Comparing the expression of integrins αvß3, αvß5, αvß6, αvß8, fibronectin and fibrinogen in human brain metastases and their corresponding primary tumors.

AIMS: To evaluate the expression of αv-series integrins in brain metastases. Inhibitors targeting these integrins are being tested for their therapeutic potential. MATERIAL AND METHOD: The extracellular regions of the αvß3, αvß5, αvß6, αvß8, the cytoplasmic domain of ß3, the αv-chain, and the ECM molecules fibronectin and fibrinogen were studied immunohistochemically in a series of 122 carcinoma and 60 melanomas metastatic to the central nervous system. In addition, 38 matched primary and metastatic tumors to the brain were compared directly. RESULTS: The αv-subunit was generally moderately to highly expressed in most tumors. αvß3 and cytoplasmic ß3 were weakly to moderately detectable in metastatic renal cell carcinomas and melanomas, αvß5 was prominently expressed in metastatic renal and colorectal carcinomas, αvß6 was most abundantly detectable in metastatic lung adenocarcinomas, but absent in melanomas. The tumor associated vessels in CNS metastases consistently expressed αvß3, αvß5, αv-, fibronectin and fibrinogen, however, mostly at low levels, while αvß6, αvß8 were lacking in vasculature. The comparative analysis of 38 matched primary tumors and brain metastases showed comparable levels of expression only for αvß3 and αvß8, while αvß6 and αvß5 were higher in primaries. CONCLUSION: We confirmed that integrin expression exhibits considerable heterogeneity according to tumor origin. αvß5 is the most promising target for integrin targeted treatment in brain metastases.

Validation and comparison of anti-αvß3 and anti-αvß5 rabbit monoclonal versus murine monoclonal antibodies in four different tumor entities.

Integrins are pivotal in cancer biology and are putative candidates for cancer therapy. The investigation of integrins has been hampered by the lack of antibodies suitable for formalin-fixed and paraffin-embedded (FFPE) tissue specimens. Here, we validated monoclonal rabbit antibodies (RabMAbs) against integrins αvß3 (EM22703) and αvß5 (EM09902) with murine monoclonal antibodies (MuMabs) LM609 (against αvß3) and P1F6 (against αvß5), in immunohistochemistry. Immunostaining was performed on sections of matching unfixed, cryoconserved (CC) and FFPE tissue from 19 colorectal, 20 lung, 17 breast, and 9 ovarian carcinomas. Sections were stained with LM609 and P1F6 and compared with the immunoreactions of the RabMAbs. The degree of concordance was assigned for staining patterns and intensity. Concordance between MuMAbs and RabMAbs ranged from weak, for anti-αvß5 antibodies, to nearly complete for anti-αvß3 antibodies. We confirmed that MuMAbs LM609 and P1F6 bound very weakly in FFPE tissue and no staining was seen. By contrast, RabMAbs EM22703 and EM09902 generally showed a high degree of agreement in staining patterns of CC and FFPE tissue. In summary, the RabMAbs had overlapping staining patterns that were generally more intense for CC when compared with FFPE material. This study suggests that EM22703 and EM09902 staining closely matches the staining of standard MuMAbs and also does so in archival FFPE tissue.

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.

Retinal pigment epithelial cells use a MerTK-dependent mechanism to limit the phagocytic particle binding activity of αvß5 integrin.

BACKGROUND INFORMATION: αvß5 integrin and Mer tyrosine kinase (MerTK) receptors reside at the apical surface of the retinal pigment epithelium (RPE) in the eye to promote the diurnal, synchronised phagocytosis of shed photoreceptor outer segment fragments (POS) that is critical for vision. Phagocytosis assays studying RPE cells in culture have defined roles for αvß5 in POS surface binding and for MerTK in engulfment of bound POS. Both receptors have thus far only been studied separately. It is therefore unknown if αvß5 integrin activity in POS binding is independent of the engulfment function of RPE cells. This study investigates how increasing αvß5 receptor levels affect POS binding and internalisation by wild-type (wt), αvß5- or MerTK-deficient RPE. RESULTS: ß5 integrin-green fluorescent protein (ß5-GFP) fusion proteins formed heterodimeric receptors with endogenous αv integrin subunits at the apical surface of mouse or rat RPE cells that co-immunoprecipitated focal adhesion kinase and redistributed with bound POS such as endogenous αvß5 receptors. In ß5(-/-) RPE cells, de novo formation of αvß5-GFP receptors restored POS binding and internalisation up to, but not, above wt POS uptake levels. In wt RPE cells, increasing levels of αvß5 surface receptors by over-expressing ß5-GFP only moderately stimulated POS binding, even if POS internalisation was inhibited pharmacologically or by lowering incubation temperatures. In contrast, the same increase in αvß5 receptor levels dramatically enhanced POS binding of RPE cells lacking MerTK. Furthermore, decreasing MerTK expression by RNA interference increased POS binding to endogenous αvß5 receptors of wt RPE cells. CONCLUSIONS: Expressing ß5-GFP is sufficient to reverse phagocytic deficiencies of RPE cells derived from ß5(-/-) mice, indicating that these cells do not irreversibly lose other components of the phagocytic machinery. RPE cells expressing the engulfment receptor MerTK control POS binding by limiting activity of endogenous αvß5 and αvß5-GFP integrins, although they reside at the apical, phagocytic surface. In contrast, RPE cells permanently or transiently losing MerTK expression lack this regulatory mechanism and bind excess POS via surface αvß5 receptors. Taken together, these data reveal a novel feedback mechanism that restricts binding of POS to surface αvß5 integrin receptors in RPE cells.

A novel PET tracer for the imaging of αvß3 and αvß5 integrins in experimental breast cancer bone metastases.

The aim of this study was the evaluation of (68)Ga-DOTA-E-[c(RGDfK)](2) as a novel PET tracer to image αvß3 and αvß5 integrins. For this purpose, DOTA-E-[c(RGDfK)](2) was labeled with (68)Ga, which was obtained from a (68)Ge/(68)Ga generator, purified by solid-phase extraction and the radiochemical purity analyzed by radio-RP-HPLC. (68) Ga-DOTA-E-[c(RGDfK)](2) was obtained reproducibly in radiochemical yields of 60 ± 6% and with an excellent radiochemical purity of >99%. In nude rats bearing bone metastases after injection of MDA-MB-231 human breast cancer cells, biodistribution studies were performed to evaluate the accumulation of the radiotracer in selected organs, blood and bone metastases 0.5, 1, 2 and 3 h post injection. A rapid uptake into the bone metastases and rapid blood clearance was observed, resulting in tumor-blood ratios of up to 26.6 (3 h post injection) and tumor-muscle ratios of up to 7.9 (3 h post injection). A blocking experiment with coinjected αvß3/αvß5 antagonist showed the tumor uptake to be receptor-specific. In an initial in vivo micro PET evaluation of the tracer using the same animal model, the bone metastasis was clearly visualized. These results suggest that (68)Ga-DOTA-E-[c(RGDfK)](2) is a promising PET tracer suitable for the imaging of αvß3 and αvß5 integrins in bone metastases. This novel PET tracer should be further evaluated concerning its usefulness for early detection of bone metastases and monitoring treatment response of these lesions.

Regulation of pulmonary inflammation and fibrosis through expression of integrins alphaVbeta3 and alphaVbeta5 on pulmonary T lymphocytes.

OBJECTIVE: Pulmonary diseases associated with fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs. These cells are thought to facilitate lung fibrosis, but the exact mechanisms of their profibrotic action are not clear. Several alphaV-containing integrins, including alphaVbeta3 and alphaVbeta5, have been shown to directly activate transforming growth factor beta (TGFbeta) and promote collagen accumulation. The aim of this study was to investigate whether pulmonary T cells express profibrotic integrins and regulate collagen accumulation. METHODS: Expression of integrins was assessed by immunohistochemical analysis of lung tissue, by flow cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18 overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to determine whether integrin-expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if so, through what possible mechanism. RESULTS: Lymphocytes and integrin-positive cells were present in the lungs, and pulmonary T cells expressed integrins alphaVbeta3 and alphaVbeta5 in patients with SSc and in the animal model. Systemic administration of neutralizing anti-integrin alphaV antibody or a genetic deficiency of integrin beta3 in the CCL18 overexpression model significantly attenuated CCL18-driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin alphaVbeta3 or integrin alphaVbeta5 in cocultures with primary pulmonary fibroblasts stimulated collagen accumulation and Smad2 nuclear translocation. Neutralizing anti-TGFbeta antibody attenuated the profibrotic effect of integrin-expressing T cells. CONCLUSION: Pulmonary infiltrating T lymphocytes may express integrins alphaVbeta3 and alphaVbeta5 that are necessary for lymphocytic infiltration and T cell-associated TGFbeta activation and collagen accumulation.

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.

Radiation sensitization of glioblastoma by cilengitide has unanticipated schedule-dependency.

We investigated whether cilengitide could amplify the antitumor effects of radiotherapy in an orthotopic rat glioma xenograft model. Cilengitide is a specific inhibitor of alphav series integrins, and acts as an antiangiogenic. U251 human glioma cells express alphavbeta3 and alphavbeta5 integrins. We used in vitro assays of adhesion and growth of tumor and endothelial cells to evaluate cytotoxicity and the potential for cilengitide to enhance radiation toxicity. Treatment was then evaluated in an orthotopic model to evaluate synergy with therapeutic radiation in vivo. In vitro, cilengitide blocked cell adhesion, but did not influence the effects of radiation on U251 cells; cilengitide strongly amplified radiation effects on endothelial cell survival. In vivo, radiotherapy prolonged the survival of U251 tumor-bearing rats from 50 to over 110 days. Cotreatment with cilengitide and radiation dramatically amplified the effects of radiation, producing survival over 200 days and triggering an enhanced apoptotic response and suppression of tumor growth by histology at necropsy. Signaling pathways activated in the tumor included NFkappab, a documented mediator of cellular response to radiation. Because cilengitide has a short plasma half-life (t((1/2)) approximately 20 min), antiangiogenic scheduling typically uses daily injections. We found that a single dose of cilengitide (4 mg/kg) given between 4 and 12 hr prior to radiation was sufficient to produce the same effect. Our results demonstrate that blockade of alphav integrins mediates an unanticipated rapid potentiation of radiation, and suggests possible clinical translation for glioma therapy.

Endometrial cancer invasion depends on cancer-derived tumor necrosis factor-alpha and stromal derived hepatocyte growth factor.

Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor-alpha expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF-antagonist/angiogenesis inhibitor)-sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin alpha(v)beta(5) as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention.

Reduced growth and integrin expression of prostate cells cultured with lycopene, vitamin E and fish oil in vitro.

Integrins are transmembrane proteins that facilitate the interaction of cells with the extracellular environment. They have also been implicated in cancer progression. The effects of nutrients thought to be involved in the prevention of prostate cancer on integrin expression have not been determined. Prostate cancer cell lines representing a range of malignancy from normal (RWPE-1) to highly invasive phenotypes (22Rv1 < LNCaP < PC-3) were cultured with or without lycopene (10 nM), vitamin E (5 microm) or fish oil (100 microm) for 48 h. Growth and integrin (alpha2beta1, alphavbeta3 and alphavbeta5) expression were assessed using Trypan Blue exclusion and monoclonal antibodies combined with flow cytometry. Vitamin E enhanced (P < 0.001) whereas fish oil reduced the growth of all the cell lines tested (P < 0.001). Lycopene had no effect on growth. All the malignant cell lines exhibited lower expression of alpha2beta1 with the addition of lycopene to culture media. Supplemental fish oil reduced alpha2beta1 in most invasive cell lines (LNCaP and PC-3). Each nutrient at physiological levels reduced integrins alphavbeta3 and alphavbeta5 in most invasive cell lines (PC-3). The results suggest that integrins may represent an additional target of bioactive nutrients and that the effects of nutrients may be dependent on the type of cell line used.

99mTc-NC100692--a tracer for imaging vitronectin receptors associated with angiogenesis: a preclinical investigation.

INTRODUCTION: Technetium 99m (99mTc)-NC100692 is being developed as a marker of vitronectin receptor expression. The purpose of this study was to confirm the binding affinity [dissociation constant (Kd)] of 99mTc-NC100692 for a range of integrin receptors including alphavbeta3 and alphavbeta5 as well as to establish the biodistribution and metabolic stability of 99mTc-NC100692 in Wistar rats. METHODS: The Kd of 99mTc-NC100692 for a range of human integrin receptors was established in an in vitro saturation binding assay. The biodistribution and metabolic stability of 99mTc-NC100692 in normal Wistar rats was investigated. RESULTS: The Kd of 99mTc-NC100692 to alphavbeta3 and alphavbeta5 was less than 1 nM. It was not possible to saturate the binding of 99mTc-NC10092 towards alphaIIbbeta3, alpha5beta1, alpha3beta1 or alpha1beta1, and as a result, accurate Kd values could not be determined. The biodistribution of 99mTc-NC100692 in male and female Wistar rats showed that radioactivity was rapidly excreted, predominantly into the urine, with very little background tissue retention apart from the liver and kidneys. Kidney and liver retention was reduced in the presence of excess NC100692 ligand. In vivo, there was little systemic metabolism of 99mTc-NC100692. CONCLUSIONS: 99mTc-NC100692 has a high affinity for the vitronectin receptors that are associated with angiogenesis. 99mTc-NC100692 is metabolically stable in the systemic circulation of rats with a biodistribution that is favourable for imaging purposes. This evidence suggests that 99mTc-NC100692 might be a useful marker of vitronectin receptor expression in vivo.

Osteoclast nuclei of myeloma patients show chromosome translocations specific for the myeloma cell clone: a new type of cancer-host partnership?

A major clinical manifestation of bone cancers is bone destruction. It is widely accepted that this destruction is not caused by the malignant cells themselves, but by osteoclasts, multinucleated cells of monocytic origin that are considered to be the only cells able to degrade bone. The present study demonstrates that bone-resorbing osteoclasts from myeloma patients contain nuclei with translocated chromosomes of myeloma B-cell clone origin, in addition to nuclei without these translocations, by using combined FISH and immunohistochemistry on bone sections. These nuclei of malignant origin are transcriptionally active and appear fully integrated amongst the other nuclei. The contribution of malignant nuclei to the osteoclast population analysed in this study was greater than 30%. Osteoclast-myeloma clone hybrids contained more nuclei than normal osteoclasts and their occurrence correlated with the proximity of myeloma cells. Similar hybrid cells were generated in myeloma cell-osteoclast co-cultures, as revealed by tracing myeloma nuclei using translocations, bromo-deoxyuridine, or the Y chromosome of male myeloma cells in female osteoclasts. These observations indicate that hybrid cells can originate through fusion between myeloma cells and osteoclasts. In conclusion, malignant cells contribute significantly to the formation of bone-resorbing osteoclasts in multiple myeloma. Osteoclast-myeloma clone hybrids reflect a previously unrecognized mechanism of bone destruction in which malignant cells participate directly. The possibility that malignant cells corrupt host cells by the transfer of malignant DNA may have been underestimated to date in cancer research.

Use of surface-enhanced Raman spectroscopy for the detection of human integrins.

Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, alphaVbeta3 and alpha5beta1, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.

Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking alphavbeta5 integrin.

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-alpha(v) integrin, but not by anti-CD36 antibody treatment. Introduction of alpha(v)beta(5) integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with alpha(v)beta(5) integrin, but not to alpha(v)beta(3) integrin in brain tissue. Fluorescent BAI1-TSR colocalized with alpha(v)beta(5) integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking alpha(v)beta(5) integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.

Cellular mechanisms of osteoclast formation and lacunar resorption in giant cell granuloma of the jaw.

BACKGROUND: Giant cell granuloma (GCG) is an osteolytic tumour of the jaw which is characterised by the presence of both mononuclear and multinucleated (osteoclast-like) giant cell components. The nature of these component cells and the pathogenesis of the extensive osteolysis associated with this lesion is uncertain. METHODS: Using cell culture techniques and immunohistochemistry, we defined the phenotypic characteristics of the mononuclear and multinucleated cells present in four cases of GCG of the jaw. We also analysed the cellular and humoral factors associated with osteoclast formation and osteolysis in these tumours and determined whether GCG stromal cells are capable of supporting osteoclast formation. RESULTS: GCG-derived giant cells expressed the phenotypic characteristics of osteoclasts (TRAP+, VNR+, and calcitonin responsive) and were capable of lacunar resorption. In addition to macrophages, the mononuclear cell population contained numerous spindle-shaped stromal cells which proliferated in culture and expressed RANKL; these GCG-stromal cells were capable of supporting human osteoclast formation from circulating monocyte precursors. CONCLUSION: Our findings indicate that the giant cells in GCG of the jaw are osteoclast-like and formed from monocyte/macrophage precursors which differentiate into osteoclasts under the influence of RANKL-expressing mononuclear stromal cells found in this lesion.

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma.

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.

Effect of corticosteroids on human osteoclast formation and activity.

Chronic corticosteroid treatment is known to induce bone loss and osteoporosis. Osteoclasts are specialised bone-resorbing cells that are formed from mononuclear phagocyte precursors that circulate in the monocyte fraction. In this study we have examined the effect of the synthetic glucocorticoid, dexamethasone, on human osteoclast formation and bone-resorbing activity. Human monocytes were cultured for up to 21 days on glass coverslips and dentine slices, with soluble receptor activator for nuclear factor kappaB ligand (RANKL; 30 ng/ml) and human macrophage-colony stimulating factor (M-CSF; 25 ng/ml) in the presence and absence of dexamethasone (10(-8) M). The addition of dexamethasone over a period of 7 and 14 days of culture of monocytes (during which cell proliferation and differentiation predominantly occurred) resulted in a marked increase in the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and an increase in lacunar resorption. The addition of dexamethasone to monocyte cultures after 14 days (when resorptive activity of osteoclasts had commenced) reduced the extent of lacunar resorption compared with cultures to which no dexamethasone had been added. The addition of dexamethasone to osteoclasts isolated from giant cell tumours of bone significantly inhibited resorption pit formation. Our findings indicate that dexamethasone has a direct effect on osteoclast formation and activity, stimulating the proliferation and differentiation of human osteoclast precursors and inhibiting the bone-resorbing activity of mature osteoclasts.

Alterations in expression of endometrial endothelial nitric oxide synthase and alpha(v)beta(3) integrin in women with endometriosis.

OBJECTIVE: To determine the expression of endometrial endothelial nitric oxide synthase (eNOS) protein and alpha(v)beta(3) integrin in patients with and without endometriosis. DESIGN: Case-control cohort study. SETTING: University-based tertiary care center. PATIENT(S): Endometrial biopsy samples were obtained from 9 fertile women with regular cycles and 30 infertile women with varying severity of endometriosis. Peritoneal fluid levels of nitric oxide were determined in 13 infertile women with a normal pelvis and 12 infertile women with endometriosis. MAIN OUTCOME MEASURE(S): Expression of eNOS and alpha(v)beta(3) integrin protein in the endometrium and peritoneal fluid levels of nitric oxide. RESULTS: In patients with endometriosis, expression of eNOS was significantly increased in the glandular and luminal epithelium, with no significant changes in the stroma. Peritoneal fluid levels of nitric oxide were unchanged, and expression of alpha(v)beta(3) integrin expression in glandular and luminal epithelium was significantly decreased compared with controls. A significant negative correlation was observed between luminal expression of eNOS and alpha(v)beta(3) integrin and between glandular expression of eNOS and luminal expression of alpha(v)beta(3) integrin. CONCLUSION(S): The nitric oxide pathway may play a role in the pathogenesis of endometriosis.

Acute cellular rejection following human heart transplantation is associated with increased expression of vitronectin receptor (integrin alphavbeta3).

The vitronectin receptor (integrin alphavbeta3), a cell-surface adhesion receptor, has been shown to play a significant role in endothelial cell migration, apoptosis, atherosclerosis, and T-lymphocyte activation. This study was undertaken to test the hypothesis that cardiac allograft rejection is associated with increased expression of alphavbeta3. We also determined whether fibronectin receptor (alpha5beta1) and tissue factor are up-regulated in the presence of acute cellular rejection. We evaluated endomyocardial biopsy specimens with histologic evidence of different degrees of acute cellular rejection (grade 0, n = 10; grade 1A, n = 10; grade 2, n = 10; grade 3A, n = 10). Biopsies were obtained 2-4weeks after cardiac transplantation. Immunoperoxidase staining was performed for alphavbeta3, tissue factor, and alpha5beta1, and protein levels were further determined by Western blot analysis. Specimens with grade 2 and grade 3A rejection showed positive staining of alphavbeta3 in lymphocytic aggregates and vascular endothelial cells. By immunoblotting, we identified significantly increased expression of alphavbeta3 in the presence of acute rejection, grade 2 (3-fold, p = 0.01) and grade 3A (3.6-fold, p = 0.005) compared to grade 0 and 1 A specimens. There was no evidence of increased expression of alpha5beta1 or tissue factor. Acute cellular rejection, a process characterized by T-lymphocyte activation and release of inflammatory cytokines, is associated with increased expression of alphavbeta3.

Human urinary bladder carcinomas express adenovirus attachment and internalization receptors.

The use of adenoviral vectors as potent gene delivery systems requires expression of the Coxsackievirus/adenovirus receptor (CVADR) on the target cell surface. This receptor is important for virus attachment to the cell surface. For effective internalization of the vector into the target cell the integrins alpha(v)beta(3) and/or alpha(v)beta(5) are needed. Since there have been reports of loss of CVADR in bladder cancer cell lines, we wanted to investigate the expression of this receptor in bladder carcinoma biopsies. Surgical biopsies, as well as five human bladder cancer cell lines, were analyzed for expression of CVADR, the integrins alpha(v)beta(3) and alpha(v)beta(5) and MHC class I. Further, we studied the ability to transduce these cell lines using adenoviral vectors. Immunohistochemistry revealed that all biopsies (27/27) were positive for CVADR. Some variation in expression was evident, and superficially growing tumors stained more strongly than invasive ones. Most human tumors expressed the integrin alpha(v)beta(5) (14/24), whereas integrin alpha(v)beta(3) was less frequently seen (3/20). The established cell lines were efficiently transduced with adenoviral vectors, and transduction could be reduced with anti-CVADR antibodies. The abundance of appropriate viral receptors on tumor biopsy cells is a further argument for using adenoviral vectors in gene therapy of bladder cancer.