Integrin-blocking Antibodies Research Articles

Blood vitronectin is a major activator of LIF and IL-6 in the brain through integrin-FAK and uPAR signaling.

We defined how blood-derived vitronectin (VTN) rapidly and potently activates leukemia inhibitory factor (LIF) and pro-inflammatory interleukin 6 (IL-6) in vitro and after vascular injury in the brain. Treatment with VTN (but not fibrinogen, fibronectin, laminin-111 or collagen-I) substantially increased LIF and IL-6 within 4 h in C6-astroglioma cells, while VTN-/- mouse plasma was less effective than that from wild-type mice. LIF and IL-6 were induced by intracerebral injection of recombinant human (rh)VTN in mice, but induction seen upon intracerebral hemorrhage was less in VTN-/- mice than in wild-type littermates. In vitro, VTN effects were inhibited by RGD, αvβ3 and αvβ5 integrin-blocking peptides and antibodies. VTN activated focal adhesion kinase (FAK; also known as PTK2), whereas pharmacological- or siRNA-mediated inhibition of FAK, but not PYK2, reduced the expression of LIF and IL-6 in C6 and endothelial cells and after traumatic cell injury. Dominant-negative FAK (Y397F) reduced the amount of injury-induced LIF and IL-6. Pharmacological inhibition or knockdown of uPAR (also known as PLAUR), which binds VTN, also reduced cytokine expression, possibly through a common target of uPAR and integrins. We propose that VTN leakage into tissues promotes inflammation. Integrin-FAK signaling is therefore a novel IL-6 and LIF regulation mechanism relevant to the inflammation and stem cell fields.

Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas.

Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration, and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. The aims were to determine how integrin interactions modulate OA-induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co-immunoprecipitation. We investigated OA-mediated activation of mitogen-activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA-mediated cell migration were determined. We assessed effects of OA knock-down on cell migration and proliferation. OA is overexpressed in OSCC cell lines, and serves as a migration-promoting adhesion molecule. OA co-localized with integrin subunits, and co-immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the β1 subunit, inhibited cell adhesion (P = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin-neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock-down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. J. Cell. Physiol. 231: 1761-1770, 2016. © 2015 Wiley Periodicals, Inc.

Epitopes in α8β1 and other RGD-binding integrins delineate classes of integrin-blocking antibodies and major binding loops in α subunits.

Identification of epitopes for integrin-blocking monoclonal antibodies (mAbs) has aided our understanding of structure-function relationship of integrins. We mapped epitopes of chicken anti-integrin-α8-subunit-blocking mAbs by mutational analyses, examining regions that harboured all mapped epitopes recognized by mAbs against other α-subunits in the RGD-binding-integrin subfamily. Six mAbs exhibited blocking function, and these mAbs recognized residues on the same W2:41-loop on the top-face of the β-propeller. Loop-tips sufficiently close to W2:41 (<25 Å) contained within a footprint of the mAbs were mutated, and the loop W3:34 on the bottom face was identified as an additional component of the epitope of one antibody, clone YZ5. Binding sequences on the two loops were conserved in virtually all mammals, and that on W3:34 was also conserved in chickens. These indicate 1) YZ5 binds both top and bottom loops, and the binding to W3:34 is by interactions to conserved residues between immunogen and host species, 2) five other blocking mAbs solely bind to W2:41 and 3) the α8 mAbs would cross-react with most mammals. Comparing with the mAbs against the other α-subunits of RGD-integrins, two classes were delineated; those binding to “W3:34 and an top-loop”, and “solely W2:41”, accounting for 82% of published RGD-integrin-mAbs.

Abstract 232: Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols

Abstract Some cancer-preventive agents such as selenium and green tea polyphenols effectively prevent cancer in some cases, but fail in other cases. It could be possible that the cancer-preventive actions of these agents may be altered by the interaction of cancer cells with the extracellular matrix (ECM) components, thus making this an important issue to study. Others have previously cloned a family of human prostate cancer cell lines with a common lineage that show the progression of characteristics from low to high degrees of malignancy and mimic different stages of tumor progression seen in human prostate cancer. For this study, we used these prostate cancer cell lines with increasing tumorigenicity and invasive ability: WPE1-NA22 showing the lowest, WPE1-NB14 and WPE1-NB11 showing intermediate, and WPE1-NB26 showing the greatest. The low tumorigenic cell line, WPE1-NA22 resembles noninvasive prostatic intra-epithelial neoplasia and represents a good target for chemoprevention in humans. Initially, we grew these cells on the surface of ECM components, laminin, collagen I, and fibronectin and determined the tumor cell sensitivity to methylseleninic acid (MSA) and epigallocatechin-3-gallate (EGCG). Poly-L-lysine was used as a control substratum. Cell growth was determined by sulforhodamine B staining assay. Then we determined whether these ECM proteins could enhance tumor cell resistance to MSA and EGCG. ECM components, but not polylysine, decreased tumor cell sensitivity to these agents. The effect of ECM components was prevented by β1 integrin-blocking antibodies suggesting that β1 integrin plays a role in mediating intracellular signaling conferring resistance against MSA and EGCG. Previously, we have shown an increase in expression of protein kinase Cε (PKCε) in advanced prostate tumor cells WPE1-NB26. A decrease in expression of PKCε by siRNA approach decreased the tumor cell resistance caused by ECM components. Furthermore, an inhibition of cell survival enzymes phosphoinositide-3-kinase (PI3K) with its specific inhibitor LY294002 (50 μM) as well as Akt with its specific inhibitor MK-2206 (1 μM) decreased the effect of ECM components on tumor cell survival against MSA and EGCG. WPE1-NB14, a low tumorigenic cell line having low PKCε and Akt activities, responded less to ECM components and showed higher sensitivity to MSA and EGCG. Conceivably, ECM components, laminin, collagen, and fibronectin induced β1 integrin signaling to activate cell survival pathways involving PKCε and PI3K-Akt, thereby decreasing prostate tumor cell sensitivity to MSA and EGCG. Thus, ECM may be one of the limiting factors for the action of cancer-preventive agents. Citation Format: Rayudu Gopalakrishna, Tiffany Fan, Ronald Deng, David Rayudu, Zachary W. Chen, William S. Tzeng, Usha Gundimeda. Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 232. doi:10.1158/1538-7445.AM2014-232

Particle size influences fibronectin internalization and degradation by fibroblasts

The application of nanotechnology for drug targeting underlines the importance of controlling the kinetics and cellular sites of delivery for optimal therapeutic outcomes. Here we examined the effect of particle size on internalization and degradation of surface-bound fibronectin by fibroblasts using polystyrene nanoparticles (NPs; 51nm) and microparticles (MPs; 1 μm). Fibronectin was strongly bound by NPs and MPs as assessed by immuno-dot blot analysis (5.1 ±0.4×10– 5pg fibronectin per μm2 of NP surface; 4.2±±0.3×10–5pg fibronectin per μm2 of MP surface; p>0.2). We estimated that ~193 fibronectin molecules bound to a MP compared with 0.6 fibronectin molecules per NP, indicating that ~40% of nanoparticles were not bound by fibronectin. One hour after incubation, fibronectin-coated NPs and MPs were rapidly internalized by Rat-2 fibroblasts. MPs and NPs were engulfed partly by receptor-mediated endocytosis as indicated by decreased uptake when incubated at 4°C, or by depletion of ATP with sodium azide. Pulse-chase experiments showed minimal exocytosis of NPs and MPs. Internalization of NPs and MPs was inhibited by jasplakinolide, whereas internalization of MPs but not NPs was inhibited by latrunculin B and by integrin-blocking antibodies. Extraction of plasma membrane cholesterol with methyl β-cyclodextrin inhibited internalization of fibronectin-coated NPs but not MPs. Biotinylated fibronectin internalized by cells was extensively degraded on MPs but not NPs. Particle size affects actin and clathrin-dependent internalization mechanisms leading to fibronectin degradation on MPs but not NPs. Thus either prolonged, controlled release or an immediate delivery of drugs can be achieved by adjusting the particle size along with matrix proteins such as FN.

Integrins modulate the infection efficiency of West Nile virus into cells

The underlying mechanisms allowing West Nile virus (WNV) to replicate in a large variety of different arthropod, bird and mammal species are largely unknown but are believed to rely on highly conserved proteins relevant for viral entry and replication. Consistent with this, the integrin αvβ3 has been proposed lately to function as the cellular receptor for WNV. More recently published data, however, are not in line with this concept. Integrins are highly conserved among diverse taxa and are expressed by almost every cell type at high numbers. Our study was designed to clarify the involvement of integrins in WNV infection of cells. A cell culture model, based on wild-type and specific integrin knockout cell lines lacking the integrin subunits αv, β1 or β3, was used to investigate the susceptibility to WNV, and to evaluate binding and replication efficiencies of four distinct strains (New York 1999, Uganda 1937, Sarafend and Dakar). Though all cell lines were permissive, clear differences in replication efficiencies were observed. Rescue of the β3-integrin subunit resulted in enhanced WNV yields of up to 90 %, regardless of the virus strain used. Similar results were obtained for β1-expressing and non-expressing cells. Binding, however, was not affected by the expression of the integrins in question, and integrin blocking antibodies failed to have any effect. We conclude that integrins are involved in WNV infection but not at the level of binding to target cells.

Fibronectin conformation regulates the proangiogenic capability of tumor-associated adipogenic stromal cells

BackgroundChanges in fibronectin (Fn) matrix remodeling contribute to mammary tumor angiogenesis and are related to altered behavior of adipogenic stromal cells; yet, the underlying mechanisms remain unclear due in part to a lack of reductionist model systems that allow the inherent complexity of cell-derived extracellular matrices (ECMs) to be deciphered. In particular, breast cancer-associated adipogenic stromal cells not only enhance the composition, quantity, and rigidity of deposited Fn, but also partially unfold these matrices. However, the specific effect of Fn conformation on tumor angiogenesis is undefined. MethodsDecellularized matrices and a conducting polymer device consisting of poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) were used to examine the effect of Fn conformation on the behavior of 3T3-L1 preadipocytes. Changes in cell adhesion and proangiogenic capability were tested via cell counting and by quantification of vascular endothelial growth factor (VEGF) secretion, respectively. Integrin-blocking antibodies were utilized to examine varied integrin specificity as a potential mechanism. ResultsOur findings suggest that tumor-associated partial unfolding of Fn decreases adhesion while enhancing VEGF secretion by breast cancer-associated adipogenic precursor cells, and that altered integrin specificity may underlie these changes. Conclusions and general significanceThese results not only have important implications for our understanding of tumorigenesis, but also enhance knowledge of cell-ECM interactions that may be harnessed for other applications including advanced tissue engineering approaches. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.

Focal Adhesion Dynamics Are Altered in Schizophrenia

Evidence from genetic association studies implicate genes involved in neural migration associated with schizophrenia risk. Neural stem/progenitor cell cultures (neurosphere-derived cells) from olfactory mucosa of schizophrenia patients have significantly dysregulated expression of genes in focal adhesion kinase (FAK) signaling, a key pathway regulating cell adhesion and migration. The aim of this study was to investigate whether olfactory neurosphere-derived cells from schizophrenia patients have altered cell adhesion, cell motility, and focal adhesion dynamics. Olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects were used. Cells were assayed for cell adhesion and cell motility and analyzed for integrins and FAK proteins. Focal adhesions were counted and measured in fixed cells, and time-lapse imaging was used to assess cell motility and focal adhesion dynamics. Patient-derived cells were less adhesive and more motile than cells derived from healthy control subjects, and their motility was reduced to control cell levels by integrin-blocking antibodies and by inhibition of FAK. Vinculin-stained focal adhesion complexes were significantly smaller and fewer in patient cells. Time-lapse imaging of cells expressing FAK tagged with green fluorescent protein revealed that the disassembly of focal adhesions was significantly faster in patient cells. The evidence for altered motility and focal adhesion dynamics in patient-derived cells is consistent with dysregulated gene expression in the FAK signaling pathway in these cells. Alterations in cell adhesion dynamics and cell motility could bias the trajectory of brain development in schizophrenia.

Effective treatment of mouse experimental colitis by alpha 2 integrin antibody: comparison with alpha 4 antibody and conventional therapy

To compare the therapeutic effect of α(2) and α(4) integrin-blocking antibodies to conventional inflammatory bowel disease drugs methotrexate, 5-aminosalicylic acid and azathioprine in the dextran sulphate sodium mouse colitis model. Colitis was induced in balb/c mice with 2.5-3.0% dextran sulphate sodium. Treatment was given daily for 7 days after the onset of colitis, by rectal installation. Clinical signs of disease were assessed daily using a disease activity index. After 19 days, all animals were killed and colon samples collected for histological grading and mRNA/protein analysis. All treatment groups were compared with an untreated control group and a treatment group receiving dextran sulphate sodium alone to monitor the potential degree of clinical remission. Treatment with anti-α(2) antibodies and methotrexate reduced the body weight loss. At the end of treatment, anti-α(2) antibodies reduced rectal bleeding, while methotrexate reduced the disease activity index score. Histological evaluation showed that anti-α(2) antibodies, methotrexate, 5-aminosalicylic acid and azathioprine treatment reduced the acute inflammation; methotrexate was the only treatment with effect on the crypt score. Compared with the dextran sulphate sodium alone group, the methotrexate group showed down-regulation of IL-1β at the mRNA level, while the anti-α(2) antibody group displayed decreased protein expression of iNOS and IL-1β. Specific blocking of extravascular trafficking of leucocytes with α(2)-antibodies could be a new beneficial drug target in inflammatory bowel disease.

239 MOLECULAR MECHANISMS OF SPECIFIC INHIBITION OF RENAL CELL CANCER (RCC) CELL MIGRATION BY THE DISINTEGRIN ECHISTATIN

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011239 MOLECULAR MECHANISMS OF SPECIFIC INHIBITION OF RENAL CELL CANCER (RCC) CELL MIGRATION BY THE DISINTEGRIN ECHISTATIN Wolfgang Jäger, Frederik Roos, Anna Becker, Elke Schneider, Joachim Thüroff, Christian Hampel, and Walburgis Brenner Wolfgang JägerWolfgang Jäger Mainz, Germany More articles by this author , Frederik RoosFrederik Roos Mainz, Germany More articles by this author , Anna BeckerAnna Becker Mainz, Germany More articles by this author , Elke SchneiderElke Schneider Mainz, Germany More articles by this author , Joachim ThüroffJoachim Thüroff Mainz, Germany More articles by this author , Christian HampelChristian Hampel Mainz, Germany More articles by this author , and Walburgis BrennerWalburgis Brenner Mainz, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.308AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastasis depends on adhesion of tumor cells to the endothelium, migration into subendothelial tissue and proliferation in secondary organs. Particularly during cell migration, binding of integrins, a group of cell adhesion molecules, to extracellular matrix is mediated by the amino acid sequence arginine-glycine-aspartate (RGD). Disintegrins, RGD containing proteins, can block integrins and thus inhibit cell migration and possibly metastasis. We analyzed the influence of disintegrins kistrin and echistatin on the migration of RCC cells. METHODS RCC-RC1 cells were pre-incubated with kistrin, echistatin and RGD containing peptides (P-RGD-M analogue to kistrin, A-RGD-D analogue to echistatin, G-RGD-S analogue to fibronectin, and RGD tripeptide). Migration was subsequently quantified in a boyden chemotaxis chamber. To analyse the binding mechanisms of these molecules, maleic anhydride activated plates (Pierce) were coated with echistatin, kistrin and the RGD containing molecules and the adherence of tumor cells, pre-incubated with á5- and 1-integrin-blocking antibodies, was quantified. The importance of the tertiary structure of the disintegrins was analysed by previous splitting of the disulfide chains. RESULTS In contrast to kistrin, echistatin significantly reduces tumor cell migration. Of the RGD containing peptides, G-RGD-S inhibited cell migration up to 45% and A-RGD-D up to 80%, whereas P-RGD-M did not show any inhibitory influence. Tumor cell adhesion was equally strong in echistatin and kistrin, and to a lower extent in fibronectin, but there was only a very weak binding to other RGD containing peptides. Previous incubation of RCC cells with integrin-blocking antibodies did not have any influence on tumor cell adhesion either. After splitting the disulfide chains of the disintegrins, the inhibitory effect of echistatin on tumor cell migration was reversed. Tumor cells also showed no adhesion to the modified echistatin. CONCLUSIONS Both disintegrins, echistatin and kistrin, contain the amino acid sequence RGD in equal amounts. After incubating RCC cells with disintegrins, only echistatin inhibited tumor cell migration. This effect seems not to be caused by a different binding capacity of these agents. After splitting the disulfide chains of echistatin and consequently destructing its proper tertiary structure, tumor cells lost adhesion capability. Subsequently inhibition of cell migration was not possible. Therefore a proper placement of the amino acid sequence RGD in echistatin is crucial for its inhibitory effects. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e97 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Wolfgang Jäger Mainz, Germany More articles by this author Frederik Roos Mainz, Germany More articles by this author Anna Becker Mainz, Germany More articles by this author Elke Schneider Mainz, Germany More articles by this author Joachim Thüroff Mainz, Germany More articles by this author Christian Hampel Mainz, Germany More articles by this author Walburgis Brenner Mainz, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ovarian cancer ascites protects from TRAIL-induced cell death through αvβ5 integrin-mediated focal adhesion kinase and Akt activation

Interactions between ovarian cancer cells and the surrounding tumor microenvironment are not well characterized. We have earlier shown that ovarian cancer ascites induces Akt activation and protect tumor cells from TRAIL-induced apoptosis. Here, we investigated the mechanism by which ascites activates Akt. The ability of ovarian cancer ascites to activate Akt and inhibit TRAIL-induced cell death and caspase activity was decreased by heat inactivation, but was retained in ascites fractions >5 kDa. The survival promoting activity of ascites was not affected by inhibitors of growth factor receptor including epidermal growth factor receptor (EGFR), VEGFR, FGFR, Her2/neu, and IGF-R1. However, this activity was inhibited by an alphavbeta5 integrin-blocking antibody, but not by blocking antibodies against alphavbeta3, beta1, or beta3 integrins. alphavbeta5 integrin-blocking antibodies also inhibited ascites-induced Akt phosphorylation and c-FLIPs up-regulation. Ovarian cancer ascites induced a rapid phosphorylation of focal adhesion kinase (FAK), which closely correlated with the phosphorylation of Akt overtime. FAK phosphorylation was strongly inhibited by alphavbeta5 integrin-blocking antibodies. Depletion of FAK content by RNA interference was also associated with inhibition of ascites-mediated Akt activation and survival. These results suggest that ovarian cancer ascites induces FAK and Akt activation in an alphavbeta5 integrin-dependent pathway, which confers protection from TRAIL-induced cell death and caspase activation.

Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation

AbstractWe show that endothelial cell (EC)–generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, α5β1, α3β1, α6β1, and α1β1, which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

RGD-labeled USPIO Inhibits Adhesion and Endocytotic Activity of αvβ3-Integrin–expressing Glioma Cells and Only Accumulates in the Vascular Tumor Compartment

To investigate the biologic effect of arginine-glycine-aspartic acid (RGD)-labeled ultrasmall superparamagnetic iron oxide (USPIO) (referred to as RGD-USPIO) on human umbilical vein endothelial cells (HUVECs), ovarian carcinoma (MLS) cells, and glioblastoma (U87MG) cells and on U87MG xenografts in vivo. All experiments were approved by the governmental review committee on animal care.USPIOs were coated with integrin-specific (RGD) or unspecific (arginine-alanine-aspartic acid [RAD]) peptides. USPIO uptake in HUVECs, MLS cells, and U87MG cells and in U87MG tumor xenografts was determined with T2 magnetic resonance (MR) relaxometry in 16 nude mice. Cells and tumors were characterized by using immunofluorescence microscopy. Trypan blue staining and lactate dehydrogenase assay were used to assess cytotoxicity. Statistical evaluation was performed by using a Mann-Whitney test or a linear mixed model with random intercept for the comparison of data from different experiments. Post hoc pairwise comparisons were adjusted according to a Tukey test. HUVECs and MLS cells internalized RGD-USPIOs significantly more than unspecific probes. Controversially, U87MG cells accumulated RGD-USPIOs to a lesser extent than USPIO. Furthermore, only in U87MG cells, free RGD and alpha(v)beta(3) integrin-blocking antibodies strongly reduced endocytosis of nonspecific USPIOs. This was accompanied by a loss of cadherin-dependent intercellular contacts, which could not be attributed to cell damage. In U87MG tumors, RGD-USPIO accumulated exclusively at the neovasculature but not within tumor cells. The vascular accumulation of RGD-USPIO caused significantly higher changes of the R2 relaxation rate of tumors than observed for USPIO. In glioma cells with unstable intercellular contacts, inhibition of alpha(v)beta(3) integrins by antibodies and RGD and RGD-USPIO disintegrated intercellular contacts and reduced endocytotic activity, illustrating the risk of inducing biologic effects by using molecular MR probes.

Recombinant Vitronectin Is a Functionally Defined Substrate That Supports Human Embryonic Stem Cell Self-Renewal via αVβ5 Integrin

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel, a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here, we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin, fibronectin, and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that alphaVbeta5 integrins mediated adhesion to vitronectin, alpha5beta1 mediated adhesion to fibronectin, and alpha6beta1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices, but in defined, nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel, supporting sustained self-renewal and pluripotency in three independent hESC lines.

Contributions of Galectin-3 and -9 to Epithelial Cell Adhesion Analyzed by Single Cell Force Spectroscopy

Galectins are widely expressed in epithelial tissues and have been implicated in a variety of cellular processes, including adhesion and polarization. Here we studied the contributions of galectins in cell adhesion and cyst formation of Madin-Darby canine kidney cells. Quantitative single cell force spectroscopy and standard adhesion assays were employed to study both early (<2 min) and long term (90 min) adhesion of cells to different extracellular matrix components. Inhibitors were used to examine the contribution of integrins and galectins in general and RNA interference to specifically address the role of two abundantly expressed galectins, galectin-3 and -9. We found that both galectin-3 and -9 were required for optimal long term cell adhesion to both collagen I and laminin-111. Early adhesion to laminin was found to be integrin-independent and was instead mediated by carbohydrate interactions and galectin-3 and -9. The opposite was observed for early adhesion to collagen. Although similar, the contributions of galectin-3 and -9 to adhesion appeared to be by distinct processes. These defects in adhesion of the two galectin knockdown cell lines may underlie the epithelial phenotypes observed in the cyst assays. Our findings emphasize the complex regulation of epithelial cell functions by galectins.

Role of Ligand-Specific Integrins in Endothelial Cell Alignment and Elongation Induced by Cyclic Strain

Ligand-specific integrins are thought to play a critical role in regulating multiple biological processes. However, the mechanisms by which ligand-specific integrins mediate external stimuli and activate intracellular signaling pathways remain to be elucidated. The aim of this study was to clarify the role of ligand-specific integrins in the morphological changes induced by cyclic strain (CS) via the p38 mitogen-activated protein kinase (p38 MAPK) pathway. Endothelial cells (ECs) were cultured on collagen (a ligand for integrin alpha 2 beta1, but not for alpha 5 and beta 4)-coated flexible plates and incubated for 24 h with or without anti-alpha2 integrin antibody (anti-alpha2), anti-alpha5, anti-beta1, or anti-beta4. ECs were then subjected to 15.6% average CS at 60 cycles/min up to 24 h. After exposure to CS, the cell shape index (defined as (4pi x cell area)/(cell perimeter)(2)), the cell orientation angle, and activation of p38 MAPK were assessed. ECs in the absence of integrin-blocking antibodies were elongated and aligned in response to CS. Anti-alpha 2 and anti-beta1 abolished both morphological changes of ECs as well as the activation of p38 MAPK. In contrast, anti-alpha 5 and anti-beta 4 inhibited neither morphological changes of ECs nor the activation of p38 MAPK. Our results indicate that ligand-specific integrins play a crucial role in the morphological changes of ECs induced by CS via the p38 MAPK pathway.

JAM-C Regulates Tight Junctions and Integrin-mediated Cell Adhesion and Migration

Junctional Adhesion Molecules (JAMs) have been described as major components of tight junctions in endothelial and epithelial cells. Tight junctions are crucial for the establishment and maintenance of cell polarity. During tumor development, they are remodeled, enabling neoplastic cells to escape from constraints imposed by intercellular junctions and to adopt a migratory behavior. Using a carcinoma cell line we tested whether JAM-C could affect tight junctions and migratory properties of tumor cells. We show that transfection of JAM-C improves the tight junctional barrier in tumor cells devoid of JAM-C expression. This is dependent on serine 281 in the cytoplasmic tail of JAM-C because serine mutation into alanine abolishes the specific localization of JAM-C in tight junctions and establishment of cell polarity. More importantly, the same mutation stimulates integrin-mediated cell migration and adhesion via the modulation of beta1 and beta3 integrin activation. These results highlight an unexpected function for JAM-C in controlling epithelial cell conversion from a static, polarized state to a pro-migratory phenotype.

Extracellular Matrix Regulates Enhanced Eotaxin Expression in Asthmatic Airway Smooth Muscle Cells

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with fibronectin and collagen are key features of asthma. Previously, we have reported these ECM proteins enhance ASM synthetic function. We compared ASM cultured from endobronchial biopsies from subjects with and without asthma to assess if asthmatic cells were hypersecretory and determined whether the underlying mechanism involved autocrine ECM production. Cells from subjects with and without asthma were cultured on plastic or in plates precoated with ECM proteins. Cytokine production was evaluated by enzyme-linked immunosorbent assay and by reverse transcriptase-polymerase chain reaction. Function-blocking integrin antibodies were used to identify integrin involvement. Baseline eotaxin and its production after stimulation with interleukin (IL)-13, IL-1beta, or tumor necrosis factor-alpha was increased (2.5- to 6.0-fold) in ASM cells cultured from subjects with asthma compared with healthy subjects. When seeded on ECM from asthmatic ASM, IL-13-dependent eotaxin release from healthy or asthmatic ASM was enhanced compared with culture on healthy ECM. The ECM substrates fibronectin and type I collagen each enhanced IL-13-dependent eotaxin release, and Western immunoblot indicated that fibronectin expression was higher in asthmatic ASM cells. Integrin-blocking antibodies revealed that alpha5beta1 was required for more than 50% of the enhanced IL-13-dependent eotaxin release by ASM cells from subjects with asthma, whereas alpha2beta1 or alphavbeta3 neutralization lacked effect. The data indicate that ASM cells cultured from subjects with asthma are hypersecretory compared with cells from healthy donors and that autocrine fibronectin secretion acting via alpha5beta1 in part underlies this effect.

α2β1 and αVβ1 integrin signaling pathways mediate amyloid-β-induced neurotoxicity

Pathological hallmarks of Alzheimer's disease are the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles, and neurodegeneration. The principal component of amyloid plaques is the amyloid-β peptide (Aβ). Accumulating evidence indicates that Aβ may play a causal role in Alzheimer's disease. In this report, we demonstrate that Aβ deposition and neurotoxicity in human cortical primary neurons are mediated through α2β1 and αVβ1 integrins using specific integrin-blocking antibodies. An aberrant integrin signaling pathway causing the neurotoxicity is mediated through Pyk2. The role of α2β1 and αVβ1 integrins can be extended to another amyloidosis using an amylin in vitro neurotoxicity model. These results indicate that the α2β1 and αVβ1 integrin signaling pathway may be critical components of neurodegeneration in Alzheimer's disease and that integrins may recognize and be activated by a shared structural motif of polymerizing amyloidogenic proteins.

Adhesion of human ovarian adenocarcinoma IGROV1 cells to endothelial cells is partly mediated by the αv integrins–vitronectin adhesive system and induces an alteration of endothelial integrity

Several lines of evidence suggest that vascularization plays an important role in the growth, local expansion and dissemination of ovarian epithelial tumours. However, the interaction of ovarian carcinoma cells with the endothelium remains poorly understood. To investigate adhesive events underlying this process, we used an in vitro model of cocultures between the IGROV1 human ovarian adenocarcinoma cell line and human umbilical vein endothelial cells (HUVECs). IGROV1 cells were shown to adhere rapidly on the HUVECs monolayer. Adhesion was inhibited by anti-alphav integrin and anti-Vn blocking antibodies, but not by anti-beta1 integrin antibodies. Anchorage of carcinoma cells led to the rupture of endothelial integrity, as revealed by the formation of holes in the monolayer and by the disappearance of the interendothelial VE-Cadherin network. Considering the ability of ovarian carcinoma to disseminate by a haematogenous way, these in vitro events could mimic a preliminary step for carcinoma cells crossing the endothelial barrier to extravasate.