophic calcifications. We suggest, that the rare simultaneous production of the specific ECM components might lead to the formation of chondroid areas in ependymomas. An abundant production of some ECM glycoproteins (tenascin and vitronectin) is present in a proportion of ependymomas and its immunohistochemical detection is of prognostic relevance.

Adolescent↗
PubMed1992

Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.

Amino Acid Sequence↗
PubMed2006

In vitro analysis of integrin expression during chondrogenic differentiation of mesenchymal stem cells and chondrocytes upon dedifferentiation in cell culture.

Tissue engineering represents a promising method for generating chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Stem cells, however, displaying unlimited self-renewal and the capacity to differentiate towards chondrocytes, might be usable after further characterization. As the interactions between the extracellular matrix and the cellular compartment can alter the cellular behaviour, we investigated the expression of integrins using microarray analysis during chondrogenic differentiation of human mesenchymal stem cells (MSC) in comparison with de-differentiating human chondrocytes (HC) harvested during septoplasty. During chondrogenic differentiation of MSC, the fibronectin-receptor (Integrin beta1alpha5), fibronectin and the GPIIb/IIIa-receptor were downregulated. The components of the vitronectin-receptor (Integrin alphavbeta3) and CD47 were constantly expressed and ILK was downregulated. Vitronectin and osteopontin were not expressed by the cells. In HC, Integrin beta1alpha5 in conjunction with the ligand fibronectin were upregulated during dedifferentiation, Integrin alphavbeta3 as well as the GBIIb/IIIa-receptor were activated on day 21 but neither vitronectin nor osteopontin were expressed by the cells. The integrins, beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7, alpha11, were not expressed at any time. ILK, CD47, and ICAP were activated with ongoing dedifferentiation. In conclusion, a candidate for signal-transmission is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphavbeta3), or their ligands do not seem to be involved in signal transmission for dedifferentiation. The GPIIb/IIIa-receptor might assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might be involved in the transduction of integrin-dependent signals.

Cell Differentiation↗
PubMed1993

Structural and functional analyses of the Arg-Gly-Asp sequence introduced into human lysozyme.

To determine the functional conformation of the Arg-Gly-Asp (RGD) sequence, we have constructed mutant proteins by inserting 4-12 amino acid residues from the RGD region of human fibronectin between Val74 and Asn75 of human lysozyme. RGDS-, GRGDSP-, TGRGDSPA-, VTGRGDSPAS-, and AVTGRGDS-PASS-introduced mutant lysozymes were expressed in yeast, purified, and designated as RGD4, -6, -8, -10, and -12, respectively. Using baby hamster kidney cells, RGD8, RGD10, and RGD12 were shown to possess high cell adhesion activity nearly equal to 10% of human vitronectin activity. RGD4 and RGD6 exhibited somewhat lower cell adhesion activity. The activities of these mutant proteins were inhibited by the addition of either GRGDSP peptide or polyclonal antibody against vitronectin receptor, as was the case for the vitronectin activity. The results suggest that the cell adhesion signals are transduced to cells through the interaction with the vitronectin receptor. The three-dimensional structures of RGD4 and RGD8 were determined at 1.8-A resolution by x-ray crystallography. A model of the inserted region in RGD4 could be built in the electron density map, but the positions of the preceding residues, Ala73-Val74, were uncertain. The inserted region in RGD8 did not demonstrate continuous electron densities. The results suggest that these RGD sequence-containing regions are highly flexible and that such flexibility could allow the conformation of the RGD regions to be induced to fit into the binding pocket of the integrin receptor.

Amino Acid Sequence↗
PubMed1996

Thrombus imaging with a technetium-99m-labeled activated platelet receptor-binding peptide.

UNLABELLED: The objective of this work was the preclinical evaluation of 99mTc-P280, a 99mTc-labeled peptide having high affinity and specificity for the GPIIb/IIIa receptor expressed on activated platelets, for use as a thrombus imaging agent. METHODS: The affinity and specificity of P280 peptide for the GPIIb/IIIa receptor was assessed by the inhibition of ADP-stimulated human platelet aggregation, the inhibition of the binding of fibrinogen to the GPIIb/IIIa receptor and the inhibition of the binding of vitronectin to the vitronectin receptor. P280 peptide was radiolabeled with 99mTc by ligand exchange using 99mTc-glucoheptonate. The ability of 99mTc-P280to detect thrombi in vivo was assessed using a canine venous thrombosis model and the biodistribution of 99mTc-P280 was determined in rats and rabbits. RESULTS: P280 peptide had an IC50 of 79 nM for the inhibition of aggregation of human platelets in platelet rich plasma, an IC50 of 6.8 nM for the inhibition of fibrinogen binding to the GPIIb/IIIa receptor and an IC50 of 13 microM for the inhibition of vitronectin binding to the vitronectin receptor, showing the high in vitro receptor binding affinity and specificity of the peptide. 99mTc-P280 was readily prepared in > or = 90% radiochemical and yield purity and provided images of femoral vein thrombin in the canine model by 1 hr postinjection (thrombus-to-blood ratio of 4.4 and thrombus-to-muscle ratio of 11 at 4 hr). Dog, rat and rabbit studies all showed rapid clearance of the radiotracer from the blood and rapid renal excretion. CONCLUSION: The combination of high in vitro receptor-binding affinity and specificity, in vivo thrombus imaging and fast clearance support the evaluation of 99mTc-280 as a clinical imaging agent.

Animals↗
PubMed1997

Reattachment of cultured human retinal pigment epithelium to extracellular matrix and human Bruch's membrane.

PURPOSE: To determine the mechanism of reattachment of harvested human retinal pigment epithelium (RPE) to RPE-derived extracellular matrix and Bruch's membrane. METHODS: Confluent first-to third-passage human RPE were harvested from tissue culture and plated onto RPE-derived extracellular matrix or human Bruch's membrane exoplants denuded of cells by treatment with 0.02 N ammonium hydroxide. The authors measured RPE reattachment to uncoated surfaces or surfaces precoated with extracellular matrix proteins (fibronectin, laminin, vitronectin, or type IV collagen), antibodies to extracellular matrix-proteins, or the synthetic peptide RGDS (arginine-glycine-aspartate-serine). Some RPE were pretreated with anti-beta 1 integrin antibodies before plating onto either substrate. RESULTS: Coating the surface of either RPE-derived extracellular matrix or Bruch's membrane with fibronectin, laminin, vitronectin, or type IV collagen increased the RPE attachment rate. Exposing RPE to anti-beta 1 integrin antibodies or RGDS or precoating the surface with antibodies to fibronectin, laminin, vitronectin, or type IV collagen decreased the RPE attachment rate to both surfaces. The RPE attachment rate to Bruch's membrane was lower when the exoplants were harvested from the macula of older (age, 70 to 90 years) versus younger (age, 30 to 40 years) persons (52.4 +/- 3.6% versus 64.3 +/- 3.5%, respectively; P < 0.05). CONCLUSIONS: The attachment of cultured human RPE cells to human Bruch's membrane or to RPE-derived extracellular matrix is mediated by an interaction between the beta 1-subunit of integrin on the RPE surface and ligands in the extracellular matrix that include laminin, fibronectin, vitronectin, and type IV collagen. The lower rate of RPE reattachment to the macula from older human cadaveric eyes may have implications for studies aimed at RPE transplantation in elderly persons.

Adhesiveness↗
PubMed2000

Regulation of alphaVbeta3 and alphaVbeta5 integrins by dexamethasone in normal human osteoblastic cells.

Long-term administration of pharmacological doses of glucocorticoids inhibits bone formation and results in osteoporosis. Since integrin-mediated cell-matrix interactions are essential for osteoblast function, we hypothesized that the detrimental effect of glucocorticoids on bone derived, at least in part, from decreased integrin-matrix interactions. Because alphavbeta3 and alphavbeta5 integrins can interact with several bone matrix proteins, we analyzed the effects of dexamethasone (Dex) on the expression of these integrins in normal human osteoblastic cells. We found adhesion of these cells to osteopontin and vitronectin to be dependent on alphavbeta3 and alphavbeta5, respectively; this ligand specificity was not altered by Dex. The effects of Dex on the adhesion of human osteoblastic cells to osteopontin and vitronectin were biphasic with an increase after 2 days, followed by a decrease after 8 days of treatment. Consistently, surface alphavbeta3 and alphavbeta5 integrins, which were increased after 2 days of Dex treatment, were decreased after 8 days. Similarly, total cellular alphav, beta3, and beta5 proteins, which were increased by Dex early in the culture, were diminished after 8 days. Metabolic labeling studies indicated that Dex exhibited biphasic regulation on the biosynthesis of alphavbeta5, with stimulation observed during the second day of treatment, followed by inhibition during the 8th day of exposure. By contrast, the biosynthesis of alphavbeta3 was inhibited by Dex on day 1 and remained inhibited on day 8. Analysis of the mRNA indicated that alphav and beta5 levels were increased by Dex during early exposure (1-3 days), followed by inhibition after prolonged exposure (>/=7 days). By contrast, Dex decreased beta3 mRNA level at all the time points analyzed. Consistently, Dex decreased beta3 promoter activity after 1 day and persisted over 8-day period. By contrast, Dex stimulated beta5 promoter activity after 1 or 2 days but had no effect after 8 days. To further evaluate mechanism(s) leading to the decreased integrin expression after prolonged Dex treatment, mRNA stability was analyzed. Dex was found to accelerate the degradation of alphav, beta3 and beta5 mRNA after an 8-day treatment. Thus, the regulation of alphavbeta3 was dependent on transcription and posttranscriptional events whereas the expression of alphavbeta5 was dependent mainly on posttranscriptional events after prolonged Dex treatment. In conclusion, Dex exhibited time-dependent regulation on the expression of alphavbeta3 and alphavbeta5 integrins in normal human osteoblastic cells. Short-term exposure to Dex increased the levels of alphavbeta3 and alphavbeta5 on the surface and cell adhesion to osteopontin and vitronectin whereas long-term exposure to Dex decreased the expression of both integrins and inhibited the cell adhesion to matrix proteins.

Antigens, CD↗
PubMed2001

Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor cells than titanium or steel.

The implant material hydroxylapatite (HA) has been shown in numerous studies to be highly biocompatible and to osseointegrate well with existing bone; however, the molecular mechanisms at work behind this osseointegration remain largely unexplored. One possibility is that the implant, exposed to the patient's blood during surgery, adsorbs known cell adhesive proteins such as fibronectin and vitronectin from the serum. Osteoblast precursors could then adhere to these proteins through integrin-mediated mechanisms. In the present study, we have used a quantitative ELISA assay to test the hypothesis that hydroxylapatite will adsorb more fibronectin and vitronectin from serum than two commonly used hard-tissue materials, commercially pure titanium, and 316L stainless steel. We further used the ELISA, as well as a standard cell adhesion assay, to test the hypothesis that increased protein adsorption will lead to better binding of purified integrins alpha5beta1 and alpha(v)beta3 and osteoblast precursor cells to the HA than to the metals. Our results show that fibronectin, vitronectin, alpha5beta1, alpha(v)beta3, and osteoblast precursor cells do indeed bind better to HA than to the metals, suggesting that improved integrin-mediated cell binding may be one of the mechanisms leading to better clinical bone integration with HA-coated implants.

Adsorption↗
PubMed1996

Disintegrin interaction with alpha V beta 3 integrin on human umbilical vein endothelial cells: expression of ligand-induced binding site on beta 3 subunit.

The effect of seven disintegrins (albolabrin, barbourin, bitistatin, echistatin, eristostatin, flavoridin, and kistrin) and the neurotoxin analogue, mambin, on the adhesion of human umbilical vein endothelial cells (HUVEC) to immobilized vitronectin and fibronectin has been studied. Adhesion to vitronectin was significantly inhibited by echistatin, kistrin, flavoridin, and mambin. Echistatin, flavoridin, and kistrin bound with high affinity to immobilized alpha V beta 3 in solid phase assay; other disintegrins bound at a much lower level. Echistatin and flavoridin had a modest inhibitory effect on HUVEC adhesion to fibronectin. HUVEC adhered to disintegrins with a high selectivity toward bitistatin, echistatin, flavoridin, kistrin, and mambin. Adhesion of HUVEC to fibronectin and vitronectin resulted in cell spreading, whereas cells adhering to immobilized echistatin remained globular and cells adhering to kistrin showed abnormal morphology. Echistatin and kistrin potently inhibited the binding of monoclonal antibody (Mab) 7E3, which recognizes the alpha V beta 3 complex, to HUVEC. Echistatin and kistrin also induced the binding to HUVEC of Mab 62, which recognizes the ligand-induced binding site (LIBS) epitope on the beta 3 subunit, enhancing HUVEC binding to immobilized Mab 62. Similar results with both antibodies were obtained in Chinese hamster ovary cells transfected with alpha V beta 3 genes. In conclusion, disintegrin interaction with HUVEC appears to be selectively mediated by alpha V beta 3 receptors, and it results in an expression of LIBS epitope that may play a role in the regulation of ligand-binding affinity and intracellular signaling.

Animals↗
PubMed1997

alphaV Integrins on HT-29 colon carcinoma cells: adhesion to fibronectin is mediated solely by small amounts of alphaVbeta6, and alphaVbeta5 is codistributed with actin fibers.

HT-29 colon carcinoma cells form liver metastases upon intrasplenic injection, and adhesion to fibronectin under the liver microvascular liver endothelium is likely to be important for metastasis formation. We have therefore studied the integrins involved in fibronectin adhesion. This was not affected by blocking antibodies against the beta1, alpha3, and alpha5 integrin subunits, but it was blocked by an RGD-containing peptide, indicating involvement of RGD-dependent non-beta1 alphaV integrins. Both alphaVbeta5 and alphaVbeta6 were detected on HT-29 cells. Blocking mAb against alphaV, but not against alphaVbeta5, abolished adhesion. From a HT-29 cell lysate, only alphaVbeta6 bound to a fibronectin-Sepharose column. Thus, alphaVbeta6 is the main fibronectin receptor on HT-29 cells, despite the very low levels of alphaVbeta6 and the much higher levels of alphaVbeta5. The HT29 cells did not spread on fibronectin in the absence of serum, not even after a three- to fourfold increase in alphaVbeta6 levels, induced by interleukin 4. The cells did spread on vitronectin. Using immunofluorescence we observed that both on vitronectin and on fibronectin alphaVbeta5 was arranged in a striped pattern, aligned with actin fibers, and not in focal adhesions. On fibronectin, but not on vitronectin, alphaVbeta6 was concentrated in a punctate pattern at the periphery of cell islands.

Actins↗
PubMed1989

(Arg-Gly-Asp)n-albumin conjugates as a model substratum for integrin-mediated cell adhesion.

We have prepared protein-peptide conjugates composed of bovine serum albumin (BSA) derivatized with short peptides containing the Arg-Gly-Asp (RGD) sequence derived from the adhesion site of fibronectin. The RGD-BSA conjugates were used to coat tissue culture plastic surfaces which then served as substrata in cell adhesion experiments. Our results indicate that the efficiency of adhesion to RGD-BSA-coated surfaces is highly dependent on the valency of the (RGD)n-BSA conjugates. For example, on surfaces with approximately equal amounts of RGD ligand, CHO cells adhered virtually 100% to the (RGD)n-BSA (n = 20.8) conjugate and not at all to the (RGD)n-BSA (n = 3.5) conjugate. Adhesion on (RGD)n-BSA-coated substrata and on fibronectin- or vitronectin-coated substrata was also examined in terms of the relationship between cell adhesion and the intermolecular distances of adsorbed proteins. It was observed that for substrata coated with relatively compact, symmetric molecules, such as RGD-BSA or vitronectin, adhesion dropped off sharply as intermolecular distances increased; by contrast, for fibronectin, a large asymmetric molecule, adhesion declined more gradually as intermolecular distances increased. Finally, we have examined the role of different cell-surface receptors in the process of adhesion to RGD-BSA substrata. Interestingly, competition and blocking experiments with antibodies and with soluble competing proteins suggest that it is the vitronectin receptor rather than the fibronectin receptor which mediates adhesion to RGD-BSA.

Adsorption↗
PubMed2006

Cell surface overexpression of alphavbeta5 integrin impedes alphavbeta3-mediated migration of the human ovarian adenocarcinoma cell line IGROV1.

Human ovarian surface epithelium and epithelial tumors express integrin alphavbeta5, which can interact with vitronectin. In addition, in vitro acquisition of cisplatin resistance by alphavbeta3-expressing IGROV1 cells is accompanied by cell-surface expression of integrin alphavbeta5. To further explore the role of alphavbeta5 in ovarian carcinoma cells, IGROV1 cells were stably transfected with a human beta5 integrin cDNA construct, and three beta5 transfectant clones were selected for the expression of alphavbeta5 integrin at their cell surface. Despite a delayed entry in the exponential phase of growth, beta5-transfectant cells kept a proliferation ability similar to that of parental cells, while their growth rate was hindered in the presence of an anti-alphavbeta5 blocking antibody. Only simultaneous blockade of alphavbeta3 and alphavbeta5 by specific antibodies impeded the adhesion to vitronectin of beta5 transfectants and of the beta5-expressing cisplatin-resistant variant IGROV1-R10, suggesting that the two heterodimers cooperated in the regulation of this process. Cell surface expression of alphavbeta5 resulted in an attenuation of alphavbeta3-mediated migration on vitronectin. Alphavbeta5 participated to migration events in the absence of exogenous growth factors only in one transfectant clone and in IGROV1-R10 cells. Finally, the response to cisplatin was not significantly modified in beta5 transfectants when compared to IGROV1 parental cells.

Adenocarcinoma↗
PubMed1999

Selective regulation of integrin--cytoskeleton interactions by the tyrosine kinase Src.

Cell motility on extracellular-matrix (ECM) substrates depends on the regulated generation of force against the substrate through adhesion receptors known as integrins. Here we show that integrin-mediated traction forces can be selectively modulated by the tyrosine kinase Src. In Src-deficient fibroblasts, cell spreading on the ECM component vitronectin is inhibited, while the strengthening of linkages between integrin vitronectin receptors and the force-generating cytoskeleton in response to substrate rigidity is dramatically increased. In contrast, Src deficiency has no detectable effects on fibronectin-receptor function. Finally, truncated Src (lacking the kinase domain) co-localizes to focal-adhesion sites with alpha v but not with beta 1 integrins. These data are consistent with a selective, functional interaction between Src and the vitronectin receptor that acts at the integrin-cytoskeleton interface to regulate cell spreading and migration.

Animals↗
PubMed1995

Ca(2+)- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils.

Chemoattractants stimulate neutrophil migration by activating signalling pathways including repeated transient increases in intracellular free calcium, [Ca2+]i. A motile neutrophil sends out many pseudopods, some of which adhere to the substrate; to continue moving forward the cell must release these attachments. Adhesion can be actively regulated, and neutrophils in which [Ca2+]i transients are inhibited become stuck on fibronectin or vitronectin, extracellular matrix proteins that neutrophils encounter in vivo. Function-blocking antibodies to beta 3 integrins or the alpha v beta 3 heterodimer restore motility on vitronectin to [Ca2+]i-buffered cells (B. Hendey, M.A.L., E. Marcantonio and F.R.M., manuscript submitted), indicating that an alpha v beta 3-like integrin is responsible for the [Ca2+]i-sensitive adhesion. We show that the density of alpha v beta 3 integrins in the adherent membrane of neutrophils migrating on vitronectin is much higher at the leading edge than at the rear, but [Ca2+]i buffering or inhibition of Ca(2+)-calmodulin-activated protein phosphatase 2B (calcineurin) leads to accumulation of alpha v beta 3 on the adherent surface at the rear of the cell. We show that the polarized distribution of alpha v beta 3 integrins in migrating neutrophils is maintained by [Ca2+]i-dependent release of adhesion followed by endocytosis of these integrins and recycling to the leading edge.

Calcineurin↗
PubMed1998

Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor?

The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity.

Amino Acid Sequence↗
PubMed2003

Functional overlap and cooperativity among alphav and beta1 integrin subfamilies during skin angiogenesis.

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.

Animals↗
PubMed2000

Integrin alpha(v)beta(3) mediates rotavirus cell entry.

Rotavirus strains differ in their need for sialic acid (SA) for initial binding to the cell surface; however, the existence of a postattachment cell receptor, common to most, if not all, rotavirus strains, has been proposed. In the present study, antibodies to the alpha(v) and beta(3) integrin subunits, and the alpha(v)beta(3) ligand, vitronectin, efficiently blocked the infectivity of the SA-dependent rhesus rotavirus RRV, its SA-independent variant nar3, and the neuraminidase-resistant human rotavirus strain Wa. Vitronectin and anti-beta(3) antibodies, however, did not block the binding of virus to cells, indicating that rotaviruses interact with alpha(v)beta(3) at a postbinding step, probably penetration. This interaction was shown to be independent of the tripeptide motif arginine-glycine-aspartic acid present in the natural ligands of this integrin. Transfection of CHO cells with alpha(v)beta(3) genes significantly increased their permissiveness to all three rotavirus strains, and the increment of virus infectivity was reverted by incubation of these cells either with antibodies to beta(3) or with vitronectin. These findings implicate alpha(v)beta(3) integrin as a cellular receptor common to neuraminidase-sensitive and neuraminidase-resistant rotaviruses, and support the hypothesis that this integrin could determine, at least in part, the cellular susceptibility to rotaviruses.

Animals↗
PubMed1996

Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.

Animals↗