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PubMed1994

Differentiation, extracellular matrix synthesis, and integrin assembly by Drosophila embryo cells cultured on vitronectin and laminin substrates.

Two contrasting substrates, Drosophila laminin and human vitronectin, caused determined primary Drosophila embryo cells to follow alternate intermediate differentiation steps without affecting the final outcome of differentiation. Integrin alpha PS2 beta PS3 was essential for the initial spreading of myocytes on vitronectin: focal contacts rich in beta PS3 integrins formed and were connected by actin- and myosin-containing stress fibers. While alpha PS2 beta PS3 was unnecessary for myotube formation on laminin, it was required for the subsequent change to a sarcomeric cytoarchitecture. The differentiating primary cultures synthesized integrins and assembled them into detergent-insoluble, cytoskeleton-associated complexes. Collagen IV, laminin, glutactin, papilin, and other extracellular matrix proteins were made primarily by hemocytes and were secreted into the medium. Further differentiation within the cultures was influenced by secreted components and by later addition of vitronectin or bovine serum. Comparison of the differentiation of various cell types on the two substrates showed that vitronectin provided a selective advantage for the differentiation of myocytes, with enrichment over epithelia, epidermal cells, and neurites.

Animals↗
PubMed2004

Vitronectin is significant in the adhesion of lens epithelial cells to PMMA polymers.

A major complication of intraocular lens surgery is diminished visual acuity caused by the regrowth of lens epithelial cells (secondary cataract). Polymethylmethacrylate (PMMA) is a commonly used intraocular lens material. This study addresses the mechanisms underlying the initial adhesion of lens epithelial cells to PMMA and a functionalized PMMA-based terpolymer known to inhibit cell proliferation. Rabbit lens epithelial cells were cultured on the test polymer surfaces in medium containing serum depleted of either fibronectin or vitronectin (or both) to identify the role of these proteins in the initial process of cell adhesion. Adherent cells were quantitated after 60 min, and the actin cytoskeleton and focal contact formation were compared in each serum treatment on both polymers. Vitronectin was significantly more effective for initial cell attachment to both polymers than fibronectin. Normal cell spreading on PMMA required vitronectin and was independent of fibronectin, whereas cell spreading on the terpolymer was abnormal and required the presence of fibronectin and vitronectin together. Together, these results help to explain the inhibition of cell proliferation previously shown on the functionalized PMMA. This work contributes to the design of a polymer for use in intraocular lenses that inhibits proliferation of the target cells.

Animals↗
PubMed1993

The cell attachment and spreading activity of vitronectin is dependent on the Arg-Gly-Asp sequence. Analysis by construction of RGD and domain deletion mutants.

The cell attachment activity of vitronectin has been ascribed to an Arg-Gly-Asp (RGD) sequence near the amino terminus. To verify the importance of the RGD sequence for cell binding, we created RAD and RGE vitronectin mutants and also deleted either the somatomedin B (delta S-rVN) or heparin (delta H-rVN) binding domains. These mutants were expressed as fusion proteins, purified using Ni+2 affinity chromatography, and assayed for cell attachment. EAhy.926 cells bound equally well to wild-type, delta S-rVN, and to delta H-rVN, but binding to RAD-rVN and RGE-rVN was inhibited by more than 90%. We therefore conclude that the RGD sequence of vitronectin is the most important cell recognition site and that neither the somatomedin B nor heparin domains contribute significantly to the cell adhesive activity of vitronectin.

Amino Acid Sequence↗
PubMed2001

Effect of hypoxia on cellular adhesion to vitronectin and fibronectin.

Cellular invasion of extracellular matrix (ECM) occurs during normal and pathological settings. For cells to invade, they must adhere to the underlying substratum, break down barrier molecules, and detach from the substratum prior to migrating through the ECM. We previously demonstrated that incubation under reduced oxygen levels increases the in vitro invasiveness of trophoblast and breast carcinoma cells, an effect linked to elevated expression of the cell surface receptor for urokinase-type plasminogen activator (uPAR). This study examined the role of oxygen, integrins and the urokinase-type plasminogen activator (uPA) system on the adhesion of trophoblast and breast carcinoma cells to the ECM molecules vitronectin and fibronectin. Compared to exposure to 20 and 5% oxygen, exposure to 1% oxygen decreased adhesion of these cells to vitronectin and fibronectin, an effect that was reversible by re-exposure to 20% oxygen. Incubation in 1% oxygen also resulted in reduced expression of surface alpha(5) integrin. Furthermore, adhesion to vitronectin and fibronectin was reduced by compounds that interfere with integrin function, such as EDTA, anti-integrin antibodies, or by antibodies that interfere with the binding of pro-uPA to uPAR, soluble uPAR, soluble vitronectin, phosphatidylinositol-specific phospholipase C, as well as plasminogen activator inhibitor-1. These findings suggest an important role for oxygen in the regulation of cellular invasion, possibly in part through its effects on integrin and uPAR-mediated mechanisms of adhesion.

Cell Adhesion↗
PubMed1989

The role of vitronectin as multifunctional regulator in the hemostatic and immune systems.

Vitronectin (= complement S-protein) belongs to the group of structurally and functionally homologous adhesive proteins (fibrinogen, fibronectin, von Willebrand factor) which are essential in the procoagulant phase of the hemostatic system, interacting with platelets and the vessel wall. In addition to a structural motif in vitronectin responsible for this interaction (cell attachment domain) other functional domains in the protein molecule exist that contribute to its multifunctional role as regulator in the immune system (complement) as well as in fibrinolysis. These various activities and the ubiquitous distribution of vitronectin in the organism are discussed with regard to structure-function relationships of the protein molecule. Vitronectin may thus provide a conceptual molecular link between cell adhesion, humoral immune response and the hemostatic system, particularly at the blood-vessel wall interphase.

Amino Acid Sequence↗
PubMed2004

Activated vitronectin as a target for anticancer therapy with human antibodies.

The formation of a provisional extracellular matrix represents an important step during tumor growth and angiogenesis. Proteins that participate in this process become activated and undergo conformational changes that expose biologically active cryptic sites. Activated matrix proteins express epitopes not found on their native counterparts. We hypothesized that these epitopes may have a restricted tissue distribution, rendering them suitable targets for therapeutic human monoclonal antibodies (huMabs). In this study, we exploited phage antibody display technology and subtractive phage selection to generate human monoclonal antibody fragments that discriminate between the activated and native conformation of the extracellular matrix protein vitronectin. One of the selected antibody fragments, scFv VN18, was used to construct a fully human IgG/kappa monoclonal antibody with an affinity of 9.3 nM. In immunohistochemical analysis, scFv and huMab VN18 recognized activated vitronectin in tumor tissues, whereas hardly any activated vitronectin was detectable in normal tissues. Iodine 123-radiolabeled huMabVN18 was shown to target to Rous sarcoma virus-induced tumors in chickens, an animal model in which the epitope for huMab VN18 is exposed during tumor development. Our results establish activated vitronectin as a potential target for tumor therapy in humans.

Animals↗
PubMed1992

Vitronectin is the major serum protein essential for NGF-mediated neurite outgrowth from PC12 cells.

The rat pheochromocytoma cell line PC12 can be induced to differentiate in response to nerve growth factor (NGF) in the presence of 1% fetal calf serum (FCS). Using a novel assay procedure we have developed a purification protocol which has allowed the isolation of the protein in serum responsible for neurite outgrowth after NGF treatment. FCS has been fractionated using four chromatographic procedures and in each case the peak of biological activity copurified with vitronectin. We have concluded, therefore, that vitronectin is the protein present in FCS which can mediate NGF-dependent neurite outgrowth in PC12 cells. Vitronectin and fibronectin from FCS have been chromatographically separated and only the former is capable of inducing neurite outgrowth. We have also shown that vitronectin utilizes the RGD amino acid sequence in binding to the surface of PC12s.

Amino Acid Sequence↗
PubMed1990

Sequence location of a putative transglutaminase cross-linking site in human vitronectin.

We attempted to locate the glutamine residue in human vitronectin, susceptible to cross-linking by transglutaminases. Vitronectin was incubated with 14C-labelled putrescine and plasma factor XIIIa and, after reduction and alkylation, the vitronectin was digested with trypsin. HPLC of the digest followed by scintillation counting revealed one major and two minor radioactivity labelled peaks. Sub-digestion with Staphylococcus aureus protease, sequence analysis and mass-spectrometry of the resulting peptides demonstrated that Gln-93 of vitronectin had incorporated putrescine. Additionally, Gln-73, Gln-84 and Gln-86 were found to be minor sites for incorporation.

Amino Acid Sequence↗
PubMed1990

The phosphorylation of the two-chain form of vitronectin by protein kinase A is heparin dependent.

In circulating blood, vitronectin occurs in two forms: a single-chain (75 kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held together by a disulfide bridge. The 75 kDa form was previously shown to be phosphorylated at Ser378 by protein kinase A, released by physiologically stimulated platelets. By contrast, at pH 7.5 the two-chain form is not phosphorylated at all. Heparin or heparan sulfate are shown here to modulate the conformation of clipped vitronectin at physiological pH, exposing Ser378 and allowing its stoichiometric phosphorylation by the kinase. At this pH the two-chain form of vitronectin in plasma exhibits a higher affinity for heparin, and behaves as a flexible molecule, which can conformationally respond to heparin and heparan sulfate, effectors involved in vitronectin function.

Amino Acid Sequence↗
PubMed1995

The interaction of vascular endothelial cells and dorsal root ganglion neurites is mediated by vitronectin and heparan sulfate proteoglycans.

The interaction of peripheral nerve and blood vessels during development was studied by using DRG explant culture plated on confluent monolayer of vascular endothelial cells (VEC). The comparison of neurite length on various substrates showed a preference of DRG neurites in the following order; thrombospondin > laminin, vitronectin > fibronectin, VEC monolayer > collagen I, rat astrocyte monolayer. On layers of fibroblasts (3T3) or gliomas (C6), neurite extension was not observed. To identify the neurite outgrowth promoting adhesion molecules on VEC surface, several antibodies and synthetic peptides were added to the culture medium of DRG. With vitronectin antibody or with peptides containing the Arg-Gly-Asp (RGD) sequence, 30-40% of neurite outgrowth was inhibited and these two effects were not additive. Therefore, a part of neurite outgrowth in this system is mediated by vitronectin in RGD dependent manner. Another molecule which promotes neurite outgrowth on VEC was identified by a new monoclonal antibody (MAb) EC1. In the Western blot analysis, the immunoreactive band which was over 400 kDa was intensified by guanidine HCl extraction. EC1 immunoreactive band disappeared after the treatment of heparitinase but not with other glycolyases, indicating that EC1 antigen is heparan sulfate proteoglycan(s). The DRG neurite outgrowth was inhibited by MAb EC1 by about 30-40%. By the combination of MAb EC1 and RGD peptide, the neurite outgrowth in explant culture was inhibited by about 50%, and in DRG dissociated culture nearly 100% inhibition was observed. Thus, for the DRG neurite elongation on VEC, vitronectin and heparan sulfate proteoglycan(s) are playing crucial roles.

Animals↗
PubMed2005

Vitronectin: a possible determinant of adenovirus type 19 tropism for human corneal epithelium.

PURPOSE: Adenoviruses typically demonstrate specific tissue tropisms, as in the association of Ad19 with epidemic keratoconjunctivitis. We sought to determine factors that might influence the apparent tropism of Ad19 for the cornea. DESIGN: Laboratory investigation. METHODS: Adenovirus serotypes Ad2, 5, 9, 10, 11, 13, and 19 were compared for their capacity to replicate in human corneal epithelial cells (HCECs) in culture. Organotypically cultured human corneas were infected with Ad19 or Ad2, and viral titers were compared after 7 days. Replication of both viruses was compared in HCECs cultured on various extracellular matrices. Western blot analysis and immunohistochemistry were applied to human donor corneas and HCECs. RESULTS: One week after infection of HCEC monolayer cultures, Ad2 titers were significantly higher than any of the other viruses tested (P <.05). In organotypic corneal cultures, Ad19 titers were significantly higher than Ad2 (P = .0003). Ad2 replication in HCECs equaled or exceeded that of Ad19 on all extracellular matrices except vitronectin, where Ad2 replication was reduced and Ad19 replication enhanced (P <.0001). Vitronectin was detected by immunohistochemistry within the corneal epithelial basement membranes of human donor corneas. Increased alpha(v) integrin expression and greater tyrosine kinase phosphorylation in HCECs cultured on vitronectin were demonstrated by Western blot analysis. CONCLUSIONS: In vitro, vitronectin enhances growth of Ad19, possibly by up-regulation of receptor alpha(v) integrins and increased activity of tyrosine kinases necessary for adenoviral internalization. We hypothesize that differential tissue tropisms for adenoviruses may derive in part from tissue-specific extracellular matrix expression.

Adenoviruses, Human↗
PubMed1986

Physicochemical, immunochemical and functional comparison of human S-protein and vitronectin. Evidence for the identity of both plasma proteins.

The comparison of the complement inhibitor s-protein, isolated from human plasma, with vitronectin, a serum spreading factor, revealed a high degree of similarity of both proteins with respect to molecular weight, band pattern in polyacrylamide gels in the presence of sodium dodecyl sulfate and amino acid composition. While radiolabeled S-protein was precipitated by antiserum against vitronectin, both proteins exhibited precipitin lines of complete identity in double immunodiffusion analysis when tested mutually against antisera of the appropriate components. The functional property of vitronectin to promote cell spreading of fibroblasts was also documented for purified S-protein. These findings indicate a high degree of similarity with respect to structural and functional properties of S-protein and vitronectin and hence may implicate that both proteins are identical.

Cell Adhesion↗
PubMed1997

Identification of novel heparin-binding domains of vitronectin.

Vitronectin is a multifunctional serum protein which provides a unique regulatory link between cell adhesion, humoral defense mechanism and the hemostatic system, and the heparin-binding properties of vitronectin are thought to have participated in various functional aspects. In addition to the carboxy-terminal glycosaminoglycan-binding motif, we report on two novel heparin-binding domains which were identified using phage display technique. One heparin-binding domain is located between amino acids Asp82 and Cys137 at the end of the connector region, while the other is in the second hemopexin-type repeat, between amino acids Lys175 and Asp219 of the vitronectin molecule. Our findings may shed new light to the activities of vitronectin and its binding to cells, which could not be explained solely on the basis of the known heparin-binding domain.

Amino Acid Sequence↗
PubMed1999

Vitronectin and its fragments purified as serum inhibitors of Staphylococcus aureus gamma-hemolysin and leukocidin, and their specific binding to the hlg2 and the LukS components of the toxins.

Staphylococcal gamma-hemolysin and leukocidin are bi-component cytolysins, consisting of LukF (or Hlg1)/Hlg2 and LukF/LukS, respectively. Here, we purified serum inhibitors of gamma-hemolysin and leukocidin from human plasma. Protein sequencing showed that the purified inhibitors of 62, 57, 50 and 38 kDa were the vitronectin fragments with truncation(s) of the C-terminal or both N- and C-terminal regions. The purified vitronectin fragments specifically bound to the Hlg2 component of gamma-hemolysin and the LukS component of leukocidin to form high-molecular-weight complexes with them, leading to inhibition of the toxin-induced lysis of human erythrocytes and human polymorphonuclear leukocytes, respectively. Intact vitronectin also showed inhibitory activity to the toxins. The ability of gamma-hemolysin and leukocidin to bind vitronectin and its fragments is a novel function of the pore-forming cytolysins.

Amino Acid Sequence↗
PubMed1999

Vitronectin receptors are expressed by macaque trophoblast cells and play a role in migration and adhesion to endothelium.

The objective of this work was to develop an in vitro system that would extend the usefulness of the macaque as a model for studying trophoblast invasion and spiral artery modification. We sought to determine whether trophoblast cells isolated from early gestation macaque placentas expressed vitronectin receptors and tested the idea that these receptors play a role in trophoblast migration and adhesion. Cytotrophoblast cells were isolated from 40-100 day macaque placentas, cultured, and characterized by immunofluorescence microscopy and flow cytometry. The cells expressed alphaV, beta3, and beta1 integrins on their surfaces. Immunohistochemical analysis of early gestation placentas and decidua basalis confirmed that intravascular trophoblast cells express alphaVbeta3/beta5. Using migration chambers we found that the trophoblast cells migrated towards vitronectin but not towards bovine serum albumin. This specific migration was blocked by preincubating the trophoblast cells with anti-vitronectin receptor (alphaVbeta3/beta5) antibodies. In other experiments, macaque trophoblast cells adhered to myometrial endothelial cells in a time-dependent manner and adhesion was significantly blocked by antibodies against alphaVbeta3/beta5 integrin. The results suggest that vitronectin receptors expressed by macaque trophoblast cells play a role in the migratory activity of these cells and may also be important in mediating attachment to endothelium.

Animals↗
PubMedDate not supplied

Evidence for sequential utilization of fibronectin, vitronectin, and collagen during fibroblast-mediated collagen contraction.

Contraction plays a major role in wound healing and is inevitably mediated through the mechanical interaction of fibroblast cytoskeleton and integrins with their extracellular matrix ligands. Cell-matrix attachment is critical for such events. In human dermal fibroblasts most such interactions are mediated by the beta1-type integrins. This study investigated the role played by key components in this system, notably fibronectin, vitronectin, and integrin subcomponents alpha2 and alpha5, which recognize collagen and fibronectin. Inhibition of adhesion through these ligands was studied either by antibody blocking or with fibronectin and/or vitronectin depletion. Functional effects of inhibition were monitored as force generation in collagen-glycosaminoglycan (IntegraTM) sponges, over 20 hours using a culture force monitor. Dose and time-course inhibition studies indicated that initial attachment and force generation (approx. 0-5 hours postseeding) was through fibronectin receptors and this was followed by vitronectin ligand and receptor utilization (4 hours onward). Utilization of the collagen integrin subcomponent alpha2 appeared to be increasingly important between 6 and 16 hours and dominant thereafter. Additionally, there was evidence for functional interdependence between the three ligand systems fibronectin, vitronectin, and collagen. We propose that there is a short cascade of sequential integrin-ligand interactions as cells attach to, extend through, and eventually contract their matrix. (WOUND REP REG 2002;10:-408)

Biocompatible Materials↗
PubMed1986

cDNA and amino acid sequences of the cell adhesion protein receptor recognizing vitronectin reveal a transmembrane domain and homologies with other adhesion protein receptors.

Cells adhere to vitronectin substrates through a cell surface receptor that recognizes an Arg-Gly-Asp sequence in vitronectin. The receptor is a glycoprotein composed of a 150-kDa alpha and a 115-kDa beta subunit. The alpha subunit consists of two disulfide-bonded chains of 125 kDa and 25 kDa. cDNA clones were isolated for the alpha subunit of the vitronectin receptor from a phage lambda gt11 expression library made with RNA from a human fibroblast cell line, IMR-90. The identity of the clones that had been selected from the library based on immunological criteria was verified by comparison of DNA and protein sequences. NH2-terminal sequences were obtained for each of the alpha-subunit chains. The sequence of the 25-kDa chain of the alpha subunit was found in a cDNA clone, and the amino acid sequence deduced from the cDNA establishes the complete amino acid sequence of the 25-kDa chain. This chain contains a membrane-spanning domain as well as a putative intracytoplasmic region that is 32 amino acids long and consists mostly of polar amino acids. Comparison of the cDNA and protein sequences shows that the 25-kDa chain is generated by proteolytic cleavage of an alpha-subunit precursor, the partial sequence of which is contained in the cDNA clones. These clones contain 1910 base pairs of open reading frame and a 3' untranslated sequence. RNA blot hybridization detected one transcript of about 7 kilobases in RNA from fibroblastic and epithelial cells. Together, the cDNA clones cover 4442 bases of this RNA. The alpha-subunit sequence showed strong homology with the sequence of the alpha subunit of fibronectin receptor. Moreover, the NH2-terminal protein sequence of the 125-kDa chain was homologous with the NH2-terminal sequences of two other cell surface proteins, lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen 1 (Mac-1), which have been implicated as receptors for adhesion proteins of leukocytes. These results establish several of the structural features in the vitronectin receptor and suggest the existence of a superfamily of receptors for cell adhesion proteins.

Amino Acid Sequence↗
PubMed2001

Vitronectin functions as a cofactor for rapid inhibition of activated protein C by plasminogen activator inhibitor-1. Implications for the mechanism of profibrinolytic action of activated protein C.

Activated protein C (APC) is a natural anticoagulant in plasma that down-regulates the coagulation cascade by degrading factors Va and VIIIa. In addition to its anticoagulant function, APC is also known to possess a profibrinolytic property. This property of APC has been attributed to its ability to neutralize PAI-1, thereby increasing the concentration of tissue plasminogen activator in plasma leading to up-regulation of the fibrinolytic cascade. This hypothesis, however, has not been well established, since the concentration of PAI-1 in plasma is low, and its reactivity with APC is very slow in a purified system. Here we demonstrate that vitronectin enhances the reactivity of PAI-1 with APC approximately 300-fold making PAI-1 the most efficient inhibitor of APC thus far reported (k(2) = 1.8 x 10(5) m(-)1 s(-)1). We further show that PAI-1 inhibition of the Glu(192) --> Gln mutant of APC is enhanced approximately 40-fold, independent of vitronectin, suggesting that vitronectin partially overcomes the inhibitory interaction of PAI-1 with Glu(192). Additionally, we show that PAI-1 inhibition of the Lys(37)-Lys(38)-Lys(39) --> Pro-Gln-Glu mutant of APC is severely impaired, suggesting that, similar to tissue plasminogen activator, the basic 39-loop of APC plays a critical role in the reaction. Together, these results suggest that vitronectin functions as a cofactor to promote the profibrinolytic activity of APC.

Amino Acid Substitution↗