Integrin Fibronectin Research Articles

Enhancement of the adhesive and spreading potentials of ovarian carcinoma RMG-1 cells due to increased expression of integrin α5β1 with the Lewis Y-structure on transfection of the α1,2-fucosyltransferase gene

Le Y antigen is known to be associated with malignant properties including metastasis and a poor prognosis of ovarian carcinomas. To clarify the mechanisms underling these properties, we established ovarian carcinoma-derived cells exhibiting enhanced expression of Le Y by transfection with α1,2-fucosyltransferase and compared their cellular properties with those of the original cells. So the human α1,2-fucosyltransferase gene was transfected into ovarian carcinoma-derived RMG-1 cells, which are known to contain Le X, a precursor of Le Y, and RMG-1-hFUT cells exhibiting enhanced expression of Le Y were established by selection with anti-Le Y antibodies, and their adhesive and spreading potentials on fibronectin-coated plates were compared with those of RMG-1 cells. Results showed that the relative expression of Le Y in RMG-1-hFUT cells was about 20-fold that in RMG-1 cells, and that of integrin α5β1 and an integrin-mediated signal transduction molecule, focal adhesion kinase, was also increased in RMG-1-hFUT cells. Interestingly, anti-Le Y antibodies were revealed to immunoprecipitate integrin α5β1, indicating that its oligosaccharides are composed of Le Y, the amounts of which was substantially elevated in RMG-1-hFUT cells. The adhesion and spreading potentials on fibronectin-coated plates of RMG-1-hFUT cells were significantly enhanced in comparison to those of RMG-1 cells, and were greatly suppressed by anti-Le Y antibodies, indicating that Le Y is involved in the integrin–fibronectin interaction. These results suggested that transfection of the α1,2-fucosyltransferase gene into ovarian carcinoma-derived cells brought about elevated expression of integrin α5β1 with Le Y, resulting in enhancement of the adhesion and spreading potentials of cells through the integrin–fibronection interaction, which was inhibited by anti-Le Y antibodies. Thus, Le Y in integrin α5β1 was thought to be involved in the enhanced cell adhesion properties of malignant ovarian carcinomas.

Mg2+modulates integrin–extracellular matrix interaction in vascular smooth muscle cells studied by atomic force microscopy

Atomic force microscopy (AFM) was used to investigate the interaction between alpha5beta1 integrin and fibronectin (FN) in the presence of divalent cations. AFM probes were labeled with FN and used to measure binding strength between alpha5beta1 integrin and FN by quantifying the force required to break single FN-integrin bonds on a physiological range of loading rates (100-10,000 pN/s). The force necessary to rupture single alpha5beta1-FN bond increased twofold over the regime of loading rates investigated. Changes in Mg(2+) and Ca(2+) concentration affected the thermodynamical parameters of the interaction and modulated the binding energy. These data indicate that the external ionic environment in which vascular smooth muscle cells reside, influences the mechanical parameters that define the interaction between the extracellular matrix and integrins. Thus, in a dynamic mechanical environment such as the vascular wall, thermodynamic binding properties between FN and alpha5beta1 integrin vary in relation to locally applied loads and divalent cations concentrations. These changes can be recorded as direct measurements on live smooth muscle cells by using AFM.

159. EXTRACELLULAR MATRIX DYNAMICS IN SCAR-FREE ENDOMETRIAL REPAIR

Tissue destruction and repair occur simultaneously within the menstruating uterus. Whilst the factors that control menstruation are increasingly understood (1) the endometrial milieu which governs repair remains elusive. The extracellular-matrix (ECM) plays a dynamic role within the repairing endometrium, with roles suggested for fibronectin and certain integrins (2). We here use both an in vivo mouse model of breakdown and repair (3) and an in vitro model of re-epithelialisation to determine factors involved in repair of this tissue. Murine uterine horns were subjected to laser-capture microdissection of the repairing luminal epithelium (LE) and immediate sub-luminal stroma. Extracted RNA was processed for pathway-focused array that revealed expression of epithelial and leukocyte expressed integrins along with their ligands, proteases and protease inhibitors and basement membrane proteins as elevated in repairing uterine horns. Immunohistochemistry confirmed expression and localisation of integrins a 5 and b 1 in addition to integrin ligands VCAM-1 and fibronectin. Additionally, we demonstrated intimate association of neutrophils with repairing/recently repaired luminal epithelium. To confirm the relevance of these ECM interactions in a human model, human endometrial luminal epithelial cell (ECC-1) monolayers were wounded and treated with inhibitors of MMPs (doxycycline, GM6001) or integrin-fibronectin interactions (RGDS). Significant inhibition of in vitro re-epithelialisation was seen following treatment of wounded ECC-1 monolayers with RGDS, doxycycline or GM6001, suggesting a requirement for these ECM components in endometrial re-epithelialisation. We subsequently demonstrated that menstrual blood derived factors may play a role in the initial endometrial re-epithelialisation. Menstrual blood and peripheral blood were collected on the same day from volunteers. Treatment of wounded ECC-1 monolayers with menstrual blood plasma enhanced the rate of repair compared with peripheral blood plasma from the same woman. Enhanced understanding of the mechanisms governing scar-free repair in the endometrium may help resolve pathological endometrial bleeding disorders and may be applied to aid healing in other tissues. (1) Jabbour et al, 2006. Endocr Rev. 27(1): 17–46.(2) Cao et al, 2007. Hum Reprod. 22(12): 3223–31(3) Brasted et al, 2003. Biol Reprod. 69(4): 1273–80.

Fibronectin–integrin mediated signaling in human cervical cancer cells (SiHa)

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin-integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin-integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-kappaB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.

Cytoprotective Effects of a Cyclic RGD Peptide in Steatotic Liver Cold Ischemia and Reperfusion Injury

The serious need for expanding the donor population has attracted attention to the use of steatotic donor livers in orthotopic liver transplantation (OLT). However, steatotic livers are highly susceptible to hepatic ischemia-reperfusion injury (IRI). Expression of fibronectin (FN) by endothelial cells is an important feature of hepatic response to injury. We report the effect of a cyclic RGD peptide with high affinity for the alpha5beta1, the FN integrin receptor, in a rat model of steatotic liver cold ischemia, followed by transplantation. RGD peptide therapy ameliorated steatotic IRI and improved the recipient survival rate. It significantly inhibited the recruitment of monocyte/macrophages and neutrophils, and depressed the expression of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and interferon (IFN)-gamma. Moreover, it resulted in profound inhibition of metalloproteinase-9 (MMP-9) expression, a gelatinase implied in leukocyte migration in damaged livers. Finally, we show that RGD peptide therapy reduced the expression of the 17-kDa active caspase-3 and the number of apoptotic cells in steatotic OLTs. The observed protection against steatotic liver IRI by the cyclic RGD peptides with high affinity for the alpha5beta1 integrin suggests that this integrin is a potential therapeutic target to allow the successful utilization of marginal steatotic livers in transplantation.

α5β1 Integrin Ligand PHSRN Induces Invasion and α5 mRNA in Endothelial Cells to Stimulate Angiogenesis

Angiogenesis requires endothelial cell invasion and is crucial for wound healing and for tumor growth and metastasis. Invasion of native collagen is mediated by the α5β1 integrin fibronectin receptor. Thus, α5β1 up-regulation on the surfaces of endothelial cells may induce endothelial cell invasion to stimulate angiogenesis. We report that the interaction of α5β1 with its PHSRN peptide ligand induces human microvascular endothelial cell invasion and that PHSRN-induced endothelial cell invasion is regulated by α4β1 integrin and requires matrix metalloproteinase 1 (MMP-1). Moreover, our results show that exposure to PHSRN causes rapid, specific up-regulation of surface levels of α5β1 integrin and significantly increases α5 integrin mRNA in microvascular endothelial cells. Consistent with these results, α5 small interfering RNA abrogates PHSRN-induced surface α5 and MMP-1 up-regulation, as well as blocking invasion induction. We also observed dose-dependent, PHSRN-induced α5β1 integrin up-regulation on endothelial cells in vivo in Matrigel plugs. We further report that the PHSCN peptide, an α5β1-targeted invasion inhibitor, blocks PHSRN-induced invasion, α5β1 up-regulation, α5 mRNA induction, and MMP-1 secretion in microvascular endothelial cells and that systemic PHSCN administration prevents PHSRN-induced α5β1 up-regulation and angiogenesis in Matrigel plugs. These results demonstrate a critical role for α5β1 integrin and MMP-1 in mediating the endothelial cell invasion and angiogenesis and suggest that PHSRN-induced α5 transcription and α5β1 up-regulation may form an important feed-forward mechanism for stimulating angiogenesis.

Formation and activation of fibroblast spheroids depend on fibronectin–integrin interaction

Clustering of fibroblasts into spheroids induces a massive proinflammatory, proteolytic and growth-factor response, named nemosis, which promotes tumor cell invasiveness and differentiation of leukemia cells. We have now sought to investigate mechanisms leading to the formation of multicellular spheroids and subsequent activation of fibroblasts (nemosis). Cell lines either lacking fibronectin expression (FN −/−) or expressing FN with a mutated integrin-binding site (FN RGE/RGE) were unable to form compact spheroids. Furthermore, inhibition of FN synthesis by siRNA or functional inhibition of FN or its integrins impaired spheroid formation (α5, β1) and quenched fibroblast activation (αV). The integrin ligand GRGDSP hexapeptide interfered with spheroid formation and induced activation of fibroblasts. Surprisingly, a 70 kDa FN fragment, which prevents deposition of FN matrix but does not interfere with FN–integrin interaction, prevented spheroid formation only marginally and did not block the activation. Our results present a new mechanism of fibroblast activation, which is initiated by interaction of FN with its integrin receptors.

Radiation-induced Nuclear Translocation of IkB-beta and Transcription of the Alpha 5 Integrin Gene may Upregulate Surface Alpha 5 Beta 1 to Cause Invasion by Human Pancreatic Cancer Cells

Ionizing radiation rapidly induces Panc-1 and BxPC-3 human pancreatic cancer cell invasion, which is mediated by the alpha 5 beta 1 integrin fibronectin receptor (FnR) and requires MMP 1 activity. Here, we report the effects of radiation on the levels and localization of alpha 5 beta 1 FnR and MMP 1 proteins, on alpha 5 integrin mRNA upregulation, and on the nuclear localization of the NFkB chaperone, IkB-beta.

Talin depletion reveals independence of initial cell spreading from integrin activation and traction

Cell spreading, adhesion and remodelling of the extracellular matrix (ECM) involve bi-directional signalling and physical linkages between the ECM, integrins and the cell cytoskeleton. The actin-binding proteins talin1 and 2 link ligand-bound integrins to the actin cytoskeleton and increase the affinity of integrin for the ECM. Here we report that depletion of talin2 in talin1-null (talin1(-/-)) cells did not affect the initiation of matrix-activated spreading or Src family kinase (SFK) activation, but abolished the ECM-integrin-cytoskeleton linkage and sustained cell spreading and adhesion. Specifically, focal adhesion assembly, focal adhesion kinase (FAK) signalling and traction force generation on substrates were severely affected. The talin1 head domain restored beta1 integrin activation but only full-length talin1 restored the ECM-cytoskeleton linkage and normal cytoskeleton organization. Our results demonstrate three biochemically distinct steps in fibronectin-activated cell spreading and adhesion: (1) fibronectin-integrin binding and initiation of spreading, (2) fast cell spreading and (3) focal adhesion formation and substrate traction. We suggest that talin is not required for initial cell spreading. However, talin provides the important mechanical linkage between ligand-bound integrins and the actin cytoskeleton required to catalyse focal adhesion-dependent pathways.

IL-1 induced shape changes mediated by ras

IL-1, an inflammatory cytokine, is known to mediate a wide range of biological activities in a number of cell lines. IL-1 receptor binding causes modulation at focal adhesions and reorganisation of the actin cytoskeleton. Further, IL-1 induced signal transduction is regulated by integrin binding and fibronectin attachment is permissive in IL-1 mediated gene regulation. Members of the p21ras superfamily of GTP-binding proteins have been shown to play a central role in mediating coregulation of structural and growth factor/cytokine mediated responses. To determine the role of GTP-binding proteins in matrix regulation of IL-1 mediated responses we have made GTPase/EGFP constructs of human Ras and the dominant negative mutant Asn17-Ras. Confocal microscopy of live cells shows that IL-1 stimulation of Ha-Ras transfected cells plated on a fibronectin matrix show relocalisation of the Ras/EGFP fusion protein to the cell membrane, and subsequent rapid and pronounced shape changes, causing cell rounding to 50–70% of original section area. No such changes were seen upon stimulation with EGF, a known activator of Ras. The shape changes correlate with a transient activation of Ras in fibronectin attached cells, comparable with that of EGF. Transfection of cells with the dominant negative Ras construct (Asn17-Ras), or plating of Ras transfected cells on bare tissue culture plastic abrogates the observed IL-1 induced changes in cell morphology and activation. Automated screening of transfected cells at low magnification gave similar results, showing that IL-1 affects approximately 50% of the transfected cell population. These data indicate that IL-1 induced shape changes are due to activation of Ras, and that it is involved in regulating IL-1 mediated responses in a matrix dependent manner. The studies are supported by the STFUSH, the Medical Research Council and the Wellcome Trust.

Expression of Integrin Subunits in the Herniated Intervertebral Disc

Integrins are a class of cell adhesion molecules that regulate interactions between cells and their extracellular matrix (ECM). Several specific integrin receptors have been indentified in intervertebral discs, including the fibronectin-binding integrin receptors α5 β1, αv β3 and the collagen-binding integrin receptors α1 β1, α2 β1, and, αv β1. But the integrins expressions in degenerated intervertebral discs are still unknown. In our study, the expressions of α1, α2, α5, αv, β1, β3 integrin subunits, collagens, and fibronectin in normal and herniated intervertebral discs of human were determined. Specimens of human lumbar intervertebral discs were divided into 3 groups: normal discs (n = 10), protrusion (n = 15), and extrusion (n = 15). Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunoprecipitation were used to evaluate the α1, α2, α5,αv, β1, and β3 integrin subunits messenger ribonucleic acid (mRNA) and protein expressions. RT-PCR was also performed to measure the mRNA level of collagen I, collagen II, and fibronectin. The expressions of α5 and β1 subunits were increased in herniated discs, especially in the discs of extrusion. But as to α1, α2, αv and β3, their expressions had no difference among the discs. Fibronectin, whose binding integrin receptor was α5 β1 was also increased. And in herniated discs, the collagen I was increased, but the collagen II was decreased. The expressions of some integrin subunits were increased in herniated discs. These results may be attributed to the interaction between cells and the ECM in the process of degeneration.

Stimulation of lung carcinoma cell growth by fibronectin–integrin signalling

Throughout many countries, lung cancer will kill more people this year than malignancies related to breast, prostate, colon, liver, kidney and melanoma combined. Despite recent advances in understanding the molecular biology of lung carcinoma and the introduction of multiple new chemotherapeutic agents for its treatment, its dismal five-year survival rate (<15%) has not changed substantially. The lack of advancement in this area reflects the limited knowledge available concerning the factors that promote oncogenic transformation and proliferation of carcinoma cells in the lung. Malignant transformation plays a key role in tumor growth and invasion; however, other factors such as the surrounding stroma, local growth factors, vascularity, and systemic hormones are important contributors as well. We believe that the composition of the lung extracellular matrix is also important due to its ability to affect malignant cell behavior in vitro. The matrix glycoprotein fibronectin, for example, is highly expressed in chronic lung disorders where most lung carcinomas are identified. This document reviews information that implicates fibronectin in the stimulation of lung carcinoma cell growth. Data available to date indicate that by binding to specific integrin receptors expressed on the surface of tumor cells, fibronectin stimulates intracellular signals implicated in the pathobiology of lung carcinogenesis and lung tumor chemoresistance including mitogen-activated protein kinases, GTPases, and the PI3-kinase/Akt/mTOR pathway. Thus, integrin-mediated signals triggered by fibronectin in tumor cells represent promising targets for the development of novel anti-cancer strategies.

Epithelial-derived Fibronectin Expression, Signaling, and Function in Intestinal Inflammation

Fibronectin (FN) is a multifunctional extracellular matrix protein that plays an important role in cell proliferation, adhesion, and migration. FN expression or its role in colitis is not known. The goal of this study is to characterize FN expression, regulation, and role during intestinal inflammation. Wild-type and transgenic mice expressing luciferase under the control of the human FN promoter, given water or 3% dextran sodium sulfate, were used as animal models of colitis. The Caco2-BBE model intestinal epithelial cell line was used for in vitro studies. FN protein is abundantly expressed by surface epithelial cells in the normal colon. Immunohistochemistry and luciferase assay in mice expressing the FN promoter linked to luciferase demonstrated that FN synthesis was up-regulated during colitis, during both the acute phase and the healing phase. In vitro experiments demonstrated that FN increased the expression of the FN integrin receptor alpha5beta1 in a dose- and time-dependent manner. FN also induced the expression and activation of NF-kappaB. Further, FN potentiated Caco2-BBE cell attachment and wound healing, which was inhibited by RGD peptide as well as NF-kappaB inhibitors MG-132 and 1-pyrrolidinecarbodithioic acid, ammonium salt. In conclusion, FN is abundantly expressed and synthesized by colonic epithelial cells. FN is transcriptionally up-regulated in epithelial cells during both the dextran sodium sulfate-induced colitic and the recovery phase. FN enhances cell attachment and wound healing, which is dependent on binding to the integrin receptor and the NF-kappaB signaling. Together our data show that epithelial-derived FN potentiates cell attachment and wound healing through epithelial-matrix interactions and that FN expression may have important implications for maintaining normal epithelial integrity as well as regulating epithelial response to injury during colitis.

Proliferation and adhesion of periodontal ligament cells on synthetic biominerals

Hydroxiapatite (HA) has been suggested as a useful biomaterial to support the regeneration of tissues. In this study, we investigated the adhesion of periodontal ligament (PDL) cells on octacalcium phosphate (OCP) and its hydrolyzed apatitic product (HL), which are known precursors of HA. Rat PDL cells were cultured on OCP or HL-coated dishes. Cell proliferation and adhesion and mRNA expression of collagen I, fibronectin integrin subunits were examined. Cell adhesion inhibition assays were carried out by GRGDSPK (Gly-Arg-Gly-Asp-Ser-Pro-Lys). In early culture period, the cell number of PDL cells was lower on OCP and HL than that on control without any coating. However, the cell number on OCP or HL caught up with control later period. mRNA expression level of collagen I and fibronectin on OCP and HL were similar among OCP HL and control, although they differed early in the culture period. Integrin subunits were expressed on both OCP and HL as well as on control. Cell adhesion was inhibited by RGD inhibitor peptide. Our findings indicated that rat PDL cells produce collagen I and fibronectin on OCP and HL, and then show increased cell numbers depending on adhesion to the matrices through integrins.

The FN13 peptide inhibits human tumor cells invasion through the modulation of αvβ3 integrins organization and the inactivation of ILK pathway

We report the effect of the stable expression of a 13 amino acid human fibronectin (FN) peptide (FN13) on the organization of the FN extracellular matrix (ECM) and of FN integrin receptors (FNRs), in relationship with the inhibition of cellular invasion, in three FN-ECM defective human tumor-derived cell lines: SK-Hep1C3, hepatoma, ACN, neuroblastoma, and SK-OV-3, ovary carcinoma. All these cell lines stably expressing the FN13 peptide, organized an FN-ECM, disorganized αvβ1 integrins and inactivated the ILK pathway, with the loss of secretion of MMP-9. This was associated with the inhibition of cell invasion in Matrigel matrix only in SK-Hep1C3 and ACN, but not in SK-OV-3 cells. Analysis of the integrin receptors organization showed that the FN13 expressing cells SK-Hep1C3 and ACN organized αvβ3 integrins, whereas SK-OV-3 organized αvβ5 dimers. The functional block of αvβ5 integrins, with an inactivating anti-αvβ5 antibody, led to the induction of αvβ3 integrins also in SK-OV-3 cells, and to the inhibition of cell invasion. These data show that in the human tumor cells studied FN13 inhibits the in vitro invasion through the dissociation of αvβ1 dimers, leading to ILK pathway inactivation, only when the organization of αvβ3 integrins is induced in the plasma membrane.

FAK‐dependent regulation of myofibroblast differentiation

Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGFbeta) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to "de-differentiate" into fibroblasts. TGFbeta induces expression of smooth muscle alpha actin (SMalphaA) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGFbeta induces the expression of fibronectin and fibronectin integrins. Fibronectin-generated signals contribute to the TGFbeta-mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGFbeta-mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK -/- MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SMalphaA in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK -/- [corrected]MEFs. We conclude that FAK does not contribute to TGFbeta-dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down-regulating expression of SMalphaA, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.

ERBB2-Mediated Transcriptional Up-regulation of the α5β1 Integrin Fibronectin Receptor Promotes Tumor Cell Survival Under Adverse Conditions

Oncogenic activation of the receptor tyrosine kinase ERBB2 is a key event in the development of a number of epithelial malignancies. In these tumors, high levels of ERBB2 are strongly associated with metastatic disease and poor prognosis. Paradoxically, an inherent cellular response to hypermitogenic signaling by ERBB2 and other oncogenes seems to be growth arrest, rather than proliferation. Molecular characterization of this yet undefined antiproliferative state in independent cell lines overexpressing either wild-type ERBB2 or the mutationally activated receptor unveiled a dramatic induction of the alpha5beta1 integrin fibronectin receptor. alpha5 Integrin up-regulation is mainly a transcriptional response mediated by the hypoxia-inducible transcription factors (HIF), leading to a massive increase in membrane-resident receptor molecules and enhanced fibronectin adhesiveness of the respective cells. Functionally, ERBB2-dependent ligation of fibronectin results in improved survival of mammary adenocarcinoma cells under adverse conditions, like serum withdrawal, hypoxia, and chemotherapy. HIF-1alpha is an independent predictor of poor overall survival in patients with breast cancer. In particular, HIF-1alpha overexpression correlates significantly with early local relapse and distant metastasis, a phenotype also highly characteristic of ERBB2-positive tumors. As HIF-1alpha is known to be stabilized by ERBB2 signaling under normoxic conditions, we propose that alpha5 integrin is a major effector in this regulatory circuit and may represent the molecular basis for the HIF-1alpha-dependent aggressiveness observed in ERBB2-overexpressing breast carcinomas. Hypermitogenic ERBB2 signaling and tumor hypoxia may act synergistically to favor the establishment of chemoresistant dormant micrometastatic cells frequently observed in patients with breast cancer. This new insight could be the basis for additional approaches complementing current cancer therapy.

Differential cell adhesion to vocal fold extracellular matrix constituents

The human vocal folds are a complex layering of cells and extracellular matrix. Vocal fold extracellular matrix uniquely contributes to the biomechanical viscoelasticity required for human phonation. We investigated the adhesion of vocal fold stellate cells, a novel cell type first cultured by our laboratory, and fibroblasts to eight vocal fold extracellular matrix components: elastin, decorin, fibronectin, hyaluronic acid, laminin and collagen types I, III and IV. Our data demonstrate that these cells adhere differentially to said substrates at 5 to 120 min. Cells were treated with hyaluronidase and Y-27632, a p160ROCK-specific inhibitor, to test the role of pericellular hyaluronan and Rho-ROCK activation in early and mature adhesion. Reduced adhesion resulted; greater inhibition of fibroblast adhesion was observed. We modulated the fibronectin affinity exhibited by both cell types using Nimesulide, an inhibitor of fibronectin integrin receptors alpha5beta1 and alphavbeta3. Our results are important in understanding vocal fold pathologies, wound healing, scarring, and in developing an accurate organotypic model of the vocal folds.

Targeting Invasion Induction as a Therapeutic Strategy for the Treatment of Cancer

The spread of cancer cells from the primary tumor to a distant site involves many of the invasive processes normally required for wound healing, including migration through the local connective tissue, invasion of the vasculature, extravasation, invasion of the connective tissue at a distant site, and angiogenesis. Thus, the abilities of tumor cells to invade the host, and to induce endothelial cell invasion and neovascularization, are central to malignant progression. The plasminogen activator system, which plays a direct role in stimulating alpha5beta1 integrin fibronectin receptor-mediated invasion during wound healing, is also very important in tumor cell invasion and metastasis, as well as in angiogenesis. Therefore, the alpha5beta1 receptor and the plasminogen activator system may be promising targets for directed anticancer therapies.

The regulation of expanded human nasal chondrocyte re-differentiation capacity by substrate composition and gas plasma surface modification

Optimizing re-differentiation of clinically relevant cell sources on biomaterial substrates in serum containing (S+) and serum-free (SF) media is a key consideration in scaffold-based articular cartilage repair strategies. We investigated whether the adhesion and post-expansion re-differentiation of human chondrocytes could be regulated by controlled changes in substrate surface chemistry and composition in S+ and SF media following gas plasma (GP) treatment. Expanded human nasal chondrocytes were plated on gas plasma treated (GP+) or untreated (GP−) poly(ethylene glycol)-terephthalate–poly(butylene terephthalate) (PEGT/PBT) block co-polymer films with two compositions (low or high PEG content). Total cellularity, cell morphology and immunofluorescent staining of vitronectin (VN) and fibronectin (FN) integrin receptors were evaluated, while post-expansion chondrogenic phenotype was assessed by collagen types I and II mRNA expression. We observed a direct relationship between cellularity, cell morphology and re-differentiation potential. Substrates supporting high cell adhesion and a spread morphology (i.e. GP+ and low PEG content films), resulted in a significantly greater number of cells expressing α 5 β 1 FN to α Vβ 3 VN integrin receptors, concomitant with reduced collagen type II/I mRNA gene expression. Substrates supporting low cell adhesion and a spherical morphology (GP− and high PEG content films) promoted chondrocyte re-differentiation indicated by high collagen type II/I gene expression and a low percentage of α 5 β 1 FN integrin expressing cells. This study demonstrates that cell–substrate interactions via α 5 β 1 FN integrin mediated receptors negatively impacts expanded human nasal chondrocyte re-differentiation capacity. GP treatment promotes cell adhesion in S+ media but reverses the ability of low PEG content PEGT/PBT substrates to maintain chondrocyte phenotype. We suggest alternative cell immobilization techniques to GP are necessary for clinical application in articular cartilage repair.