VLA-4 Research Articles

476 The selective alpha5beta1 integrin antagonist, CLT-28643, inhibits tumors angiogenesis and growth

476 The selective alpha5beta1 integrin antagonist, CLT-28643, inhibits tumors angiogenesis and growth

Eriodictyol decreases very late antigen-4 (VLA-4) expression, cellular adhesion, and migration through an NFκB-dependent pathway in monocytes

Very late antigen-4 (VLA-4)-mediated monocyte adhesion and transendothelial migration are important events during immune surveillance and in the pathogenesis of inflammatory diseases, such as atherosclerosis. Under physiological conditions, circulating monocytes that are in various states of maturation enter tissue microenvironments and participate in immune responses by interacting with other leukocytes. The effects and regulatory mechanisms of eriodictyol on VLA-4 expression, cell adhesion, and migration in U937 cells were investigated. Eriodictyol lowered VLA-4 expression in a dose- and time-dependent manner in U937 cells. Moreover, VLA-4- and vascular adhesion molecular-1 (VCAM-1)-mediated adhesion and migration were decreased by eriodictyol. Upon treatment with eriodictyol, NF-κB translocated to the nucleus; the NF-κB inhibitor JSH-23 inhibited this translocation and prevented the decrease of VLA-4 expression by eriodictyol. Thus, eriodictyol regulates immune responses by modulating VLA-4 expression, cellular migration, and VLA-4-mediated adhesion in monocytes – evidence that eriodictyol regulates adhesion molecules during immune responses.

The PhScN Peptide as a Potent Agent for Targeting Both Invasion and Survival and for Increasing Radiosensitivity in Human Prostate Cancer

Purpose/Objectives(s): Metastatic invasion is caused by constitutive activation of the alpha5 beta1 integrin fibronectin receptor (FnR), and is blocked by the PHSCN peptide (Ac-PHSCN-NH2) in both preclinical models and in Phase 1 clinical trial. An endoproteinase-resistant derivative of Ac-PHSCN-NH2 was made by including D-isomers of Histidine and Cysteine to form the PhScN peptide: Ac-PhScN-NH2. Ac-PhScN-NH2 is a highly potent inhibitor of alpha5 beta1 integrin-mediated invasion and survival of human prostate cancer cells, both in vitro and in vivo.

Physical Characterization of TRK-720 Hydrate, the Very Late Antigen-4 (VLA-4) Inhibitor, as a Solid Form for Inhalation: Preparation of the Hydrate by Solvent Exchange among Its Solvates and Mechanistical Considerations

TRK-720 has been under development as a dry powder inhaler (DPI) for treating bronchial asthma. DPI is a drug formulation of an active pharmaceutical ingredient (API) supported by carrier particles. On inhalation, the API particles dissociate from the carriers through physical stimulation and reach the lung through the humid upper airways, therefore, the API has to remain physically and chemically stable despite contact with carriers and low relative humidity. Also it should be pulverized into an appropriate size (1-10 microm) for reaching the target region. To fulfill these requirements, API in crystal form is needed. Polymorphic screening using 42 solvents produced 9 solvates but not the target hydrate or ansolvate crystal. When the solvent was removed from each solvate, only methanolate could reproducibly be converted into hydrate by water vapor substitution. As the hydrate satisfied the above requirements for DPI, it was selected as a solid form for development. Also, the results of single crystal X-ray structural analyses and calculations of crystal packing energies, for five of the solvates, indicated that only the methanolate can be converted into the hydrate through solvent substitution.

Endothelial α3β1-Integrin Represses Pathological Angiogenesis and Sustains Endothelial-VEGF

Integrin alpha3beta1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of alpha3beta1 can affect angiogenesis either positively or negatively, either a direct in vivo role for alpha3beta1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of alpha3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where alpha3 is deleted specifically in endothelial cells (ec-alpha3-/-). Here we show that ec-alpha3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that alpha3beta1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial alpha3beta1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.

FUNCTIONAL COUPLING OF VALVULAR INTERSTITIAL CELLS AND COLLAGEN VIA α2β1 INTEGRINS IN THE MITRAL LEAFLET.

Once considered passive flaps, we now understand that mitral leaflets are dynamic structures with their own vasculature and innervation that actively remodel and even generate force in response to their environments. Valvular interstitial cells (VICs) are contractile and could underlie mitral leaflet force generation, but the exact mechanisms for VICs in mitral leaflet force generation are not understood. This study tested the hypothesis that actin-mediated VIC force generation coupled to collagen via alpha2beta1 integrins is necessary for force generation in the mitral leaflet. High magnification fluorescent imaging of freshly excised porcine mitral leaflets revealed VIC cytoplasm tightly conforming to collagen fibers, with actin within VIC cytoplasmic processes appearing to attach to the collagen fibers. Functional studies of isometric force development demonstrated that while control samples developed force in response to KCl, either blocking alpha2beta1 integrins or blocking actin polymerization via cytochalasin abolished KCl-induced force development (p<0.001). These results strongly suggest that VIC-collagen coupling, mediated by alpha2beta1 integrins, is necessary for KCl-induced force generation in the mitral leaflet. This functional coupling between collagen and VICs via alpha2beta1 integrins may play a role for in vivo mitral valve function.

Novel poly(ethylene‐co‐acrylic acid) nanofibrous biomaterials for peptide synthesis and biomedical applications

Poly(ethylene-co-acrylic acid) (PE-co-AA) fibers in sizes of 200-500 nm were prepared by using a novel melt-extrusion-extraction fabrication process. The thermoplastic nanofibers could be controllably dispersed and reassembled by a novel solvent exchange filtration method. The dispersed PE-co-AA nanofibers possess active surface areas and could directly conduct chemical reactions on surfaces. Surface modifications and organic synthesis on the nanofibers were proven effective and controllable after the dispersion. Multistep synthesis of biomolecules, such as peptide ligand HWRGWV against Fc portion of human IgG, was successful. The surface-anchored ligand has shown bioactivity through selective binding to and staining by human IgG-alkaline phosphatase conjugate. Another peptide, LXY3, a selective cyclic peptide ligand against alpha3beta1 integrin of MDA-MB-231 breast cancer cells, was also prepared on the surfaces of the dispersed nanofibers. The results showed that MDA-MB-231 cells were able to specifically bind to and grow on surfaces of the nanofibers that were functionalized with LXY3.

Mesenchymal Stem Cells, Used As Bait, Disclose Tissue Binding Sites: A Tool in the Search for the Niche?

We developed an ex vivo approach characterizing renal mesenchymal stem cell (MSC) adhesion to kidney sections. Specificity of MSC adhesion was confirmed by demonstrating a) 3T3 cells displayed 10-fold lower adhesion, and b) MSC adhesion was CXCR4/stromal-derived factor-1 (SDF-1)-dependent. MSC adhesion was asymmetrical, with postischemic sections exhibiting more than twofold higher adhesion than controls, and showed preference to perivascular areas. Pretreating kidney sections with cyclic arginine-glycine-aspartic acid peptide resulted in increased MSC adhesion (by displacing resident cells), whereas blockade of CXCR4 with AMD3100 and inhibition of alpha4beta1(VLA4) integrin or vascular cellular adhesion molecule-1, reduced adhesion. The difference between adhered cells under cyclic arginine-glycine-aspartic acid peptide-treated and control conditions reflected prior occupancy of binding sites with endogenous cells. The AMD3100-inhibitable fraction of adhesion reflected CXCR4-dependent adhesion, whereas maximal adhesion was interpreted as kidney MSC-lodging capacity. MSC obtained from mice overexpressing caveolin-1 exhibited more robust adhesion than those obtained from knockout animals, consistent with CXCR4 dimerization in caveolae. These data demonstrate a) CXCR4/SDF-1-dependent adhesion increases in ischemia; b) CXCR4/SDF-1 activation is dependent on MSC surface caveolin-1; and c) occupancy of MSC binding sites is decreased, while d) capacity of MSC binding sites is expanded in postischemic kidneys. In conclusion, we developed a cell-bait strategy to unmask renal stem cell binding sites, which may potentially shed light on the MSC niche(s) and its characteristics.

Myosin IIb Regulates Actin Dynamics during Synaptic Plasticity and Memory Formation

Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.

Divalent Cations Modulate α2β1 Integrin-Mediated Malignancy in a Novel 3-Dimensional In Vitro Model of Pancreatic Cancer

We previously showed that divalent cations regulate alpha2beta1 integrin-mediated pancreatic cancer cell interactions with type I collagen in 2 dimensions (2D), including cell adhesion, migration, and proliferation. Presently, we examined divalent cation-dependent alpha2beta1 integrin-mediated pancreatic cancer cell adhesion and proliferation on type I collagen in a novel 3D in vitro model.Cell attachment, proliferation, and antibody inhibition assays on type I collagen in both 2D and 3D, and microscopy and immunoblotting were used for these studies.As in 2D, cell attachment on type I collagen in 3D is Mg-dependent and inhibited by Ca. Proliferation in 3D is also Mg-dependent, but maximal when Mg is present at concentrations that promote maximal cell adhesion and Ca is present at concentrations less than Mg. Immunoblotting studies demonstrate that the divalent cation-dependent changes in cell-cell adhesion observed on type I collagen in both 2D and 3D are associated with the changes in E-cadherin and beta-catenin expression. Antibody inhibition assays indicate further that the alpha2beta1 integrin specifically mediates proliferation on type I collagen in 3D under altered divalent cation conditions.Divalent cation shifts could activate alpha2beta1 integrin-mediated malignancy in the type I collagen-rich 3D tumor microenvironment of pancreatic cancer.

Alpha2beta1 integrin is the major collagen‐binding integrin expressed on human Th17 cells

Growing evidence indicates that collagen-binding integrins are important costimulatory molecules of effector T cells. In this study, we demonstrate that the major collagen-binding integrin expressed by human Th17 cells is alpha2beta1 (α2β1) or VLA-2, also known as the receptor for collagen I on T cells. Our results show that human naïve CD4(+) T cells cultured under Th17 polarization conditions preferentially upregulate α2β1 integrin rather than α1β1 integrin, which is the receptor for collagen IV on T cells. Double staining analysis for integrin receptors and intracellular IL-17 showed that α2 integrin but not α1 integrin is associated with Th17 cells. Cell adhesion experiments demonstrated that Th17 cells attach to collagen I and collagen II using α2β1 integrin but did not attach to collagen IV. Functional studies revealed that collagens I and II but not collagen IV costimulate the production of IL-17A, IL-17F and IFN-γ by human Th17 cells activated with anti-CD3. These results identify α2β1 integrin as the major collagen receptor expressed on human Th17 cells and suggest that it can be an important costimulatory molecule of Th17 cell responses.

α5β1 Integrin-Fibronectin Interactions Specify Liquid to Solid Phase Transition of 3D Cellular Aggregates

BackgroundTissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM) connections, regulated by integrins. Integrin α5β1 and soluble fibronectin (sFN) are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin α5β1 and sFN and its influence on tissue mechanical properties and cell sorting behavior.Methodology/Principal FindingsWe generated a series of cell lines varying in α5β1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin α5β1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as α5β1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high α5β1 levels. We also show that differential expression of α5β1 integrin can promote phase-separation between cells.Conclusions/SignificanceThe interplay between α5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a tunable level of control between integrins and the ECM that can influence tissue cohesion and other mechanical properties, which may translate to the specification of tissue structure and function. These studies provide insights into important biological processes such as embryonic development, wound healing, and for tissue engineering applications.

MMP-9, uPAR and Cathepsin B Silencing Downregulate Integrins in Human Glioma Xenograft Cells In Vitro and In Vivo in Nude Mice

BackgroundInvolvement of MMP-9, uPAR and cathepsin B in adhesion, migration, invasion, proliferation, metastasis and tumor growth has been well established. In the present study, MMP-9, uPAR and cathepsin B genes were downregulated in glioma xenograft cells using shRNA plasmid constructs and we evaluated the involvement of integrins and changes in their adhesion, migration and invasive potential.Methodology/Principal FindingsMMP-9, uPAR and cathepsin B single shRNA plasmid constructs were used to downregulate these molecules in xenograft cells. We also used MMP-9/uPAR and MMP-9/cathepsin B bicistronic constructs to evaluate the cumulative effects. MMP-9, uPAR and cathepsin B downregulation significantly inhibits xenograft cell adhesion to several extracellular matrix proteins. Treatment with MMP-9, uPAR and cathepsin B shRNA of xenografts led to the downregulation of several alpha and beta integrins. In all the assays, we noticed more prominent effects with the bicistronic plasmid constructs when compared to the single plasmid shRNA constructs. FACS analysis demonstrated the expression of αVβ3, α6β1 and α9β1 integrins in xenograft cells. Treatment with bicistronic constructs reduced αVβ3, α6β1 and α9β1 integrin expressions in xenograft injected nude mice. Migration and invasion were also inhibited by MMP-9, uPAR and cathepsin B shRNA treatments as assessed by spheroid migration, wound healing, and Matrigel invasion assays. As expected, bicistronic constructs further inhibited the adhesion, migration and invasive potential of the xenograft cells as compared to individual treatments.Conclusions/SignificanceDownregulation of MMP-9, uPAR and cathespin B alone and in combination inhibits adhesion, migration and invasive potential of glioma xenografts by downregulating integrins and associated signaling molecules. Considering the existence of integrin inhibitor-resistant cancer cells, our study provides a novel and effective approach to inhibiting integrins by downregulating MMP-9, uPAR and cathepsin B in the treatment of glioma.

The disintegrin contortrostatin in combination with docetaxel is a potent inhibitor of prostate cancer in vitro and in vivo

There are few available treatments for hormone refractory prostate cancer. Through the inhibition of integrins, contortrostatin (CN) effects tumor cell growth directly as well as through the inhibition of angiogenesis. The effect of CN in combination with docetaxel on prostate cancer cell lines in vitro and in vivo is evaluated in the present study. FACS analysis of integrin expression, assessment of CN and docetaxel exposure on viability of plated cancer cells, and scratch test migration analysis were performed on PC-3 prostate cancer cells. CN and docetaxel inhibition of both PC-3 and CWR-22 prostate cancer cell lines were evaluated in a mouse xenograft bone model. Angiogenic activity in tumors were assessed using IHC with antibodies to CD31. Cell culture experiments indicate that the combination of docetaxel and CN inhibits growth in an additive fashion. FACS analysis of PC-3 cells shows expression of alpha5beta1 and alphavbeta5 integrins, but little expression of the alphavbeta3. CN showed complete inhibition of PC-3 migration in cultures grown on matrigel plates. In mice xenograft bone models, CN with docetaxel showed increased inhibition of both PC-3 and CWR-22 derived tumors. Analysis of treated xenograft tumors showed significantly decreased expression of CD31 indicating suppression of angiogenesis.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.

VIP Promotes Resistance in thePseudomonas aeruginosa–Infected Cornea by Modulating Adhesion Molecule Expression

This study tested the hypothesis that the neuropeptide vasoactive intestinal peptide (VIP) regulates adhesion molecule expression, reduces inflammatory cell migration and infiltration into the Pseudomonas aeruginosa-infected cornea of susceptible B6 mice, and promotes corneal healing and resistance. B6 mice received daily intraperitoneal (IP) injections of VIP from -1 through 5 days after infection. Control mice were similarly injected with sterile phosphate-buffered saline (PBS). Transcript levels of adhesion molecules were determined by PCR array, then select molecules were tested individually by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) or immunofluorescent staining with confocal laser scanning microscopy at various time points after infection to assess the effects of VIP treatment in the regulation of adhesion molecule expression. Injection of B6 mice with VIP compared with PBS resulted in significant downregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, platelet-endothelial cell adhesion molecule-1, and P-selectin and L-selectin mRNA expression. Protein levels for ICAM-1 and VCAM-1, detected by ELISA, supported the mRNA data at similar time points. Immunofluorescence staining further confirmed the effects of VIP treatment, showing reduced corneal expression of ICAM-1/leukocyte function-associated antigen (LFA-1) and VCAM-1/very late antigen-4 (VLA-4) at select time points compared with PBS-treated animals. VIP treatment downregulates the production of adhesion molecules integral to the transmigration process of host inflammatory cells (polymorphonuclear neutrophils, macrophages) into the infected cornea. This results directly in reduced cellular infiltration, less stromal destruction, and better disease outcome.

α5β1 integrin antagonists reduce chemotherapy‐induced premature senescence and facilitate apoptosis in human glioblastoma cells

The alpha5beta1 integrin represent a new therapeutic target for glioblastoma, which are malignant brain tumors difficult to cure with conventional therapies. Glioblastoma are known to be highly resistant to chemotherapy. We, therefore, investigated whether blocking alpha5beta1 integrin with specific nonpeptidic antagonists concomitantly with chemotherapy (ellipticine and temozolomide) may impact the response to chemotherapy of human glioblastoma. Here we show that inhibiting alpha5beta1 integrin with 2 selective ligands (SJ749 and K34c) decreases chemotherapy-induced premature senescence and facilitates cell apoptosis in a functional p53 background (U87MG cells). When p53 is mutated and inactive (U373 cells), chemotherapy induces p53-independent cell apoptosis instead of senescence that is not improved by integrin antagonists. Silencing p53 in U87MG cells with siRNA as well as evaluating HCT116 p53+/+ and p53-/- colon carcinoma cell behavior support the hypothesis of an as yet unknown effect of alpha5beta1 integrin antagonists on the control of chemotherapy-induced premature senescence and apoptosis. alpha5beta1 integrin antagonists modulate the p53 signaling induced by chemotherapy. Our results highlight a new role of the alpha5beta1 integrin in the control of glioblastoma aggressiveness and responsiveness to chemotherapy, which may have a crucial impact in the clinical management of patients suffering from brain tumors.

The Tetraspanins CD9 and CD81 Regulate CD9P1-Induced Effects on Cell Migration

CD9P-1 is a cell surface protein with immunoglobulin domains and an unknown function that specifically associates with tetraspanins CD9 and CD81. Overexpression of CD9P-1 in HEK-293 cells induces dramatic changes in cell spreading and migration on various matrices. Experiments using time-lapse videomicroscopy revealed that CD9P-1 expression has led to higher cell motility on collagen I but lower motility on fibronectin through a β1-integrins dependent mechanism. On collagen I, the increase in cell motility induced by CD9P-1 expression was found to involve integrin α2β1 and CD9P-1 was observed to associate with this collagen receptor. The generation of CD9P-1 mutants demonstrated that the transmembrane and the cytoplasmic domains are necessary for inducing effects on cell motility. On the other hand, expression of tetraspanins CD9 or CD81 was shown to reverse the effects of CD9P-1 on cell motility on collagen I or fibronectin with a concomitant association with CD9P-1. Thus, the ratio of expression levels between CD9P-1 and its tetraspanin partners can regulate cell motility.

Precursor of Advanced Glycation End Products Mediates ER-Stress-Induced Caspase-3 Activation of Human Dermal Fibroblasts through NAD(P)H Oxidase 4

BackgroundThe precursor for advanced glycation end products, 3-deoxyglucosone (3DG) is highly upregulated in skin explants of diabetic cutaneous wounds, and has been shown to negatively impact dermal fibroblasts, which are crucial in wound remodeling. 3DG induces apoptosis however; the mechanisms involved in the apoptotic action of 3DG in the pathogenesis of diabetic chronic wounds are poorly understood. Therefore, we sought to delineate novel mechanisms involved with the 3DG-collagen induced apoptosis.Methodology/Principal FindingsUsing human dermal fibroblasts, we demonstrated that 3DG-modified collagen induces oxidative stress and caspase-3 activation. Oxidative stress was found to be dependent on the upregulation of NAD(P)H oxidase 4 (Nox4), a reactive oxygen species (ROS) Nox homologue, triggering endoplasmic reticulum (ER) stress, as assessed by the ER stress-induced apoptosis marker Growth Arrest and DNA Damage-inducible gene 153 (GADD153). We demonstrated that 3DG-collagen activated GADD153 via phosphorylation of p38 mitogen activated protein kinase (MAPK), and this was dependent on upstream ROS. Inhibition of ROS and/or p38 MAPK abrogated 3DG-collagen induced caspase-3 activation. Our investigations also demonstrated that 3DG-collagen-induced caspase-3 activation did not signal through the canonical receptor for advanced glycation end products (RAGE) but through integrin α1β1. To further verify the role of integrins, neutralization of integrins α1β1 prevented 3DG-collagen-induced upregulation of ROS, GADD153, and caspase-3 activation; suggesting that 3DG-collagen signaling to the fibroblast is dependent on integrins α1β1.Conclusions/SignificanceTaken together, these findings demonstrate for the first time that a RAGE independent mechanism is involved in 3DG-collagen-induced apoptosis. Moreover, the ER stress pathway through activation of Nox4 by integrins α1β1 plays a key role in 3DG-collagen-induced caspase-3 activation, which may play an important role in the pathogenesis of diabetic wounds.

511 Caveolin-1, TGFβ/Smad2 and Alpha5 Beta1 integrins connection in human glioblastoma

Mature Endothelial-Monocyte Activating Polypeptide (mEMAP) II functions as a potent antiangiogenic peptide. Although the anti-tumor effect of mEMAP II has been described, little is known regarding its mechanism of action. Observations that mEMAP II induced apoptosis only in a subset of migrating and proliferating endothelial cells (EC) suggests a targeted effect on cells engaged in angiogenic activities which are known to rely upon cell adhesion and migration. Indeed, we demonstrate that mEMAP II inhibited fibronectin (FN) dependent microvascular EC (MEC) adhesion and spreading and we show that this depends upon the alpha5 beta1 integrin. Immunofluorescence analysis demonstrated that mEMAP II-dependent blockade of FN–alpha5 beta1 interactions was associated with disassembly of both actin stress fiber networks and FN matrix. These findings suggest that mEMAP II blocks MEC adhesion and spreading on fibronectin, via a direct interaction with the integrin alpha5 beta1, thus implicating that alpha5 integrin may be a mediator of mEMAP II's antiangiogenic function.