Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.     

Endothelial cell migration on fibronectin is regulated by syntaxin 6-mediated alpha5beta1 integrin recycling

The α5β1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for α5β1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and α5β1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of β1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated α5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of α5β1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.     

The opsonin MFG-E8 is a ligand for the alphavbeta5 integrin and triggers DOCK180-dependent Rac1 activation for the phagocytosis of apoptotic cells

Opsonization of apoptotic cells facilitates recognition by phagocytes for the rapid clearance of potentially inflammatory cellular material. The secreted glycoprotein Milk Fat Globule Factor-E8 (MFG-E8) is a member of this family of bridging molecules and is believed to bind phosphatidylserine (PS) on the dying cell, linking it to integrin receptors on the phagocyte. Here we report the characterization of a functional signaling module involving MFG-E8, alphavbeta5 integrin, and DOCK180 for the activation of Rac1. We show that MFG-E8 and DOCK180 are both expressed in phagocytic-competent primary immature dendritic cells (DCs) and DC2.4 cells, and are potently down-regulated upon DC maturation, consistent with their role in phagocytosis and antigen capture. Coexpression of MFG-E8 with alphavbeta5 integrin potentiated integrin-mediated Rac1 activation, which was abrogated by mutagenesis in the RGD motif in MFG-E8. Moreover, expression of antisense DOCK180 abrogated MFG-E8-alphavbeta5-mediated Rac activation and impaired the phagocytosis of apoptotic cells. These data demonstrate a biochemical link between an opsonin of apoptotic cells, the alphavbeta5 integrin, and the Crk-DOCK180-Rac1 pathway, and importantly, show that MFG-E8 and DOCK180 are expressed according to the functional status of the phagocyte.     

Signalling mechanisms of the leukocyte integrin αMβ2: Current and future perspectives

Integrins are a family of heterodimeric cell adhesion receptors expressed on most cells and are involved in many cellular functions including phagocytosis, a process by which professional phagocytes recognise, bind and internalise foreign materials larger than 0.5 µm in diameter. An example of a phagocytic integrin receptor is αMβ2, and this review seeks to provide fresh insights into the current knowledge of this subject. Key areas that this review will emphasise include, the classical understanding of bi‐directional signalling to and from αMβ2 (aka inside‐out and outside‐in signalling, respectively). For inside‐out signalling, we will review the involvement of the small GTPase, Rap1, FERM‐containing proteins such as talin and kindlin‐3, some of the kinases, and the GEF, cytohesin‐1 and vasodilator‐stimulated phosphoprotein (VASP). We also summarise studies into outside‐in signalling, focussing on the roles of RhoA and RhoG, and activation of Rac1 through the complex comprising TIAM, 14‐3‐3 and β2. We will also consider non‐classical signalling processes, which include integrin clustering and membrane ruffling. Through this review, we hope to highlight the importance of αMβ2 signalling mechanisms and their relevance to other integrin‐mediated events.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.     

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the ‘wrapping’ pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D.     

Cell surface overexpression of alphavbeta5 integrin impedes alphavbeta3-mediated migration of the human ovarian adenocarcinoma cell line IGROV1

Human ovarian surface epithelium and epithelial tumors express integrin alphavbeta5, which can interact with vitronectin. In addition, in vitro acquisition of cisplatin resistance by alphavbeta3-expressing IGROV1 cells is accompanied by cell-surface expression of integrin alphavbeta5. To further explore the role of alphavbeta5 in ovarian carcinoma cells, IGROV1 cells were stably transfected with a human beta5 integrin cDNA construct, and three beta5 transfectant clones were selected for the expression of alphavbeta5 integrin at their cell surface. Despite a delayed entry in the exponential phase of growth, beta5-transfectant cells kept a proliferation ability similar to that of parental cells, while their growth rate was hindered in the presence of an anti-alphavbeta5 blocking antibody. Only simultaneous blockade of alphavbeta3 and alphavbeta5 by specific antibodies impeded the adhesion to vitronectin of beta5 transfectants and of the beta5-expressing cisplatin-resistant variant IGROV1-R10, suggesting that the two heterodimers cooperated in the regulation of this process. Cell surface expression of alphavbeta5 resulted in an attenuation of alphavbeta3-mediated migration on vitronectin. Alphavbeta5 participated to migration events in the absence of exogenous growth factors only in one transfectant clone and in IGROV1-R10 cells. Finally, the response to cisplatin was not significantly modified in beta5 transfectants when compared to IGROV1 parental cells.     

Identification of integrin alpha1 as an interacting protein of protein tyrosine phosphatase PRL-3

PRL-3 is a newly identified protein tyrosine phosphatase associated with tumor metastasis. It is over-expressed in various cancers, such as colorectal cancer, gastric cancer, and ovarian cancer, and is correlated with the progression and survival of cancers. Although PRL-3 plays a causative role in promoting cancer cell invasion and metastasis, the molecular mechanism is unknown. To investigate PRL-3's roles in tumorigenesis and signal transduction pathway, we screened the human placenta brain cDNA library with the bait of PRL-3 in yeast two-hybrid system. Then we identified integrin alpha1 as a PRL-3-interacting protein for the first time, and verified this physical association with pull-down and co-immunoprecipitation assays. Furthermore, we found that PRL-3 could down-regulate the tyrosine-phosphorylation level of integrin beta1 and increased the phosphorylation level of Erk1/2. Our present discovery will provide new clues for elucidating the molecular mechanism of PRL-3 in promoting cancer invasion and metastasis.     

TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis

Gene deletion experiments have shown that the three TGFβ isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins αvβ6 and αvβ8 activate latent forms of TGFβ1 and TGFβ3. This raises the possibility that TGFβ1 and TGFβ3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFβ1 activation (Tgfb1^RGE/RGE) that were also homozygous for a null mutation in the TGFβ3 gene. Tgfb1^RGE/RGE; Tgfb3^−/− mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking αvβ8. Some Tgfb1^RGE/RGE; Tgfb3^+/− and Tgfb1^RGE/RGE; Tgfb3^+/+ mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFβ1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3^−/− mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFβ1 and TGFβ3 during development based on a shared mechanism of activation.     

Matrin 3 as a key regulator of endothelial cell survival

Matrin 3 is an integral component of nuclear matrix architecture that has been implicated in interacting with other nuclear proteins and thus modulating the activity of proximal promoters. In this study, we evaluated the contribution of this protein to proliferation of endothelial cells. To selectively modulate matrin 3 expression, we used siRNA oligonucleotides and transfection of cells with a pEGFP-N1-Mtr3. Our data indicate that downregulation of matrin 3 is responsible for reduced proliferation and leads to necrosis of endothelial cells. This conclusion is supported by observations that reducing matrin 3 expression results in (a) producing signs of necrosis detected by PI staining, LDH release, and scatter parameters in flow cytometry, (b) affecting cell cycle progression. It does not cause (c) membrane asymmetry of cells as indicated by lack of Annexin V binding as well as (d) activation of caspase 3 and cleavage of PARP. We conclude that matrin 3 plays a significant role in controlling cell growth and proliferation, probably via formation of complexes with nuclear proteins that modulate pro- and antiapoptotic signaling pathways. Thus, degradation of matrin 3 may be a switching event that induces a shift from apoptotic to necrotic death of cells.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Tensile strain induces integrin β1 and ILK expression higher and faster in 3D cultured rat skeletal myoblasts than in 2D cultures

According to previous research, integrin β1 and ILK play an important role in the extracellular matrix (ECM)–integrin–cytoskeleton pathway for mechanotransduction. The aim of this study was to investigate strain induced integrin β1 and ILK expression in three-dimensional (3D) and in two-dimensional (2D) cultured rat skeletal myoblasts. Sprague–Dawley (SD) rat skeletal myoblasts were isolated and seeded on the PLGA-collagen composite scaffolds. The 3D cultured and the conventionally 2D monolayer cultured myoblasts were loaded 2000 μstrain tensile strain at 0.5 Hz for 2 h, 4 h, 8 h, 12 h and 24 h, respectively with the self-made four-point bending system. The expressions of integrin β1 and ILK mRNA were measured by RT-PCR and the different changes between the 3D and 2D cultures were compared. The mRNA expression levels of both integrin β1 and ILK were up regulated after mechanical loading (P < 0.05), meanwhile, it was higher and peaked faster in 3D cultures than in the 2D cultures. It can be concluded that the ECM–integrin–cytoskeleton pathway responds to tensile strain by elevated expression of integrin β1 and ILK, and the response is stronger in 3D cultures than in conventional 2D monolayer cultures.     

Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser‐Asp‐Val (SDV) sequence and integrin αvβ3. P11 (HSDVHK) is a novel antagonistic peptide of integrin αvβ3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin‐binding site of integrin αvβ3. The Arg‐Gly‐Asp (RGD)‐binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin–vitronectin. P11 showed a strong antagonism against αvβ3‐GRGDSP interaction with an IC₅₀ value of 25.72±3.34 nM, whereas the value of GRGDSP peptide was 1968.73±444.32 nM. The binding‐free energies calculated from the docking simulations for each P11 and RGD peptide were ?3.99 and ?3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5‐fold lower K_i value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in αvβ3. The analyzed docked poses suggest that a divalent metal–ion coordination was a common driving force for the formation of both SDV/αvβ3 and RGD/αvβ3 complexes. This is the first report on the specific recognition of the RGD‐binding site of αvβ3 by a non‐RGD containing peptide using a computer‐assisted proteomic approach.     

Synthesis and discovery of a novel pyrazole derivative as an inhibitor of apoptosis through modulating integrin beta4, ROS, and p53 levels in vascular endothelial cells

Recently, pyrazole derivatives as high affinity and selective A2A adenosine receptor antagonists have been reported. But, so far, there are no reports about the inhibitory effects of multi-substituted pyrazole derivatives on apoptosis of vascular endothelial cells (VECs). In this study, we synthesized six pyrazole derivatives and characterized the structures of the compounds by IR, ¹H NMR, mass spectroscopy, and element analysis. The biology assay showed that a novel pyrazole derivative, ethyl 3-(o-chlorophenyl)-5-methyl-1-phenyl-1H-pyrazole-4-carboxylate (MPD) at low concentration (25 μM) increased VECs viability and inhibited VECs apoptosis induced by deprivation of serum and FGF-2. During this process, the levels of integrin β4, reactive oxygen species (ROS), and p53 were depressed obviously. The data suggested that MPD was a potential inhibitor of apoptosis associated with the signal pathway mediated by integrin β4, ROS, and p53 in VECs.     

Anti-β1 integrin antibody inhibits schwann cell meylination

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypased by adding a purifed basal lamina component, laminin, to SC/neuron cocultures. We have examined the role of laminin receptors, Namely, the β1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the β1 subunit in association with the α1 or α6 subunit. In co-cultures of myelinating SCs, α1β1 is no longer present, α6β1 is still present but at reduced levels, and α6β4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that β1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of β1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-β1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an α1β1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the β1 subfamily of integrins other than α1β1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell. 1994 John Wiley & Sons, Inc.     

DDX3 acts as a tumor suppressor in colorectal cancer as loss of DDX3 in advanced cancer promotes tumor progression by activating the MAPK pathway

Objective: The treatment and prognosis of patients with advanced colorectal cancer (CRC) remain a difficult problem. Herein, we investigated the role of DEAD (Asp-Glu-Ala-Asp) box helicase 3 (DDX3) in CRC and proposed potential therapeutic targets for advanced CRC.Methods: The expression of DDX3 in CRC and its effect on prognosis were explored by databases and CRC tissue microarrays. Stable DDX3 knockdown and overexpression cell lines were established with lentiviral vectors. The effects of DDX3 on CRC were investigated by functional experiments in vitro and in vivo. The molecular mechanism of DDX3 in CRC was explored by western blotting. Molecular-specific inhibitors were further used to explore potential therapeutic targets for advanced CRC.Results: The expression of DDX3 was decreased in advanced CRC, and patients with low DDX3 expression had a poor prognosis. In vitro and in vivo experiments showed that low DDX3 expression promoted the proliferation, migration and invasion of CRC. DDX3 loss regulated E-cadherin and β-catenin signaling through the mitogen-activated protein kinase (MAPK) pathway as shown by western blotting. In addition, the MEK inhibitor, PD98059, significantly reduced the increased cell proliferation, migration and invasion caused by knockdown of DDX3.Conclusions: DDX3 acts as a tumor suppressor gene in CRC. DDX3 loss in advanced cancer promotes cancer progression by regulating E-cadherin and β-catenin signaling through the MAPK pathway, and targeting the MAPK pathway may be a therapeutic approach for advanced CRC.     

TGFBIp/betaig-h3 protein: a versatile matrix molecule induced by TGF-beta

TGFBIp/βig-h3 protein is an extracellular matrix molecule initially cloned from human adenocarcinoma cells treated with TGF-β. Its precise function remains obscure but a number of studies have demonstrated it to be an intriguingly versatile molecule role in a wide range of physiological and pathological conditions. To date, the most extensively studied and reported action of TGFBIp/βig-h3 protein is in corneal dystrophy and several excellent reviews are available on this. Work from various laboratories on this molecule has compiled a tremendous amount of information over the past decade and a half. Here we review the current understanding on TGFBIp/βig-h3 protein and its functions in morphogenesis, extracellular matrix interactions, adhesion/migration, corneal dystrophy, tumorigenesis, angiogenesis, nephropathies, osteogenesis, wound healing and inflammation.