Osteopontin stimulates invasion of NCI-h295 cells but is not associated with survival in adrenocortical carcinoma.

Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin alphavbeta3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin alphavbeta3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI-h295 cells (>six-fold increase, p < 0.001). Transfection with integrin alphavbeta3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti-integrin beta3 antibody, indicating a functional relationship of OPN and integrin alphavbeta3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan-Meier analysis including 111 patients with primary tumours graded for OPN staining and follow-up data available. In conclusion, our in vitro data indicate that OPN and integrin alphavbeta3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue.

The efficacy of forceful ankle and toe exercises to increase venous return: A comprehensive Doppler ultrasound study.

Objective Venous stasis is a risk factor for venous thromboembolism. We aimed to determine the efficacy of forceful foot exercises for actuation of the calf muscle pump to counteract stasis. Methods We examined 20 seated healthy subjects. The peak systolic velocity at the level of the popliteal vein was assessed by Doppler ultrasound. Results The mean peak systolic velocity measurements (in cm/s) were as follows: baseline = 5.6; ankle plantar flexion with toe flexion = 91.0; toe touch heel lift = 107.4; ankle dorsiflexion with toe extension = 193.6; isolated flexion of all toes = 118.8; ankle plantarflexion with 100 and 250 Newton forefoot force = 89.9 and 154.5, respectively. Conclusion All exercises achieved significant increases in peak systolic velocity compared to baseline. Ranking showed that forceful ankle dorsiflexion, plantarflexion with 250 Newtons and forceful flexion of all toes yielded the highest mean peak systolic velocity values (193.6, 154.5, and 118.8 cm/s, respectively).

Expression pattern of the CCAAT/enhancer-binding proteins C/EBP-alpha, C/EBP-beta and C/EBP-delta in the human placenta.

The human trophoblast has the capacity to invade maternal tissue in a controlled fashion and to produce a wide range of hormones. The transcription factors belonging to the CCAAT/enhancer-binding protein (C/EBP) family are regulators of intracellular processes and mediators of hormone action. C/EBP binding sites have been described in the promoters of several placenta-expressed target genes. In the present study, we used immunohistochemistry and Western-blot analysis to investigate the expression pattern of the three most important members of this family, C/EBP-alpha, -beta, and -delta, in the normal human placenta as well as in isolated trophoblast cell populations. We found C/EBP-alpha and C/EBP-beta expression in the villous syncytiotrophophoblast (ST) and the extravillous (intermediate) trophoblast (EVT), but it was absent from the villous cytotrophoblast (CT). Interestingly, expression of C/EBP-beta continued to be very strong up to the third trimester of pregnancy, especially in the ST. C/EBP-delta showed overall lower expression levels, stronger only in the EVT, while CT/ST showed very low/negative expression. These data show for the first time the expression pattern and tissue localization of C/EBP factors in the human placenta, indicating that these factors (especially C/EBP-beta) may play important roles in the regulation of placenta-specific genes and processes.

Expression of the high-mobility group protein HMGI(Y) in human trophoblast: potential role in trophoblast invasion of maternal tissue.

The high-mobility group protein HMGI(Y) is a member of a family of non-histone chromosomal proteins, which have been implicated in the regulation of inducible gene transcription, integration of retroviruses into chromosomes and induction of neoplastic transformation and metastatic progression in cancer cells. The human trophoblast is a tissue that shares proliferation capacity and invasiveness with neoplastic tissues, but in which these processes are tightly regulated. In the present study, we analyzed the expression of HMGI(Y) in the human placenta using immunohistochemistry. We found expression of HMGI(Y), with nuclear localization, in the villous cytotrophoblast (vCT), which is a highly proliferative cell type. In contrast, the majority of the nuclei of the villous syncytiotrophoblast, a terminally differentiated tissue, was negative. Interestingly, expression of HMGI(Y) was strongest in anchoring villi at the implantation site and in extravillous (intermediate) trophoblast (EVT) invading the maternal decidua. As vCT cells differentiate to become EVT, the HMGI(Y) protein appears to switch from a nuclear to a cytoplasmic localization. Expression of HMGI(Y) in isolated trophoblast populations in primary cell culture was also confirmed using Western-blot analysis. This study shows for the first time expression and localization of HMGI(Y) in the subpopulations of placental tissue.

[Mesenterial lymphangiolipoma - a rare finding in an asymptomatic patient]. / Mesenteriales Lymphangiolipom - eine Rarität als Zufallsbefund.

BACKGROUND: Lymphangioma is an uncommon tumor, an intraperitoneal lymphangiolipoma is exceedingly rare. These tumors are principally benign, but lead to complications due to their size and localization. CASE REPORT: A 46 year old male patient presented for a regular medical check up. Apart from a hearing loss 2006 and 2008 he reported no previous or chronic diseases. An extensive health examination had been performed two years ago and had been without pathological results. Abdominal ultrasound revealed a large polycystic lesion in the right middle and lower abdomen, approximately 12x10x7 cm in size. There was no vascularisation in the septae. In MRI, the tumor appeared cystic as well without communication to the intestinal wall. Laboratory values including echinococcus serology was without pathological results. An explorative laparotomy was done with right hemicolectomy and subsequent ileotransversostomy. Histologically, a lymphangiolipoma was diagnosed, as well as a chronic appendicitis and chronic lymphangitis of the ileocolic lymph nodes. Postoperatively, the patient recovered without any complications. CONCLUSION: Lymphangiomas, especially lymphangiolipomas, are an extremely rare differential diagnosis of intraabdominal cystic tumors. Potential complications included ileus, intussusception or an immuring growth. Abdominal ultrasound can reveal important pathological findings even in symptom- free patients.

Expression pattern of osteopontin in endometrial carcinoma: correlation with expression of the adhesion molecule CEACAM1.

Osteopontin (OPN) and CEACAM1 have diverse biological functions in the uterus and placenta throughout the estrous cycle and pregnancy and have been shown to interact with integrin beta3. OPN is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. Recently we showed the expression pattern of OPN in gestational trophoblastic tumors. CEACAM1 is an adhesion molecule of the carcinoembryonic antigen family that we have recently found to be expressed in endometrial cancer and that has been shown to be down-regulated in colorectal, prostate, and breast cancer. In this study, immunohistochemistry and immunofluorescence with specific antibodies were performed on a series of 20 normal endometrial samples, 17 endometrial hyperplasias, and 43 endometrial carcinomas (28 endometrioid, 10 serous, and 5 clear cell carcinomas) to investigate the expression pattern and cell-type specific localization of OPN and to correlate it with the expression of CEACAM1. In addition, Western blot was performed on normal human endometrium and endometrial neoplasia. Strong OPN expression with a consistent cytoplasmic localization in epithelial glandular cells was observed in the normal human endometrium in 80% of the samples of the proliferative and secretory phase (score 8-12). Similar results could be found in endometrial hyperplasias. Strong expression of OPN could be observed in 29 (67.4%) of the 43 analyzed endometrial carcinomas. Of the 43 analyzed tumors, 18 (41.8%) were in the high score (8-12) category with a strong OPN expression level; 11 of 43 (25.5%) showed a moderate score (4-7) category. In endometrioid carcinoma with increasing malignancy grade, increasing areas with low OPN expression level or complete loss of OPN expression could be observed. In contrast, serous tumors showed a strong OPN expression level. Similar results could be found in Western blot analysis. CEACAM1 showed similar results and could be found to be coexpressed with OPN in normal human endometrium and in endometrial neoplasia as we showed using immunofluorescence. In this study, the different expression patterns of OPN in endometrial tumors could additionally support the biological diversity of endometrioid and serous carcinomas together with other markers. We suggest that OPN might play a different role in the pathogenesis of endometrial cancer (possibly as a functional complex with CEACAM1) and could be relevant for invasive growth of such lesions.

Corticotropin-releasing hormone modulates human trophoblast invasion through carcinoembryonic antigen-related cell adhesion molecule-1 regulation.

Abnormalities in the process of trophoblast invasion may result in abnormal placentation. Both the embryonic trophoblast and maternal decidua produce corticotropin-releasing hormone (CRH), which promotes implantation. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is expressed in extravillous trophoblasts (EVTs) of normal human placenta, may also function in tro-phoblast/endometrial interactions. We investigated whether locally produced CRH plays a role in trophoblast invasion, primarily by regulating CEACAM1 expression. We examined cultures of freshly isolated human EVTs, which express CEACAM1, and an EVT-based hybridoma cell line, which is devoid of endogenous CEACAM1. CRH inhibited EVT invasion in Matrigel invasion assays, and this effect was blocked by the CRH receptor type 1 (CRHR1)-specific antagonist antalarmin. Additionally, CRH decreased CEACAM1 expression in EVTs in a dose-dependent manner. After transfection of the hybridoma cell line with a CEACAM1 expression vector, the invasiveness of these cells was strongly enhanced. This effect was inhibited by addition of blocking monoclonal antibody against CEACAM1. Furthermore, blocking of endogenous CEACAM1 in EVTs inhibited the invasive potential of these cells. Taken together these findings suggest that CRH inhibits trophoblast invasion by decreasing the expression of CEACAM1 through CRHR1, an effect that might be involved in the pathophysiology of clinical conditions, such as preeclampsia and placenta accreta.

Association of plasma resistin levels with coronary heart disease in women.

OBJECTIVE: To examine the association between plasma resistin levels and the presence of coronary heart disease (CHD) in women. RESEARCH METHODS AND PROCEDURES: Plasma resistin levels were measured in a case-control study including 185 women with angiographically confirmed CHD and 227 population-based female controls from the Coronary Risk Factors for Atherosclerosis in Women (CORA) study. RESULTS: After adjustment for age, smoking, family history of myocardial infarction, retirement, education, physical activity, menopausal status, hormone replacement use, BMI, hypertension, diabetes, and dyslipidemia, the odds ratio for CHD for women in the highest compared with lowest quintile of plasma resistin levels was 3.19 (95% confidence interval, 1.44 to 7.10; p log trend, 0.001). After additional adjustment for plasma C-reactive protein levels, this association was substantially attenuated and no longer significant (odds ratio, 1.80; 95% confidence interval, 0.69 ti 4.69; p trend = 0.23). DISCUSSION: These results suggest that plasma resistin levels are significantly associated with the presence of CHD in women; however, this association can largely be explained by concomitant inflammatory processes. Further studies are needed to determine the causal role of resistin in the development of CHD in humans.

Regulation of the human leukemia inhibitory factor gene by ETS transcription factors.

OBJECTIVES: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine mainly produced by activated T lymphocytes. We previously demonstrated that human Jurkat T lymphoma cells represent a valid model of LIF gene expression. This study was designed to identify regions critical for LIF promoter activation in Jurkat cells. METHODS: Luciferase constructs under the control of different portions of the human LIF promoter were transfected into Jurkat cells, and promoter activity was determined by luminometry. Similar experiments were performed with constructs bearing mutations in the putative ETS binding regions in the LIF promoter. RT-PCR, Western blot and gelshift experiments were performed to study expression and DNA binding of ETS factors in lymphoid cells. RESULTS: With the exception of the shortest construct not including the putative ETS binding sites, all wildtype LIF promoter constructs were strongly inducible by phorbol ester/ionomycin. In contrast, the mutant constructs were significantly less inducible. Cotransfection of the wild-type constructs with ETS expression vectors resulted in significant enhancement of promoter activity. ets-1 and ets-2 mRNA and protein were shown to be expressed in Jurkat cells. Gelshift experiments revealed that proteins present in nuclear extracts from Jurkat cells specifically bind to both artificial ETS consensus sites and ETS binding sites present in the LIF promoter. CONCLUSIONS: We conclude that binding of ETS transcription factors to the ETS binding sites in the human LIF promoter is critical for its inducibility in response to T cell activators. ETS transcription factors thus play an important functional role within the endocrine-immune network.

Expression pattern of the activating protein-1 family of transcription factors in the human placenta.

The human placenta is a tissue with a unique capacity for rapid, but--as opposed to malignant tumours--tightly controlled proliferation and invasion capacity. The members of the activating protein-1 (AP-1) family of transcription factors are key regulators of cellular proliferation, differentiation and invasion processes in many systems and could, thus, play an important role in regulating these processes in the human placenta as well. In the present study, we used immunohistochemistry with specific antibodies against all members of the AP-1 family (c-Jun, JunB, JunD and c-Fos, FosB, Fra-1, Fra-2) to investigate their expression pattern and tissue localization in the human placenta. With the exception of c-Jun, which was expressed in a small fraction of villous cytotrophoblast nuclei and JunD, expressed in some syncytiotrophoblast nuclei, all other members of the AP-1 family were completely absent from villous cyto- and syncytiotrophoblast. Interestingly, most AP-1 factors were expressed in the intermediate (extravillous) trophoblast, with expression being strongest for JunD and Fra2 (100% of nuclei showing strong expression), followed by c-Jun (80% positive nuclei), c-Fos and FosB (50% positive nuclei). This was true for samples of both first trimester and later pregnancy. These data show that, in the human placenta, the AP-1 transcription factors are specifically expressed in the intermediate (extravillous) trophoblast, were they could be implicated in regulating proliferation, differentiation and/or expression of invasion-specific molecules, such as matrix metalloproteinases, which have been shown to be regulated by AP-1 in vitro and are expressed by the invasive trophoblast.

The role of the AP-1 transcription factors c-Fos, FosB, Fra-1 and Fra-2 in the invasion process of mammary carcinomas.

Members of the Fos family of AP-1 transcription factors (c-Fos, FosB, FosB2, Fra-1 and Fra-2) are able to form dimers with Jun proteins which bind to the regulatory sequences of target genes. As many proteases involved in tumor invasion are AP-1-regulated, we assumed that Fos family members might be important for invasion of mammary carcinomas. Therefore, we performed transient transfections with expression vectors for c-Fos, FosB, FosB2, Fra-1 and Fra-2, followed by matrigel invasion assays. Fra-1 transfection resulted in a 2-4-fold increase of invasive cells in both cell lines. In a less degree, the invasive potential of MDA-MB231 cells was stimulated by Fra-2, whereas MCF7 invasion was enhanced by c-Fos and FosB. By double-labelling immunocytochemistry, PAI-1 up-regulation was observed in cells transfected with c-Fos, Fra-1 and Fra-2 expression vectors, whereas MMP1 and MMP9 expression was not affected. Results of cotransfection with a MMP9 promoter construct and AP-1 expression vectors do not indicate a direct up-regulation of MMP9 expression by Fos proteins except a positive effect of c-Fos in MCF7 cells. In parallel, expression of Fos family members as determined by Western Blot analysis in 75 mammary carcinomas was correlated with MMP1, MMP9, PAI-1 and uPAR protein levels in the tumors. Interestingly, high FosB levels were significantly associated with MMP1 overexpression, whereas expression of c-Fos and phosphorylated Fra-1 correlated with MMP9 protein levels. Strong Fra-2 expression correlated with high levels of MMP9, PAI-1, the uPA/PAI-1 complex and early recurrence. These data indicate that Fos proteins, especially Fra-1, c-Fos and Fra-2, might be involved in invasion of breast cancer cells.