Upregulation of the N-formyl Peptide receptors in scleroderma fibroblasts fosters the switch to myofibroblasts.

Systemic sclerosis (SSc) is characterized by chronic inflammation and fibrosis. N-Formyl peptide (fMLF) receptors (FPRs) are chemotactic receptors involved in inflammation. Three FPRs have been identified: FPR1, FPR2, and FPR3. We have examined, by RT-PCR, Western blot and immunohistochemistry, FPRs expression in skin fibroblasts from 10 normal subjects and 10 SSc patients, showing increased expression in SSc fibroblasts. Several functions of FPRs occur through the interaction with a region of the urokinase-type plasminogen activator receptor (uPAR88-92), able to interact with FPRs and to mediate urokinase (uPA) or fMLF-dependent cell migration. Soluble uPAR84-95 peptide can act as a direct ligand of FPRs. Furthermore, uPA or its aminoterminal fragment (ATF) can promote the exposure of the uPAR88-92 region. The WKYMVm peptide is a FPRs pan-agonist. We investigated the functional effects of these agonists on normal and SSc fibroblasts. ATF, uPAR84-95, and WKYMVm regulated adhesion, migration, and proliferation of normal fibroblasts. Despite FPR overexpression, the response of SSc fibroblasts to the same agonists was greatly reduced, except for the proliferative response to ATF. SSc fibroblasts showed increased α-smooth muscle actin expression and improved capability to induce wound closure. Indeed, they overexpressed a cleaved uPAR form, exposing the uPAR88-92 region, and vitronectin, both involved in fibrosis and in the fibroblast-to-myofibroblast transition. FPR stimulation promoted α-smooth muscle actin expression in normal fibroblasts as well as motility, matrix deposition, αvß5 integrin expression, and radical oxygen species generation in normal and SSc fibroblasts. This study provides evidence that FPRs may play a role in fibrosis and in the fibroblast-to-myofibroblast transition.

Colorectal Cancer Metastases Settle in the Hepatic Microenvironment Through α5ß1 Integrin.

Colorectal cancer (CRC) metastasis dissemination to secondary sites represents the critical point for the patient's survival. The microenvironment is crucial to cancer progression, influencing tumour cell behaviour by modulating the expression and activation of molecules such as integrins, the cell-extracellular matrix interacting proteins participating in different steps of the tumour metastatic process. In this work, we investigated the role of α5ß1 integrin and how the microenvironment influences this adhesion molecule, in a model of colon cancer progression to the liver. The culture medium conditioned by the IHH hepatic cell line, and the extracellular matrix (ECM) proteins, modulate the activation of α5ß1 integrin in the colon cancer cell line HCT-116, and drives FAK phosphorylation during the process of cell adhesion to fibronectin, one of the main components of liver ECM. In these conditions, α5ß1 modulates the expression/activity of another integrin, α2ß1, involved in the cell adhesion to collagen I. These results suggest that α5ß1 integrin holds a leading role in HCT-116 colorectal cancer cells adhesion to the ECM through the modulation of the intracellular focal adhesion kinase FAK and the α2ß1 integrin activity. The driving role of the tumour microenvironment on CRC dissemination, here detected, and described, strengthens and adds new value to the concept that α5ß1 integrin can be an appropriate and relevant therapeutic target for the control of CRC metastases.

Integrins αvß3 and αvß5 as prognostic, diagnostic, and therapeutic targets in gastric cancer.

BACKGROUND: We investigated the expression of two αv integrins, αvß3 and αvß5, in gastric cancer (GC) by testing the following hypotheses: that these molecules are expressed in GC; that they are implicated in GC biology; that they help to distinguish between the two major histological subtypes of GC, according to Laurén; and that they are prognostically relevant. METHODS: Formalin-fixed and paraffin-embedded tissue samples from 482 GC samples were stained immunohistochemically using rabbit monoclonal antibodies directed against αvß3 (EM22703) and αvß5 (EM09902). Immunostaining of tumor, stroma, and endothelial cells was evaluated separately by the quantity and intensity, generating an immunoreactivity score. The immunoreactivity score of both antibodies was correlated with clinicopathology data and patient survival. RESULTS: Each integrin was expressed in at least one tumor component in all GCs. Both were expressed significantly more often in the intestinal phenotype according to Laurén. Moreover, patients who grouped as "positive" for expression of αvß3 on endothelial cells, and patients with an intestinal type GC, grouped as "negative" for expression of αvß5 on stroma cells, had significantly longer survival. The expression of αvß5 on stroma cells was confirmed to be an independent prognostic factor of intestinal-type GC. CONCLUSION: The expression of αvß3 and αvß5 in at least one tumor component in all GC samples is an interesting new result that should form a basis for further investigations; for example, regarding selective integrin antagonists and the value of αvß3 and αvß5 as putative prognostic biomarkers. Moreover, both markers might be helpful in the routine classification of GC subtypes.

Human limbal epithelial progenitor cells express αvß5-integrin and the interferon-inducible chemokine CXCL10/IP-10.

Stem cell (SC) therapy is the main treatment modality for patients with limbal stem cell deficiency. If limbal epithelial stem cells (LESC) can be more readily identified, isolated and maintained ex vivo, patients could be treated with better quality grafts. With prior knowledge that vitronectin (VN) is present within the LESC niche and that it supports LESC in vitro, we postulated that VN receptors (integrins αvß3/5) are expressed by, and can be used to identify and isolate LESC. Immunolocalization studies were conducted on human corneas. Corneas were also used to expand limbal epithelial cells from either biopsies or enzyme-dissociated tissue and αvß3/5 expression determined by flow cytometry. Integrin expressing cells were isolated by magnetic activated cell sorting then assessed by immunocytology, colony forming efficiency, RT-PCR and microarray analysis. Integrin αvß5(+) cells co-localized to N-cadherin(+)/CK-15(+) putative LESC. αvß5 was restricted to less than 4% of the total limbal epithelial cells, which expressed higher levels of CK-15 and formed more colonies compared to αvß5(-) cells. Transcriptional profiling of αvß5(+/-) cells by microarray identified several highly expressed interferon-inducible genes, which localize to putative LESC. Integrin αvß5 is a candidate LESC marker since its expression is restricted to the limbus and αvß5(+) limbal epithelial cells have phenotypic and functional properties of LESC. Knowledge of the niche's molecular composition and the genes expressed by its SC will facilitate isolation and maintenance of these cells for therapeutic purposes.

The PEA-15/PED protein regulates cellular survival and invasiveness in colorectal carcinomas.

The PEA-15/PED (phosphoprotein enriched in astrocytes 15kD/phosphoprotein enriched in diabetes) protein is a multifunctional phosphoprotein involved in various signaling pathways which determine survival, proliferation, and migration of cancer cells. Here, we investigated the expression and cellular functions of PEA-15 in colorectal carcinoma (CRC). PEA-15 is expressed in the majority of human CRC, predominantly in well differentiated tumor areas. A tissue microarray analysis of 1262 human CRC specimens from the DACHS study showed that PEA-15 expression is significantly associated with a low pT stadium as defined by limited invasion into the bowel wall. Moreover, patients with PEA-15-positive CRC exhibited a significantly longer tumor-specific survival time. To investigate the functional relevance of PEA-15 expression on a cellular level, we over-expressed PEA-15 in several CRC cell lines. Increased expression of PEA-15 resulted in a strong inhibition of clonogenicity, proliferation, and invasiveness of CRC cells. These effects were associated with a PEA-15-dependent down-regulation of integrin αvß5 as well as with elevated levels of the phosphorylated MAP kinase ERK1/2. Moreover, expression of PEA-15 resulted in significant protection from cell death induced by cytotoxic drugs (5-FU, cisplatin), by the death ligand TRAIL, or by serum withdrawal. In conclusion, the PEA-15 protein regulates invasiveness, proliferation, and apoptosis resistance in CRC cells. PEA-15 might play an important role in chemoresistance, progression and metastasis in CRC.

A continuous bovine kidney cell line constitutively expressing bovine αvß6 integrin has increased susceptibility to foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the αVß6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the αV and ß6 bovine integrin subunits. This stable cell line (LFBK-αVß6) showed ß6 expression and enhanced susceptibility to FMDV infection for ≥ 100 cell passages. LFBK-αVß6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK-αVß6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation.

Elevated expression of integrin αv and ß5 subunit in laryngeal squamous-cell carcinoma associated with lymphatic metastasis and angiogenesis.

In the past several years, the αv integrin subfamily has been repeatedly found to be involved in tumor progression and angiogenesis. The aim of this study was to investigate the expression of the integrin αv subfamily in laryngeal squamous cell carcinoma (LSCC), and to correlate the expression rate with tumor biological behavior and angiogenesis of the LSCC. The integrin αv subfamily, including αv, ß1, ß3, ß5, ß6 and ß8 subunits, was immunohistochemically found to be expressed in 64 patients with LSCC, and we analyzed the relationship between the expression rate and the clinicopathological stage of this cancer. Immunohistochemical staining for CD105 was carried out in the same group of the patients. The intratumoral microvessel density (IMVD) of the LSCC was calculated by CD105 staining, and the correlation between the IMVD and αv subfamily expression was discussed. The results showed that all members of the integrin αv subfamily could be detected in the LSCC. The expression rate of integrin αv and ß5 subunits in primary cancer was significantly higher than in normal tissue, and their expression rate in the group with lymphatic metastasis was significantly higher than in the group without metastasis. The IMVD of the group with positive expression of αv and ß5 subunits was significantly higher than in the group with negative expression, but there were no significant effects on the ß1, ß3, ß6 and ß8 subunits in these biological processes. In conclusion, the expressions of integrin αv and ß5 subunits were significantly associated with lymphatic metastasis and angiogenesis of the LSCC. Among the members of integrin αv subfamily, integrin αvß5 might play an important role in invasion and metastases of the LSCC, and it may become a valuable marker for the evolution of the LSCC.

Differences in integrin expression and signaling within human breast cancer cells.

BACKGROUND: Integrins are used as prognostic indicators in breast cancer. Following engagement with extracellular matrix proteins, their signaling influences numerous cellular processes including migration, proliferation, and death. Integrin signaling varies between cell types through differential expression of integrin subunits, and changes within a given cell upon exposure to a cell agonist or through changes in its surroundings. These variations in signaling can profoundly affect the phenotypic, tumorogenecity and metastatic properties of cancer cells. In the present study, we investigated if there were differences in the expression of integrins, integrin structures, and integrin co-receptors within three breast cancer cells and if these differences effected integrin signaling. METHODS: Expression of integrins, urokinase receptor and vascular endothelial cell growth factor receptor (VEGFR) in metastatic MDA-MB-435 and MDA-MB-231, non-metastatic MCF7 and non-breast cancer Hek-293 cells was measured by flow cytometry. Cell adhesion was assessed using collagen, fibrinogen, fibronectin and vitronectin coated plates. Changes in kinase levels following PMA stimulation, and cell adhesion-induced activation of kinases were determined by western blot analysis. Distribution of actin stress fibers and focal adhesions was assessed by immunocytochemistry. RESULTS: All cells expressed αv integrins, while high ß5 and αvß5 expression was restricted to the cancer cells and high ß3 and αvß3 expression was restricted to MDA-MB-435 cells. The two metastatic cells were the least adhesive, but all cells adhered well to most proteins in the absence of PMA. All proliferating cells expressed activated pSrc, but only proliferating metastatic cells expressed high pMEK levels. PMA treatment resulted in time-dependent changes in activated kinase levels, and only MDA-MB-231 cells constitutively expressed high levels of activated pMEK. MDA-MB-435 cells formed more stress fibers and focal adhesions and only exhibited adhesion-induced activation of pMEK and pFAK. All cells expressed the urokinase receptor, but MCF7 cells had markedly higher VEGFR expression. Adhesion induced differential expression of pFAK, pMEK and pERK. CONCLUSIONS: This study demonstrates that breast cancers vary in their expression of integrins, their capacity to form focal adhesion and to signal through integrins. These differences likely contribute to phenotypic variations between cancer lines and account for some of the heterogeneity of breast cancer.

The expression of receptivity markers in the fallopian tube epithelium.

Pinopodes represent the morphological and integrins, the biomolecular markers of endometrial receptivity. We studied using scanning electron microscopy, the expression of pinopodes on tubal samples and their corresponding endometria, from 21 women of reproductive age (7 from proliferative phase, 7 from day LH +5 and 7 from day LH +7). In addition, we examined the immunohistochemical staining of integrins alpha v beta 3, alpha v beta 5 and their ligands, fibronectin (FN) and osteopontin (OPN) in the same tubal epithelium samples. Pinopodes were detected on the tubal epithelium exclusively during day LH +7, coincident with their formation in the endometrium and synchronous to alpha v beta 3 sharp increase in the oviduct epithelium, suggesting a regulation similar to the endometrium. In contrast, alpha v beta 5, FN and OPN remained unchanged during the cycle. These results show for the first time the formation of pinopodes in the tubal epithelium at the time of endometrial receptivity and correlate it with the upregulation of the intact dimmer alpha v beta 3 in the tubes.

CYR61 and alphaVbeta5 integrin cooperate to promote invasion and metastasis of tumors growing in preirradiated stroma.

Radiotherapy is widely used to treat human cancer. Patients locally recurring after radiotherapy, however, have increased risk of metastatic progression and poor prognosis. The clinical management of postradiation recurrences remains an unresolved issue. Tumors growing in preirradiated tissues have an increased fraction of hypoxic cells and are more metastatic, a condition known as tumor bed effect. The transcription factor hypoxia inducible factor (HIF)-1 promotes invasion and metastasis of hypoxic tumors, but its role in the tumor bed effect has not been reported. Here, we show that tumor cells derived from SCCVII and HCT116 tumors growing in a preirradiated bed, or selected in vitro through repeated cycles of severe hypoxia, retain invasive and metastatic capacities when returned to normoxia. HIF activity, although facilitating metastatic spreading of tumors growing in a preirradiated bed, is not essential. Through gene expression profiling and gain- and loss-of-function experiments, we identified the matricellular protein CYR61 and alphaVbeta5 integrin as proteins cooperating to mediate these effects. The anti-alphaV integrin monoclonal antibody 17E6 and the small molecular alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 suppressed invasion and metastasis induced by CYR61 and attenuated metastasis of tumors growing within a preirradiated field. These results represent a conceptual advance to the understanding of the tumor bed effect and identify CYR61 and alphaVbeta5 integrin as proteins that cooperate to mediate metastasis. They also identify alphaV integrin inhibition as a potential therapeutic approach for preventing metastasis in patients at risk for postradiation recurrences.

Three-dimensional MR mapping of angiogenesis with alpha5beta1(alpha nu beta3)-targeted theranostic nanoparticles in the MDA-MB-435 xenograft mouse model.

Our objectives were 1) to characterize angiogenesis in the MDA-MB-435 xenograft mouse model with three-dimensional (3D) MR molecular imaging using alpha(5)beta(1)(RGD)- or irrelevant RGS-targeted paramagnetic nanoparticles and 2) to use MR molecular imaging to assess the antiangiogenic effectiveness of alpha(5)beta(1)(alpha(nu)beta(3))- vs. alpha(nu)beta(3)-targeted fumagillin (50 mug/kg) nanoparticles. Tumor-bearing mice were imaged with MR before and after administration of either alpha(5)beta(1)(RGD) or irrelevant RGS-paramagnetic nanoparticles. In experiment 2, mice received saline or alpha(5)beta(1)(alpha(nu)beta(3))- or alpha(nu)beta(3)-targeted fumagillin nanoparticles on days 7, 11, 15, and 19 posttumor implant. On day 22, MRI was performed using alpha(5)beta(1)(alpha(nu)beta(3))-targeted paramagnetic nanoparticles to monitor the antiangiogenic response. 3D reconstructions of alpha(5)beta(1)(RGD)-signal enhancement revealed a sparse, asymmetrical pattern of angiogenesis along the tumor periphery, which occupied <2.0% tumor surface area. alpha(5)beta(1)-targeted rhodamine nanoparticles colocalized with FITC-lectin corroborated the peripheral neovascular signal. alpha(5)beta(1)(alpha(nu)beta(3))-fumagillin nanoparticles decreased neovasculature to negligible levels relative to control; alpha(nu)beta(3)-targeted fumagillin nanoparticles were less effective (P>0.05). Reduction of angiogenesis in MDA-MB-435 tumors from low to negligible levels did not decrease tumor volume. MR molecular imaging may be useful for characterizing tumors with sparse neovasculature that are unlikely to have a reduced growth response to targeted antiangiogenic therapy.

Adenoviral receptor expression of normal bladder and transitional cell carcinoma of the bladder.

OBJECTIVE: The insertion of absent or underexpressed genes into cancer cells to alter their malignant phenotype is an important potential application of available gene therapy technology. One of the more common viral vector systems that has been extensively studied for this purpose are the replication-deficient adenoviruses (Ad). Adenoviral infection of cells is mediated through a complex pathway, initiated following viral-cell attachment. Adenoviral-cell attachment occurs following interactions with a 46-kDa transmembrane protein with high affinity for both the Coxsackie and adenovirus, designated the CAR (Coxsackie and adenoviral receptor). Additional important cell-viral interactions that occur involve the alpha(v)-based integrins, specifically alpha(v)beta3 and alpha(v)beta5. The purpose of the present study was to determine the extent of expression and localization of the known Ad receptor proteins (CAR, alpha(v)beta3, and alpha(v)beta5) in normal and cancerous human bladders. MATERIAL AND METHODS: Frozen tissue samples of normal bladder and invasive transitional cell cancers of the bladder were evaluated. Tissue blocks containing muscle-invasive transitional cell carcinoma (TCC) were obtained following radical cystectomy, which were performed at our institution. Thirty-two invasive transitional cell bladder tumors were evaluated, each with a matched sample of histologically normal-appearing bladder used as a control. Four additional samples of normal bladder were obtained from patients with no evidence of disease of the bladder and served as further controls. Three additional cases of invasive bladder cancer with no matching normal tissue were also evaluated. Identification of the CAR receptor was performed using the anti-CAR mouse monoclonal antibody designated RmBC. The integrins alpha(v)beta3 and alpha(v)beta5 were identified using the mouse monoclonal antibodies designated LM609 and P1F6 respectively. All slides were evaluated by two of the authors (M.B., B.B.) without knowledge of the clinical and pathological data. RESULTS: Normal bladder: Normal bladder mucosa demonstrated a marked positivity for CAR in 29/35 (82.8%) cases. In contrast, normal transitional epithelial cells were uniformly negative when tested for the integrins alpha(v)beta3 and alpha(v)beta5. Subepithelial tissues, specifically the connective tissue components of the lamina propria and deep muscle wall of the bladder, were positive for alpha(v)beta3 and for alpha(v)beta5 in 61 and 75% of samples, respectively. Endothelial cells associated with the various layers throughout the bladder uniformly expressed both integrins and served as a consistent internal control for both antibodies. An almost identical staining pattern of the endothelium was observed using LM609 and P1F6 in all samples tested. Bladder transitional cell carcinoma: CAR immunoreactivity against TCC cells was uniformly decreased compared to normal transitional cells. Nine tumors exhibited a weak positivity for CAR while the remaining samples were negative. In some cases, the absence of CAR positivity was associated with histological evidence of carcinoma in situ. In 6 cases, it led to the identification of small regions of carcinoma in situ that were not noted on primary pathological evaluation. Peritumoral connective tissue expressed both integrins in the majority of cases, similar to the pattern described above for normal bladder. Transitional cell cancers demonstrated a similar pattern of expression of alpha(v)beta5, in which all tumor cells exhibited minimal or no staining. CONCLUSIONS: The success of all viral-mediated gene therapy strategies relies on the ability of the vector to efficiently deliver its genetic material to a target cell population. In the current study, we demonstrate that the bladder epithelial layer consistently expresses high levels of CAR. Deeper layers of the epithelium also express CAR, including the basal layer cells. A decrease in the expression of CAR appears as an early event in bladder carcinogenesis. We observed that both alpha(v)beta3 and alpha(v)beta5 are strongly expressed in muscle cells surrounding the neoplastic cells, as well as within the peritumoral connective tissue. In cases of invasive bladder cancer that have lost CAR expression, an adenoviral vector may still be utilized through the less efficient interactions with the integrins. Bladder tumor tissue may be less susceptible to an adenoviral-mediated gene therapy approach in which a significant percentage of tumor cells require transduction. Adenoviral uptake by tumor or peritumoral cells with subsequent gene transfer could be predicted by the level of CAR and alpha(v)-based integrin expression. This would enhance our ability to identify those patients whose tumors would be more susceptible to Ad-mediated gene delivery as part of an antitumor treatment.

Novel and selective alpha(v)beta3 receptor peptide antagonist: design, synthesis, and biological behavior.

Among RGD-dependent integrins, the alpha(v)beta3 receptor has recently received increasing attention as a therapeutic target because of its critical role in tumor-induced angiogenesis and metastasis formation. Here, we describe a new peptide antagonist of alpha(v)beta3 receptor, designed on the basis of the crystal structure of integrin alpha(v)beta3 in complex with c(RGDf[NMe]V) and the NMR structure of echistatin. Cell adhesion assays have demonstrated that the peptide is a potent and selective antagonist of the alpha(v)beta3 receptor.

Efficient targeting of adenoviral vectors to integrin positive vascular cells utilizing a CAR-cyclic RGD linker protein.

Vascular smooth muscle (VSMC) and endothelial cells (EC) are particularly resistant to infection by type 5 adenovirus (Ad) vectors. To overcome this limitation and target Ad vectors to ubiquitously expressed alpha(V)beta(3/5) integrins, we have generated a linker protein consisting of the extracellular domain of the coxsackie adenovirus receptor (CAR) connected via avidin to a biotinylated cyclic (c) RGD peptide. After optimization of CAR to cRGD and to Ad coupling, infection of mouse heart endothelial cells (H5V) could be augmented significantly, as demonstrated by 600-fold increased transgene expression levels. In EOMAs, a hemangioendothelioma-derived cell line, the fraction of infected cells was enhanced 4- to 6-fold. Furthermore, the fraction of infected primary mouse VSMC was increased from virtually 0% to 25%. Finally, in human umbilical vein endothelial cells, the number of GFP positive cells was enhanced from 2% to 75%. In conclusion, CAR-cRGD is a versatile and highly efficient construct to target Ad vectors to both transformed and primary VSMC and EC.

Involvement of alphavbeta5 integrin-mediated activation of latent transforming growth factor beta1 in autocrine transforming growth factor beta signaling in systemic sclerosis fibroblasts.

OBJECTIVE: To confirm the involvement of alphavbeta5 in the self-activation system in systemic sclerosis (SSc) fibroblasts. METHODS: Levels of alphavbeta5 expression were analyzed by immunoprecipitation. The promoter activity of the human alpha2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor beta (TGFbeta) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis. RESULTS: Levels of alphavbeta5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-alphavbeta5 antibody or beta5 antisense oligonucleotide significantly reduced human alpha2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFbeta1 antisense oligonucleotide, the exogenous latent TGFbeta1 stimulation significantly increased human alpha2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-alphavbeta5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with beta5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of SSc fibroblasts. CONCLUSION: Up-regulated expression of alphavbeta5 contributes to the establishment of autocrine TGFbeta signaling in SSc fibroblasts through activation of endogenous latent TGFbeta1.

Switch from alphavbeta5 to alphavbeta6 integrin expression protects squamous cell carcinomas from anoikis.

Stratified squamous epithelia express the alphavbeta5 integrin, but in squamous cell carcinomas (SCCs) there is down-regulation of alphavbeta5 and up-regulation of alphavbeta6. To investigate the significance of this finding, we transduced an alphav-negative human SCC line with retroviral vectors encoding alphav integrins. alphavbeta5-expressing cells underwent suspension-induced apoptosis (anoikis), whereas alphav-negative cells and cells expressing alphavbeta6 did not. Resistance to anoikis correlated with PKB/Akt activation in suspension, but not with changes in PTEN or p110alpha PI3 kinase levels. Anoikis was induced in parental and alphavbeta6-expressing cells by inhibiting PI3 kinase. Conversely, activation of Akt or inhibition of caspases in alphavbeta5-expressing cells suppressed anoikis. Caspase inhibition resulted in increased phosphoAkt, placing caspase activation upstream of decreased Akt activation. Anoikis required the cytoplasmic domain of beta5 and was independent of the death receptor pathway. These results suggest that down-regulation of alphavbeta5 through up-regulation of alphavbeta6 may protect SCCs from anoikis by activating an Akt survival signal.

Increased expression levels of integrin alphavbeta5 on scleroderma fibroblasts.

Integrin alphavbeta5 is a receptor for vitronectin, a plasma glycoprotein that is also distributed in extracellular matrix of various tissues. Matrix-bound vitronectin has the potential to stabilize the active form of plasminogen activator inhibitor-1, resulting in the inhibition of the plasmin-mediated pericellular proteolytic cascade. In this study, we compared the levels of alphavbeta5 and matrix-bound vitronectin between normal and scleroderma fibroblasts and investigated the association with fibrosis. We demonstrated that alphavbeta5 was up-regulated on scleroderma fibroblasts. The up-regulated alphavbeta5 contributed to the increase in vitronectin-binding ability in scleroderma fibroblasts, which led to the vitronectin-dependent activation of plasminogen activator inhibitor-1. In immunohistochemistry, the alphav and beta5 subunits were stained strongly on scleroderma fibroblasts and the amount of vitronectin was increased in the pericellular matrix of those cells. The transient overexpression of alphavbeta5 on normal fibroblasts enhanced the human alpha2(I) collagen promoter activity through Sp-1 and Smad3 as well as the vitronectin-dependent plasminogen activator inhibitor-1 activity. This effect on the promoter activity was also observed in the absence of vitronectin and completely disappeared in the presence of anti-alphavbeta5 antibody. These results indicate that the up-regulated alphavbeta5 may contribute to the phenotypical alteration of scleroderma fibroblasts, while at the same time suppressing the plasmin-mediated pericellular proteolytic cascade.

Centrifugation facilitates transduction of green fluorescent protein in human monocytes and macrophages by adenovirus at low multiplicity of infection.

Due to their phagocytic and poorly proliferative nature, it has been difficult to transfect human monocytes and macrophages. Adenoviral vectors have recently allowed transduction of a high percentage of human macrophages, but only after CSF upregulation of the integrins, alphavbeta3 or alphavbeta5, during culture for 48 h, a time allowing significant monocyte to macrophage differentiation. In our hands, after 24-h incubation with M-CSF (20 ng/ml) and a further 24-h incubation with an adenoviral vector encoding green fluorescent protein (AdV-GFP) [multiplicity of infection (MOI)=50:1], only 35% of CD14-positive cells express GFP. We report that centrifugation of these cells with AdV-GFP at 2000 x g for 1 h at 37 degrees C significantly enhanced the number of cells expressing GFP (to 65%) and the level of GFP expression per transduced cell (fivefold). The viability of the cells was not compromised (<5 % CD14-positive cells were 7-aminoactinomycin D (7AAD)-positive after 24 h AdV-GFP exposure at MOI=50:1). Centrifugation allowed efficient transduction of monocytes and macrophages with an MOI at least tenfold lower than otherwise required and did not activate the transduced cells or affect their ability to produce TNFalpha or IL-1beta in response to lipopolysaccharide (LPS). This methodology was also suitable for transducing large numbers of in vitro monocyte-derived macrophages (MDMac) and macrophages isolated from synovial fluids with up to 75-80% of CD14-positive cells transduced after 24-h exposure to AdV-GFP (50:1) and centrifugation (2000 x g). This methodology should provide significant expression of transgenes in human monocytes and macrophages.

Overexpression of platelet-type 12-lipoxygenase promotes tumor cell survival by enhancing alpha(v)beta(3) and alpha(v)beta(5) integrin expression.

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.