ShlA toxin of Serratia induces P2Y2- and α5ß1-dependent autophagy and bacterial clearance from host cells.

Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.

Integrin αVß1 regulates procollagen I production through a non-canonical transforming growth factor ß signaling pathway in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor ß (TGF-ß), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVß1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-ß1-activated primary human HSCs. Unexpectedly, either a selective αVß1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-ß1-activated procollagen I production in primary human HSCs, in which the role of ß1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-ß1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-ß-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVß1 integrin is involved in non-canonical TGF-ß signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-ß1 induced phosphorylation of ERK1/2 and decreased TGF-ß1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-ß1 induced procollagen I production. Taken together, current data indicate that αVß1 integrin can regulate TGF-ß signaling independent of its reported role in activating latent TGF-ß. Our data further support that αVß1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.

Gefitinib induces EGFR and α5ß1 integrin co-endocytosis in glioblastoma cells.

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

Dual antagonists of α5ß1/αvß1 integrin for airway hyperresponsiveness.

Inhibition of integrin α5ß1 emerges as a novel therapeutic option to block transmission of contractile forces during asthma attack. We designed and synthesized novel inhibitors of integrin α5ß1 by backbone replacement of known αvß1 integrin inhibitors. These integrin α5ß1 inhibitors also retain the nanomolar potency against αvß1 integrin, which shows promise for developing dual integrin α5ß1/αvß1 inhibitor. Introduction of hydrophobic adamantane group significantly boosted the potency as well as selectivity over integrin αvß3. We also demonstrated one of the inhibitors (11) reduced airway hyperresponsiveness in ex vivo mouse tracheal ring assay. Results from this study will help guide further development of integrin α5ß1 inhibitors as potential novel asthma therapeutics.

Adhesion to fibronectin via α5ß1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.

EM2D9, A monoclonal antibody against integrin α5ß1, has potent antitumor activity on endometrial cancer in vitro and in vivo.

Endometrial cancer, a type of primary epithelial malignant tumor in the endometrium, is one of the three most common malignant tumors of the female reproductive system. While the incidence of endometrial cancer has been recently rising, its etiology remains unclear. In this study we found that EM2D9, an independently developed monoclonal antibody, specifically recognized endometrial cancer cells; we further determined that EM2D9 target protein was α5ß1. In vitro and in vivo experiments showed that EM2D9 inhibited the migration of endometrial cancer cells. Real-time quantitative PCR results showed that the expression of CD151 mRNA in endometrial carcinoma cells significantly decreased after EM2D9 treatment. We also found that EM2D9 affected the FAK signaling pathway. Collectively, these results shed light on a new mechanism for the development of endometrial carcinoma.

Cartilage Oligomeric Matrix Protein Negatively Influences Keratinocyte Proliferation via α5ß1-Integrin: Potential Relevance of Altered Cartilage Oligomeric Matrix Protein Expression in Psoriasis.

In psoriasis, nonlesional skin shows alterations at the dermal-epidermal junction compared with healthy skin. Cartilage oligomeric matrix protein (COMP) is part of the papillary dermis of healthy skin, and its expression has not yet been studied in psoriatic skin. In this study, we found that COMP localization extended deeper into the dermis and formed a more continuous layer in psoriatic nonlesional skin compared with healthy skin, whereas in psoriatic lesions, COMP showed a partially discontinuous deposition at the dermal-epidermal junction. COMP and ß1-integrin showed strong colocalization in nonlesional skin, where the laminin layer within the basement membrane is discontinuous. In in vitro models, the presence of exogenous COMP decreased the proliferation rate of keratinocytes, and this proliferation-suppressing effect was diminished by blocking α5ß1-integrin. Our results suggest that COMP can interact with α5ß1-integrin of basal keratinocytes through the disrupted basement membrane, and this interaction might stabilize the epidermis in the nonlesional state by contributing to the suppression of keratinocyte proliferation. The antiproliferative effect of COMP is likely to be relevant to other skin diseases in which chronic nonhealing wounds are coupled with massive COMP accumulation.

Integrin α5ß1 inhibition by ATN-161 reduces neuroinflammation and is neuroprotective in ischemic stroke.

Stroke remains a leading cause of death and disability with limited therapeutic options. Endothelial cell ß1 integrin receptors play a direct role in blood-brain barrier (BBB) dysfunction through regulation of tight junction proteins and infiltrating leukocytes, potentially mediated by ß1 integrins. Following tandem transient common carotid artery/middle cerebral artery occlusion on wild-type mice, we administered the integrin a5b1 inhibitor, ATN-161, intraperitoneal (IP) injection at 1 mg/kg acutely after reperfusion, on post-stroke day (PSD)1 and PSD2. Systemic changes (heart rate, pulse distension, and body temperature) were determined. Additionally, infarct volume and edema were determined by 2,3-triphenyltetrazolium chloride and magnetic resonance imaging, while neurological changes were evaluated using an 11-point Neuroscore. Brain immunohistochemistry was performed for claudin-5, α5ß1, IgG, and CD45 + cells, and quantitative polymerase chain reaction (qPCR) was performed for matrix metalloproteinase-9 (MMP-9), interleukin (IL)-1ß, collagen IV, and CXCL12. ATN-161 significantly reduced integrin α5ß1 expression in the surrounding peri-infarct region with no systemic changes. Infarct volume, edema, and functional deficit were significantly reduced in ATN-161-treated mice. Furthermore, ATN-161 treatment reduced IgG extravasation into the parenchyma through conserved claudin-5, collagen IV, CXCL12 while reducing MMP-9 transcription. Additionally, IL-1ß and CD45 + cells were reduced in the ipsilateral cortex following ATN-161 administration. Collectively, ATN-161 may be a promising novel stroke therapy by reducing post-stroke inflammation and BBB permeability.

Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

Cell proliferation and invasion are regulated differently by EGFR and MRP1 in T-DM1-resistant breast cancer cells.

We recently reported that T-DM1-resistant JIMT1 (T-DM1R-JIMT1) cells exhibited high invasive activity via EGFR and integrin cooperated pathways and gained cross-resistance to doxorubicin. Here, we show that EGFR positively coordinates with MRP1 in T-DM1R-JIMT1 cells to contribute to cross-resistance to doxorubicin. Downregulating EGFR and MRP1 inhibits T-DM1R-JIMT1 cell growth and re-sensitizes T-DM1R cells to doxorubicin, suggesting that dual targeting EGFR and MRP1 could serve as a therapeutic approach to overcome T-DM1 resistance. However, it increases cell invasion activity of T-DM1R-JIMT1 cells with molecular and cellular phenotypes similar to the breast cancer cells that express low levels of HER2 (MDA-MB-231 and BT-549 cells). Importantly, the invasion activity of MDA-MB-231 and BT-549 cells is also significantly increased after chronically exposed to T-DM1 although cell growth of MDA-MB-231 and BT-549 cells is not inhibited by T-DM1. These results highlight the importance of HER2 heterogenicity in HER-positive breast cancers treated with T-DM1. Our study also provides evidence demonstrating that proliferation and invasion activities of T-DM1R-JIMT1, and MDA-MB-231 and BT-549 cells are regulated by different mechanisms and that different aspects of cancer cell behaviors affected by targeted-therapeutics should be fully characterized in order to overcome T-DM1-resistant disease and to prevent cancer metastasis.

Cyclic hydrostatic compress force regulates apoptosis of meniscus fibrochondrocytes via integrin alpha5beta1.

Meniscus is a semilunar fibrocartilaginous tissue, serving important roles in load buffering, stability, lubrication, proprioception, and nutrition of the knee joint. The degeneration and damage of meniscus has been proved to be a risk factor of knee osteoarthritis. Mechanical stimulus is a critical factor of the development, maintenance and repair of the meniscus fibrochondrocytes. However, the mechanism of the mechano-transduction process remains elusive. Here we reported that cyclic hydrostatic compress force (CHCF) treatment promotes proliferation and inhibits apoptosis of the isolated primary meniscus fibrochondrocytes (PMFs), via upregulating the expression level of integrin ?5ß1. Consequently, increased phosphorylated-ERK1/2 and phosphorylated-PI3K, and decreased caspase-3 were detected. These effects of CHCF treatment can be abolished by integrin ?5ß1 inhibitor or specific siRNA transfection. These data indicate that CHCF regulates apoptosis of PMFs via integrin ?5ß1-FAK-PI3K/ERK pathway, which may be an important candidate approach during meniscus degeneration.

The Psoriasis Therapeutic Potential of a Novel Short Laminin Peptide C16.

Psoriasis is a chronic inflammatory skin disease characterized by excessive growth of keratinocytes and hyperkeratosis in the epidermis. An abnormality of the non-lesional epidermis at an early stage of psoriasis is involved in triggering inflammatory cell infiltration into the dermis. Integrin α5ß1 acts as a receptor for fibronectin and has been found to be overexpressed in non-lesional psoriatic epidermis. To investigate whether α5ß1 integrin has a potential as a drug target for psoriasis treatment, the α5ß1 integrin-binding peptide, C16, was used to obstruct the HaCat keratinocyte cellular responses induced by fibronectin (Fn) in culture and psoriasis-like skin inflammation induced in mice by imiquimod (IMQ). The C16 exhibited antagonistic activity against α5ß1 integrin in HaCat cells, with evidence of suppression of the Fn-mediated proliferative, cytoskeletal, and inflammatory responses. Topical treatment with C16 greatly reduced the IMQ-induced epidermal hyperplasia, infiltration of neutrophils/macrophages, and expression of pro-inflammatory mediators in mouse skin. The C16SP (C16-derived short peptide; DITYVRLKF) also exhibited antagonistic activity, suppressing α5ß1 integrin activity in culture, and reducing IMQ-induced skin inflammation. Taken together, this study provides the first evidence that α5ß1 integrin may be a potential drug target for psoriasis. The synthetic C16 peptide may serve as an agent for psoriasis therapy.

ATN-161 reduces virus proliferation in PHEV-infected mice by inhibiting the integrin α5ß1-FAK signaling pathway.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a typical neurotropic virus that can cause obvious nerve damage. Integrin α5ß1 is a transmembrane macromolecular that closely related to neurological function. We recently demonstrated that integrin α5ß1 plays a critical role in PHEV invasion in vitro. To determine the function and mechanism of integrin α5ß1 in virus proliferation in vivo, we established a mouse model of PHEV infection. Integrin α5ß1-FAK signaling pathway was activated in PHEV-infected mice by qPCR, Western blotting, and GST pull-down assays. Viral proliferation and integrin α5ß1-FAK signaling pathway were significantly inhibited after intravenous injection of ATN-161, an integrin α5ß1 inhibitor. Through a histological analysis, we found that ATN-161-treated mice only showed pathological changes in neuronal cytoplasmic swelling at 5 day post-infection. In summary, our results provide the first evidence that ATN-161 inhibits the proliferation of PHEV in mice and explores its underlying mechanisms of action.

A collagen IV-derived peptide disrupts α5ß1 integrin and potentiates Ang2/Tie2 signaling.

The angiopoietin (Ang)/Tie2 signaling pathway is essential for maintaining vascular homeostasis, and its dysregulation is associated with several diseases. Interactions between Tie2 and α5ß1 integrin have emerged as part of this control; however, the mechanism is incompletely understood. AXT107, a collagen IV-derived peptide, has strong antipermeability activity and has enabled the elucidation of this previously undetermined mechanism. Previously, AXT107 was shown to inhibit VEGFR2 and other growth factor signaling via receptor tyrosine kinase association with specific integrins. AXT107 disrupts α5ß1 and stimulates the relocation of Tie2 and α5 to cell junctions. In the presence of Ang2 and AXT107, junctional Tie2 is activated, downstream survival signals are upregulated, F-actin is rearranged to strengthen junctions, and, as a result, endothelial junctional permeability is reduced. These data suggest that α5ß1 sequesters Tie2 in nonjunctional locations in endothelial cell membranes and that AXT107-induced disruption of α5ß1 promotes clustering of Tie2 at junctions and converts Ang2 into a strong agonist, similar to responses observed when Ang1 levels greatly exceed those of Ang2. The potentiation of Tie2 activation by Ang2 even extended to mouse models in which AXT107 induced Tie2 phosphorylation in a model of hypoxia and inhibited vascular leakage in an Ang2-overexpression transgenic model and an LPS-induced inflammation model. Because Ang2 levels are very high in ischemic diseases, such as diabetic macular edema, neovascular age-related macular degeneration, uveitis, and cancer, targeting α5ß1 with AXT107 provides a potentially more effective approach to treat these diseases.

Intrinsic Surface Effects of Tantalum and Titanium on Integrin α5ß1/ ERK1/2 Pathway-Mediated Osteogenic Differentiation in Rat Bone Mesenchymal Stromal Cells.

BACKGROUND/AIMS: Accumulating evidence demonstrates the superior osteoinductivity of tantalum (Ta) to that of titanium (Ti); however, the mechanisms underlying these differences are unclear. Thus, the objective of the present study was to examine the effects of Ta and Ti surfaces on osteogenesis using rat bone mesenchymal stromal cells (rBMSCs) as a model. METHODS: Ta and Ti substrates were polished to a mirror finish to minimize the influences of structural factors, and the intrinsic surface effects of the two materials on the integrin α5ß1/mitogen-activated protein kinases 3 and 1 (ERK1/2) cascade-mediated osteogenesis of rBMSCs were evaluated. Alkaline phosphatase (ALP) activity, Alizarin Red staining, real-time polymerase chain reaction, and western blot assays of critical osteogenic markers were conducted to evaluate the effects of the two substrates on cell osteogenesis. Moreover, the role of the integrin α5ß1/ERK1/2 pathway on the osteoinductive performance of Ta and Ti was assessed by up- and down-regulation of integrin α5 and ß1 with RNA interference, as well as through ERK1/2 inhibition with U0126. RESULTS: Osteogenesis of rBMSCs seeded on the Ta surface was superior to that of cells seeded on the Ti surface in terms of ALP activity, extracellular matrix calcification, and the expression of integrin α5, integrin ß1, ERK1/2, Runt-related transcription factor 2, osteocalcin, collagen type I, and ALP at both the mRNA and protein levels. Moreover, down-regulation of integrin α5 or integrin ß1, or ERK1/2 inhibition severely impaired the osteoblastic differentiation on the Ta surface. By contrast, over-expression of integrin α5 or integrin ß1 improved osteogenesis on the Ti substrates, while subsequent ERK1/2 inhibition abrogated this effect. CONCLUSION: The integrin α5ß1/ERK1/2 pathway plays a crucial role in regulating rBMSCs osteogenic differentiation; thus, the greater ability of a Ta surface to trigger integrin α5ß1/ERK1/2 signaling may explain its better osteoinductivity. The different effects of Ta and Ti surfaces on rBMSC osteogenesis are considered to be related to the conductive behaviors between integrin α5ß1 and the oxides spontaneously formed on the two metals. These results should facilitate the development of engineering strategies with Ta and Ti surfaces for improved osteogenesis in endosteal implants.

IL-1ß promotes transendothelial migration of PBMCs by upregulation of the FN/α5ß1 signalling pathway in immortalised human brain microvascular endothelial cells.

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain. The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the ß1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether ß1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1ß (IL-1ß)-induced inflammatory in vitro BBB model. We demonstrate that IL-1ß increases cell-matrix adhesion and induces a redistribution of active ß1 integrins to the basal surface. In particular, binding of α5ß1 integrin to its ligand fibronectin is enhanced and α5ß1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5ß1 integrin reduces the IL-1ß-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs). These data imply that IL-1ß-induced inflammation not only destabilizes tight junctions but also increases α5ß1 integrin-dependent cell-matrix adhesion to fibronectin.

The Protective Effect of a Long-Acting and Multi-Target HM-3-Fc Fusion Protein in Rheumatoid Arthritis.

Current treatment of rheumatoid arthritis (RA) is limited by relative shortage of treatment targets. HM-3 is a novel anti-RA polypeptide consisting of 18 amino acids with integrin αVß3 and α5ß1 as targets. Previous studies confirmed that HM-3 effectively inhibited the synovial angiogenesis and the inflammatory response. However, due to its short half-life, the anti-RA activity was achieved by frequent administration. To extend the half-life of HM-3, we designed a fusion protein with name HM-3-Fc, by combination of modified Fc segment of immunoglobulin 4 (IgG4) with HM-3 polypeptide. In vitro cell experiments demonstrated that HM-3-Fc inhibited the proliferation of splenic lymphocytes and reduced the release of TNF-α from macrophages. The pharmacodynamics studies on mice paw in Collagen-Induced Arthritis (CIA) model demonstrated that HM-3-Fc administered once in 5 days in the 50 and 25 mg/kg groups, or once in 7 days in the 25 mg/kg group showed a better protective effect within two weeks than the positive control adalimumab and HM-3 group. Preliminary pharmacokinetic studies in cynomolgus confirmed that the in vivo half-life of HM-3-Fc was 15.24 h in comparison with 1.32 min that of HM-3, which demonstrated that an Fc fusion can effectively increase the half-life of HM-3 and make it possible for further reduction of subcutaneous injection frequency. Fc-HM-3 is a long-acting active molecule for RA treatment.

ATN-161 as an Integrin α5ß1 Antagonist Depresses Ocular Neovascularization by Promoting New Vascular Endothelial Cell Apoptosis.

BACKGROUND ATN-161 (Ac-PHSCN-NH2), an antagonist of integrin α5ß1, has shown an important influence in inhibiting tumor angiogenesis and metastasis of other tumor types. However, the mechanism of action of ATN-161 and whether it can inhibit ocular neovascularization (NV) are unclear. This study investigated the role of ATN-161 in regulating ocular angiogenesis in mouse models and explored the underlying signaling pathway. MATERIAL AND METHODS An oxygen-induced retinopathy (OIR) mouse model and a laser-induced choroidal neovascularization (CNV) mouse model were used to test integrin a5b1 expression and the effect of ATN-161 on ocular NV by immunofluorescence staining, Western blot analysis, and flat-mount analysis. The activation of nuclear factor-κB (NF-κB), matrix metalloproteinase-2/9 (MMP-2/9), and cell apoptosis were detected by immunofluorescence staining, Western blot, real-time RT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The cell proliferation was detected by BrdU labeling. RESULTS In OIR and CNV mice, the protein expression level of integrin α5ß1 increased compared with that in age-matched controls. The mice given ATN-161 had significantly reduced retinal neovascularization (RNV) and CNV. Blocking integrin a5b1 by ATN-161 strongly inhibited nuclear factor-κB (NF-κB) activation and matrix metalloproteinase-2/9 (MMP-2/9) expression and promoted cell apoptosis, but the effect of ATN-161 on proliferation in CNV mice was indirect and required the inhibition of neovascularization. Inhibiting NF-κB activation by ammonium pyrrolidinedithiocarbamate (PDTC) reduced RNV and promoted cell apoptosis in ocular NV. CONCLUSIONS Blocking integrin α5ß1 by ATN-161 reduced ocular NV by inhibiting MMP-2/MMP-9 expression and promoting the cell apoptosis of ocular NV.

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.

Simultaneous Targeting of RGD-Integrins and Dual Murine Double Minute Proteins in Glioblastoma Multiforme.

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin α5ß1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein, we present the discovery of compound 7, which inhibits both MDM2/4 and α5ß1/αvß3 integrins. A lead optimization campaign was carried out on 7 with the aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4 and α5ß1/αvß3 blocker. In p53-wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of α5ß1 integrin.