The E8 subfragment of laminin promotes locomotion of myoblasts over extracellular matrix.

The locomotion of murine myoblasts over the extracellular matrix components laminin and fibronectin was analyzed using quantitative videomicroscopy, and the organization of the cytoskeleton was observed in parallel immunofluorescence studies. Cells plated on the laminin-nidogen complex locomoted twice as fast as on laminin alone. The main form of translocation on laminin was a jerky cycle of prolonged lamellipod extension followed by rapid (approximately 200- less than 500 microh h-1) movement of the cell body into the extended lamellipod. The locomotion-stimulating activity of laminin resides in the elastase digestion fragment E8, part of the laminin long arm, while the E1-4 fragment containing the three short arms is inactive. Myoblasts moved poorly over fibronectin irrespective of whether high, intermediate, or low coating concentrations were used (approximately 5,000- approximately 10 fmol cm-2). In contrast, the locomotory responses both to laminin and to E8 peaked sharply at coating concentrations approximately 20-50 fmol cm-2 and decreased at higher concentrations. This response corresponds to that expected for a haptotactic stimulant. When cells locomoted over a mixed substrate of laminin and fibronectin, the fibronectin effects appeared to predominate. The cytoskeleton has been implicated in many cellular motile processes. Within 6 h on fibronectin many cells expressed vinculin-containing focal contacts, elaborated stress fibers and had periodically organized alpha actinin, whereas on laminin, most cells showed diffuse vinculin and alpha actinin and a fine meshlike actin cytoskeleton. We conclude that the poor locomotion of cells over fibronectin is because of the cytoskeletal stabilization it induces.

Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading.

Two subfragments of laminin, E8, a major part of the long arm, and E1-4, the three short arms, promote cell adhesion and spreading. Three distinct types of adhesive behavior are seen in short term (1 h) assays, typified by secondary murine fibroblasts, adherent only on fibronectin; secondary murine myoblasts, adherent on fibronectin, laminin, and the E8 fragment; and Rugli human glioblastoma cells, adherent on fibronectin, laminin, E8, and E1-4. E8-specific polyclonal antibodies block myoblast adhesion to E8 and to laminin with identical concentration dependence; Rugli binding to E8 but not to laminin is also totally blocked by these antibodies. Heating of E8 and laminin to approximately 60 degrees C abolishes cell attachment-promoting activity for myoblasts. Adhesion of Rugli cells to E8 is also lost, but on laminin the attachment-promoting activity remains constant. This is due to an increase in the activity of E1-4 fragment as it is heated. Thus, major sites for initial cell adhesion to and spreading on laminin lie within the E8 and E1-4 fragments, but not all cells binding to laminin will bind to both fragments. These data may tentatively be explained by the existence of more than one type of receptor for laminin at the cell surface; one is needed for each fragment.

Multiple cell surface receptors for the short arms of laminin: alpha 1 beta 1 integrin and RGD-dependent proteins mediate cell attachment only to domains III in murine tumor laminin.

Cell surface molecules that interact with the cross formed by the three short arms of murine tumor laminin were studied using thermal perturbation, antibody and peptide blocking, and affinity chromatography. Several potential receptors for the laminin short arms were revealed that differed from those mediating cell attachment to the E8 (long arm) fragment. Two cell lines, Rugli and L8 attached well to E1-X (short arm) fragments of laminin. This attachment was blocked by antibodies against alpha 1 integrin chains. Other cells were unable to attach strongly to E1-X, but attached to P1. This attachment was unaffected by anti-beta 1 integrin antibodies, but specifically blocked by the peptide GRGDS. By contrast, binding of Rugli cells was RGD independent and blocked by anti-beta 1 integrin antibodies. G7 and C2C12 myoblasts were very sensitive to GRGDS (ID50 approximately 2 micrograms.ml-1) for attachment to P1 which implied that a non-beta 1 series integrin, possibly alpha v beta 3, was involved. On heat denaturation of P1(3) attachment remained sensitive to RGDS and ID50 was unchanged. On heat denaturation of E1-X, attachment remained sensitive to RGDS but the ID50 increased to approximately 200 micrograms.ml-1. Cellular beta 1 integrins were retained on laminin affinity columns. A beta 1 integrin with an approximately 190 kD alpha-chain could be isolated from Rugli cells whose attachment could be blocked by anti-alpha 1 antibodies and not from cells blocked by RGDS peptides. Anti-alpha 1 antibodies blocked Rugli attachment to native laminin, but only when the E8 cell binding sites on laminin were also blocked. Thus, a receptor related to alpha 1 beta 1 integrin can function simultaneously with a receptor for E8. Anti-alpha 1 also blocked attachment to heated laminin, suggesting that the heat-stable attachment activity in laminin involved the E1-X binding site. Thus, at least two putative receptors mediate attachment to the short arms of laminin. One, related to alpha 1 beta 1 integrin, recognizes RGDS-independent sites in E1-X defined by P1 (within domains III, IIIa, IIIb), and one is an RGD-dependent molecule recognizing sites in P1, and is not a beta 1 integrin.

Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin.

It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti-Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity.

Crystal structure of the extracellular segment of integrin alpha Vbeta3.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Decreased angiogenesis and arthritic disease in rabbits treated with an alphavbeta3 antagonist.

Rheumatoid arthritis (RA) is an inflammatory disease associated with intense angiogenesis and vascular expression of integrin alphavbeta3. Intra-articular administration of a cyclic peptide antagonist of integrin alphavbeta3 to rabbits with antigen-induced arthritis early in disease resulted in inhibition of synovial angiogenesis and reduced synovial cell infiltrate, pannus formation, and cartilage erosions. These effects were not associated with lymphopenia or impairment of leukocyte function. Furthermore, when administered in chronic, preexisting disease, the alphavbeta3 antagonist effectively diminished arthritis severity and was associated with a quantitative increase in apoptosis of the angiogenic blood vessels. Therefore, angiogenesis appears to be a central factor in the initiation and persistence of arthritic disease, and antagonists of integrin alphavbeta3 may represent a novel therapeutic strategy for RA.

Noninvasive imaging of alpha(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography.

The alpha(v)beta3 integrin is an important cell adhesion receptor involved in tumor-induced angiogenesis and tumor metastasis. Here we describe the 18F-labeling of the RGD-containing glycopeptide cyclo(-Arg-Gly-Asp-D-Phe-Lys(sugar amino acid)-) with 4-nitrophenyl 2-[18F]fluoropropionate and the evaluation of this compound in vitro and in tumor mouse models. Binding assays with isolated immobilized alpha(v)beta3, alpha(v)beta5, and alpha(IIb)beta3 as well as in vivo studies using alpha(v)beta3-positive and -negative murine and xenotransplanted human tumors demonstrated receptor-specific binding of the radiolabeled glycopeptide yielding high tumor:background ratios (e.g., 120 min postinjection: tumor:blood, 27.5; tumor:muscle, 10.2). First imaging results using a small animal positron emission tomograph suggest that this compound is suitable for noninvasive determination of the alpha(v)beta3 integrin status and therapy monitoring.

Different cellular receptors for human placental laminin and murine EHS laminin.

Human placental laminin purified without the use of proteases promotes similar levels of cell attachment to murine EHS laminin. The major HT1080 cell binding site of human placental laminin is thermally less labile than that of EHS laminin. Monoclonal antibody GOH3, which recognises the integrin alpha 6 subunit, inhibits HT1080 and B16 cell attachment to EHS laminin but not to human placental laminin, indicating that the cells use different receptors to bind to these two types of laminin. Antibodies P1E6 and P1B5, which recognise the integrin alpha 2 and alpha 3 subunits respectively, do not affect HT1080 cell binding to either type of laminin.

Integrins, cations and ligands: making the connection.

Integrins are cell adhesion receptors that couple extracellular divalent cation-dependent recognition events with intracellular mechanical and biochemical responses and vice versa, thus affecting every function of nucleated cells. The structural basis of this bidirectional signaling and its dependency on cations has been the focus of intensive study over the past three decades. Significant progress made recently in elucidating the three-dimensional structure of the extracellular and cytoplasmic segments of integrins is giving valuable new insights into the tertiary and quaternary changes that underlie activation, ligand recognition and signaling by these receptors.

Solid-phase synthesis of a nonpeptide RGD mimetic library: new selective alphavbeta3 integrin antagonists.

The solid-phase synthesis of a low molecular weight RGD mimetic library is described. Activities of the compounds in inhibiting the interaction of ligands, vitronectin and fibrinogen, with isolated immobilized integrins alphavbeta3 and alphaIIbbeta3 were determined in a screening assay. Highly active and selective nonpeptide alphavbeta3 integrin antagonists with regard to orally bioavailability were developed, based on the aza-glycine containing lead compound 1. An important variation is the substitution of the aspartic amide of 1 by an aromatic residue. Furthermore, different guanidine mimetics have been incorporated to improve the pharmacokinetic profile. Exchange of the beta-amino acid NH by a methylene moiety in one set of RGD mimetics leads to the azacarba analogue compounds representing a novel peptidomimetic approach, which should increase the metabolic stability.

N-Methylated cyclic RGD peptides as highly active and selective alpha(V)beta(3) integrin antagonists.

The alpha(V)beta(3) integrin receptor plays an important role in human tumor metastasis and tumor-induced angiogenesis. The in vivo inhibition of this receptor by antibodies or by cyclic peptides containing the RGD sequence may in the future be used to selectively suppress these diseases. Here we investigate the influence of N-methylation of the active and selective alpha(V)beta(3) antagonist cyclo(RGDfV) (L1) on biological activity. Cyclo(RGDf-N(Me)V-) (P5) was found to be even more active than L1 and is one of the most active and selective compounds in inhibiting vitronectin binding to the alpha(V)beta(3) integrin. Its high-resolution, three-dimensional structure in water was determined by NMR techniques, distance geometry calculations, and molecular dynamics calculations, providing more insight into the structure-activity relationship.

Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics.

UNLABELLED: The alpha(v)beta3 integrin plays an important role in metastasis and tumor-induced angiogenesis. Targeting with radiolabeled ligands of the alpha(v)beta3 integrin may provide information about the receptor status and enable specific therapeutic planning. Previous studies from our group resulted in tracers that showed alpha(v)beta3-selective tumor uptake. However, these first-generation compounds predominantly revealed hepatobiliary excretion with high radioactivity found in the liver. In this report, the synthesis and biological evaluation of the first glycosylated RGD-containing peptide (RGD-peptide) for the noninvasive imaging of alpha(v)beta3 expression are described. METHODS: Peptides were assembled on a solid support using fluorenylmethoxycarbonyl-coupling protocols. The precursor cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP1 was synthesized by coupling 3-acetamido-2,6-anhydro-4,5,7-tri-O-benzyl-3-deoxy-beta-D-glycero-D-gulo-heptonic acid (SAA(Bn3)) with cyclo(-Arg(Mtr)-Gly-Asp(OtBu)-D-Tyr(tBu)-Lys-) and subsequent removal of the protection groups. Iodine labeling was performed by the Iodo-Gen method (radiochemical yield > 50%). The in vitro binding assays were performed using purified immobilized alpha(IIb)beta3, alpha(v)beta5, and alpha(v)beta3 integrins. For in vivo experiments, nude mice bearing xenotransplanted melanomas and mice with osteosarcomas were used. RESULTS: The glycosylated peptide 3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Lys(SAA)-) GP2 showed high affinity and selectivity for alpha(v)beta3 in vitro (50% inhibitory concentration = 40 nmol/L). Pretreatment studies indicate specific binding of [125I]GP2 on alpha(v)beta3-expressing tumors in vivo. Comparison of the pharmacokinetics of [125I]GP2 and [125I]-3-iodo-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) [125I]P2 revealed for [125I]GP2 an increased activity concentration in the blood (e.g., 3.59 +/- 0.35 percentage injected dose [%ID]/g vs. 1.72 +/- 0.44 %ID/g at 10 min postinjection) and a significantly reduced uptake in the liver (e.g., 2.59 +/- 0.24 %ID/g vs. 21.96 +/- 2.78 %ID/g at 10 min postinjection). Furthermore, a clearly increased activity accumulation in the tumor was found (e.g., 3.05 +/- 0.31 %ID/g vs. 0.92 +/- 0.16 %ID/g at 240 min postinjection), which remained almost constant between 60 and 240 min postinjection. This resulted in good tumor-to-organ ratios for the glycosylated tracer (e.g., 240-min postinjection osteosarcoma model: tumor-to-blood = 16; tumor-to-muscle = 7; tumor-to-liver = 2.5), which were confirmed by the first gamma-camera images of osteosarcoma-bearing mice at 240 min postinjection. CONCLUSION: This study demonstrates that the introduction of a sugar moiety improves the pharmakokinetic behavior of a hydrophobic peptide-based tracer. Additionally, this alpha(v)beta3-selective glycosylated radioiodinated second-generation tracer GP2 shows high tumor uptake and good tumor-to-organ ratios that allow noninvasive visualization of alpha(v)beta3-expressing tumors and monitoring therapy with alpha(v)beta3 antagonists. Finally, the favorable biokinetics make the glycosylated RGD-peptide a promising lead structure for tracers to quantify the alpha(v)beta3 expression using PET.

Three-dimensional extracellular matrix textured biomaterials.

Clinical and experimental investigations have reported that manufactured surface topographies have significant effects on cell adhesion and tissue integration. However, essentially all previously examined topographies bear little relation to cell adhesion substrates found in biological tissues. In vivo, many cells are adherent to extracellular matrices (ECM), which have an extremely complex 3-D topography in the micrometre to nanometre range. In addition, many studies indicate that micro- and nano-scale mechanical stresses generated by cell-matrix adhesion have significant effects on cellular phenotypic behaviour. In this report we describe methodology for the fabrication of topographic replicas of the subendothelial ECM topography with a biomedical polyurethane. Using three-dimensional high resolution scanning electron microscopy, accurate replication of subendothelial ECM topography from the macroscopic to the macromolecular scale is demonstrated. Bovine aortic endothelial cells cultured on the ECM replicas spread more rapidly and had a three-dimensional appearance and spread areas at confluence which appeared more like endothelial cells in native arteries, compared with cells cultured on untextured control surfaces. Since the fabrication process may be used with many different types of materials, including polymers of synthetic and biological origin, these biomimetic ECM-textured surfaces may find both research and clinical applications.

Effects of synthetic micro- and nano-structured surfaces on cell behavior.

Topographical cues, independent of biochemistry, generated by the extracellular matrix may have significant effects upon cellular behavior. Studies have documented that substratum topography has direct effects on the ability of cells to orient themselves, migrate, and produce organized cytoskeletal arrangements. Basement membranes are composed of extracellular matrix proteins and found throughout the vertebrate body, serving as substrata for overlying cellular structures. The topography of basement membranes is a complex meshwork of pores, fibers, ridges, and other features of nanometer sized dimensions. Synthetic surfaces with topographical features have been shown to influence cell behavior. These facts lead to the hypothesis that the topography of the basement membrane plays an important role in regulating cellular behavior in a manner distinct from that of the chemistry of the basement membrane. This paper describes the topography of the basement membrane and reviews the fabrication of synthetic micro- and nano-structured surfaces and the effects of such textured surfaces on cell behavior.

Nanoscale topography of the corneal epithelial basement membrane and Descemet's membrane of the human.

PURPOSE: Quantitatively define and compare the nanoscale topography of the corneal epithelial basement membrane (anterior basement membrane) and Descemet's membrane (posterior basement membrane) of the human. METHODS: Human corneas not suitable for transplantation were obtained from the Wisconsin Eye Bank. The corneas were placed in 2.5 mM EDTA for 2.5 h or 30 min. for removal of the epithelium or endothelium, respectively. After removal of the overlying cells, specimens were fixed in 2% glutaraldehyde and either examined in this state by atomic force microscopy only or dehydrated through an ethanol series and prepared for transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). RESULTS: The subepithelial and subendothelial basement membrane surfaces have a similar appearance that consists of an interwoven meshwork of fibers and pores. Topographic feature sizes were found to be in the nanometer size range with the epithelial basement membrane features larger and less densely packed than Descemet's membrane features. The topographic features are fractile in nature and increase surface area for cell contact. CONCLUSION: With the use of the TEM, SEM, and AFM, a detailed description of the surface topography of corneal epithelial basement membrane and Descemet's membrane of the human cornea are provided. The significance of differences in corneal basement membrane topography may reflect differences in function of the overlying cells or may be related to differences in cell migration and turnover patterns between the epithelium and endothelium.

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy.

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.