Receptor Alphavbeta3 Research Articles

Deglycosylated Human Monoclonal Antibody Against HPA-1a Prevents Anti-HPA-1a Alloantibodies Mediated Endothelial Apoptosis

Foetal/neonatal alloimmune thrombocytopenia (FNAIT) is caused by the destruction of fetal platelets by maternal platelet alloantibodies which crossed through the placenta during pregnancy. Alloantibodies (aabs) against Human Platelet Antigen-1a (HPA-1a) residing on the b-subunit of the fibrinogen receptor (alphaIIbbeta3 integrin) on platelets are responsible for intracranial hemorrhage (ICH) in severe FNAIT cases. Recently, we could demonstrate that the deglycosylated mouse mab SZ21 against HPA-1a is able to cross through placenta, and prevents the destruction of platelets in mouse model (Bakchoul et al, Blood 2013). Meanwhile, this mab is humanized as chimeric antibody (mab 813). It is known that the beta3-subunit is abundantly expressed as vitronectin receptor (alphavbeta3 integrin) on endothelial surface. In this study, we sought to investigate the effect of deglycosylated mab 813 (deg-813) towards endothelial function.Deg-813 was prepared and characterized as previously described (Bakchoul et al, Blood 2013). Flow cytometry analysis showed similar binding activity of intact 813 and deg-813 with platelets and endothelial cells. This result could be confirmed by surface plasmon resonance technology; native and deg-813 interacted equally with purified alphavbeta3 immobilized on the sensor chip (Kd 7.43x10-11 and 6.67x10-11, respectively). To study the influence of mab 813 on endothelial function, apoptosis experiment with HUVEC was performed using Tunel and Caspase-3/7 assays. In both assays, endothelial apoptosis was not observed with intact 813 and deg-813. In contrast, inhibitory mabs against alphavbeta3 receptor (clones 23C6, LM609) as well as cyclic Arg-Gly-Asp peptide caused significant apoptosis of these cells. Similar results were obtained with brain endothelial cells (hCMEC/D3). Accordingly, deg-813 did not inhibit the adhesion of HUVEC on vitronectin matrix when compared to alphavbeta3 specific mabs.Furthermore, we asked the question whether maternal anti-HPA-1a aabs may induce endothelial apoptosis. Anti-HPA-1a aabs derived from FNAIT mothers giving birth of babies with ICH (n = 3) were purified by absorption/elution method using HUVEC. Analysis of the eluted material by antigen capture assay showed specific binding to alphavbeta3, but not to alphaIIbbeta3. This result could be confirmed by immunoprecipitation analysis. When alphavbeta3-reactive anti-HPA-1a antibodies were tested with endothelial cells, significant apoptosis was observed. In the control experiment, anti-alphaIIbbeta3 complex specific antibodies did not cause endothelial apoptosis. Finally, incubation of HUVEC with deg-813 prevented cell death mediated by anti-HPA-1a antibodies.Taking together, our observation indicates that deg-813 can protect not only anti-HPA-1a aabs mediated platelet destruction but also endothelial disturbance without altering endothelial function. These results suggest that deg-813 may represent a novel drug to prevent ICH in severe FNAIT during pregnancy. DisclosuresNo relevant conflicts of interest to declare.

Epigallocatechin-3-gallate (EGCG) downregulates gelatinase-B (MMP-9) by involvement of FAK/ERK/NFκB and AP-1 in the human breast cancer cell line MDA-MB-231

Epigallocatechin-3-gallate (EGCG) is effective against the initiation, progression, and invasion of carcinogenesis.Matrix-metalloproteinases (MMPs) are a family of endopeptidases that hydrolyze the majority of extracellular proteins. MMP-9 is one of the most important members of the family and we observed the effect of EGCG on MMP-9 in the human breast cancer cell line, MDA-MB-231.The effect of EGCG on MMP-9 was studied by gelatin zymography, western blot, quantitative and semiquantitative real-time RT-PCR, immunoflourescence, cell adhesion assay, enzyme-linked immunosorbent assay,and electrophoretic mobility shift assay. EGCG treatment reduced the activity, protein, and mRNA expression ofMMP-9 and enhanced the expression of the tissue inhibitor of MMP 1 (TIMP-1). EGCG downregulated the activation of focal adhesion kinase (FAK) and extracellular regulated kinase (ERK), reduced the adhesion of MDA-MB-231 cells to fibronectin and vitronectin, and reduced the mRNA expression of the integrin receptors alpha5beta1 and alphavbeta3. The expression of the nuclear factor kappa B (NFjB), and the DNA binding activity of NFjB and activator protein 1 (AP1)to MMP-9 promoter were noticeably reduced on EGCG treatment. Upregulation of TIMP-1 and disruption of the functional status of integrin receptors may indicate decreased MMP-9 activation; inhibition of FAK andERK activation might indicate disruption in the FAK/ERK-induced MMP-9 secretion and induction. Decreased DNA binding activity of NFjB and AP1 to MMP-9 promoter might indicate transcriptional deregulation of MMP-9 gene on EGCG treatment. We propose EGCG as a potential inhibitor of the expression and activity of MMP-9 by a process involving FAK/ERK and transcription factorsin MDA-MB-231.

Expression of estrogen receptor, progesterone receptor and integrin alphaVbeta3 in endometrium of patients with luteinized unruptured follicle.

To investigate endometrium receptivity in patients with luteinized unruptured follicle (LUF) by measuring the expression of estrogen receptor (ER), progesterone receptor (PR) and integrin alphaVbeta3 in the endometrium. From May 2007 to Nov. 2007, 17 infertile women with LUF were selected as LUF group matched with 13 infertile cases with normal ovulation as control group. They all underwent frozen-thawed embryo transfer in Reproductive Medicine Center, Renmin Hospital of Wuhan University. Endometrial tissue in anterior and posterior wall of uterus of LUF group and control group were biopsied by a small curettage between 7 and 11 days after luteinizing hormone (LH) surge. The expression of ER, PR and integrin alphaVbeta3 in endometrium were detected by immunohistochemistry staining. The level of estrogen and progesterone were measured by chemiluminescence assay. Then, the relationship between alphaVbeta3 expression in endometrium and the level of estrogen/progesterone were analyzed in LUF patients. (1) There was no remarkable difference in the level of estrogen between LUF [(656 +/- 299) pmol/L] and control group [(727 +/- 275) pmol/L, P > 0.05]. However, the level of progesterone were (23 +/- 8) nmol/L in LUF group and (35 +/- 10) nmol/L in control group, which reached statistical difference (P < 0.01). (2) The expression of ER, PR in endometrium of LUF patients were 183.9 +/- 2.4 and 168 +/- 3, which were significantly higher than 109.4 +/- 6.3 and 106 +/- 4 in control group (P < 0.01). The expression of integrin alphaVbeta3 in endometrium of 115 +/- 11 in LUF group were significantly lower than 191 +/- 9 in control group (P < 0.01). (4) In LUF group, the expression of alphaVbeta3 in endometrium was correlated positively with the level of progesterone (r = 0.77, P < 0.01) and irrelevant with the level of estrogen (r = 0.01, P > 0.05). The higher expression of estrogen and progesterone and lower expression of integrin alphaVbeta3 might confer impaired receptivity of endometrium and interfere with embryo implantation.

Nectins and nectin-like molecules: roles in contact inhibition of cell movement and proliferation

Nectins and nectin-like molecules (Necls) are immunoglobulin-like transmembrane cell adhesion molecules that are expressed in various cell types. Homophilic and heterophilic engagements between family members provide cells with molecular tools for intercellular communications. Nectins primarily regulate cell-cell adhesions, whereas Necls are involved in a greater variety of cellular functions. Recent studies have revealed that nectins and NECL-5, in cooperation with integrin alphavbeta3 and platelet-derived growth factor receptor, are crucial for the mechanisms that underlie contact inhibition of cell movement and proliferation; this has important implications for the development and tissue regeneration of multicellular organisms and the phenotypes of cancer cells.

Modulation of integrin antagonist signaling by ligand binding of the heparin‐binding domain of vitronectin to the αVβ3 integrin

The interaction between the arginine glycine and aspartic acid motif (RGD) of integrin ligands such as vitronectin and the integrin receptor alphaVbeta3 in mediating cell attachment has been well described. Similarly, the ability of disintegrins, small RGD containing peptides, to inhibit cell attachment and other cellular processes has also been studied extensively. Recently, we characterized a second site of interaction between vitronectin and its integrin partner. We determined that amino acids within the heparin-binding domain of vitronectin bind to a cysteine loop (C-loop) region of beta3 and that this interaction is required for the positive effects of alphaVbeta3 ligand occupancy on IGF-I signaling in smooth muscle cells. In this study we examine the signaling events activated following ligand binding of disintegrins to the alphaVbeta3 and the ability of these signals to be regulated by binding of the heparin-binding domain of vitronectin. We demonstrate that disintegrin ligand binding activates a series of events including the sequential activation of the tyrosine kinases c-Src and Syk. This leads to the activation of calpain and the cleavage of the beta3 cytoplasmic tail. Addition of vitronectin or a peptide homologous to the heparin-binding domain inhibited activation of this pathway. Our results suggest that the signaling events that occur following ligand binding to the alphaVbeta3 integrin reflects a balance between the effects mediated through the RGD binding site interaction and the effects mediated by the heparin binding site interaction and that for intact vitronectin the effect of the heparin-binding domain predominates.

Novel Near-Infrared Fluorescent Integrin-Targeted DFO Analogue

Desferrioxamine (DFO), a siderophore initially isolated from Streptomyces pilosus, possesses extraordinary metal binding properties with wide biomedical applications that include chelation therapy, nuclear imaging, and antiproliferation. In this work, we prepared a novel multifunctional agent consisting of (i) a near-infrared (NIR) fluorescent probe-cypate; (ii) an integrin alpha vbeta3 receptor (ABIR)-avid cyclic RGD peptide, and (iii) a DFO moiety, DFO-cypate-cyclo[RGDfK(approximately)] (1, with approximately representing the cypate conjugation site at the side chain of lysine; f is d-phenylalanine). Compound 1 and two control compounds, cypate-cyclo[RGDfK(approximately)] ( 2) and cypate-DFO ( 3), were synthesized by modular assembly of the corresponding protected RGD peptide cyclo[R(Pbf)GD(OBut)fK] and DFO on the dicarboxylic acid-containing cypate scaffold in solution. The three compounds exhibited similar UV-vis and emission spectral properties. Metal binding analysis shows that DFO as well as 1 and 3 exhibited relatively high binding affinity with Fe(III), Al(III), and Ga(III). In contrast to Ga(III), the binding of Fe to 1 and 3 quenched the fluorescence emission of cypate significantly, suggesting an efficient metal-mediated approach to perturb the spectral properties of NIR fluorescent carbocyanine probes. In vitro, 1 showed a high ABIR binding affinity (10 (-7) M) comparable to that of 2 and the reference peptide cyclo(RGDfV), indicating that both DFO and cypate motifs did not interfere significantly with the molecular recognition of the cyclic RGD motif with ABIR. Fluorescence microscopy showed that internalization of 1 and 2 in ABIR-positive A549 cells at 1 h postincubation was higher than 3 and cypate alone, demonstrating that incorporating ABIR-targeting RGD motif could improve cellular internalization of DFO analogues. The ensemble of these findings demonstrate the use of multifunctional NIR fluorescent ABIR-targeting DFO analogues to modulate the spectral properties of the NIR fluorescent probe by the chelating properties of DFO and visualize intracellular delivery of DFO by receptor-specific peptides. These features provide a strategy to explore the potential of 1 in tumor imaging and treatment as well as some molecular recognition processes mediated by metal ions.

Radiolabeled Divalent Peptidomimetic Vitronectin Receptor Antagonists as Potential Tumor Radiotherapeutic and Imaging Agents

The integrin receptor alphavbeta3 is overexpressed on the endothelial cells of growing tumors and on some tumor cells themselves. A radiolabeled alphavbeta3 antagonists belonging to the quinolin-4-one class of peptidomimetics (TA138) was previously shown to exhibit high affinity for integrin alphavbeta3 and high selectivity versus other integrin receptors. 111In-TA138 exhibited high tumor uptake in the c-neu Oncomouse mammary adenocarcinoma model and produced excellent scintigraphic images. This study describes the synthesis of eight divalent versions of TA138 and their evaluation as potential tumor radiotherapeutic agents. The two main variables in this study were the length of the spacer bridging the biotargeting moieties and the total negative charge of the molecules imparted by the cysteic acid pharmacokinetic modifiers. Receptor affinity was evaluated in a panel of integrin receptor affinity assays, and biodistribution studies using the 111In-labeled derivatives were carried out in the c-neu Oncomouse model. All divalent agents maintained the high receptor affinity and selectivity of TA138, and six of the eight 111In derivatives exhibited blood clearance that was faster than 111In-TA138 at 24 h postinjection (PI). All divalent agents exhibited tumor uptake and retention at 24 h PI that was higher than 111In-TA138. Tumor/organ ratios were improved for most of the divalent agents at 24 h PI in critical nontarget organs marrow, kidney, and liver, with the agents having intermediate-length spacers (29-43 A) showing the largest improvement. As an example, 111In-15 showed tumor uptake of 14.3% ID/g at 24 h PI and tumor/organ ratios as follows: marrow, 3.24; kidney, 7.29; liver, 8.51. A comparison of therapeutic indices for 90Y-TA138 and 177Lu-15 indicate an improved therapeutic index for the divalent agent. The implications for radiotherapeutic applications and the mechanism of this multivalent effect are discussed.

Synthesis and Characterization of PAMAM Dendrimer-Based Multifunctional Nanodevices for Targeting αvβ3 Integrins

We have synthesized a stable and clinically relevant nanodevice (cRGD-BT-ND; ND for short) that exhibits superior binding to the biologic target alphavbeta3 integrins, when either compared to the same free cRGD peptide or to the biotinylated nanodevice without covalently attached peptides (BT-ND). Selective targeting of alphavbeta3 integrins was achieved by coupling cyclic cRGD peptides to the nanodevice (ND) surface, while biotin groups (BT) were used for amplified detection of bound cRGD-BT-ND by anti-biotin antibody or avidin linked to horseradish peroxidase after binding. The synthesis involved the following steps: the amino-terminated ethylenediamine core generation 5 poly(amidoamine) (PAMAM_E5.NH2) dendrimer was first partially acetylated and then biotinylated, and residual primary amine termini were converted to succinamic acid groups (SAH), some of which finally were conjugated with cRGD peptide residues through the amino group of the lysine side chain. The starting material and all derivatives were extensively characterized by polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), potentiometric acid-base titration, MALDI-TOF, and NMR. Cytotoxicity of all dendrimer derivatives was examined in B16F10 melanoma cell cultures using the XTT colorimetric assay for cellular viability. Binding of nanodevices to the biological target was determined using plates coated with human alphavbeta3 integrin and alphavbeta3 receptor expressing human dermal microvascular endothelial cells (HDMECs). The PAMAM_E5.(NHAc)72(NHBT)8(NHSAH)35(NHSA-cR GD)4 nanodevice is nontoxic within physiologic concentration ranges and specifically binds to the alphavbeta3 integrins, apparently much stronger than the cyclic cRGD peptide itself.

Use of Antibody as Carrier of Oligomers of Peptidomimetic αvβ3 Antagonist to Target Tumor-Induced Neovasculature

Sulfhydryl selective reactions were explored to conjugate oligomers of a peptidomimetic integrin alphavbeta3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonylamino-beta-alanine (IA) to monoclonal antibody (MoAb) to increase integrin alphavbeta3 receptor-binding avidity. To generate sulfhydryl groups, N-succinimidyl-S-acetylthioacetate (SATA) was conjugated to both MoAb and IA. Sulfhydryl groups were then generated upon the deacetylation of the protecting acetyl group from the S-acetylthioacetato (ATA) moiety of MoAb-(ATA)n or IA-ATA with 0.02 M hydroxylamine in the presence of 1 mM EDTA at pH 7.2. The major focus was on optimizing the reaction concentrations, molar ratios, and reaction pH to conjugate high levels of IA-(A-SH) to MoAb-(A-SH)n without causing the inter- and intramolecular cross-linking of MoAb. Stepwise reactions of MoAb-(A-SH)n (15 microM MoAb) with a homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol (BM[PEO]2) at a >50x molar excess to the -SH, followed by the reaction of the purified product MoAb-(A-S-succinimidomaleimido-[PEO]2)n with IA-(A-SH) at pH 7.2 afforded monomeric MoAb-(A-S-succinimido-[PEO]2-succinimido-S-A-IA)n with <10% high molecular weight oligomeric MoAb. Monomeric MoAb-(A-S-S-[PEO]2-S-S-A-IA)10 (MoAb-IA10) radiolabeled with 111In using 2-(p-isothiocyanatobenzyl)cyclohexyl-DTPA and with 125I using the Iodogen method showed >70% bindability to 0.4 microM alphavbeta3. When injected iv to nude mice with the receptor-positive M21 tumor, MoAb-IA10 radiolabeled with both 111In and 125I accumulated rapidly and was retained in the tumor for a 44 h period while the radioactivity cleared rapidly from the blood, thereby resulting in increasing tumor-to-blood ratios over time. The tumor uptake was similar between the 125I label and the 111In label for a 44 h period. In contrast, the blood radioactivity was lower, but liver and other organ uptakes were much higher for the 111In label than for the 125I. The 111In label produced higher tumor-to-blood ratios but much lower tumor-to-organ ratios than the 125I. The rapid blood clearance, a short peak tumor uptake time, and a low peak tumor uptake value with prolonged tumor retention of this macromolecule appear to support a hypothesis that MoAb-IA10 primarily binds to alphavbeta3 receptors on angiogenic vessels, but not on the tumor. This hypothesis was substantiated by the fluorescence microscopic analysis of FITC-MoAb-IA10, which showed that FITC-MoAb-IA10 outlined neovasculatures but not tumor cells at 4 and 21 h ex vivo. Additional proof was observed when blood vessels outlined with rhodamine-lectin, which specifically binds to blood vessels, were superimposable on neovasculatures outlined with FITC-MoAb-IA10.

Antitumor effect of RGD-4C-GG-D(KLAKLAK)2 peptide in mouse B16(F10) melanoma model.

Vasculature targeting agents have been tested as cancer therapeutics for the past few years. Such therapy could be accomplished using, for example, bifunctional (two-domain) peptides. RGD-4C-GG-D(KLAKLAK)2, a peptide designed by Ellerby and coworkers (1999) (full sequence: ACDCRGDCFCGGKLAKLAKKLAKLAK), binds selectively to alphaVbeta3 integrin receptors expressed in tumor neovasculature and, after internalization, effectively induces apoptosis of endothelial cells. The aim of this study was to examine if RGD-4C-GG-D(KLAKLAK)2 would efficiently target cells, among them B16(F10), that overexpress alphaVbeta3 receptors, and whether it would be suitable for therapeutic treatment of primary B16(F10) murine melanoma tumors. Thus, the peptide would target two distinct tumor compartments: that formed by endothelium of blood vessels and that made up of neoplastic cells. The therapeutic peptide was recognized and did induce apoptosis in B16(F10) cell line. Tumor growth inhibition was observed following direct intratumoral administration. However, cessation of peptide administration led to rapid tumor growth and death of the animals.

Discovery of Small Molecule Integrin αvβ3 Antagonists as Novel Anticancer Agents

Integrin alphavbeta3 has been implicated in multiple aspects of tumor progression and metastasis. Many tumors have high expression of alphavbeta3 that correlates with tumor progression. Therefore, alphavbeta3 receptor is an excellent target for drug design and delivery. We have discovered a series of novel alphavbeta3 antagonists utilizing common feature pharmacophore models. Upon validation using a database of known alphavbeta3 receptor antagonists, a highly discriminative pharmacophore model was used as a 3D query. A search of a database of 600 000 compounds using the pharmacophore Hypo5 yielded 832 compounds. On the basis of structural novelty, 29 compounds were tested in alphavbeta3 receptor specific binding assay and four compounds showed excellent binding affinity. A limited SAR analysis on the active compound 26 resulted in the discovery of two compounds with nanomolar to subnanomolar binding affinity. These small-molecule compounds could be conjugated to paclitaxel for selective delivery to alphavbeta3 positive metastatic cancer cells.

Nectins and nectin‐like molecules: Roles in cell adhesion, polarization, movement, and proliferation

Nectins and nectin-like molecules (Necls) are immunoglobulin-like cell adhesion molecules that constitute families containing four and five members, respectively. All members, except for Necl-5, trans-interact homophilically. Furthermore, all members, including Necl-5, trans-interact heterophilically with their respective specific partners among the members. Necl-5 regulates cell movement and proliferation cooperatively with integrin alphavbeta3 and growth factor receptors. Nectins function as cell-cell adhesion molecules at a variety of cell-cell junctions, including adherens junctions, and regulate the initial step of cell-cell junction formation. Nectins and integrin alphavbeta3 are further involved in the cross-talk between cell-matrix and cell-cell junctions. Thus, both nectin and Necl family members play important roles in fundamental cellular functions, including cell adhesion, polarization, movement, and proliferation.

L’ostéopontine, une molécule aux multiples facettes

Osteopontin (OPN) was initially isolated from bovine bone cortex, as a complex syalilated phospho-glyco-protein of around 60 kDa, with many postranslational modifications. It has been long considered a structural bone protein linking bone cells to the bone extracellular matrix (osteo : bone, pontin : bridge). It has been cloned for the first time in 1986. Since then, it was established that it is part of a protein family called SIBLINGs, which genes share common expression in bone and tooth, and encode among others a RGD motif. OPN is an intracellular as well as secreted protein, which binds to multiple organic or mineral ligands, like the integrin receptor alphaVbeta3, CD44, factor H and hydroxyapatite, depending on its final configuration (phosphorylation state). Pleiotropic functions of osteopontin have been demonstrated, and the osteopontin knock out phenotype in mice gave some new insight on the implication of the molecule in vivo. Osteopontin inhibits mineralization in bone and urine. Besides, it is a strong chemoattractive and proinflammatory molecule, implicated in tumors, like breast or prostate cancers, and in the defense against various infectious agents like tuberculosis, listeria or herpes. More recently, its key implication in TH1 mediated autoimmune diseases like multiple sclerosis and its animal model experimental autoimmune encephalomyelitis has been demonstrated. Osteopontin is a valuable therapeutic target in the animal model, and a biological tool correlating with clinical disease activity in humans. Structural, functional and pathological aspects of osteopontin are reviewed, as well as the osteopontin deficient phenotype in mouse.

Engineering conditionally replication-competent adenoviral vectors carrying the cytosine deaminase gene increases the infectivity and therapeutic effect for breast cancer gene therapy

We constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug-activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor-specific promoter. In order to overcome the low infectivity of the adenoviral vectors for breast cancer cells, and to increase the safety and efficacy for cancer gene therapy, this vector was further modified on a transductional level by simultaneously ablating the native tropism of the vector to the primary CAR receptor and inserting a RGD-4C peptide into the HI loop of the fiber, which allows the vector to use the alphavbeta3 and alphavbeta5 receptors as alternative receptors. The resulting vector was named Ad.Lp-CD-IRES-E1A(MRGD). The transduction efficiency of the vector for breast cancer cell lines which have low expression level of CAR was increased both in vitro and in vivo. The Ad.Lp-CD-IRES-E1A(MRGD) vector produces a higher vector particle yield and a greater cytotoxic effect in tumor cells which have a low expression level of CAR, than did the Ad.Lp-CD-IRES-E1A(control) vector. Intratumoral injection of the Ad.Lp-CD-IRES-E1A(MRGD) vector following the intraperitoneal injection of 5FC into xenotransplanted human breast cancer cell lines which have low expression level of CAR led to greater degree of tumor regression in vivo than did the intratumoral injection of control adenoviral vectors not so modified.

Non Peptidic &amp;#945;v&amp;#946;3 Antagonists: Recent Developments

The alphavbeta3 receptor, which are members of the group of the cellular adhesion molecules (CAM), are heterodimeric transmembrane glycoprotein receptors involved in processes such as cell-cell and cell-matrix adhesion, cell migration and signaling. Integrin alphavbeta3 receptor is expressed on almost all cells originating from the mesenchyme and seem to mediate several biological processes, including adhesion of osteoblasts to the bone matrix, migration of vascular smooth muscle cells, and angiogenesis. Many efforts were done in the last 10 years to individuate inhibitors for alphavbeta3 receptors, due to their involvement in important pathophysiological functions. In fact, selective alphavbeta3 antagonists offer new therapeutic opportunities for the treatment of several human pathologies like osteoporosis, restenosis and diseases involving neovascularization such as rheumatoid arthritis, tumor induced angiogenesis and metastasis. Purpose of this account is to summarize the recent developments in the field of non-peptidic alphavbetav antagonists.

A novel CYR61-triggered ‘CYR61-αvβ3 integrin loop’ regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway

The angiogenic inducer CYR61 is differentially overexpressed in breast cancer cells exhibiting high levels of Heregulin (HRG), a growth factor closely associated with a metastatic breast cancer phenotype. Here, we examined whether CYR61, independently of HRG, actively regulates breast cancer cell survival and chemosensitivity, and the pathways involved. Forced expression of CYR61 in HRG-negative MCF-7 cells notably upregulated the expression of its own integrin receptor alphavbeta3 (>200 times). Small peptidomimetic alphavbeta3 integrin antagonists dramatically decreased cell viability of CYR61-overexpressing MCF-7 cells, whereas control MCF-7/V remained insensitive. Mechanistically, functional blockade of alphavbeta3 specifically abolished CYR6-induced hyperactivation of ERK1/ERK2 MAPK, whereas the activation status of AKT did not decrease. Moreover, CYR61 overexpression rendered MCF-7 cells significantly resistant (>10-fold) to Taxol-induced cytotoxicity. Remarkably, alphavbeta3 inhibition converted the CYR61-induced Taxol-resistant phenotype into a hypersensitive one. Thus, the augmentation of Taxol-induced apoptotic cell death in the presence of alphavbeta3 antagonists demonstrated a strong synergism as verified by the terminal transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometric analysis for DNA content. Indeed, functional blockade of alphavbeta3, similarly to the pharmacological MAPK inhibitor U0126, synergistically increased both the proportion of CYR61-overexpressing breast cancer cells in the G2 phase of the cell cycle and the appearance of sub-G1 hypodiploid (apoptotic) cells caused by Taxol. Strikingly, CYR61 overexpression impaired the accumulation of wild-type p53 following Taxol exposure, while inhibition of alphavbeta3 or ERK1/ERK2 MAPK signalings completely restored Taxol-induced upregulation of p53. Moreover, antisense downregulation of CYR61 expression abolished the anchorage-independent growth of breast cancer cells engineered to overexpress HRG, and significantly increased their sensitivity to Taxol. Our data provide evidence that CYR61 is sufficient to promote breast cancer cell proliferation, cell survival, and Taxol resistance through a alphavbeta3-activated ERK1/ERK2 MAPK signaling. The identification of a 'CYR61-alphavbeta3 autocrine loop' in the epithelial compartment of breast carcinoma strongly suggests that targeting alphavbeta3 may simultaneously prevent breast cancer angiogenesis, growth, and chemoresistance.

Oncolytic herpes simplex virus mutants are more efficacious than wild‐type adenovirus Type 5 for the treatment of high‐risk neuroblastomas in preclinical models

High-risk neuroblastoma (Nb) is incurable using current treatment regimens in the majority of patients. Oncolytic virotherapy is a novel approach being tested for several types of adult cancers. To compare the susceptibility of Nb tumor models to oncolytic adenovirus and HSV mutants and delineate the mechanisms of resistance or sensitivity. Human Nb cell lines were used to determine susceptibility to adenovirus type 5 wild-type and HSV1 mutant (NV1066) infection, adenovirus receptor expression, support of NV1066 replication, and induction of apoptosis. Human xenograft tumors in immunodeficient mice were evaluated for histological effects and tumor response to intratumoral injection of an oncolytic HSV mutant. All eight Nb cell lines tested in culture were relatively resistant to infection with wild type and attenuated adenoviruses. Cells expressed the cocksackie-adenovirus attachment receptor (CAR) but had low or absent expression of the internalization receptors (alphavbeta3, alphavbeta5 integrins). In contrast, all cells were uniformly sensitive to infection with the attenuated HSV mutant, NV1066. Productive virus replication and induction of apoptosis were observed in HSV-infected cells. CHLA-20 and LAN-5 xenograft tumors injected with a single dose of NV1066 showed a significant antitumor response, and the animals had a prolonged survival post infection in comparison to the PBS-treated control group. HSV injected tumors showed extensive areas of necrosis and morphologic evidence of apoptosis. Nb tumor models are resistant to adenovirus mediated oncolysis but highly sensitive to HSV mediated oncolysis. Further studies of HSV virotherapy as a novel treatment for Nb are warranted.

Regulation of Macrophage Foam Cell Formation by αVβ3 Integrin: Potential Role in Human Atherosclerosis

The accumulation of macrophage foam cells in atherosclerotic lesions is associated with both initiation and progression of this disease. Scavenger receptors CD36 and SRA are the primary receptors responsible for conversion of macrophages into foam cells. Integrin alphaVbeta3 plays a role in the differentiation of several cell types, but its involvement in the transition of macrophages into foam cells and the potential role of this receptor in atherosclerosis have not been examined. Using an in vitro model of single surface receptor activation by binding with an immobilized monoclonal antibody specific to alphaVbeta3 integrin we show that ligation of alphaVbeta3 integrin prevents differentiation of blood monocytes and macrophages into the foam cell phenotype via coordinate down-regulation of CD36 and SRA. This effect of alphaVbeta3 integrin ligation can be reproduced by contact with endothelial cells, whereas the inhibition of alphaVbeta3 receptor ligation restores the uptake of oxidized low-density lipoprotein. Moreover, we found that alphaVbeta3 integrin is readily detected in situ on macrophages in early and advanced atherosclerotic lesions and that in vitro exposure to oxidized low-density lipoprotein up-regulates alphaVbeta3 integrin expression. We hypothesize that alphaVbeta3 integrin regulates macrophage functional maturation into foam cells in a persistent manner, and therefore, by targeting alphaVbeta3 receptor it could potentially be possible to regulate progression of atherosclerosis in humans.

Enterovirus infection in human pancreatic islet cells, islet tropism in vivo and receptor involvement in cultured islet beta cells.

It is thought that enterovirus infections cause beta-cell damage and contribute to the development of Type 1 diabetes by replicating in the pancreatic islets. We sought evidence for this through autopsy studies and by investigating known enterovirus receptors in cultured human islets. Autopsy pancreases from 12 newborn infants who died of fulminant coxsackievirus infections and from 65 Type 1 diabetic patients were studied for presence of enteroviral ribonucleic acid by in situ hybridisation. Forty non-diabetic control pancreases were included in the study. The expression and role of receptor candidates in cultured human islets were investigated with receptor-specific antibodies using immunocytochemistry and functional assays. Enterovirus-positive islet cells were found in some of both autopsy specimen collections, but not in control pancreases. No infected cells were seen in exocrine tissue. The cell surface molecules, poliovirus receptor and integrin alphavbeta3, which act as enterovirus receptors in established cell lines, were expressed in beta cells. Antibodies to poliovirus receptor, human coxsackievirus and adenovirus receptor and integrin alphavbeta3 protected islets and beta cells from adverse effects of poliovirus, coxsackie B viruses, and several of the arginine-glycine-aspartic acid motifs containing enteroviruses and human parechovirus 1 respectively. No evidence was found for expression of the decay-accelerating factor which acts as a receptor for several islet-cell-replicating echoviruses in established cell lines. The results show a definite islet-cell tropism of enteroviruses in the human pancreas. Some enteroviruses seem to use previously identified cell surface molecules as receptors in beta cells, whereas the identity of receptors used by other enteroviruses remains unknown.

Induction of cyclooxygenase-1 and -2 modulates angiogenic responses to engagement of alphavbeta3.

Prostaglandins and cyclooxygenase (COX) have been implicated in the angiogenesis that occurs around tumours, but how they are induced is unclear. Prostaglandin formation is regulated by the availability of arachidonic acid and/or COX activity that in turn are controlled by activation of G-protein-coupled receptors or kinase receptors. Adhesion receptors provide another potential level of control as they transduce a variety of "outside-in" signals implicated in inflammation. We examined whether engagement of the vitronectin receptor (alphavbeta3) modulated prostacyclin (PGI2) formation in human umbilical vein endothelial cells (EC). Engagement of EC alphavbeta3 by vitronectin (versus fibronectin or gelatin) or by monoclonal antibodies (mAbs) LM609 and LIBS6, enhanced PGI2 generation and also induced expression of both COX-1 and -2 isoforms. Alphavbeta3 engagement also led to vascular endothelial cell growth factor (VEGF) generation and EC proliferation that was attenuated by inhibition of both COX-1 and COX-2. COX-1 inhibition also prevented new vessel formation in an in vitro model of angiogenesis that is alphavbeta3 dependent. Inhibition of angiogenesis by the COX-1 inhibitor was partially reversed by removal of the inhibitor or by addition of the stable analogue of PGI2, iloprost. These findings strongly indicate that alphavbeta3-mediated angiogenesis is partly due to induction of both isoforms of COX.