Soluble CD40L activates soluble and cell-surface integrin αvß3, α5ß1, and α4ß1 by binding to the allosteric ligand-binding site (site 2).

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.

Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS.

Angiopoietin-2 (Ang2), a ligand of the endothelial Tie2 tyrosine kinase, is involved in vascular inflammation and leakage in critically ill patients. However, the role of Ang2 in demyelinating central nervous system (CNS) autoimmune diseases is unknown. Here, we report that Ang2 is critically involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis. Ang2 expression was induced in CNS autoimmunity, and transgenic mice overexpressing Ang2 specifically in endothelial cells (ECs) developed a significantly more severe EAE. In contrast, treatment with Ang2-blocking Abs ameliorated neuroinflammation and decreased spinal cord demyelination and leukocyte infiltration into the CNS. Similarly, Ang2-binding and Tie2-activating Ab attenuated the development of CNS autoimmune disease. Ang2 blockade inhibited expression of EC adhesion molecules, improved blood-brain barrier integrity, and decreased expression of genes involved in antigen presentation and proinflammatory responses of microglia and macrophages, which was accompanied by inhibition of α5ß1 integrin activation in microglia. Taken together, our data suggest that Ang2 provides a target for increasing Tie2 activation in ECs and inhibiting proinflammatory polarization of CNS myeloid cells via α5ß1 integrin in neuroinflammation. Thus, Ang2 targeting may serve as a therapeutic option for the treatment of CNS autoimmune disease.

Integrin activation by the lipid molecule 25-hydroxycholesterol induces a proinflammatory response.

Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5ß1 and αvß3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvß3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.

Phase I study of the anti-α5ß1 monoclonal antibody MINT1526A with or without bevacizumab in patients with advanced solid tumors.

PURPOSE: MINT1526A is a monoclonal antibody that blocks the interaction of integrin alpha 5 beta 1 (α5ß1) with its extracellular matrix ligands. This phase I study evaluated the safety and pharmacokinetics of MINT1526A with or without bevacizumab in patients with advanced solid tumors. METHODS: MINT1526A was administered every 3 weeks (Q3W) as monotherapy (arm 1) or in combination with bevacizumab 15 mg/kg, Q3W (arm 2). Each arm included a 3 + 3 dose-escalation stage and a dose-expansion stage. RESULTS: Twenty-four patients were enrolled in arm 1 (dose range 2-30 mg/kg) and 30 patients were enrolled in arm 2 (dose range 3-15 mg/kg). Monocyte α5ß1 receptor occupancy was saturated at a dose of 15 mg/kg. No dose-limiting toxicities were observed, and the maximum tolerated dose was not reached in either arm. The most common adverse events, regardless of causality, included abdominal pain (25%), diarrhea (25%), nausea (21%), vomiting (21%), and fatigue (21%) in arm 1 and nausea (40%), fatigue (33%), vomiting (30%), dehydration (30%), headache (30%), and hypertension (30%) in arm 2. No grade ≥ 3 bleeding events were observed in either arm. No confirmed partial responses (PR) were observed in arm 1. In arm 2, one patient with thymic carcinoma experienced a confirmed PR and two patients with hepatocellular carcinoma (HCC) experienced durable minor radiographic responses. CONCLUSIONS: MINT1526A, with or without bevacizumab, was well-tolerated. Preliminary evidence of combination efficacy, including in patients with HCC, was observed, but cannot be distinguished from bevacizumab monotherapy in this phase I study.

Frontline Science: Functionally impaired geriatric CAR-T cells rescued by increased α5ß1 integrin expression.

Chimeric antigen receptor expressing T cells (CAR-T) are a promising form of immunotherapy, but the influence of age-related immune changes on CAR-T production remains poorly understood. We showed that CAR-T cells from geriatric donors (gCAR-T) are functionally impaired relative to CAR-T from younger donors (yCAR-T). Higher transduction efficiencies and improved cell expansion were observed in yCAR-T cells compared with gCAR-T. yCAR-T demonstrated significantly increased levels of proliferation and signaling activation of phosphorylated (p)Erk, pAkt, pStat3, and pStat5. Furthermore, yCAR-T contained higher proportions of CD4 and CD8 effector memory (EM) cells, which are known to have enhanced cytolytic capabilities. Accordingly, yCAR-T demonstrated higher levels of tumor antigen-specific cytotoxicity compared with gCAR-T. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin and granzyme B. yCAR-T had increased α5ß1 integrin expression, a known mediator of retroviral transduction. We found that treatment with M-CSF or TGF-ß1 rescued the impaired transduction efficiency of the gCAR-T by increasing the α5ß1 integrin expression. Neutralization of α5ß1 confirmed that this integrin was indispensable for CAR expression. Our study suggests that the increase of α5ß1 integrin expression levels enhances CAR expression and thereby improves tumor killing by gCAR-T.

An Integrin-Targeting RGDK-Tagged Nanocarrier: Anticancer Efficacy of Loaded Curcumin.

Herein we report the design and development of α5 ß1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against αv ß3 , αv ß5 , and α5 ß1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α5 ß1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors.

Blocking α5ß1 Integrin Attenuates sCD40L-Mediated Platelet Activation.

The soluble form of CD40L (sCD40L) is a platelet-derived mediator that links inflammation, hemostasis, and vascular dysfunction. Indeed, blockade of CD40L by neutralizing antibodies or genetic disruption in mice prevents atherosclerosis and atherothrombosis. Until recently, it was believed that CD40 and αIIbß3 were the only receptors on platelets responsible for binding sCD40L, leading to platelet activation and initiation of thrombotic events. Recent findings showed α5ß1 integrin as a novel platelet sCD40L receptor, with an unknown function. For the first time, using anti-α5ß1 blocking antibodies, we show that sCD40L/α5ß1 interaction leads to platelet activation as evaluated in the human whole blood. Establishing α5ß1 integrin's role in platelet activation, and therefore thrombosis will help further shed light on the etiology of thrombotic disease.

Cerebral ischemia-induced angiogenesis is dependent on tumor necrosis factor receptor 1-mediated upregulation of α5ß1 and αVß3 integrins.

BACKGROUND: The pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), is expressed in ischemic tissue and is known to modulate angiogenesis; however, the role of the two distinct TNF-α receptors, TNFR1 and TNFR2, in mediating angiogenic signaling after cerebral ischemic stroke is relatively unknown. METHODS: C57BL6 mice were subject to 90 min of ischemia by temporary occlusion of the middle cerebral artery (MCAO) and given daily intra-cerebroventricular injections of antibodies against TNFR1, TNFR2 or control IgG (doses of 10, 50, and 100 ng/day) for 4 days following 90 min MCAO. Vascular remodeling and α5ß1 and αVß3 integrin expression were then examined in the brains of these mice after 4, 7, and 14 days post-ischemia. In parallel in vitro studies, flow cytometry was used to determine the influence of TNF-α on proliferation and integrin expression of human brain microvascular endothelial cells (HBMECs). RESULTS: The post-ischemic cerebral angiogenic response was inhibited by antibodies against TNFR1 but not TNFR2, and this correlated with reduced endothelial proliferation and decreased α5ß1 and αVß3 integrin expression after 4 and 7 days post-ischemia. Consistent with these findings, in vitro studies showed that TNF-α induced endothelial proliferation and upregulation of α5ß1 and αVß3 integrins was abrogated by anti-TNFR1 but not anti-TNFR2 antibodies in cultured HBMECs. In addition, blocking antibodies to α5ß1 and αVß3 integrins significantly inhibited TNF-α-induced HBMEC proliferation. CONCLUSIONS: Our results suggest that TNFR1-mediated signaling plays a critical role in triggering angiogenic integrins and subsequent angiogenic responses following cerebral ischemia. These novel findings could form a platform for future therapeutic strategies aimed at stimulating angiogenesis following cerebral ischemia.

Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN α5ß1-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION.

We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5ß1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-ß1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-ß1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5ß1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking.

Angiopoietin-like protein 2 promotes inflammatory conditions in the ligamentum flavum in the pathogenesis of lumbar spinal canal stenosis by activating interleukin-6 expression.

PURPOSE: Chronic inflammation is thought to cause ligamentum flavum (LF) degeneration and hypertrophy in lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is highly expressed in hypertrophied LF. Because Angptl2 regulates interleukin-6 (IL-6) expression in various tissues, we investigated whether IL-6 is expressed in hypertrophied LF and, if so, does Angptl2 induce IL-6 expression in LF fibroblasts. METHODS: LF tissue was obtained from LSCS patients and non-LSCS patients. Polymerase chain reaction (PCR) for Angptl2 and IL-6 genes and immunohistochemistry for IL-6 protein were performed in LF tissue. Fibroblasts from LF tissue were used for in vitro experiments. Expression of integrin α5ß1 (an Angptl2 receptor) and Angptl2 binding to receptors on LF fibroblasts were examined by fluorescence-activated cell sorter analysis and cell adhesion assays. After Angptl2 recombinant protein treatment, NF-κB activation and IL-6 expression in LF fibroblasts were investigated by immunocytochemistry, PCR, and enzyme-linked immunosorbent assay. RESULTS: IL-6 mRNA expression was increased in hypertrophied LF tissue from LSCS patients and positively correlated with LF thickness and Angptl2 mRNA expression. IL-6 protein was highly expressed in LF fibroblasts in hypertrophied LF tissue. In vitro experiments demonstrated integrin α5ß1 expression on LF fibroblasts and Angptl2 binding to cells via receptors. Angptl2 stimulation promoted NF-κB nuclear translocation and induced IL-6 expression and secretion in LF fibroblasts. CONCLUSIONS: Angptl2 promotes inflammation in LF tissue by activating IL-6 expression, leading to LF degeneration and hypertrophy.

Interaction of CD154 with different receptors and its role in bidirectional signals.

In addition to its classical receptor, CD40, it is now well established that CD154 also binds αIIbß3, α5ß1, and αMß2 integrins. Although these integrins are all members of the same family, they bind CD154 differently. The current investigation aims to analyze the interaction of CD154 with α5ß1 and αMß2 and investigate its role in bidirectional signals in various human cell lines. Results obtained herein indicate that the CD154 residues involved in the interaction with α5ß1 are N151 and Q166, whereas those involved in αMß2 binding are common to residues required for CD40, namely Y145 and R203. Soluble CD40/CD154 or αMß2/CD154 complexes do not interfere with the binding of CD154 to α5ß1-positive cells, but inhibit the binding of CD154 to CD40- or αMß2-positive cells, respectively. Ligation of CD154 on CD154-positive cells with soluble CD40, αIIbß3, α5ß1, or αMß2 stimulates intracellular signaling, including MAPK phosphorylation. Given that CD154 exists as a trimer, our data strongly suggest that CD154 may bind concomitantly to two receptors of the same or different family, and biologically activate cells expressing both receptors. The characterization of CD154/receptor interactions helps the identification of new therapeutic targets for the prevention and/or treatment of CD154-associated autoimmune and inflammatory diseases.

A first-in-human study of the anti-α5ß1 integrin monoclonal antibody PF-04605412 administered intravenously to patients with advanced solid tumors.

PURPOSE: A first-in-human clinical trial of a fully human, Fc-engineered IgG1 monoclonal antibody targeting integrin α5ß1 was conducted to evaluate tolerability, maximum tolerated dose, pharmacokinetics, pharmacodynamics and preliminary anti-tumor activity. METHODS: Escalating doses of PF-04605412 were given IV on day 1, 28 and every 2 weeks thereafter to patients with advanced solid tumors until disease progression or unacceptable toxicity. Sequential dose cohorts were evaluated based on a modified 3 + 3 dose-escalation design. The starting dose was 7.5 mg based on preclinical data. RESULTS: Thirty-three patients were enrolled to six dose levels (7.5, 11.25, 16.9, 34, 68 and 136 mg). Twenty-three patients were evaluable for the primary endpoint (determination of the maximum tolerated dose). Five patients required permanent drug discontinuation due to acute infusion-related reactions, which occurred as grade 3 events in two patients. PK analysis indicated that the targeted drug exposure based on preclinical models was not achieved by the tolerated doses and PK modeling suggesting that doses at least fivefold higher would be necessary. No anti-tumor activity was observed. CONCLUSION: Based on the safety data, the risks associated with the likelihood of significant cytokine-mediated infusion reactions at higher doses, the projected high dose necessary to affect on the biological target and the lack of anti-tumor activity at the doses explored, the trial was prematurely terminated without determining a formal maximum tolerated dose. Further clinical development of PF-04605412 has been discontinued.

Stimulation through very late antigen-4 and -5 improves the multifunctionality and memory formation of CD8⁺ T cells.

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T-cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH-296, we demonstrated that stimulation via very late Ag (VLA)-4 and VLA-5 in human and BALB/c mouse CD8(+) T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR-transgenic mouse-derived CD8(+) T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma-derived tumor Ag, we showed that stimulation by CH-296 improved the ability of tumor-specific CD8(+) T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3(+) CD4(+) Treg cells in tumors. These results suggest that stimulation via VLA-4 and VLA-5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.

Microglia and macrophages in malignant gliomas: recent discoveries and implications for promising therapies.

Malignant gliomas are the most common primary brain tumors. Their deadliest manifestation, glioblastoma multiforme (GBM), accounts for 15% of all primary brain tumors and is associated with a median survival of only 15 months even after multimodal therapy. There is substantial presence of microglia and macrophages within and surrounding brain tumors. These immune cells acquire an alternatively activated phenotype with potent tumor-tropic functions that contribute to glioma growth and invasion. In this review, we briefly summarize recent data that has been reported on the interaction of microglia/macrophages with brain tumors and discuss potential application of these findings to the development of future antiglioma therapies.

Phagocytized beads reduce the α5ß1 integrin facilitated invasiveness of cancer cells by regulating cellular stiffness.

Cell invasion through the extracellular matrix (ECM) of connective tissue is an important biomechanical process, which plays a prominent role in tumor progression. The malignancy of tumors depends mainly on the capacity of cancer cells to migrate and metastasize. A prerequisite for metastasis is the invasion of cancer cells through connective tissue to targeted organs. Cellular stiffness and cytoskeletal remodeling dynamics have been proposed to affect the invasiveness of cancer cells. Here, this study investigated whether highly invasive cancer cells are capable of invading into dense 3D-ECMs with an average pore-size of 1.3 or 3.0 µm when phagocytized beads (2.7 and 4.5 µm diameter) increased their cellular stiffness and reduced their cytoskeletal remodeling dynamics compared to weakly invasive cancer cells. The phagocytized beads decreased the invasiveness of the α5ß1(high) cancer cells into 3D-ECMs, whereas the invasiveness of the α5ß1(low) cancer cells was not affected. The effect of phagocytized beads on the highly invasive α5ß1(high) cells was abolished by specific knock-down of the α5 integrin subunit or addition of an anti-α5 integrin blocking antibody. Furthermore, the reduction of contractile forces using MLCK and ROCK inhibitors abolished the effect of phagocytized beads on the invasiveness of α5ß1(high) cells. In addition, the cellular stiffness of α5ß1(high) cells was increased after bead phagocytosis, whereas the bead phagocytosis did not alter the stiffness of α5ß1(low) cells. Taken together, the α5ß1 integrin dependent invasiveness was reduced after bead phagocytosis by altered biomechanical properties, suggesting that the α5ß1(high) cells need an appropriate intermediate cellular stiffness to overcome the steric hindrance of 3D-ECMs, whereas the α5ß1(low) cells were not affected by phagocytized beads.

Phase Ib safety and pharmacokinetic study of volociximab, an anti-α5ß1 integrin antibody, in combination with carboplatin and paclitaxel in advanced non-small-cell lung cancer.

BACKGROUND: This phase Ib study evaluated volociximab, an anti-α5ß1 integrin antibody, in combination with carboplatin (Eli Lilly and Co., Indianapolis, IN) and paclitaxel (Taxol) in advanced, untreated non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Three cohorts were treated with volociximab (10, 20, or 30 mg/kg) for up to six 3-week cycles in combination with carboplatin-paclitaxel chemotherapy and continued as maintenance therapy for patients with stable disease (SD) or better. Dose-limiting toxic effects, adverse events (AEs), pharmacokinetics, and anti-volociximab antibodies were assessed. RESULTS: A maximum tolerated dose was not reached up to the maximum planned dose of 30 mg/kg. In 29 patients who received volociximab, the most common grade≥3 AEs were neutropenia (24%), hyponatremia (17%), and fatigue (10%). Three patients experienced volociximab-related serious AEs. No hemorrhages were observed. Of 33 patients enrolled, 8 (24%) achieved a partial response and 17 (52%) had SD. The median progression-free survival was 6.3 months (95% confidence interval 5.5-8.1). Levels of potential biomarkers of angiogenesis or metastasis were reduced following six cycles of treatment. CONCLUSIONS: Volociximab combined with carboplatin and paclitaxel was generally well-tolerated and showed preliminary evidence of efficacy in advanced NSCLC.

Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.

Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains.

Periostin activates integrin α5ß1 through a PI3K/AKT­dependent pathway in invasion of cholangiocarcinoma.

Periostin (PN) is mainly produced from stromal fibroblasts in cholangiocarcinoma (CCA) and shows strong impact in cancer promotion. This work aimed to investigate the mechanism that PN uses to drive CCA invasion. It was found that ITGα5ß1 and α6ß4 showed high expression in non-tumorigenic biliary epithelial cells and in almost all CCA cell lines. PN had preferential binding to CCA cells via ITGα5ß1 and blocking this receptor by either neutralizing antibody or siITGα5 could attenuate PN-induced invasion. After PN-ITGα5ß1 binding, intracellular pAKT was upregulated whereas there was no change in pERK. Moreover, PN could not activate AKT in condition of treatment with a PI3K inhibitor. These data provide evidence that PN-activated invasion of CCA cells is through the ITGα5ß1/PI3K/AKT pathway. Strategies aimed to inhibit this pathway may, thus, provide therapeutic benefits.

Antibody-mediated inhibition of integrin α5ß1 blocks neurotoxic prion peptide PrP106-126-induced activation of BV2 microglia.

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The identification of cell surface molecules that mediate the prion protein (PrP) synthetic peptide interaction with microglia is of great significance as it represents potential target molecules to modulate the events leading to the pathophysiology of prion diseases. Here, we carried out in vitro experiments to investigate the involvement of α5ß1 integrin in neurotoxic prion peptide PrP(106-126)-induced activation of BV2 microglia. The results showed that the exposure to PrP(106-126) upregulated the mRNA expression of proinflammatory factors (IL-1 ß, IL-6, and iNOS) and NALP3 inflammasome components (NALP3 and ASC), increased the release of iNOS and its product nitric oxide, and stimulated NF-κB activation. Blockade of α5ß1 integrin with monoclonal antibody BMC5 prior to PrP(106-126) treatment abrogated the upregulation of the mRNA expression of IL-1 ß, IL-6, iNOS, and ASC, but had no effect on the mRNA expression of NALP3, blocked the release of iNOS and nitric oxide, and inhibited NF-κB activation. These results suggest that α5ß1 integrin is involved in the PrP(106-126)-induced microglial activation through the participation in the activation of NF-κB and NALP3/ASC inflammasome. Our study unveils a previously unidentified role of α5ß1 integrin as an intermediate signaling molecule in neurotoxic prion peptides-microglia interactions and identifies a potential molecular target for the modulation of prion-induced microglial activation.

Effect of ischemia duration on autoantibody response in rats undergoing retinal ischemia-reperfusion.

Both the innate and the adaptive immune systems are involved in the pathogenic processes following ischemia-reperfusion injury. We analyzed the possible correlation between the duration of ischemia and autoantibody diversification in a model of ocular ischemia. Rats were subjected to 30, 45, or 90 min of ischemia, and retinal ganglion cell (RGC) density and antibody reactivity were analyzed via customized protein microarray slides. After ocular ischemia, significant alterations in antibody response were observed, while increasing exposure caused more severe RGC damage. Distinct antibody responses after ischemia were detected; these alterations comprised decreased reactivities against cyclophilin A and glyceraldehyde-3-phosphate dehydrogenase, possibly due to increased binding of circulating antibodies to debris material. Other antibodies, like those against α(5)ß(1)-integrin or ß(2) -adrenergic receptor, were upregulated after ischemia.