Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target.

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvß5 (ITGαvß5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB-OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

The Interaction of CD154 with the α5ß1 Integrin Inhibits Fas-Induced T Cell Death.

CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbß3, αMß2 and α5ß1. Given the role attributed to integrins and particularly the ß1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5ß1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5ß1-dependent manner. Binding of soluble CD154 to α5ß1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs), phosphoinositide 3 kinase (PI-3K), and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5ß1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin.

CCN4 induces IL-6 production through αvß5 receptor, PI3K, Akt, and NF-κB singling pathway in human synovial fibroblasts.

INTRODUCTION: Osteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells. METHODS: CCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65. RESULTS: Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvß5 but not α5ß1 and αvß3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor (LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation. CONCLUSIONS: Our results suggest that CCN4 activates αvß5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.

Expression, purification, and characterization of RGD-mda-7, a His-tagged mda-7/IL-24 mutant protein.

RGD peptide (Arg-Gly-Asp tripeptide) binds to integrin αVß(3) and αVß(5), which is selectively expressed in tumor neovasculature and on the surface of some tumor cells. Some studies showed that coupling the RGD peptides to anticancer drugs yielded compounds with increased efficiency against tumors and lowered toxicity to normal tissues. The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, we inserted a glycine residue into the wild type (mda-7/IL-24) between (164)Arg and (165)Asp to form a RGD peptide, named RGD-mda-7, then expressed RGD-mda-7 in Escherichia coli. Herein, we describe the expression and purification of RGD-mda-7. We detected the characterizations of immunostimulatory activity, tumor targeting, potent cytopathic effect, and apoptosis inducing exploited by RGD-mda-7 in tumor cells, and also compared these characterizations with wtmda-7/IL-24. The data showed that RGD-mda-7 had more potent tumor targeting and apoptosis-inducing effects than wtmda-7/IL-24.

Thrombin stimulates integrin ß1-dependent adhesion of human pancreatic cancer cells to vitronectin through protease-activated receptor (PAR)-1.

BACKGROUND/AIMS: The aim of this study was to investigate the effect of thrombin and the thrombin receptor protease-activated receptor (PAR)-1 on adhesion of human pancreatic cancer cell lines to extracellular matrices (ECMs) and to identify related integrins with these effects. METHODOLOGY: Human pancreatic cancer cell lines SUIT-2 and its four sublines, and Panc- 1, AsPC-1 and MiaPaCa-2 were treated with thrombin, PAR-1 agonist TRAP-6, PAR-1 antagonist SCH79797, or anti-integrin ±vß3, ±vß5 and ß1 monoclonal antibodies. Cells were incubated for 45 minutes on micro titer plates that were pre-coated with ECMs (fibronectin, laminin, vitronectin, type IV collagen). The number of adherent cells was measured by the MTT method. RESULTS: Eight human pancreatic cancer cell lines expressed PAR-1. Thrombin significantly enhanced adhesion of SUIT-2 and its sublines and MiaPaCa-2 to vitronectin, especially in the SUIT-2 subline S2-007. We obtained similar results on S2-007 cells through treatment with TRAP-6. However, SCH79797 inhibited the effect of thrombin. Furthermore, anti-integrin ß1 antibody conspicuously inhibited 1U/mL thrombin-induced enhancement of adhesion to vitronectin. CONCLUSIONS: Thrombin significantly enhanced adhesion of pancreatic cancer cells to vitronectin through PAR- 1 depending on the presence of integrin ß1. Suppression of thrombin action by anti-integrin ß1 antibody will become a useful therapy against pancreatic cancer.

Cyr61 increases matrix metalloproteinase-3 expression and cell motility in human oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvß5 or α6ß1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvß3 or α6ß1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.

CTGF inhibits cell motility and COX-2 expression in oral cancer cells.

Oral squamous cell carcinoma (SCC) has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity and COX-2 expression in human oral cells is mostly unknown. Here we found that CTGF reduced the migration and expression of COX-2 in human oral cancer cells. αvß5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002 and wortmannin) and Akt inhibitor reversed the CTGF-inhibited the migration and COX-2 down-regulation of oral cancer cells. CTGF stimulation decreased the phosphorylation of focal adhesion kinase (FAK), PI3K and Akt. In addition, c-Jun siRNA also antagonized the CTGF-inhibited migration and COX-2 expression. Moreover, CTGF decreased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Taken together, our results indicated that CTGF inhibits the migration of oral cancer cells by decreasing COX-2 expression through the αvß5 integrin receptor, FAK, PI3K, Akt, c-Jun and AP-1 signal transduction pathways.

CCN3 increases cell motility and MMP-13 expression in human chondrosarcoma through integrin-dependent pathway.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN3, also called nephroblastoma overexpressed gene (NOV), regulates proliferation and differentiation of cancer cells. However, the effect of CCN3 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). αvß3 or αvß5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase (PI3K) inhibitors (Ly294002 and wortmannin) and Akt inhibitor inhibited the CCN3-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. CCN3 stimulation increased the phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. In addition, NF-κB inhibitors also suppressed the cell migration and MMP-13 expression enhanced by CCN3. Moreover, CCN3 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-13 promoter. Taken together, our results indicate that CCN3 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the αvß3/αvß5 integrin receptor, FAK, PI3K, Akt, p65, and NF-κB signal transduction pathway.

Preclinical evaluation of monoclonal antibody 14C5 for targeting pancreatic cancer.

The use of radiolabeled antibodies that are able to target primary tumors as well as metastatic tumor sites with minimal reactivity to normal tissues is a promising approach for treating pancreatic cancer. In this study, the integrin alpha(v)beta(5) is studied as a target for the diagnosis of and potential therapy for human pancreatic cancer by using the radiolabeled murine monoclonal antibody (mAb) 14C5. Biopsy specimens from human pancreatic tumors were examined for the expression of the integrin alpha(v)beta(5). The pancreatic tumor cell line Capan-1 was used to test the in vitro targeting potency of mAb 14C5 labeled with 125/131-iodine and 111-indium. Internalization, retention, and metabolism were investigated in cellular radioimmunoassays. Biodistribution and tumor-targeting characteristics were studied in Capan-1 xenografts. All tumor sections were positive for the integrin alpha(v)beta(5), with an extensive positive staining of the stroma. Saturation binding experiments showed high affinity with comparable K(d)s. In vitro internalization experiments showed a longer intracellular retention of (111)In-p-benzyl isothiocyanate-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA)-14C5 in comparison to (125)I-14C5 and (111)In-p-isothiocyanatobenzyl diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA)-14C5. In vivo radioisotope tumor uptake was maximum at 48-72 hours, with the uptake of (111)In-p-SCN-Bz-DOTA-14C5 (35.84 +/- 8.64 percentage of injected dose per g [%ID/g]) being 3.9- and 2.2-folds higher than (131)I-14C5 (12.16 +/- 1.03%ID/g) and (111)In-p-SCN-Bz-DTPA-14C5 (14.30 +/- 3.76%ID/g), respectively. Planar gamma imaging with mAb 14C5 indicated clear localization of the pancreatic tumors versus minimal normal tissue uptake. mAb 14C5 is a promising new antibody for targeting the integrin alpha(v)beta(5) for the diagnosis of and potential therapy for pancreatic cancer.

Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin.

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived, patient-specific pluripotent cells for use in cell-based regenerative therapies. However, current methods of cell culture are tedious and expensive, and the mechanisms underlying cell proliferation are not understood. In this study, we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion, survival, and proliferation. We show that iPSC lines generated using Oct-3/4, Sox-2, Nanog, and Lin-28 express a repertoire of integrins similar to that of hESCs, with prominent expression of subunits alpha5, alpha6, alphav, beta1, and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin, in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel, as shown by immunological blockade experiments. On vitronectin, the integrin alphavbeta5 was required for initial attachment, but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore, iPSCs cultured on vitronectin for 9 passages retained normal karyotype, pluripotency marker expression, and capacity to differentiate in vitro. These studies suggest that vitronectin, or derivatives thereof, might substitute for Matrigel in a more defined system for iPSC culture.

Integrin alphavbeta1 promotes infection by human metapneumovirus.

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for alphavbeta1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with alphav or beta1 cDNAs confers hMPV infectivity, whereas reduction of alphav and beta1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that alphavbeta1 integrin is a functional receptor for hMPV.

Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and alpha3beta1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with alpha3beta1 and alphaVbeta3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-alphaV and -beta1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against alphaVbeta3 and alphaVbeta5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble alpha3beta1, alphaVbeta3, and alphaVbeta5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that alphaVbeta3 and alphaVbeta5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin alphaVbeta5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas alpha3beta1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and alphaVbeta3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of alphaVbeta5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with alpha3beta1 and KSHV. Preincubation of KSHV with soluble heparin and alpha3beta1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (alphaVbeta3, alpha3beta1, and alphaVbeta5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.

[Tumor cell-tumor endothelial cell adhesion mediated by alphavbeta3 and alphavbeta5 molecules].

OBJECTIVE: To investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro. METHODS: The expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture. RESULTS: The expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5. CONCLUSION: alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.

Spontaneous or Mycobacterium tuberculosis-induced apoptotic neutrophils exert opposite effects on the dendritic cell-mediated immune response.

Polymorphonuclear neutrophils (PMN) modulate the adaptive immune response through interactions with immature dendritic cells (iDC) while spontaneous apoptotic neutrophils PMNapo (PMNapo) may have an inhibitory effect on DC functions. We investigate the effect exerted by PMNapo in DC maturation and the role of Mycobacterium tuberculosis (Mtb)-induced PMNapo in the cross-presentation of mycobacterial antigens. We demonstrate that Mtb triggers the maturation of iDC while it is impaired by the presence of PMNapo, which abrogate Mtb-induced expression of costimulatory and HLA class II molecules, reducing IL-12 and IFN-gamma release by DC and partially inhibiting Mtb-driven lymphocyte proliferation. This inhibitory effect is not observed in already Mtb-matured DC, and it involves a direct interaction between DC and PMNapo, as supernatants from PMNapo cultures do not reveal this effect. Although PMNapo do not alter Mtb/DC-SIGN interaction, they affect the intracellular signals leading to DC maturation without requiring their entry into DC. Phagocytosis of Mtb-induced PMNapo by iDC leads to lymphoproliferation, which is significantly reduced by blocking CD36 and not DC-SIGN on iDC. Therefore, cross-presentation of Mtb antigens is taking place. Our findings suggest that the inflammatory milieu is subjected to a fine balance between non-infected and Mtb-induced PMNapo: non-infected PMNapo limiting inflammation and Mtb-induced PMNapo generating a specific immune activity.

The P2Y2 nucleotide receptor requires interaction with alpha v integrins to access and activate G12.

The P2Y2 nucleotide receptor (P2Y2R) interacts with alpha v integrins to activate G(o) and induce chemotaxis in human 1321N1 astrocytoma cells. In this study, it was determined that the P2Y2R also requires interaction with alpha v integrins to activate G12 and associated signaling pathways that control chemotaxis in 1321N1 cells. Mutation of the Arg-Gly-Asp (RGD) integrin-binding sequence in the first extracellular loop of the human P2Y2R to Arg-Gly-Glu (RGE), which prevents integrin interaction, did not inhibit G(q) or ERK1/2 signaling by the P2Y2R agonist UTP but completely inhibited activation of G12 and G12-mediated events, including Rho activation, cofilin and myosin light chain-2 phosphorylation, stress fiber formation and chemotaxis towards UTP. The involvement of G12 in all these events was verified by using a dominant negative G alpha12 construct. G12 activation by the P2Y2R also was inhibited by anti-alpha v beta5 integrin antibodies and alpha v integrin antisense oligonucleotides, suggesting that alpha v integrin activity and expression are required for the P2Y2R to activate G12. Co-immunoprecipitation experiments confirmed that G alpha12 protein associates with the wild-type P2Y2R and with alpha v integrins but not with the RGE mutant P2Y2R or with alpha3 integrins. Collectively, these results suggest that alpha v integrin complexes provide the P2Y2R with access to G12, thereby allowing activation of this heterotrimeric G protein that controls actin cytoskeletal rearrangements required for chemotaxis.

Role of alphavbeta5 integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells.

Crocidolite, containing 27% iron by weight, is the most carcinogenic form of asbestos. Crocidolite fibers are endocytized by alphavbeta5 integrin receptors in rabbit pleural mesothelial cells. We show here that crocidolite fibers are endocytized in human lung epithelial (A549) cells and in primary small airway epithelial (SAEC) cells. Presence of the integrin alphavbeta5 blocking antibody, P1F6, significantly reduced the uptake of crocidolite fibers in A549 cells. Thus, the integrin alphavbeta5 receptor is involved in endocytosis of crocidolite fibers in A549 cells as well. Previously, it has been observed that asbestos fibers lead to changes in the intracellular redox environment, i.e. a marked decrease in intracellular glutathione concentrations and an increase in the extracellular glutathione in A549 cells. In addition, the decrease in intracellular glutathione was found to be largely independent of iron present on the surface of the fiber. A549 cells were treated with crocidolite in the presence of endocytosis inhibitor cytochalasin D. Our data indicate that, upon preventing endocytosis, we were able to reverse the decrease in total intracellular glutathione. The decrease in total intracellular glutathione could also be prevented in the presence of the monoclonal antibody P1F6. Thus, we observed that endocytosis of crocidolite fibers via integrin alphavbeta5 receptor is linked to the marked decrease in total intracellular glutathione in A549 cells.

Lipid-mediated delivery of brain-specific angiogenesis inhibitor 1 gene reduces corneal neovascularization in an in vivo rabbit model.

Corneal neovascularization, which occurs in many pathologic states of the cornea, reduces the visual acuity. Recently, we found that the extracellular region of brain-specific angiogenesis inhibitor 1 (BAI1-ECR) has antiproliferative activity through functional blocking of alpha(v)beta(5) integrin in endothelial cells. In this study, we investigated the effects of lipid-mediated subconjunctival injection of the BAI1-ECR gene on corneal angiogenesis induced by epithelial debridement by heptanol in the rabbit. When a pEGFP-BAI1-ECR plasmid was given subconjunctivally 1 week after epithelial debridement, green fluorescence was detected in the corneal stroma with expression persisting for 7 days. To test the effect of BAI1-ECR on neovascularization, rabbits were injected with the BAI1-ECR gene or empty vector two or three times at 1-week intervals beginning 1 week after debridement. When measured with biomicroscopy at 1 or 2 weeks after two weekly injections, BAI1-delivered eyes had significantly less neovascularized corneal area than vector-injected ones in both time periods. Similar microscopic results were obtained after three weekly injections of BAI1-ECR. In quantitative histological examination, the BAI1-receiving eyes showed significantly less neovascular area and number of vessels than vector-injected ones. Also, after two weekly injections, BAI1-delivered eyes had decreased neovascularized corneal area equivalent to that of anti-VEGF antibody-injected ones. These results indicate that BAI1-ECR gene delivery effectively reduces experimental corneal neovascularization and suggest that the BAI1-ECR protein can be used as an angiogenesis suppressor in the eye.

Enamel matrix derivative is a potent inhibitor of breast cancer cell attachment to bone.

This study examined whether enamel matrix derivative (EMD) inhibits the adhesion of cancer cells to bone. A typical breast cancer cell line, MCF-7, was used. Conditioned human osteosarcoma cell (Saos-2) medium was used as extracellular bone matrix (ECBM) to measure cell attachment. MCF-7 cells were incubated on ECBM-coated culture plates with or without soluble EMD, Arg-Gly-Asp (RGD) sequence blocking peptides, recombinant bone sialoprotein (rBSP), or specific integrin antibodies, and the attached cells were quantified using toluidine blue staining. EMD markedly reduced the attachment of MCF-7 cells to ECBM in a dose-dependent manner. An RGD peptide (GRGDSP) and recombinant BSP inhibited cell attachment to the same degree as EMD. Similarly, anti-alphavbeta3 integrin antibody strongly reduced cell attachment, whereas anti-alphavbeta5 and anti-beta1 integrin antibodies had less marked effects on cell attachment. These results show that EMD inhibits MCF-7 cell attachment to a bone matrix and that it might be useful as an anti-adhesive agent for breast cancer cells to bone in vivo.

Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins and promotes cell motility.

Periostin (PN) is a secreted protein that shares a structural homology to the axon guidance protein fasciclin I in insects. Previously, we reported that PN expression is up-regulated in epithelial ovarian tumors. We further examined the role of PN in ovarian cancer. PN is expressed in several normal tissues but not in normal ovaries and has a tendency for higher expression in fetal tissues. Ovarian cancer cells secrete PN, which can accumulate in malignant ascites of ovarian cancer patients. Purified recombinant PN supports adhesion of ovarian epithelial cells that can be inhibited by monoclonal antibodies against alpha(V)beta(3) or alpha(V)beta(5) integrin, but not by anti-beta(1) integrin antibody. Furthermore, alpha(V)beta(3) integrin, but not beta(1) integrins, colocalizes to the focal adhesion plaques formed on PN. Cells plated on PN form fewer stress fibers and are more motile compared with those plated on fibronectin. We propose PN functions as a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins to support adhesion and migration of ovarian epithelial cells.

Acute vascular rejection is associated with up-regulation of vitronectin receptor (alphavbeta3), increased expression of tissue factor, and activation of the extracellular matrix metalloproteinase induction system.

BACKGROUND: A cascade of inflammatory reactions characterize acute vascular rejection after heart transplantation. This study was undertaken to test the hypothesis that acute vascular rejection is associated with up-regulation of vitronectin receptor (alphavbeta3), increased expression of tissue factor, and activation of the extracellular matrix metalloproteinase induction system. METHODS: Acute vascular rejection developed in 14 heart transplant recipients within 2 weeks of transplantation, confirmed by immunofluorescence (AVR group). We compared these patients with 10 transplant recipients who had no evidence of acute vascular rejection or peritransplant ischemic injury (control group). We evaluated endomyocardial biopsy specimens for alphavbeta3, tissue factor, and extracellular matrix metalloproteinase inducer (EMMPRIN). RESULTS: Compared with the control group, the AVR group demonstrated evidence of significantly increased expression of alphavbeta3 (1.9-fold, p < 0.001), tissue factor (1.8-fold, p < 0.001), and EMMPRIN (1.5-fold, p < 0.001). All patients in the AVR group received plasmapheresis; 11 of 14 patients had evidence of ischemic necrosis on biopsy specimens, and 3 of 14 patients experienced hemodynamic compromise and graft dysfunction and died within 3 weeks of transplant. Another patient died at 10 months after transplant. CONCLUSIONS: Acute vascular rejection is associated with up-regulation of alphavbeta3, tissue factor, and activation of the matrix metalloproteinase induction system, which may contribute to the lethal morbidity associated with this disease.