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PubMed1995

Membrane attack complex deposits in cutaneous lesions of dermatomyositis.

BACKGROUND AND DESIGN: The presence of membrane attack complex of complement (MAC) deposits in the intramuscular vasculature of biopsy specimens taken from patients with dermatomyositis (DM) has been implicated in the pathogenesis of this myopathy. The purpose of this study was to investigate the presence of MAC deposition in the skin lesions of patients with DM. Using immunohistochemical methods, we examined 22 biopsy specimens from lesional skin, six biopsy specimens from uninvolved skin, and 12 muscle biopsy specimens from patients with DM for the presence of MAC and vitronectin and CD59, two regulatory proteins of complement. RESULTS: The deposition of MAC was demonstrated in a large percentage of biopsy specimens obtained from the lesional skin of patients with DM. Deposits were found along the dermoepidermal junction in 19 (86%) of 22 biopsy specimens. Deposits on the vessel walls of the dermis were found in 17 (77%) of 22 biopsy specimens; but only in six of these biopsy specimens (27%) were deposits present in more than 10% of blood vessels. In contrast, deposits along the dermoepidermal junction and the vessel walls of the dermis were absent in specimens from uninvolved skin. In 12 muscle biopsy specimens obtained simultaneously from these patients, MAC deposits were found on the vessel walls in nine (75%), but only in six (50%) were deposits found in more than 10% of the intramuscular vessels. The pattern of vitronectin immunoreactivity in skin and muscle biopsy specimens obtained from patients with DM was similar to MAC deposits. The expression of CD59 was normal in all skin and muscle biopsy specimens. CONCLUSIONS: The deposition of MAC was found in a high percentage of biopsy specimens from the lesional skin of patients with DM; it was absent in uninvolved skin. These findings suggest that the complement system may be involved in the pathogenesis of the skin lesions of DM.

Adolescent↗
PubMed1999

The characterization of macrophages and osteoclasts in tissues harvested from revised total hip prostheses.

The differentiation and maturation of macrophages and osteoclasts at the prosthetic interface in cases of implant loosening are poorly understood. Using histochemical and immunohistochemical staining methods, we compare macrophage differentiation in tissues from revised hip replacements in patients with specific clinical-radiological appearances. Periprosthetic tissues were harvested from 12 cemented acetabular and 12 cemented femoral components in 24 patients undergoing revision hip replacement. The prostheses were all radiographically and clinically loose. Six acetabular and six femoral components demonstrated radiographic ballooning osteolysis. Serial 6 microm frozen sections of the periprosthetic tissues were processed with hematoxylin and eosin for general tissue morphology, and analyzed for the presence of tartrate resistant acid phosphatase (TRAP, an osteoclast marker). Immunoperoxidase staining using monoclonal antibodies to CD68 (macrophages and osteoclasts) and CD51 (the alpha chain of the vitronectin receptor, an osteoclast marker) was also performed. Approximately 8-30% of the total cells in the tissues were positive for TRAP and the vitronectin receptor, and comprised a subset of the CD68 positive macrophages and macrophage polykaryons. However, there were no statistically significant differences between specific groups (femoral vs. acetabular, osteolysis vs. no osteolysis) for the numbers or percentages of macrophages or osteoclast-like cells. Once prosthetic loosening has occurred, few differences in the macrophage-osteoclast profile of tissues from different periprosthetic locations, with and without osteolysis, are noted. This suggests a final common biologic pathway for periprosthetic bone resorption, once implant loosening has transpired.

Acid Phosphatase↗
PubMed1999

Hydrophobicity, surface tension, and zeta potential measurements of glass-reinforced hydroxyapatite composites.

Wettability and zeta potential studies were performed to characterize the hydrophobicity, surface tension, and surface charge of P2O5-glass-reinforced hydroxyapatite composites. Quantitative phase analysis was performed by the Rietveld method using GSAS software applied to X-ray diffractograms. Surface charge was assessed by zeta potential measurements. Protein adsorption studies were performed using vitronectin. Contact angles and surface tensions variation with time were determined by the sessile and pendent drop techniques, respectively, using ADSA-P software. The highest (-18.1 mV) and lowest (-28.7 mV) values of zeta potential were found for hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), respectively, with composite materials presenting values in between. All studied bioceramic materials showed similar solid surface tension. For HA and beta-TCP, solid surface tensions of 46.7 and 45.3 mJ/m2, respectively, were obtained, while composites presented intermediate surface tension values. The dispersive component of surface tension was the predominant one for all materials studied. Adhesion work values between the vitronectin solution and HA and beta-TCP were found to be 79.8 and 88.0 mJ/m2, respectively, while the 4.0 wt % glass composites showed slightly lower values than the 2.5 wt % ones. The presence of beta-TCP influenced surface charge, hydrophobicity, and protein adsorption of the glass-reinforced HA composites, and therefore indirectly affected cell-biomaterial interactions.

Adsorption↗
PubMed1996

Role of alpha v beta 5 and alpha v beta 6 integrin glycosylation in the adhesion of a colonic adenocarcinoma cell line (HT29-D4).

We have previously characterized the expression of the alpha nu beta 5 and alpha nu beta 6 integrins as major receptors for the human colonic adenocarcinoma cell line (HT29-D4), on vitronectin and fibronectin, respectively [Lehmann et al. (1994): Cancer Res 54:2102-2107]. In the present work we investigated the glycosylation role of these integrins in their adhesive functions. To this end, we used glycohydrolases to show that cell surface integrins were N-glycosylated and sialylated, and that only the alpha v subunit carried some immature oligosaccharide side chains. To alter the glycosylation state of the cell surface alpha v beta 5 and alpha v beta 6 integrins, we used two oligosaccharide-processing inhibitors: 1-deoxymannojirimycin (dMNJ) and tunicamycin (TM). Following treatment of HT29-D4 cells with dMNJ, cell surface alpha v beta 5 and alpha v beta 6 carried only high-mannose-type sugar chains, while TM-treated cells expressed de-N-glycosylated integrins. Neither alpha/beta heterodimers assembly nor cell surface expression were impaired in the presence of the drugs. Finally, we established that adhesion of dMNJ- or TM-treated cells was altered on both vitronectin and fibronectin substrata, whereas the adhesion of these cells on laminin or collagen type I was virtually unchanged.

1-Deoxynojirimycin↗
PubMed1992

Colocalization of thrombospondin and syndecan during murine development.

Thrombospondin is an adhesive glycoprotein that is thought to play a role in tissue genesis and repair. We have used a monoclonal anti-thrombospondin antibody, designated 5G11, to localize thrombospondin in paraformaldehyde fixed, paraffin-embedded sections of developing mouse embryos. Thrombospondin expression is observed in uterine smooth muscle, endometrial glands, the decidua, and trophoblastic giant cells during the initial phase of post-implantation development in the embryo. Cardiac myocytes and neuroepithelial cells show positive staining for thrombospondin at day 8.5 of gestation, and this expression continues throughout the development of the myocardium and central nervous system. Strong staining for thrombospondin is seen in developing bone and in the liver. Thrombospondin is also observed in developing smooth muscle and skeletal muscle, as well as in a variety of epithelia, including the epidermis, small intestinal epithelium, lens epithelium, renal tubular epithelium, and the epithelium of the developing tooth. Comparison of thrombospondin staining with that of two known cell surface receptors for thrombospondin, syndecan and the vitronectin receptor, reveals remarkable colocalization of thrombospondin and syndecan in all tissues, but almost no coexpression with the vitronectin receptor. Coexpression of thrombospondin and syndecan may play an important role in cell-cell or cell-matrix interactions during development.

Animals↗
PubMed1995

Posterior extension of the chick nephric (Wolffian) duct: the role of fibronectin and NCAM polysialic acid.

The nephric duct of the chick embryo starts to form at about stage 10 of Hamburger and Hamilton ([1951] J. Morphol. 88:49-92) and extends posteriorly, fusing with the cloaca at about the end of the third day of incubation (HH stage 17). Evidence from the literature suggests that the extension involves active migration of the posterior tip. This investigation concerned some molecules that might control this migration: fibronectin, vitronectin, the beta 1 integrin receptor, and NCAM polysialic acid. The concentration of fibronectin in the extracellular matrix was found by immunocytochemistry to be negligible at the posterior end of the duct; treatment of the living embryo with GRGDS failed to halt further extension of the duct; SEM examination of embryos treated with the synthetic peptides of fibronectin GRGDS, GRDGS, SDGR, and GRGES, or with vitronectin, revealed negligible morphological effects on the duct. It is concluded that there is yet no evidence that fibronectin is an important factor in duct migration. NCAM polysialic acid had a similar distribution to fibronectin, but treatment of the living embryo with Endo-N caused cessation of extension of the duct. Endo-N is an enzyme that specifically degrades PSA without affecting the NCAM polypeptide itself. It is suggested therefore that PSA may play an important role in duct extension. The synthetic peptides of fibronectin each produced distinctive patterns of blebbing on the surfaces of cells in trunk mesoderm, but the duct cells were unaffected. GRGES and SDGR caused blebbing on cells in the somites and the anterior segmental plate, though not on cells in the posterior segmental plate. This suggests that integrin receptors change in the anterior segmental plate as the mesoderm forms somites from somitomeres.

Amino Acid Sequence↗
PubMed1997

The selective engulfment of apoptotic bodies by dendritic cells is mediated by the alpha(v)beta3 integrin and requires intracellular and extracellular calcium.

Dendritic cells derived in vitro from monocytes are known to be poor phagocytes. Here we show that, unlike macrophages, monocyte-derived dendritic cells indeed fail to take up opsonized particles or necrotic cells; however, apoptotic bodies are efficiently engulfed by dendritic cells. The temperature dependence and the sensitivity to cytochalasin D indicate that the apoptotic body engulfment is representative of early stages of phagocytosis. Inhibition studies with ligands for surface molecules involved in recognition of apoptotic bodies, such as vitronectin receptor, CD36 and phosphatidylserine receptor, revealed that apoptotic body engulfment by dendritic cells is mediated preferentially by the vitronectin receptor alpha(v)beta3, while all the receptors, with different efficiency, are engaged in phagocytosis of apoptotic bodies by macrophages. The interaction between apoptotic bodies and dendritic cells elicits a rise in intracellular free calcium concentration ([Ca2+]i) which is essential for the process of engulfment. Either intra- or extracellular Ca2+ buffering inhibits apoptotic body engulfment by dendritic cells and [Ca2+]i increases, indicating the involvement of both intra- and extracellular Ca2+. In contrast, Ca2+ mobilization is dispensable for macrophage phagocytosis of apoptotic bodies. The different requirements of Ca2+ in macrophages and dendritic cells is possibly due to the differential usage of phagocytic receptors (CD36 vs. alpha(v)beta3) and might reflect different fates of apoptotic bodies in the two cell types.

Apoptosis↗
PubMed2005

Identification of a new marker of hepatocellular carcinoma by serum protein profiling of patients with chronic liver diseases.

Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a proteomic technique that enables the profiling of proteins present in any biological material studied. We used this approach to identify new biomarkers of hepatocellular carcinoma (HCC) in the sera of patients with cirrhosis. Sera from 82 patients with cirrhosis, either without (n = 38) or with (n = 44) HCC, were analyzed by SELDI-TOF MS, and the results of the two groups were compared. The most efficient protein peaks leading to discrimination of patients with HCC were selected (receiver operative characteristic curves). The highest-scoring peak combination was established in a first group of serum samples (multinomial regression) and was tested in an independent group. The protein corresponding to the highest discrimination was purified and characterized further. The intensity of 30 protein peaks significantly differed between cirrhotic patients with and without HCC. An algorithm including the six highest-scoring peaks allowed correct classification (presence or absence of HCC) of 92.5% of patients in the test sample set and 90% in the validation sample set. The highest discriminating peak (8900 Da) was purified further and was characterized as the C-terminal part of the V10 fragment of vitronectin. An in vitro study suggested that the increase of the 8900-Da fragment in the serum of patients with HCC may proceed from the cleavage of native vitronectin with metalloproteases, a family of enzymes whose activity is enhanced in HCC. In conclusion, global protein profiling is an efficient approach that enabled us to identify a catalytic fragment ofvitronectin as a new serum marker of HCC in patients with chronic liver diseases.

Aged↗
PubMed2003

Prinomastat, a hydroxamate inhibitor of matrix metalloproteinases, has a complex effect on migration of breast carcinoma cells.

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alphavbeta3 integrin have been directly implicated in tumor cell dissemination and metastasis. We have demonstrated that in the case of breast carcinoma MCF7 cells co-expressing MT1-MMP and alphavbeta3 integrin, the proteinase processes the pro-alphav integrin subunit, thus facilitating alphavbeta3 integrin maturation and cell migration on vitronectin. Our findings show that cell surface MT1-MMP is a short-lived protein with a life span in the range of several hours. In contrast, turnover of alphavbeta3 integrin is much slower. The half-life of alphavbeta3 heterodimer is about 24 hr. This large difference in life span allowed us to distinguish between the effects of MT1-MMP on cell migration brought by matrix proteolysis from those imposed through alphavbeta3 integrin maturation. We then modulated the enzyme's activity by a potent hydroxamate MMP inhibitor, Prinomastat (AG3340), to analyze the divergent effects of MT1-MMP on cell migration. Although Prinomastat immediately blocked MT1-MMP-mediated matrix degradation, the pool of MT1-MMP-modified alphavbeta3 integrin molecules was still capable of mediating cell-matrix interactions. To our considerable surprise, inhibition of MT1-MMP-dependent vitronectin proteolysis by Prinomastat allowed a several-fold increase in migration of MCF7 cells co-expressing MT1-MMP and alphavbeta3 integrin. In contrast, long-term Prinomastat inhibition of MT1-MMP-dependent pro-alphav cleavage and thus alphavbeta3 integrin maturation strongly inhibited cell motility. Our studies suggest that MT1-MMP could actually promote cell migration via modification of the cell surface receptors, including alphavbeta3 integrin, rather than facilitate cell migration through direct cleavage of the matrix proteins.

Antineoplastic Agents↗
PubMed1985

Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion.

cDNA clones coding for human S-protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S-protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S-protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of S-protein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino-terminal residues 42 are identical with the so-called somatomedin B peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S-protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways.

Amino Acid Sequence↗
PubMed2002

Hydroxylapatite with substituted magnesium, zinc, cadmium, and yttrium. II. Mechanisms of osteoblast adhesion.

The present in vitro study investigated osteoblast adhesion on hydroxylapatite (HA) doped with either cadmium (Cd), zinc (Zn), magnesium (Mg), or yttrium (Y). Compared with any other dopant tested in the present study, osteoblast adhesion was significantly (p < 0.05) greater on HA doped with Y after 4 h; in addition, osteoblast adhesion increased with concentration (2-7 mol%) of Y in HA. The findings that HA doped with greater amounts of Y adsorbed higher concentrations of calcium and, subsequently, of vitronectin and collagen (proteins known to mediate osteoblast adhesion), but not of albumin, laminin, and fibronectin, may explain the observed enhanced adhesion of osteoblasts on these substrates. Interactions (i.e., adsorption and configuration/bioactivity) of vitronectin and collagen may have been promoted by increased porosity of doped HA. Through doping with Y, the present study provided the first evidence that HA can be synthesized and processed with improved cytocompatibility properties for osteoblast adhesion, and thus offered essential information for the design of novel proactive bioceramics. Proactive bioceramics which elicit specific, timely, and desired responses from surrounding cells and tissues are necessary for improving bonding of orthopaedic/dental implants to juxtaposed bone; such osseointegration will, undoubtedly, enhance implant efficacy.

Adsorption↗
PubMed1994

Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro.

The interaction of cells with extracellular matrix is essential for their anchorage, proliferation, migration, and differentiation. In bone matrix there are multiple glycoproteins that contain the integrin-binding RGD sequence: fibronectin (FN), thrombospondin (TSP), osteopontin (OPN), bone sialoprotein (BSP), type I collagen (COLL I), and vitronectin (VN). In this study, the localization of TSP, FN, VN, and several integrins within developing human long bone using immunohistochemical methods was examined, as was the effect of all bone RGD proteins on the adhesion of human osteoblastic cells. Thrombospondin, fibronectin, and vitronectin showed distinct localization patterns within bone tissue. TSP was found mainly in osteoid and the periosteum; VN appeared to be present mainly in mature bone matrix. FN was present in the periosteum as well as within both mature and immature bone matrix. Using a panel of antiintegrin antibodies we found that bone cells in vivo and in vitro express alpha 4, alpha v, alpha 5 beta 1, alpha v beta 3, and beta 3/beta 5 integrins, and these receptors are for the most part expressed on all bone cells at different stages of maturation with quantitative rather than qualitative variations, with the exception of alpha 4, which is expressed mainly by osteoblasts. Cell attachment assays were performed using primary human cells of the osteoblastic lineage under serum-free conditions. COLL I, TSP, VN, FN, OPN, and BSP promoted bone cell attachment in a dose-dependent manner and were equivalent in action when used in equimolar concentrations. In the presence of GRGDS peptide in the medium, the adhesion to BSP, OPN, and VN was almost completely blocked (10, 10, and 15% of control, respectively), and attachment to FN, COLL I, and TSP was only slightly decreased (80, 75, and 55%, respectively). These results suggest that human bone cells may use RGD-independent mechanisms for attachment to the latter glycoproteins.

Alkaline Phosphatase↗
PubMed1992

Basolateral distribution of fibronectin matrix assembly sites on vascular endothelial monolayers is regulated by substratum fibronectin.

Endothelial cells exhibit binding sites for the amino terminus of fibronectin that participate in subendothelial fibronectin matrix assembly. These binding sites, termed matrix assembly sites, are localized on the basolateral surface of confluent endothelial monolayers (Kowalczyk et al. Blood, 75:2335, 1990). The present study investigates the role of cell-cell and cell-substratum interactions in the localization of matrix assembly sites to the basal surface of endothelial cells. Cells were cultured in Transwell culture inserts and matrix assembly sites were detected by binding assays using an iodinated 70 Kd amino-terminal fibronectin fragment. Integrity of intercellular junctions was monitored by measuring protein flux across Transwell filters. Time course experiments demonstrated that matrix assembly site expression on the basolateral cell surface preceded intercellular junction formation. Transfer of confluent monolayers to calcium-free medium resulted in the loss of junctions and in an increase in 125I-70 kD binding from the apical medium. The increased 125I-70 kD binding resulted from increased access of 125I-70 kD to basolateral matrix assembly sites and not from the relocation of binding sites to the apical membrane. To determine the effect of matrix composition on matrix assembly site expression and localization, cells were seeded onto vitronectin- or fibronectin-coated substrates. Fibronectin increased the expression of matrix assembly sites on the apical surface within 24 hours. By 48 hours, matrix assembly sites were located only on the basolateral surface. Vitronectin had no effect on the expression or localization of matrix assembly sites. These results indicate that the expression and localization of matrix assembly sites on the surface of vascular endothelial cells can be regulated by substratum fibronectin.

Animals↗
PubMedDate not supplied

Adenovirus type 5 uptake by lung adenocarcinoma cells in culture correlates with Ad5 fibre binding is mediated by alpha(v)beta1 integrin and can be modulated by changes in beta1 integrin function.

BACKGROUND: Recombinant adenoviruses (Ad) have been employed as vectors for a wide variety of gene therapy applications, but their use has been hindered by problems relating to efficacy and safety. The efficiency of Ad-mediated gene transfer depends on the interaction of the fibre and penton base proteins with their corresponding cell receptors. Ad infection is initiated by the formation of a high affinity complex between the fibre protein and a host cell protein that for most Ad serotypes is CAR (the coxsackie B virus and Ad receptor). A second molecule, the MHC class I, may also be involved in Ad type 2 and Ad type 5 uptake. Ad internalization results from the interaction of the penton base protein with cell surface integrins alpha(v)beta3 and alpha(v)beta5. In this study, we addressed the interaction between Ad type 5 (Ad5) and its receptors on lung derived adenocarcinoma cells in culture. METHODS: Using flow cytometry, we determined the level of expression of attachment and internalization receptors that are expressed on the cell surface of A549, H322 and H441 lung-derived adenocarcinoma cells in culture. The level of alpha(v)beta1 cell surface integrin was assessed by immunoprecipitation. Measuring the level of luciferase gene expression at different viral titres quantitated Ad5 uptake by these cells. The kinetics of binding of Ad5 fibre knobs to A549, H322 and H441 cells was assessed in direct binding studies using 125I labelling of purified recombinant Ad5 fibre-knob domains. In order to assess the functionality of integrins, adhesion assays were performed in the presence or absence of activators of integrin function. In competition experiments, prior to exposure to the virus, the cells were pre-incubated with purified recombinant Ad5 fibre-knob domains, function blocking anti-integrin antibodies, or integrin activating agents, prior to the introduction of luciferase expressing Ad5. RESULTS: We found that Ad5-mediated gene transfer in A549, H322 and H441 adenocarcinoma cells in culture is highly variable and that this variation correlates with specific binding of Ad5 fibre-knob domain binding to the cell surface. We also found, for the first time, that Ad5 infection is mediated by integrin alpha(v)beta1 and that functional activation of beta1 integrin by means of the specific anti-beta1 monoclonal antibody, TS2/16, induced increased A549 cell adhesion to fibronectin and vitronectin and also enhanced Ad5 uptake by these cells. CONCLUSIONS: Our studies demonstrate that the Ad5 fibre-knob domain interaction with CAR represents a major determinant of Ad5-mediated gene transfer to lung-derived adenocarcinoma cells in culture. The finding that integrin alpha(v)beta1 is involved in Ad5 infection has implications for the use of recombinant Ad5 vectors for cancer gene therapy, since alpha(v)beta1 is expressed at high levels and acts as an alternative vitronectin receptor in many epithelial and some melanoma tumours which express no alpha(v)beta3 and constant low levels of alpha(v)beta5. The fact that the beta1 integrin-activating antibody TS2/16 can enhance alpha(v)beta1-mediated Ad5 infection suggests that the efficacy of Ad5-mediated gene transfer might be influenced not only by the level of cell surface expression of integrins but also by their state of activation.

Adenocarcinoma↗
PubMed1999

Integrin subunits responsible for adhesion of human osteoblast-like cells to biomimetic peptide surfaces.

We have identified the integrin subunits responsible for the initial adhesion of human osteoblast-like cells to peptide-modified surfaces. Biomimetic peptide surfaces containing homogenous RGD (Arg-Gly-Asp), homogenous FHRRIKA (Phe-His-Arg-Arg-Ile-Lys-Ala), and a mixed ratio of FHRRIKA:RGD (25:75) were used to assess integrin-mediated adhesion. The RGD and FHRRIKA peptides were selected from the cell-binding and putative heparin-binding domains of bone sialoprotein. A panel of monoclonal antibodies against human alpha1, alpha2, alpha3, alpha4, alpha5, beta1, alpha(v), and alpha(v)beta3 was used to identify the subunits most dominant in mediating short-term (10 or 30 minutes) and long-term (4 hours) cell adhesion to the peptide surfaces. Anti-alpha2, anti-beta1, and anti-alpha(v) significantly (p < 0.05) diminished cell attachment to homogenous RGD surfaces following 30 minutes of incubation. After 4 hours of incubation on RGD-grafted surfaces, immunostaining of these integrin subunits revealed discrete localization of the alpha(v) subunit at the periphery of the cell (similar to focal contact points), whereas the alpha2 and beta1 subunits stained very diffusely throughout the cell. A radial-flow apparatus was used to determine the effect of anti-integrin antibodies on strength of cell detachment following 10 minutes of incubation on peptide-grafted surfaces. The strength of detachment from surfaces containing RGD was significantly reduced (p < 0.05) in the presence of anti-alpha2, anti-alpha(v), or anti-beta1 compared with controls (presence of preimmune mouse IgG). None of the antibodies significantly influenced cell attachment to homogenous FHRRIKA-grafted surfaces. These results demonstrate that initial (30 minutes) attachment of human osteoblast-like cells to homogenous RGD surfaces was mediated by the collagen receptor alpha2beta1 and the vitronectin receptor alpha(v)beta3, whereas only the vitronectin receptor governed longer term (longer than 30 minutes) adhesion (localization to focal contacts). The importance of distinct integrins in mediating the attachment of bone cells to RGD-immobilized surfaces indicates a strategy for engineering orthopaedic implants with a built-in surface specificity for cell adhesion.

Amino Acid Sequence↗
PubMed2000

Contortrostatin, a homodimeric disintegrin, binds to integrin alphavbeta5.

Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.

Agkistrodon↗
PubMed1994

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human lung carcinoma cells of different histotypes.

Structural and functional analyses of several integrin heterodimers were performed in non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines. The expression of beta 1, beta 3, beta 4, and beta 5 heterodimers was evaluated at protein and mRNA levels. By flow cytometry and immunoprecipitation experiments we demonstrate that NSCLC cells (A549 adenocarcinoma and DG 3 large cell carcinoma) coexpress integrin heterodimers composed of beta 1, beta 3, beta 4, and beta 5 subunits, whereas SCLC cells (AE2 and H69) express only beta 1 integrin heterodimers. Northern blot experiments confirmed immunochemical analysis: SCLC cells in contrast to NSCLC cells express only the mRNA coding for the beta 1 subunit. These data indicate that in lung carcinoma cells the diversity in the integrin repertoire depends upon differential gene expression. The functionality of integrin receptors has been studied using antibody blocking experiments. Data reported demonstrate that the alpha 6 beta 1 integrin is a laminin receptor in either SCLC or NSCLC cells. An antibody to the beta 4 subunit partially inhibits the adhesion of adenocarcinoma cells to lamin but does not block lamin adhesion of large cell carcinoma cells, even though alpha 6 beta 4 complexes are expressed on both cell types. Two antisera to vitronectin receptors inhibit the adhesion of NSCLC cels to both vitronectin and fibronectin. The same antisera inhibit the adhesion of SCLC cells only to laminin, indicating that the alpha v beta 1 integrin might function in these cells as laminin receptor.

Carcinoma, Non-Small-Cell Lung↗
PubMed1994

Formation of focal adhesions by osteoblasts adhering to different substrata.

In this study, the adhesion of an osteoblast cell line to glass and titanium surfaces coated with different extracellular matrix components has been examined. On uncoated glass or titanium surfaces, osteoblasts attached but failed to spread. On these surfaces the cells did not develop focal adhesions or stress fibers. Precoating glass coverslips or titanium disks with fibronectin enhanced spreading and resulted in the rapid formation of focal adhesions and their associated stress fibers. Osteoblasts also spread and developed focal adhesions and stress fibers on glass or titanium coated with serum. In this situation the spreading was less rapid than that on fibronectin. Following incubation with serum, we demonstrated that the surfaces became coated with vitronectin. On the vitronectin-coated surfaces, it was shown that the osteoblast focal adhesions contained integrins of the beta 3 family. In contrast, osteoblasts adhering to fibronectin-coated surfaces accumulated beta 1 integrins within their focal adhesions. The enhanced formation of focal adhesions by osteoblasts plated on surfaces coated with extracellular matrix proteins suggests that precoating titanium and other implant materials with extracellular matrix proteins may improve osseointegration.

3T3 Cells↗