Alphavbeta3 Integrin Research Articles

Evaluation of invasion patterns and their correlation with integrin alphavbeta expression in brain metastases of solid cancers.

2059 Background: Understanding the pathobiology of brain metastases (BM) could guide the establishment of new targeted therapies. Methods: We collected 57 autopsy specimens of BM (primary tumor: 27 lung cancer, 6 breast cancer, 8 melanoma, 1 kidney cancer, 2 colorectal cancer, 13 other) and histologically evaluated the patterns of invasion into the surrounding brain parenchyma. Expression of the following integrins was evaluated using immunohistochemistry: with novel antibodies for αv subunit, αvβ3, αvβ5, αvβ6 and αvβ8 integrin. Results: We observed three main invasion patterns: well-demarcated (29/57, 51%), vascular co-option (10/57, 18%) and diffuse infiltration (18/57, 32%). There was no association of invasion pattern with primary tumor type, although vascular co-option was most common in melanomas (4/10, 40%). αv subunit expression was lowest in the vascular co-option group (p = 0.05, t-test). αvβ6 levels were higher in the well-demarcated group than in the vascular co-option group (p = 0.025; t-test) and were higher in lung cancer BM than in melanoma BM (0.01, t-test). αvβ3 and αvβ5 were frequently expressed in tumoral (αvβ3: 30/57, 53%; αvβ5: 55/57, 97%) and peritumoral (αvβ3: 29/57, 51%, αvβ5: 54/57 (95%) vascular structures and 27/57 (47%) specimens showed avb5 and 6/57 (11%) αvβ3 expression on tumor cells. Prior radio- or chemotherapy did not correlate with invasion pattern or integrin expression. Conclusions: We delineate three distinct invasion patterns of BM into the brain parenchyma: well-demarcated growth, vascular co-option and diffuse infiltration. Integrin expression is frequent on tumor and vascular cells in BM and associated with distinct invasion patterns. Anti-integrin therapy could be a valid treatment option in patients with BM.

Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Abstract 131: Alphavbeta3 Integrins Mediate Shear Stress-Induced Inflammation and Early Atherogenesis

Rational The frictional shear stress generated by blood flow activates integrins on the endothelial cell surface, and signaling downstream of newly activated integrins regulates several flow-induced responses including inflammation and permeability. Signaling through transitional matrix binding integrins (α5β1, αvβ3 and αvβ5) drives shear stress-induced NF-κB activation and proinflammatory gene expression; however the specific contribution of each of these integrins remains unclear. Objective To address which transitional matrix-binding integrin mediates shear stress-induced endothelial cell. Methods and Results We now show that the small molecule αv integrin inhibitor S247, but not the α5 inhibitor ATN-161, abolished flow-induced proinflammatory signaling (PAK, NF-κB). Blocking αv expression using siRNA similarly prevents flow-induced activation of PAK and NF-κB, whereas siRNA-mediated knock-down of α5 did not. Furthermore, αv and β3 knockdown but not α5 and β5 knockdown inhibited flow-induced ICAM-1 and VCAM-1 expression (both mRNA and protein) following either short term (5 hr) laminar and long term oscillatory (24 hr) shear stress, indicating αvβ3 is the mediator for flow induced endothelial cell inflammation. In contrast, αv knockdown did not affect TNFα-induced ICAM-1 and VCAM-1 expression, suggesting the anti-inflammatory effect of αv inhibition is specific to shear stress. Integrin inhibition did not affect all shear stress responses, as neither α5 nor αv siRNA affected shear stress-induced extracellular-signal regulated kinase (ERK1/2) activation. Lastly, inhibiting αv integrins using the small molecule inhibitor S247 reduces atherosclerotic plaque formation in ApoE knockout mice. Conclusion Taken together, these studies suggest that αvβ3 integrins mediate shear stress-induced proinflammatory responses associated with the transitional matrix in vitro and early atherosclerotic plaque formation in vivo .

Abstract 4890: Periostin signaling regulates breast cancer stem cells.

Abstract Recent evidence suggests that the molecular heterogeneity inherent to breast cancer, which underlies metastasis, resistance to treatment and disease recurrence, can be driven by a distinct subpopulation of tumor cells referred to as cancer stem cells (CSCs). However, the extracellular cues and intracellular pathways that CSCs rely on remain unclear. Working with a breast cancer progression model system we found the invasive breast cancer cell line to exhibit numerous stem-like characteristics. Gene expression profiling, along with a careful review of the literature, identified a secreted extracellular matrix molecule, periostin (POSTN), which we hypothesized to be required for the maintenance of a cancer stem cell state. POSTN expression was increased in numerous basal-like breast cancer cell lines that contain an expanded CSC population, as well as in cells grown as mammospheres and in the CD44+/CD24- fraction of tumor cells. Furthermore, CSC-like lines displayed increased surface levels of the alpha-v beta-3 integrin heterodimer, a known receptor for POSTN, suggesting that hyperactivation of a POSTN signaling axis could potentially regulate the CSC state. Using stable cell lines expressing an shRNA directed at POSTN we have found that POSTN is required for efficient mammosphere formation and for the maintenance of an ALDH-positive CSC population. In line with this, we found these cells had impaired Erk and Wnt signaling and expressed less IL-6 and IL-8, known regulators of CSCs. Furthermore, high POSTN expression correlates with a worse clinical prognosis in basal-like breast cancer patients. Therefore, we suggest that POSTN signaling through integrin receptors may enrich for highly tumorigenic breast CSCs in a subset of basal-like tumors, leading to aggressive and recurrent malignancies. More broadly, our results imply that a supportive tumor niche can be established and maintained by cancer cells, and raises the possibility of targeting components of this extracellular environment to eradicate breast CSCs. Citation Format: Arthur W. Lambert, Sait Ozturk, Panos Papageorgis, Hamid Abdolmaleky, Sam Thiagalingam. Periostin signaling regulates breast cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4890. doi:10.1158/1538-7445.AM2013-4890

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

A9.1 αvβ3 Integrin Inhibition with Cilengitide Both Prevents and Treats Collagen Induced Arthritis

Background Rheumatoid arthritis (RA) is characterised by synovial inflammation and osteoclast (OC) mediated bone erosions. AlphaVbeta3 (αvβ3) integrin is highly expressed in OCs. Avβ3 blocking antibodies reduce bone resorption and mice lacking β3 are osteopetrotic. Objectives Efficacy testing of the αvβ3 inhibitor cilengitide, a synthetic Arginine-Glycine-Asparagine amino acid peptide (RGD peptide), on osteoclastogenesis and the collagen induced arthritis (CIA) model for human RA. Materials and Methods In vitro mouse bone marrow-derived cells (BMCs) were differentiated into tartrate resistant acid phosphatase positive (TRAP+) mononuclear OC precursor cells (pre-OCs) and TRAP+ multinucleated mature OCs with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). Cilengitide, was added in increasing concentrations (2 nM to 20 µM) to the culture. These osteoclastogenesis assays were performed on plates coated with RGD containing matrixes osteopontin, fibronectin and fibrinogen but also on Poly-D-lysine to assess αvβ3 independent adhesion. In vivo CIA was induced in 6–8 week old male DBA/1 mice by immunisation with bovine type II collagen at day 1, followed by boosting at day 21. For CIA prevention mice received subcutaneously (s.c.) 1.5 mg/kg cilengitide (n = 15) or placebo (n = 15), 5 days per week, 1 day prior to CIA induction until day 53. For CIA treatment, mice with arthritis were randomised and received 1.5 mg/kg (low dose, n = 19) or 75 mg/kg (high dose, n = 7) cilengitide or placebo (n = 21), until day 59. Preventive and treatment effects were evaluated by assessing paw thickness and grip strength. Results In vitro increasing concentrations of cilengitide (IC50: 250 nM) dose-dependently reduced pre-OCs on all coatings, indicating early inhibition at the pre-OC proliferation stage. OCs were reduced above 200 nM, followed by complete disappearance above 2 µM. At 200 nM an intriguing morphological difference with reduction in OC size suggested that cilengitide may disrupt spreading and fusion capacity at the early pre-OC stage. In vivo CIA prevention with cilengitide effectively reduced incidence (92.8% versus 40%) and severity of arthritis as evidenced by reduction of clinical disease activity scores. Low and high dose cilengitide effectively inhibited progression of established arthritis. Conclusions Osteoclastogenesis requires intact αvβ3 integrin function. Systemic αvβ3 integrin inhibition with cilengitide potently prevents and treats experimental CIA. Cilengitide may be a novel therapeutic target in RA.

The integrin alphav beta3 increases cellular stiffness and cytoskeletal remodeling dynamics to facilitate cancer cell invasion

The process of cancer cell invasion through the extracellular matrix (ECM) of connective tissue plays a prominent role in tumor progression and is based fundamentally on biomechanics. Cancer cell invasion usually requires cell adhesion to the ECM through the cell-matrix adhesion receptors integrins. The expression of the αvβ3 integrin is increased in several tumor types and is consistently associated with increased metastasis formation in patients. The hypothesis was that the αvβ3 integrin expression increases the invasiveness of cancer cells through increased cellular stiffness, and increased cytoskeletal remodeling dynamics. Here, the invasion of cancer cells with different αvβ3 integrin expression levels into dense three-dimensional (3D) ECMs has been studied. Using a cell sorter, two subcell lines expressing either high or low amounts of αvβ3 integrins (αvβ3high or αvβ3low cells, respectively) have been isolated from parental MDA-MB-231 breast cancer cells. αvβ3high cells showed a threefold increased cell invasion compared to αvβ3low cells. Similar results were obtained for A375 melanoma, 786-O kidney and T24 bladder carcinoma cells, and cells in which the β3 integrin subunit was knocked down using specific siRNA. To investigate whether contractile forces are essential for αvβ3 integrin-mediated increased cellular stiffness and subsequently enhanced cancer cell invasion, invasion assays were performed in the presence of myosin light chain kinase inhibitor ML-7 and Rho kinase inhibitor Y27632. Indeed, cancer cell invasiveness was reduced after addition of ML-7 and Y27632 in αvβ3high cells but not in αvβ3low cells. Moreover, after addition of the contractility enhancer calyculin A, an increase in pre-stress in αvβ3low cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase, STAT3 or Rac1 strongly reduced the invasiveness of αvβ3high cells, whereas the invasiveness of β3 specific knock-down cells and αvβ3low cells was not altered. In summary, these results suggest that the αvβ3 integrin enhances cancer cell invasion through increased cellular stiffness and enhanced cytoskeletal remodeling dynamics, which enables the cells to generate and transmit contractile forces to overcome the steric hindrance of 3D ECMs.

Action of the Disintegrin Contortrostatin on Breast Cancer Cell Primary Cultures

Integrins mediate cell adhesion to the extracellular matrix (ECM). In particular, integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site on various extracellular molecules of the extracellular matrix. Integrin aphavbeta3 has been associated with high malignant potential in breast cancer cells, and has signalized the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including snake venom disintegrins, have been used as potential anti-cancer agents. In the present work, the effect of contortrostatin, a disintegrin isolated from the venom of the snake Agkistrodon contortrix, was studied on primary cultures of human breast cancer cell. Scanning and transmission electron microscopy were employed in order to examine alterations in cell morphology and fluorescent microscopy and visualize changes in distribution of integrin alphavbeta3 and talin. Fluorescent localization of caspase 8 was made in order to visualize any sign of proapoptotosis and western immunoblotting of integrin, talin and annexin was undertaken in order to identify changes. The results suggest that the snake venom contortrostatin seriously affects cell morphology, adhesion and mobility and induces breast cancer cells to apoptosis.

RGD Binding to Integrin Alphavbeta3 Affects Cell Motility and Adhesion in Primary Human Breast Cancer Cultures

Integrins mediate cell adhesion to the extracellular matrix. Integrin alphavbeta3 recognizes the RGD motif as a ligand-binding site and has been associated with high malignant potential in breast cancer cells, signaling the onset of widespread metastasis. In recent years, several antagonists of integrin alphavbeta3, including RGD peptides, have been used as potential anti-cancer agents. In the present work, the effect of the linear RGD hexapeptide GRGDSP was studied, for the first time, on breast tumor explants, as well as on well-spread human breast cancer cells from primary cultures, using the explant technique, to clarify the role of this peptide in the suppression of breast cancer cell migration. The results showed that incubation of breast tumor explants with RGD peptide at the beginning of culture development inhibited completely the migration of cancer cells out of the tissue fragment as revealed by electron microscopy. RGD incubation of well-spread breast cancer cells from primary culture resulted in rounding and shrinkage of the cells accompanied by altered distribution of integrin alphavbeta3 and concomitant F-actin cytoskeletal disorganization, as revealed by immunofluorescence. Electron immunocytochemistry showed aggregation of integrin alphavbeta3 at the cell periphery and its detection in noncoated vesicles. However, Western immunoblotting showed no change in beta3 subunit expression, despite the altered distribution of the integrin alphavbeta3. In light of the above, it appears that the RGD peptide plays an important role in the modulation of cell motility and in the perturbation of cell attachment affecting the malignant potential of breast cancer cells in primary cultures.

Integrin alphavbeta6 is involved in measles protein-induced airway immune suppression

Background and aimsMeasles infection causes immune suppression that contributes to morbidity and mortality of the patients; the mechanism is poorly understood. Regulatory T cells (Treg) play a critical role in immune suppression. Integrin alphavbeta6 (avb6) is associated with Treg’s function. This study aims to investigate into the mechanism by which measles C protein (MVP)-induced avb6 contributes the generation of Tregs in the lung. MethodMVP was introduced to mouse lung by nasal drops. The expression of avb6 by lung tissue was examined by reverse transcription polymerase chain reaction and Western blotting. The development of tolerogenic dendritic cells (DC) and Tregs in the lung and their functions was examined by flow cytometry. The suppressor function of MVP-induced Tregs was examined by cell culture models. ResultsThe exposure to MVP markedly increased the expression of avb6 in the lung epithelial cells. Administration of MVP significantly suppressed the levels of IL-4 and IFNγ as well as increases in Tregs in lung tissue. DCs captured the MVP in the lung and differentiate into tolerogenic DCs; the latter has the ability to induce Treg development in the lung. Activation of MVP-induced Tregs powerfully suppressed polarized CD4+ T cells. ConclusionsExposure to MVP can induce Treg development in the lung that plays an important role in the suppression of CD4+ T cell function.

Abstract 5191: Periostin signaling in breast cancer stem cells

Abstract Recent evidence suggests that the molecular heterogeneity inherent to breast cancer, which underlies metastasis, resistance to treatment and disease recurrence, can be driven by a distinct subpopulation of tumor cells referred to as cancer stem cells. Breast cancer stem cells are associated with epithelial-mesenchymal transition (EMT) and the basal-like molecular subtype of tumors, exhibit a CD44+/CD24- expression profile and have an enhanced capacity to form mammospheres. However, the extracellular cues and intracellular pathways that cancer stem cells rely on remain unclear. Using a cell line model of breast cancer progression we found a carcinoma line that was enriched for cancer stem cells. Using microarray expression profiling we identified periostin, a secreted extracellular matrix protein, to be overexpressed in this cell line and numerous other basal-like cell lines. Periostin was also overexpressed in populations that were enriched for cancer stem cells through other means such as cell sorting or growth as mammospheres. Furthermore, cancer stem cell-like populations displayed increased surface levels of the alpha-v beta-3 integrin heterodimer, a known receptor for periostin, suggesting that hyperactivation of periostin signaling may be a common feature of the cancer stem cell state. In support of this, cells with high levels of alpha-v beta-3 integrin form significantly more mammospheres and exhibit an enhanced capacity for growth on soft agar, a surrogate marker of tumorigenesis. Conversely, treatment of cells with an alpha-V beta-3 inhibitory antibody decreased mammosphere formation in a dose-dependent manner. Knockdown of periostin expression does not seem to alter the proportion of CD44+/CD24- cells but it does influence their ability to grow as mammospheres and the activity of aldehyde dehydrogenase (ALDH), another marker of cancer stem cells. Finally, as periostin is a TGF-beta inducible gene, we are currently testing the role of periostin in TGF-beta induced EMT and have seen evidence for an attenuation of this process. Ongoing studies are aimed at characterizing the intracellular pathways regulated by periostin and determining the effect of periostin expression on chemotherapeutic resistance and metastasis, two properties which may be enhanced by the presence of cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5191. doi:1538-7445.AM2012-5191

Chemotherapy-Associated Angiogenesis in Neuroblastoma Tumors

The influences of cytotoxic drugs on endothelial cells remain incompletely understood. Herein, we examined the effects of chemotherapeutic agents in experimental angiogenesis models and analyzed vessel densities in clinical neuroblastoma tumor samples. Cisplatin (20 to 500 ng/mL), doxorubicin (4 to 100 ng/mL), and vincristine (0.5 to 4 ng/mL), drugs commonly involved in neuroblastoma therapy protocols, induced pro-angiogenic effects in different angiogenesis models. They enhanced endothelial cell tube formation, endothelial cell sprouting from spheroids, formation of tip cells in the sprouting assay, expression of αvβ3 integrin, and vitronectin binding. All three drugs increased global cellular kinase phosphorylation levels, including the angiogenesis-relevant molecules protein kinase Cβ and Akt. Pharmacological inhibition of protein kinase Cβ or Akt upstream of phosphatidylinositol 3-kinase reduced chemotherapy-induced endothelial cell tube formation. Moreover, the investigated chemotherapeutics dose dependently induced vessel formation in the chick chorioallantoic membrane assay. Tumor samples from seven high-risk patients with neuroblastoma were analyzed for vessel density by IHC. Results revealed that neuroblastoma samples taken after chemotherapy consistently showed an enhanced microvessel density compared with the corresponding samples taken before chemotherapy. In conclusion, our data show that chemotherapy can activate endothelial cells by inducing multiple pro-angiogenic signaling pathways and exert pro-angiogenic effects in vitro and in vivo. Moreover, we report a previously unrecognized clinical phenomenon that might, in part, be explained by our experimental observations: chemotherapy-associated enhanced vessel formation in tumors from patients with neuroblastoma.

Targeted Deletion of Jun/AP-1 in Alveolar Epithelial Cells Causes Progressive Emphysema and Worsens Cigarette Smoke–Induced Lung Inflammation

Chronic obstructive pulmonary disease appears to occur slowly and progressively over many years, with both genetic factors and environmental modifiers contributing to its pathogenesis. Although the c-Jun/activator protein 1 transcriptional factor regulates cell proliferation, apoptosis, and inflammatory responses, its role in lung pathogenesis is largely unknown. In this study, we report decreased expression levels of c-Jun mRNA and protein in the lung tissues of patients with advanced chronic obstructive pulmonary disease, and the genetic deletion of c-Jun specifically in alveolar epithelial cells causes progressive emphysema with lung inflammation and alveolar air space enlargement, which are cardinal features of emphysema. Although mice lacking c-Jun specifically in lung alveolar epithelial cells appear normal at the age of 6 weeks, when exposed to long-term cigarette smoke, c-Jun-mutant mice display more lung inflammation with perivascular and peribronchiolar infiltrates compared with controls. These results demonstrate that the c-Jun/activator protein 1 pathway is critical for maintaining lung alveolar cell homeostasis and that loss of its expression can contribute to lung inflammation and progressive emphysema.

Staphylococcal enterotoxin B compromises the immune tolerant status in the airway mucosa

The breakdown of immune tolerance plays a critical role in allergic disorders; the mechanism of breaching immune tolerance remains largely unknown. The present study aimed to investigate the role of Staphylococcal enterotoxin B (SEB) in the interference of the immune tolerance in the nasal mucosa. The immune tolerant components, tolerogenic dendritic cells (TolDC) and regulatory T cells (Treg), were assessed in the surgically removed nasal mucosa from patients with allergic rhinitis (AR) or non-AR chronic rhinitis. The contents of SEB and integrin alphavbeta6 (avb6) in the nasal epithelium were assessed using enzyme-linked immunoassay. The ability of avb6 on TolDC induction and the effect of SEB on suppression of avb6 in nasal epithelial cells were observed in cell culture. Compared with that in the non-AR nasal mucosa, the frequencies of TolDCs/Tregs were lower, the contents of SEB were higher and the contents of avb6 were lower in the AR nasal mucosa. Avb6 had the ability to induce the development of TolDCs in vitro; the latter had the ability to induce Treg development. The expression of avb6 was detected in nasal epithelial cells in culture that could be suppressed by SEB. The components of immune tolerance machinery, TolDCs and Tregs were suppressed in the AR nasal mucosa. The increases in SEB and decreases in avb6 in nasal epithelium are associated with the compromises of immune tolerance in the nasal mucosa. SEB has the ability to suppress the expression of avb6 in nasal epithelial cells.

Integrin alphavbeta6 promotes tumor tolerance in colorectal cancer.

Tumor immune tolerance plays a critical role in tumor cell survival; the establishment of tumor immune tolerance is incompletely understood yet. Integrin alphavbeta6 (avb6) is involved in tumor growth and metastasis. This study aimed to observe the effect of avb6 on the development of tumor tolerance in colorectal cancer (CRC). In this study, 28 CRC patients were recruited. The frequencies of tolerogenic dendritic cells (TolDC), regulatory T cells (Treg), and CD8+ T cells in surgically removed CRC tissue were assessed by flow cytometry. The levels of avb6 in CRC tissue were measured by enzyme-linked immunoassay (ELISA). The effect of avb6 on inducing TolDCs and Tregs was evaluated with the cell culture model. The results showed that in surgically removed CRC tissue, we detected higher frequencies of TolDC and Tregs, lower frequency CD8+ T cells and high levels of avb6 as compared with non-CRC tissue. CRC protein extracts could induce TolDC development that could be blocked by anti-avb6 antibody. CRC-derived DCs could convert naïve CD4+ T cells to Tregs. Peripheral CD8+ T cells from CRC patients still retained the ability to produce granzyme B and to proliferate in response to CRC tumor antigen in culture that was abolished by the presence of CRC-derived Tregs. We conclude that CRC-derived avb6 is involved in the establishment of tumor immune tolerance in local tissues.

Uterine Histotroph and Conceptus Development: Select Nutrients and Secreted Phosphoprotein 1 Affect Mechanistic Target of Rapamycin Cell Signaling in Ewes1

Interferon tau (IFNT), the pregnancy recognition signal in ruminants, abrogates the uterine luteolytic mechanism to ensure maintenance of function for the corpora lutea to produce progesterone (P4). IFNT also suppresses expression of classical IFN-stimulated genes by uterine lumenal epithelium (LE) and superficial glandular (sGE) epithelium but, acting in concert with progesterone, affects expression of a multitude of genes critical to growth and development of the conceptus. The LE and sGE secrete proteins and transport nutrients into the uterine lumen necessary for conceptus development, pregnancy recognition signaling, and implantation. Secretions include arginine and secreted phosphoprotein 1 (SPP1). Arginine can be metabolized to nitric oxide and to polyamines or act directly to activate the mechanistic target of rapamycin cell signaling pathway to stimulate proliferation, migration, and mRNA translation in trophectoderm cells. SPP1 binds alphavbeta3 and alpha5beta1 integrins to induce focal adhesion assembly, adhesion, and migration of conceptus trophectoderm cells during implantation. Thus, arginine and SPP1 mediate growth, migration, cytoskeletal remodeling, and adhesion of trophectoderm essential for pregnancy recognition signaling and implantation. This minireview focuses on components of histotroph that affect conceptus development in the ewe.

Adult Mesenchymal Stem Cells and Cell Surface Characterization - A Systematic Review of the Literature

Human adult mesenchymal stem cells (MSCs) were first identified by Friedenstein et al. when observing a group of cells that developed into fibroblastic colony forming cells (CFU-F). Ever since, the therapeutic uses and clinical applications of these cells have increased research and interest in this field. MSCs have the potential to be used in tissue engineering, gene therapy, transplants and tissue injuries. However, identifying these cells can be a challenge. Moreover, there are no articles bringing together and summarizing the cell surface markers of MSCs in adults. The purpose of this study is to summarize all the available information about the cell surface characterization of adult human MSCs by identifying and evaluating all the published literature in this field. We have found that the most commonly reported positive markers are CD105, CD90, CD44, CD73, CD29, CD13, CD34, CD146, CD106, CD54 and CD166. The most frequently reported negative markers are CD34, CD14, CD45, CD11b, CD49d, CD106, CD10 and CD31. A number of other cell surface markers including STRO-1, SH2, SH3, SH4, HLA-A, HLA-B, HLA-C, HLA-DR, HLA-I, DP, EMA, DQ (MHC Class II), CDIO5, Oct 4, Oct 4A, Nanog, Sox-2, TERT, Stat-3, fibroblast surface antigen, smooth muscle alpha-actin, vimentin, integrin subunits alpha4, alpha5, beta1, integrins alphavbeta3 and alphavbeta5 and ICAM-1 have also been reported. Nevertheless, there is great discrepancy and inconsistency concerning the information available on the cell surface profile of adult MSCs and we suggest that further research is needed in this field to overcome the problem.

Identification and adhesion experiment of microbubbles targeted to angiogenesis

Objective To identify microbubbles targeted (MBt) to alpha(v)beta(3) (αvβ3) via biotin-avidin bridge and evaluate the adhesion to human umbilical vein endothelial cells (HUVECs) in vitro.Methods MBt produced via biotin-avidin bridge were validated using fluorescence in vitro.Adhesion of αvβ3-integrin targeted MBt (MBαvβ3) to HUVECs was tested using the parallel plate flow chamber (PPFC) test.Results Bright green fluorescence was observed on the biotinylated microbubbles(MBB) incubated with fluorescein isothiocyanate labeled streptavidin (FITC-SA) and on MBB-SA incubated with FITC labeled biotin.There was no fluorescence seen on non-targeted control microbubbles,MBB incubated with FITC labeled protein A and MBB-SA incubated with FITC labeled protein A. The adherent rate of MBαvβ3 was significantly higher than MBt with non-specific antibody (MBN) in PPFC test,with 9.9±3.1 of MBαvβ3 and 0.8±0.3 of MBN adhered to HUVECs,respectively(P<0.05).Conclusions Avβ3 targeted microbubbles using biotin-avidin bridging method is highly efficient and reliable for HUVECs. Key words: Ultrasonography; Microbubbles; Integrin alphavbeta 3; Endothelial cells

Isthmin exerts pro-survival and death-promoting effect on endothelial cells through alphavbeta5 integrin depending on its physical state

Isthmin (ISM) is a 60 kDa secreted-angiogenesis inhibitor that suppresses tumor growth in mouse and disrupts vessel patterning in zebrafish embryos. It selectively binds to alphavbeta5 (αvβ5) integrin on the surface of endothelial cells (ECs), but the mechanism of its antiangiogenic action remains unknown. In this work, we establish that soluble ISM suppresses in vitro angiogenesis and induces EC apoptosis by interacting with its cell surface receptor αvβ5 integrin through a novel ‘RKD' motif localized within its adhesion-associated domain in MUC4 and other proteins domain. ISM induces EC apoptosis through integrin-mediated death (IMD) by direct recruitment and activation of caspase-8 without causing anoikis. On the other hand, immobilized ISM loses its antiangiogenic function and instead promotes EC adhesion, survival and migration through αvβ5 integrin by activating focal adhesion kinase (FAK). ISM unexpectedly has both a pro-survival and death-promoting effect on ECs depending on its physical state. This dual function of a single antiangiogenic protein may impact its antiangiogenic efficacy in vivo.

Abstract 424: Interference with alphaVbeta3 integrins via cyclic RGD peptides affects breast cancer cell behavior

Abstract Integrin alphavbeta3 is mainly expressed in stimulated endothelial cells, thus it has recently become the focus of intense research mainly to interfere with tumorangiogenesis. However, alphavbeta3 integrins are also expressed in a variety of tumor cells, such as melanoma, prostate cancer, breast cancer, cervix carcinoma or pancreatic cancer. While inhibition of integrin alphavbeta3 activity in endothelial cells has been studied extensively, any direct effects on integrin alphavbeta3 expressing tumor cells is hitherto unknown. In this study we examined alphavbeta3 integrin expression in breast cancer cell lines, where by MDA MB 231 revealed high integrin alphavbeta3 as well as VEGFR-2 and VEGF expression. While adhesion of MDA MB231 cells to an alphavbeta3 specific matrix such as fibrinogen was blocked by the addition of cyclic RGD peptides, sequentially addition of cyclic RGD peptides to already adherent MDA MB231 cells led to a diminished spreading in a time and dose dependent manner, starting at 20nM. Furthermore, tumor cell migration as well as transmigration through an EC monolayer upon VEGF stimulation was blocked whenever alphavbeta3 integrins were targeted by RGD mimetic peptide. Our data give first functional insights into the previously described effects of alphavbeta3 targeting small molecules, which led to a reduced breast cancer metastasis in vivo. Thus interfering with alphavbeta3 integrins on tumor cells might represent a promising therapeutic lead in certain malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 424. doi:10.1158/1538-7445.AM2011-424