Anti-integrin αv therapy improves cardiac fibrosis after myocardial infarction by blunting cardiac PW1+ stromal cells.

There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1+ cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1+ cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1+ cells (93% ± 1%), predominantly as the αVß1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFß at the surface of cardiac PW1+ cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1+CD51+ cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.

Pivotal role for αV integrins in sustained Tfh support of the germinal center response for long-lived plasma cell generation.

CD4+ follicular helper T cells (Tfh) are essential for germinal center (GC) reactions in the lymph node that generate high-affinity, long-lived plasma cells (LLPCs). Temporal GC analysis suggests B memory cells (Bmem) are generated early, while LLPCs are generated late in the GC reaction. Distinct roles for Tfh at these temporally different stages are not yet clear. Tfh entry into the GC is highly dynamic and the signals that maintain Tfh within the GC for support of late LLPC production are poorly understood. The GC is marked by inflammation-induced presentation of specific ECM components. To determine if T cell recognition of these ECM components played a role in Tfh support of the GC, we immunized mice with a T cell-restricted deletion of the ECM-binding integrin αV (αV-CD4 cKO). T cell integrin αV deletion led to a striking defect in the number and size of the GCs following immunization with OVA protein in complete Freund's adjuvant. The GC defect was not due to integrin αV deficiency impeding Tfh generation or follicle entry or the ability of αV-CD4 cKO Tfh to contact and support B cell activation. Instead, integrin αV was essential for T cell-intrinsic accumulation within the GC. Altered Tfh positioning resulted in lower-affinity antibodies and a dramatic loss of LLPCs. Influenza A infection revealed that αV integrin was not required for Tfh support of Bmem but was essential for Tfh support of LLPCs. We highlight an αV integrin-ECM-guided mechanism of Tfh GC accumulation that selectively impacts GC output of LLPCs but not Bmem.

Transforming growth factor (TGF) ß1 acted through miR-130b to increase integrin α5 to promote migration of colorectal cancer cells.

Transforming growth factor (TGF)-ß1 is a significant stimulator of tumor invasion and metastasis. More recently, it has been found that TGF-ß1 acts through microRNAs to regulate their target genes to promote cancer progresses. However, such similar regulation is rarely reported in colorectal cancer (CRC). Here, we observed a decrease in TGF-ß1 expression in CRC specimens, compared with matched adjacent normal tissues. In parallel, there was an increase in miR-130b characterized in the same samples by microarray assay. Further, treatment of CRC cells with TGF-ß1 caused a significant decrease in the expression of miR-130b and an increased CRC cell migration. Luciferase reporter assay revealed that miR-130b directly targeted the 3' untranslated region (3'UTR) region of integrin α5 gene, which encodes a key molecule involved in cell motility. Subsequently, in the overexpression of miR-130b CRC cells, we observed a decreased level of integrin α5 protein. The regulation of integrin α5 by miR-130b was further shown using the miR-130b mimics and inhibitor of miR-130b. And, knockdown miR-130b with inhibitor in the overexpression of miR-130b CRC cells recovered integrin α5 expression and integrin α5-mediated cell motility. Moreover, the inverse relevance between miR-130b and integrin α5 was also observed in CRC specimens. At last, the enhancement of integrin α5 in TGF-ß1-treated cells can be reversed partly when rescuing miR-130b expression. Together, our findings suggested that TGF-ß1 acted through miR-130b to promote integrin α5 expression, resulting in the enhanced migration of CRC cells.

Loss of epithelial p53 and αv integrin cooperate through Akt to induce squamous cell carcinoma yet prevent remodeling of the tumor microenvironment.

Most of the squamous cell carcinomas (SCCs) of the skin and head and neck contain p53 mutations. The presence of p53 mutations in premalignant lesions suggests that they represent early events during tumor progression and additional alterations may be required for SCC development. Here we show that codeletion of the p53 and αv integrin genes in mouse stratified epithelia induced SCCs in 100% of the mice, more frequently and with much shorter latency than deletion of either gene alone. The SCCs that lacked p53 and αv in the epithelial tumor cells exhibited high Akt activity, lacked multiple types of infiltrating immune cells, contained a defective vasculature and grew slower than tumors that expressed p53 or αv. These results reveal that loss of αv in epithelial cells that lack p53 promotes SCC development, but also prevents remodeling of the tumor microenvironment and delays tumor growth. We observed that Akt inactivation in SCC cells that lack p53 and αv promoted anoikis. Thus, tumors may arise in these mice as a result of the increased cell survival induced by Akt activation triggered by loss of αv and p53, and by the defective recruitment of immune cells to these tumors, which may allow immune evasion. However, the defective vasculature and lack of a supportive stroma create a restrictive microenvironment in these SCCs that slows their growth. These mechanisms may underlie the rapid onset and slow growth of SCCs that lack p53 and αv.

Targeting of alpha-v integrins reduces malignancy of bladder carcinoma.

Low survival rates of metastatic cancers emphasize the need for a drug that can prevent and/or treat metastatic cancer. αv integrins are involved in essential processes for tumor growth and metastasis and targeting of αv integrins has been shown to decrease angiogenesis, tumor growth and metastasis. In this study, the role of αv integrin and its potential as a drug target in bladder cancer was investigated. Treatment with an αv integrin antagonist as well as knockdown of αv integrin in the bladder carcinoma cell lines, resulted in reduced malignancy in vitro, as illustrated by decreased proliferative, migratory and clonogenic capacity. The CDH1/CDH2 ratio increased, indicating a shift towards a more epithelial phenotype. This shift appeared to be associated with downregulation of EMT-inducing transcription factors including SNAI2. The expression levels of the self-renewal genes NANOG and BMI1 decreased as well as the number of cells with high Aldehyde Dehydrogenase activity. In addition, self-renewal ability decreased as measured with the urosphere assay. In line with these observations, knockdown or treatment of αv integrins resulted in decreased metastatic growth in preclinical in vivo models as assessed by bioluminescence imaging. In conclusion, we show that αv integrins are involved in migration, EMT and maintenance of Aldehyde Dehydrogenase activity in bladder cancer cells. Targeting of αv integrins might be a promising approach for treatment and/or prevention of metastatic bladder cancer.

The role of integrin αv in proliferation and differentiation of human dental pulp cell response to calcium silicate cement.

INTRODUCTION: It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and ß-tricalcium phosphate (TCP) cement. METHODS: In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed. RESULTS: The results indicate that CS releases Si ion-increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. CONCLUSIONS: These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface.

Characterization of a novel periodontal ligament-specific periostin isoform.

Periostin is a mesenchymal cell marker predominantly expressed in collagen-rich fibrous connective tissues, including heart valves, tendons, perichondrium, periosteum, and periodontal ligament (PDL). Knockdown of periostin expression in mice results in early-onset periodontitis and failure of cardiac healing after acute myocardial infarction, suggesting that periostin is essential for connective tissue homeostasis and regeneration. However, its role(s) in periodontal tissues has not yet been fully defined. In this study, we describe a novel human isoform of periostin (PDL-POSTN). Isoform-specific analysis by reverse-transcription polymerase chain-reaction (RT-PCR) revealed that PDL-POSTN was predominantly expressed in the PDL, with much lower expression in other tissues and organs. A PDL cell line transfected with PDL-POSTN showed enhanced alkaline phosphatase (ALPase) activity and calcified nodule formation, compared with cells transfected with the full-length periostin isoform. A neutralizing antibody against integrin-αv inhibited both ALPase activity and calcified nodule formation in cells transfected with PDL-POSTN. Furthermore, co-immunoprecipitation assays revealed that PDL-POSTN bound to integrin αvß3 more strongly than the common isoform of periostin, resulting in strong activation of the integrin αvß3-focal adhesion kinase (FAK) signaling pathway. These results suggest that PDL-POSTN positively regulates cytodifferentiation and mineralization in PDL cells through integrin αvß3.

Ascites-induced shift along epithelial-mesenchymal spectrum in ovarian cancer cells: enhancement of their invasive behavior partly dependant on αv integrins.

At least one-third of patients with epithelial ovarian cancer (OC) present ascites at diagnosis and almost all have ascites at recurrence. The presence of ascites, which acts as a dynamic reservoir of active molecules and cellular components, correlates with the OC peritoneal metastasis and is associated with poor prognosis. Since epithelial-mesenchymal transition (EMT) is involved in different phases of OC progression, we have investigated the effect of the unique ascitic tumor microenvironment on the EMT status and the behavior of OC cells. The exposure of three OC cell lines to ascites leads to changes in cellular morphologies. Within ascites, OC cells harboring an initial intermediate epithelial phenotype are characterized by marked dislocation of epithelial markers (E-cadherin, ZO-1 staining) while OC cells initially harboring an intermediate mesenchymal phenotype strengthen their mesenchymal markers (N-cadherin, vimentin). Ascites differentially triggers a dissemination phenotype related to the initial cell features by either allowing the proliferation and the formation of spheroids and the extension of colonies for cells that present an initial epithelial intermediate phenotype, or favoring the migration of cells with a mesenchymal intermediate phenotype. In an ascitic microenvironment, a redeployment of αv integrins into cells was observed and the ascites-induced accentuation of the two different invasive phenotypes (i.e. spheroids formation or migration) was shown to involve αv integrins. Thus, ascites induces a shift toward an unstable intermediate state of the epithelial-mesenchymal spectrum and confers a more aggressive cell behavior that takes on a different pathway based on the initial epithelial-mesenchymal cell features.

Integrin αv in the mechanical response of osteoblast lineage cells.

Although osteoblast lineage cells, especially osteocytes, are thought to be a primary mechanosensory cell in bone, the identity of the mechano-receptor and downstream mechano-signaling pathways remain largely unknown. Here we show using osteoblastic cell model of mechanical stimulation with fluid shear stress that in the absence of integrin αv, phosphorylation of the Src substrate p130Cas and JNK was impaired, culminating in an inhibition of nuclear translocation of YAP/TAZ and subsequent transcriptional activation of target genes. Targeted deletion of the integrin αv in osteoblast lineage cells results in an attenuated response to mechanical loading in terms of Sost gene expression, indicative of a role for integrin αv in mechanoreception in vivo. Thus, integrin αv may be integral to a mechanosensing machinery in osteoblastic cells and involved in activation of a Src-JNK-YAP/TAZ pathway in response to mechanical stimulation.

Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers.

Integrin αv mediates contractility whereas integrin α4 regulates proliferation of human bladder smooth muscle cells via FAK pathway under physiological stretch.

PURPOSE: The requirement of integrins for mechanotransduction has been recognized for some time. We investigated the role of integrin subunits and their pathway in the physiological stretch induced contractility and proliferation of human bladder smooth muscle cells. MATERIALS AND METHODS: Human bladder smooth muscle cells were seeded on silicone membrane and subjected to stretch, simulating bladder cycles of various stretches and times, as controlled by customized software on a modified BioDynamic bioreactor. Cell proliferation, viability and cycle were determined by BrdU incorporation assay, the Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Haimen, People's Republic of China) and flow cytometry, respectively. Cell contractility was determined using a collagen gel contraction assay. RESULTS: Physiological stretch increased cell contractility, proliferation and viability. Knockdown of integrin αv but not α4 in the cells disrupted the enhanced contractility induced by stretch. Under physiological stretch conditions, the integrin αv level and phospho-FAK/FAK ratio correlated positively with cell stretch induced enhanced contractility. Further examination revealed that contractile marker expression was associated with integrin αv activation through the FAK pathway. At the same time integrin α4 but not integrin αv mediated stretch induced cell proliferation and viability. CONCLUSIONS: These data revealed that different integrins have different roles in the contractility and proliferation of human bladder smooth muscle cells under physiological stretch. This suggests that different integrins may become specific therapeutic targets in patients with voiding dysfunction. They may also be used to design a specific microenvironment for optimal bladder tissue regeneration.

αV integrin induces multicellular radioresistance in human nasopharyngeal carcinoma via activating SAPK/JNK pathway.

BACKGROUND: Tumor cells acquire the capacity of resistance to chemotherapy or radiotherapy via cell-matrix and cell-cell crosstalk. Integrins are the most important cell adhesion molecules, in which αV integrin mainly mediating the tight contact between tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of αV integrin in multi-cellular radioresistance (MCR) of human nasopharyngeal carcinoma (NPC), we performed immunohistochemistry and Western blotting to find that the expression of αV integrin in the tumor tissue of radioresistant patients is much higher than that in radiosensitive patients. In vitro, we cultured human NPC cell line CNE-2 cells as multi-cellular spheroids (MCSs) or as monolayer cells (MCs), and found that the expression of αV integrin in MCSs is significantly higher than that in MCs. MTT, flow cytometry and clonogenic survival assays showed that MCSs are less sensitive to X-ray irradiation than MCs while blocking of αV integrin in MCSs dramatically reversed their radioresistance. Furthermore, as detected by Western blotting, MCSs displayed sustained activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway in presence of irradiation. Blocking of αV integrin in MCSs decreased the expression of phosphorylated JNK. Additionally, blocking of SAPK/JNK signaling pathway synergistically induced apoptosis of MCSs exposed to irradiation by increasing the expression of cleaved caspase-3. In vivo, we found that irradiation combined with αV integrin blocking treatment significantly enhanced the radiosensitivity of NPC xenografts. CONCLUSIONS: Our results indicate a novel role of αV integrin in multi-cellular radioresistance of NPCs.

Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits.

Hydroxyapatite (HA) has been widely used as a bone substitute in dental, maxillofacial and orthopaedic surgery and as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. The aim of the present study was to investigate the osteoblastic adhesion strength on HA substrata with different surface topography and biochemistry (pre-adsorption of fibronectin) after blocking of specific integrin subunits with monoclonal antibodies. Stoichiometric HA was prepared by precipitation followed by ageing and characterized by SEM, EDX, powder XRD, Raman spectroscopy, TGA, and specific surface area analysis. Human bone marrow derived osteoblasts were cultured on HA disc-shaped substrata which were sintered and polished resulting in two surface roughness grades. For attachment evaluation, cells were incubated with monoclonal antibodies and seeded for 2 h on the substrata. Cell detachment strength was determined using a rotating disc device. Cell detachment strength was surface roughness, fibronectin preadsorption and intergin subunit sensitive.

[Chondrocyte metabolism may be regulated through the interaction with the extracellular matrix].

Articular chondrocytes are the sole type of cell that resides within articular cartilage. The chondrocytes are very sensitive to the matrix around the cells. This is best illustrated by the dramatic change in cell shape and metabolism that occurs during monolayer culture. Although this change, or dedifferentiation, has been known for decades, an underlying mechanism (s) for the change has not yet been determined. Recently, we have reported thatαvß5 integrin is significantly involved in the change through the activation of ERK signaling. This review describes our recent findings about the dedifferentiation of monolayer-cultured chondrocytes.

SPARC promotes pericyte recruitment via inhibition of endoglin-dependent TGF-ß1 activity.

Pericytes migrate to nascent vessels and promote vessel stability. Recently, we reported that secreted protein acidic and rich in cysteine (SPARC)-deficient mice exhibited decreased pericyte-associated vessels in an orthotopic model of pancreatic cancer, suggesting that SPARC influences pericyte behavior. In this paper, we report that SPARC promotes pericyte migration by regulating the function of endoglin, a TGF-ß1 accessory receptor. Primary SPARC-deficient pericytes exhibited increased basal TGF-ß1 activity and decreased cell migration, an effect blocked by inhibiting TGF-ß1. Furthermore, TGF-ß-mediated inhibition of pericyte migration was dependent on endoglin and αV integrin. SPARC interacted directly with endoglin and reduced endoglin interaction with αV integrin. SPARC deficiency resulted in endoglin-mediated blockade of pericyte migration, aberrant association of endoglin in focal complexes, an increase in αV integrins present in endoglin immunoprecipitates, and enhanced αV integrin-mediated activation of TGF-ß. These results demonstrate that SPARC promotes pericyte migration by diminishing TGF-ß activity and identify a novel function for endoglin in controlling pericyte behavior.

Alpha (v) integrins license regulatory T cells to apoptotic cells and self-associated antigens.

Defects in apoptotic cell clearance are thought to contribute to autoimmunity by failure to induce tolerance, coupled with accumulation of immunogenic material. However, little is known about the contribution of apoptosis to immune responses at mucosal sites, where regulatory T cells (T(reg) cells) and other immune cells play an essential active role in maintaining tolerance to self-associated antigens. In recent studies, we have found that α(v) integrins have an important role in apoptotic cell phagocytosis and induction of T(reg) cells in the intestine, and deletion of α(v) from myeloid cells causes colitis associated with failed apoptotic cell removal and loss of T(reg) cells. Our data show that activation of transforming growth factor (TGF)-ß by α(v) ß(8) on dendritic cells (DCs) is essential for generating T(reg) cells and inducing mucosal tolerance. These results provide a mechanism by which tolerance to apoptotic cell-derived and -associated antigens is maintained by DC "licensing" at sites of high TGF-ß expression.

JNK mediates insulin-like growth factor binding protein 2/integrin alpha5-dependent glioma cell migration.

We have previously shown that a molecular interaction between insulin-like growth factor binding protein 2 (IGFBP2) and integrin alpha5 is necessary for the enhancement of cell migration in IGFBP2-overexpressing gliomas. In the present study, we examined the mechanism through which the IGFBP2/integrin alpha5 interaction mediates enhanced glioma cell migration. Although both ERK and JNK MAP kinases were activated, JNK was specifically involved in IGFBP2-mediated migration as shown by inhibitor analysis of IGFBP2-overexpressing cells. Because gliomas are solid tumors that require contact with a surface (e.g., other cells, extracellular matrix) for migration, we used the extracellular matrix (ECM) protein fibronectin, which is the sole ligand of the alpha5beta1 integrin receptor, to show that integrin alpha5 is an important mediator of JNK activation. In addition, we found the IGFBP2/integrin alpha5 pathway to be activated in a significantly shorter interval in cells seeded onto fibronectin-coated surfaces compared to cells seeded onto plastic alone. The activation of JNK was downstream of the IGFBP2/integrin alpha5 interaction, as shown by alpha5 knockdown experiments using IGFBP2-overexpressing cells. Based on these data we propose that the interaction between IGFBP2 and integrin alpha5 accelerates cell adhesion, and this, in turn, enhances JNK-mediated glioma cell migration.

Endothelial alpha5 and alphav integrins cooperate in remodeling of the vasculature during development.

Integrin cell adhesion receptors and fibronectin, one of their extracellular matrix ligands, have been demonstrated to be important for angiogenesis using functional perturbation studies and complete knockout mouse models. Here, we report on the roles of the alpha5 and alphav integrins, which are the major endothelial fibronectin receptors, in developmental angiogenesis. We generated an integrin alpha5-floxed mouse line and ablated alpha5 integrin in endothelial cells. Unexpectedly, endothelial-specific knockout of integrin alpha5 has no obvious effect on developmental angiogenesis. We provide evidence for genetic interaction between mutations in integrin alpha5 and alphav and for overlapping functions and compensation between these integrins and perhaps others. Nonetheless, in embryos lacking both alpha5 and alphav integrins in their endothelial cells, initial vasculogenesis and angiogenesis proceed normally, at least up to E11.5, including the formation of apparently normal embryonic vasculature and development of the branchial arches. However, in the absence of endothelial alpha5 and alphav integrins, but not of either alone, there are extensive defects in remodeling of the great vessels and heart resulting in death at ~E14.5. We also found that fibronectin assembly is somewhat affected in integrin alpha5 knockout endothelial cells and markedly reduced in integrin alpha5/alphav double-knockout endothelial cell lines. Therefore, neither alpha5 nor alphav integrins are required in endothelial cells for initial vasculogenesis and angiogenesis, although they are required for remodeling of the heart and great vessels. These integrins on other cells, and/or other integrins on endothelial cells, might contribute to fibronectin assembly and vascular development.

Initial formation of IGROV1 ovarian cancer multicellular aggregates involves vitronectin.

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/alphav integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and alphav integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and alphav integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-alphav integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/alphav integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.

Vitronectin receptors, alpha v integrins, are recognized by several non-RGD-containing echoviruses in a continuous laboratory cell line and also in primary human Langerhans' islets and endothelial cells.

Previously published data suggest that the RGD-recognizing integrin, alphavbeta3, known as the vitronectin receptor, acts as a cellular receptor for RGD-containing enteroviruses, coxsackievirus A9 (CAV-9) and echovirus 9 (E-9), in several continuous cell lines as well as in primary human Langerhans' islets. As this receptor is also capable of binding the ligands by a non-RGD-dependent mechanism, we investigated whether vitronectin receptors, alpha v integrins, might act as receptors for other echoviruses that do not have the RGD motif. Blocking experiments with polyclonal anti-alphavbeta3 antibody showed that both primary human islets and a continuous laboratory cell line of green monkey kidney origin (GMK) are protected similarly from the adverse effects of several non-RGD-containing echovirus (E-7, -11, -25, -30, -32) infections. In contrast, corresponding studies on primary human endothelial cells showed that the receptor works only for E-25, E-30, E-32 and CAV-9. The inhibitory effect of the antibody was not restricted to prototype strains of echoviruses, as GMK cells infected with several field isolates of the corresponding serotypes were also protected from virus-induced cytopathic effects. Co-localization of virus particles with the receptor molecules in both GMK and primary human endothelial cells was demonstrated by live-cell stainings and confocal microscopy. Remarkably, in spite of similar virus-receptor co-localization and a comparable protective effect of the alphavbeta3 antibody, the entry pathways of the studied virus strains seemed to be divergent.