Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Av, B, Ca, Ch, H, Mk, Po, Rb||FC, IF, IHC, IP, FUNC||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Material Size||100 µg|
|Reference overview||Application||Species||Pub Med ID|
|Endothelial destabilization by angiopoietin-2 via integrin β1 activation.|
Hakanpaa, L; Sipila, T; Leppanen, VM; Gautam, P; Nurmi, H; Jacquemet, G; Eklund, L; Ivaska, J; Alitalo, K; Saharinen, P
Nature communications 6 5962 2015
Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell-cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2.
|H-CRRETAWAC-OH, a lead structure for the development of radiotracer targeting integrin α5β1?|
Haubner, R; Maschauer, S; Einsiedel, J; Eder, IE; Rangger, C; Gmeiner, P; Virgolini, IJ; Prante, O
BioMed research international 2014 243185 2014
Imaging of angiogenic processes is of great interest in preclinical research as well as in clinical settings. The most commonly addressed target structure for imaging angiogenesis is the integrin α(v)β(3). Here we describe the synthesis and evaluation of [(18)F]FProp-Cys(*)-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys(*)-OH, a radiolabelled peptide designed to selectively target the integrin α(5)β(1). Conjugation of 4-nitrophenyl-(RS)-2-[(18)F]fluoropropionate provided [(18)F]FProp-Cys(*)-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys(*)-OH in high radiochemical purity (greater than 95%) and a radiochemical yield of approx. 55%. In vitro evaluation showed α(5)β(1) binding affinity in the nanomolar range, whereas affinity to α(v)β(3) and α(IIb)β(3) was greater than 50 μM. Cell uptake studies using human melanoma M21 (α(v)β(3)-positive and α(5)β(1)-negative), human melanoma M21-L (α(v)β(3)-negative and α(5)β(1)-negative), and human prostate carcinoma DU145 (α(v)β(3)-negative and α(5)β(1)-positive) confirmed receptor-specific binding. The radiotracer was stable in human serum and showed low protein binding. Biodistribution studies showed tumour uptake ranging from 2.5 to 3.5% ID/g between 30 and 120 min post-injection. However, blocking studies and studies using mice bearing α(5)β(1)-negative M21 tumours did not confirm receptor-specific uptake of [(18)F]FProp-Cys(*)-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys(*)-OH, although this radiopeptide revealed high affinity and substantial selectivity to α(5)β(1) in vitro. Further experiments are needed to study the in vivo metabolism of this peptide and to develop improved radiopeptide candidates suitable for PET imaging of α(5)β(1) expression in vivo.
|Identification of platelet function defects by multi-parameter assessment of thrombus formation.|
de Witt, SM; Swieringa, F; Cavill, R; Lamers, MM; van Kruchten, R; Mastenbroek, T; Baaten, C; Coort, S; Pugh, N; Schulz, A; Scharrer, I; Jurk, K; Zieger, B; Clemetson, KJ; Farndale, RW; Heemskerk, JW; Cosemans, JM
Nature communications 5 4257 2014
Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)greater than GPIbgreater than α6β1, αIIbβ3greater than α2β1greater than CD36, α5β1, αvβ3. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann's thrombasthenia, Hermansky-Pudlak syndrome, May-Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.
|Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation.|
Klingberg, F; Chow, ML; Koehler, A; Boo, S; Buscemi, L; Quinn, TM; Costell, M; Alman, BA; Genot, E; Hinz, B
The Journal of cell biology 207 283-97 2014
Integrin-mediated force application induces a conformational change in latent TGF-β1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-β1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-β1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.
|Cyp1b1 mediates periostin regulation of trabecular meshwork development by suppression of oxidative stress.|
Zhao, Y; Wang, S; Sorenson, CM; Teixeira, L; Dubielzig, RR; Peters, DM; Conway, SJ; Jefcoate, CR; Sheibani, N
Molecular and cellular biology 33 4225-40 2013
Mutation in CYP1B1 has been reported for patients with congenital glaucoma. However, the underlying mechanisms remain unknown. Here we show increased diurnal intraocular pressure (IOP) in Cyp1b1-deficient (Cyp1b1(-/-)) mice. Cyp1b1(-/-) mice presented ultrastructural irregular collagen distribution in their trabecular meshwork (TM) tissue along with increased oxidative stress and decreased levels of periostin (Postn). Increased levels of oxidative stress and decreased levels of Postn were also detected in human glaucomatous TM tissues. Furthermore, Postn-deficient mice exhibited TM tissue ultrastructural abnormalities similar to those of Cyp1b1(-/-) mice. Administration of the antioxidant N-acetylcysteine (NAC) restored structural abnormality of TM tissue in Cyp1b1(-/-) mice. In addition, TM cells prepared from Cyp1b1(-/-) mice exhibited increased oxidative stress, altered adhesion, and decreased levels of Postn. These aberrant cellular responses were reversed in the presence of NAC or by restoration of Cyp1b1 expression. Cyp1b1 knockdown or inhibition of CYP1B1 activity in Cyp1b1(+/+) TM cells resulted in a Cyp1b1(-/-) phenotype. Thus, metabolic activity of CYP1B1 contributes to oxidative homeostasis and ultrastructural organization and function of TM tissue through modulation of Postn expression.
|Integrin control of the transforming growth factor-β pathway in glioblastoma.|
Roth, P; Silginer, M; Goodman, SL; Hasenbach, K; Thies, S; Maurer, G; Schraml, P; Tabatabai, G; Moch, H; Tritschler, I; Weller, M
Brain : a journal of neurology 136 564-76 2013
Transforming growth factor-β is a central mediator of the malignant phenotype of glioblastoma, the most common and malignant form of intrinsic brain tumours. Transforming growth factor-β promotes invasiveness and angiogenesis, maintains cancer cell stemness and induces profound immunosuppression in the host. Integrins regulate cellular adhesion and transmit signals important for cell survival, proliferation, differentiation and motility, and may be involved in the activation of transforming growth factor-β. We report that αvβ3, αvβ5 and αvβ8 integrins are broadly expressed not only in glioblastoma blood vessels but also in tumour cells. Exposure to αv, β3 or β5 neutralizing antibodies, RNA interference-mediated integrin gene silencing or pharmacological integrin inhibition using the cyclic RGD peptide EMD 121974 (cilengitide) results in reduced phosphorylation of Smad2 in most glioma cell lines, including glioma-initiating cell lines and reduced transforming growth factor-β-mediated reporter gene activity, coinciding with reduced transforming growth factor-β protein levels in the supernatant. Time course experiments indicated that the loss of transforming growth factor-β bioactivity due to integrin inhibition likely results from two distinct mechanisms: an early effect on activation of preformed inactive protein, and second, major effect on transforming growth factor-β gene transcription as confirmed by decreased activity of the transforming growth factor-β gene promoter and decreased transforming growth factor-β(1) and transforming growth factor-β(2) messenger RNA expression levels. In vivo, EMD 121974 (cilengitide), which is currently in late clinical development as an antiangiogenic agent in newly diagnosed glioblastoma, was a weak antagonist of pSmad2 phosphorylation. These results validate integrin inhibition as a promising strategy not only to inhibit angiogenesis, but also to block transforming growth factor-β-controlled features of malignancy including invasiveness, stemness and immunosuppression in human glioblastoma.
|The differentiation of pancreatic tumor-initiating cells by vitronectin can be blocked by cilengitide.|
Cabarcas, SM; Sun, L; Mathews, L; Thomas, S; Zhang, X; Farrar, WL
Pancreas 42 861-70 2013
Pancreatic cancer is a leading cancer type and its molecular pathology is poorly understood. The only potentially curative therapeutic option available is complete surgical resection; however, this is inadequate as most of the patients are diagnosed at an advanced or metastatic stage. Tumor-initiating cells (TICs) constitute a subpopulation of cells within a solid tumor that sustain tumor growth, metastasis, and chemo/radioresistance. Within pancreatic cancer, TICs have been identified based on the expression of specific cell surface markers.We use a sphere formation assay to enrich putative TICs and use human serum as a driver of differentiation. We demonstrate by using specific blocking reagents that we can inhibit the differentiation process and maintain TIC-associated markers and genes.We can induce differentiation of pancreatospheres with the addition of human serum, and we identified vitronectin as an inducer of differentiation. We inhibit differentiation by human serum using an arginine-glycine-aspartate-specific peptide, which is Cilengitide; hence, demonstrating this differentiation is mediated via specific integrin receptors.Overall, our studies further the definition of pancreatic TICs and provide further insight into both the maintenance and differentiation of this lethal population.
|Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αVβ3 and RhoA/ROCK-mediated mechanisms.|
Riches, K; Franklin, L; Maqbool, A; Peckham, M; Adams, M; Bond, J; Warburton, P; Feric, NT; Koschinsky, ML; O'Regan, DJ; Ball, SG; Turner, NA; Porter, KE
The international journal of biochemistry & cell biology 45 1776-83 2013
Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin αVβ3-dependent, but transforming growth factor β-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, αVβ3 neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.
|Efficient downregulation of VEGF in retinal pigment epithelial cells by integrin ligand-labeled liposome-mediated siRNA delivery.|
Chen, CW; Yeh, MK; Shiau, CY; Chiang, CH; Lu, DW
International journal of nanomedicine 8 2613-27 2013
The purpose of this study was to demonstrate the effectiveness of an integrin peptide ligand-labeled liposomal delivery system loaded with vascular endothelial growth factor (VEGF)-siRNA in a model study of gene therapy for retinopathy using human retinal pigment epithelial cells.Arg(R)-Gly(G)-Asp(D) motif peptide conjugating polyethylene glycol modified (RGD-PEGylated) liposomes were prepared using a thin-film hydration method and optimized for surface charge, particle size, small interfering RNA (siRNA) load, and entrapment efficiency. Reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine VEGF levels in retinal pigment epithelial cells. Cytotoxicity was determined using the 3-[4, 5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry.Physicochemical properties, including particle size, zeta potential, and siRNA load, of the prepared RGD-PEGylated liposomes and their entrapment efficiency were determined to be within the following ranges: 123.8-234.1 nm, 17.31-40.09 m V, 5.27%-6.33%, and greater than 97%, respectively. RGD-PEGylated liposome-mediated fluorescent-labeled siRNA delivery demonstrated significantly enhanced cellular uptake, and 3 mol% RGD-PEGylated liposomes (having 3β-[N-(N', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol) DSPE and DSPE-PEG(2000)-RGD with molar ratio of 50/47/3) were shown to have better efficacy with regard to specificity for retinal pigment epithelial cells, reduced cytotoxicity, and knockdown of the target molecule.By integrin receptor-mediated endocytosis, 3 mol% RGD-PEGylated liposomes were shown to be a suitable vector when loaded with VEGF-siRNA for efficient downregulation of VEGF in retinal pigment epithelial cells at both the protein and gene levels. This integrin ligand-modified liposomal delivery system has therapeutic potential for ocular gene therapy.
|The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus.|
Hahn, AS; Kaufmann, JK; Wies, E; Naschberger, E; Panteleev-Ivlev, J; Schmidt, K; Holzer, A; Schmidt, M; Chen, J; König, S; Ensser, A; Myoung, J; Brockmeyer, NH; Stürzl, M; Fleckenstein, B; Neipel, F
Nature medicine 18 961-6 2012
Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma(1), a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies(2,3). A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells(4). We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA(2) and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry(5,6). Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.
|MOUSE ANTI-HUMAN INTEGRIN alphaVbeta3 (VITRONECTIN RECEPTOR)|
|Does LM609 work in western blots?||No, the epitope recognized by LM609 is to the complex of Av and B3 together, thus when run on gels and denatured that epitope is physically destroyed, thus non-reactive.|
|Does this product react with paraffin embedded tissues?||Some researchers have gotten LM609 to react with traditionally fixed paraffin tissue, however Millipore cannot endorse the use of paraffin tissues with LM609 at this time.|
|Does LM609 react with rat or mouse?||LM609 traditionally has not worked with rat or mouse tissues, however one can find reports of LM609 working with rat under some special conditions. At this time Millipore does not endorse the use of LM609 with mouse or rat tissues, however.|
|What fixation works best for LM609?||Because the epitope of LM609 is fragile acetone is the preferred fixation, however short fixations in paraformaldehyde usually presents no serious difficulties.|