High‐level expression of periostin is significantly correlated with tumour angiogenesis and poor prognosis in osteosarcoma

Periostin (PN), originally named as osteoblast‐specific factor‐2 (OSF‐2), has been involved in regulating adhesion and differentiation of osteoblasts. Recently many studies have shown that high‐level expression of PN is correlated significantly with tumour angiogenesis and prognosis in many kinds of human cancer. However, whether and how periostin expression influences prognosis in osteosarcoma remains unknown. This study aimed to examine the expression of PN in patients with osteosarcoma and explore the relationship of PN expression with clinicopathologic factors, tumour angiogenesis and prognosis. Immunohistochemistry was performed to determine the expression of PN in osteosarcoma and osteochondroma respectively. Vascular endothelial growth factor (VEGF) and CD34 were also examined in tissues from the osteosarcoma patients mentioned above. The results showed that PN expression was significantly (P < 0.05) higher in osteosarcoma (80.9%) than in osteochondroma (14.7%). Increased PN protein expression was associated with histological subtype (P = 0.000), Enneking stage (P = 0.027) and tumour size (P = 0.009). The result also showed that high expression of PN correlated with VEGF expression (r = 0.285; P = 0.019) and that tumours with PN‐positive expression significantly had higher microvessal density (44.6 ± 13.7 vs. 20.6 ± 6.5; P = 0.000) compared to those in normal bone tissues. Additionally, the expression of PN was found to be an independent prognostic factor in osteosarcoma patients. In conclusion, our findings suggest that PN may have an important role in tumour progression and may be used as a prognostic biomarker for patients with osteosarcoma.     

Protocadherin-10 is involved in angiogenesis and methylation correlated with multiple myeloma

Protocadherin-10 (PCDH10) which is located at 4q28.3, is a member of the cadherin superfamily of cell adhesion molecules. PCDH10 is broadly expressed in normal adult, but nearly undetectable in multiple myeloma (ΜΜ) tissues and cell lines. Its promoter methylation was detected in virtually all the silenced or downregulated cell lines. The silencing of PCDH10 could be reversed by pharmacological demethylation, indicating a methylation-mediated mechanism. In the current study, we investigated 44 patients (23 females, 21 males), 77.27% (34/44) of whom presented high methylation of PCDH10. We found no associations between promoter hypermethylation and gender or age at the time of initial diagnosis. We also examined the role of PCDH10 as a mediator of MM cell proliferation, cell cycle progression, and its involvement in angiogenesis. Our results demonstrate that the PCDH10 gene is a target for epigenetic silencing in MM and provide a link between the dysregulation of angiogenesis and DNA methylation.     

Differential expression of Yes-associated protein is correlated with expression of cell cycle markers and pathologic TNM staging in non-small-cell lung carcinoma

Yes-associated protein, a downstream effector of the Hippo signaling pathway, has been linked to progression of non-small-cell lung carcinoma. The aim of this study was to investigate expression of Yes-associated protein in lung adenocarcinoma and squamous cell carcinoma. Associations of Yes-associated protein expression with clinicopathologic parameters, expression of cell cycle-specific markers, and epidermal growth factor receptor gene amplification were also analyzed. In a univariate analysis of the 66 adenocarcinomas, high nuclear expression of Yes-associated protein was significantly correlated with expression of cyclin A and mitogen-activated protein kinase. Multivariate analysis, including age and sex, showed that cyclin A expression was independently correlated with nuclear expression of Yes-associated protein in adenocarcinomas. Furthermore, high nuclear expression of Yes-associated protein was also a significant predictor of epidermal growth factor receptor gene amplification for adenocarcinoma. For the 102 squamous cell carcinomas, univariate analysis revealed that high cytoplasmic expression of Yes-associated protein was correlated with the low pathologic TNM staging (stage I) and histologic grading. Multivariate analysis, including age and sex, showed that cytoplasmic expression of Yes-associated protein was an independent predictor of low pathologic TNM staging. These results indicate that nuclear overexpression of Yes-associated protein contributes to pulmonary adenocarcinoma growth and that high cytoplasmic expression of Yes-associated protein is an independent predictor of low pathologic TNM staging and histologic grading. The differential effects of Yes-associated protein expression patterns in adenocarcinomas and squamous cell carcinomas suggest that Yes-associated protein may play important roles in different pathways in distinct tumor subtypes. These observations may, therefore, lead to new perspectives on therapeutic targeting of these tumor types.     

Bacterial binding to extracellular matrix proteins -- in vitro adhesion.

A binding assay was developed and used to study the binding of oral streptococcus to immobilized human fibronectin, laminin, vitronectin, fibrinogen, heparin, and collagen IV. The protein binding was dependent on the broth used for bacterial growth. The binding after growth in brain heart infusion broth, trypticase soy broth, Todd-Hewitt broth, and Dulbecco's modified Eagle's medium was examined. Most of the strains were able to bind to immobilized fibronectin and laminin, and to a minor extent vitronectin. Binding was not observed on immobilized fibrinogen, collagen IV, or heparin. Measured surface hydrophobicity correlated well with the bacterial binding strength to the proteins. Streptococcal incubation with putative inhibitors indicates multiple binding mechanisms of a lectin-like and protein nature, possibly involving protein receptors.     

The expression of fatty acid metabolism‐associated proteins is correlated with the prognosis of meningiomas

The expression of fatty acid metabolism‐associated proteins is correlated with the prognosis of meningiomas. Meningioma is a common tumor of the nervous system; however, reliable prognostic markers for meningioma are currently insufficient. High fatty acid synthase (FAS) expression occurs in many tumors, and is associated with tumor progression and grade. Few studies have previously investigated fatty acid metabolism in meningioma; thus, in this study, we investigated the expression of FAS and brain fatty acid‐binding protein (BFABP) proteins in all grades of meningioma and determined the association to meningioma grade, invasiveness, recurrence, and progression. We determined expression levels of FAS and BFABP in all grade meningiomas by immunohistochemical analysis in 314 patients diagnosed with meningioma. The expression levels of FAS and BFABP increased significantly in correlation with meningioma grade (p < 0.01). Compared with benign meningioma, the expression levels of FAS and BFABP were significantly higher in brain invasive meningioma (p < 0.01). Compared with nonrecurrent meningioma (benign meningioma), the expression of FAS was also increased in recurrent meningioma (p < 0.01). The expression of fatty acid metabolism‐associated proteins potentially correlates with meningioma grade, invasiveness, aggressiveness, and recurrent status and provides evidence for a novel therapeutic target for meningioma.     

Cytoplasmic phospholipase A2 alpha overexpression in stromal cells is correlated with angiogenesis in human colorectal cancer.

In colorectal cancer, cyclooxygenase-2 (COX-2) overexpression in stromal cells induces angiogenesis through EP2 prostaglandin E2 receptor signaling. Cytoplasmic phospholipase A2 (PLA2) alpha preferentially hydrolyses arachidonic acid, which is the limiting substrate for prostaglandin production, from membrane phospholipids. We therefore investigated a possible relationship between cytoplasmic PLA2 and COX-2 overexpression in stromal cells, angiogenesis and microsatellite instability in 48 human colorectal adenocarcinomas. Cytoplasmic PLA2 and COX-2 expression in stromal cells and vascular endothelial growth factor (VEGF) expression in tumor cells were evaluated by immunohistochemistry. Microvessel density was assessed in 10 x 400 fields after CD31 staining. Microsatellite instability was evaluated by PCR and immunohistochemistry. A total of 16 tumors had microsatellite instability. We found an overexpression of cytoplasmic PLA2 in superficial stromal cells. These cells corresponded to fibroblasts and myofibroblasts. There was an association between the number of cytoplasmic PLA2 and COX-2-expressing cells (P=0.006). Cytoplasmic PLA2-positive stromal cells usually also expressed COX-2. A high number of cytoplasmic PLA2-positive stromal cells was correlated with a high microvessel density (P=0.002), a strong VEGF (P=0.01) and the absence of microsatellite instability (P=0.001). The coordinate overexpression of cytoplasmic PLA2 and COX-2 in stromal cells could lead to an important prostaglandin production. These results suggest that cytoplasmic PLA2 overexpression in these cells regulates COX-induced angiogenesis probably by providing arachidonic acid, which is the limiting factor for prostaglandin production. The lower number of cytoplasmic PLA2-positive stromal cells in carcinomas with microsatellite instability could be related to their lower microvessel density and VEGF expression.     

The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor is correlated to angiogenesis in breast cancer

It has been shown that human thymidine phosphorylase (TP) Is Identical to platelet-derlved endothelial cell growth factor and has angiogenlc actlvhy. In the present study, the expression of TP was examined In 139 mammary carclnomas and 35 benign mammary disorders using biochemical and lmmunohlstochemlcal methods. Moreover, In order to evaluate the significance of TP expression in mammary carcinomas, the relationship between vascular density and various cllnicopathological factors, including age and menopausal status of patients with a mammary carcinoma, were compared wtth the size, nodal status, expression of estrogen receptor (ER), progesterone receptor (PgR), c-erbB-2, p53 and TP of a mammary carcinoma. Thymidine phosphorylase expression Increased in both the nuclei and cytoplasm of mammary carclnoma cells in comparison to mammary benign disorder cells. The number of mlcroves-sels In mammary carcinomas was generally correlated to the number of tumor cells with TP expression in cytoplasm. The number of cells with TP expression in cytoplasm was significantly large In tumors that measured 34 cm In diameter, compared wtth tumors measuring 1–2 and 5–6 cm in diameter. In mammary tumors of 1–4 cm diameter, TP expression and vessel denslty were slgnlficantly high in tumors negative for ER or positive for cerbB2 and In tumors positive for TP or cerbB2, respectively; whereas tumors of 5–6 cm In diameter were not modified by any cllnlcopathological factors. The results lndlcated that TP plays an Important anglogenetic role In mammary carcinomas, especially tumors with a certain progression.     

Differential expression of Trypanosoma cruzi neuraminidase in intra- and extracellular trypomastigotes.

The developmentally regulated expression of Trypanosoma cruzi neuraminidase in culture cells was monitored by immunofluorescence with a monoclonal antibody (TCN-2) against the enzyme. The results showed that TCN-2 reacted with all intracellular trypomastigote forms (NA+) but not with amastigotes. Immunoprecipitation of radiolabeled neuraminidase confirmed TCN-2 reactivity with trypomastigotes and the specificity of the antibody binding. During exiting from the host cells, all trypomastigotes were still NA+. However, when free in the extracellular environment, the relative proportion of NA+ parasites declined from 100% to about 20%, thereby establishing a subpopulation of trypomastigotes which did not express enzyme (NA-). The expression of neuraminidase in all intracellular trypomastigotes and in only a subpopulation of the extracellular counterpart suggests that the enzyme may play a role in parasite exiting from infected cells.     

Intrahepatic MxA expression is correlated with interferon-alpha expression in chronic and fulminant hepatitis

Interferon-alpha (IFN-alpha) has potent pro-inflammatory and anti-viral functions. It exerts its effects by inducing intracellular proteins such as MxA. To analyse the role of intrahepatic interferon activation, IFN-alpha and MxA expression was studied by immunohistochemistry in explant livers of 20 patients with fulminant hepatic failure (FHF), 41 patients with chronic liver disease (CLD), and ten normal controls (NCs). In NCs only small numbers of Kupffer cells, but no hepatocytes, showed IFN-alpha and MxA expression. In contrast, significantly enhanced numbers of IFN-alpha- and MxA-positive Kupffer cells, along with small numbers of MxA-positive and larger numbers of IFN-alpha-positive lymphocytes, were found in CLD and in FHF. MxA protein was also expressed on hepatocytes and bile ducts in the vicinity of IFN-alpha-positive inflammatory infiltrates (hepatocytes: NCs: 0%, CLD: 8%, FHF: 68%; bile ducts: NCs: 19%, CLD: 46%, FHF: 83%). A significant correlation was found between the numbers of IFN-alpha- and MxA-positive cells (r=0.67, p<0.001). Thus, large amounts of IFN-alpha are released in the livers of patients with FHF, which is likely to contribute to immune-mediated liver cell damage. Intrahepatic MxA expression corresponds to IFN-alpha produced particularly by infiltrating inflammatory cells, rather than by hepatocytes themselves.     

Elevated extracellular potassium is associated with a reduced extracellular space in rat neural lobe in vitro

Summary Increased neural activity of neurosecretory cells is accompanied by large increases in extracellular K⁺. The possibility that elevations of this ion might involve fluid redistribution and thus affect the size of the extracellular space and the relationship between pituicytes and axons in the rat neural lobe was explored using rapid freezing and freeze-substitution. Neural lobes were incubated for 15 min before freezing either in a normal medium or one containing a 10 mM increase in KCl (high KCl), a 10 mM increase in KCl balanced by an equimolar reduction in NaCl (high KCl-low NaCl), or only a 10 mM reduction in NaCl (low NaCl). A quantitative assessment of the region of good fixation was made to determine the relative fractions occupied by axons, pituicytes and the extracellular space near the neurohaemal contact zone. In addition, the percentage of basal lamina contacted by pituicytes and axons was calculated, as was the degree of enclosure of axons by pituicytes.In neural lobes incubated in normal medium, the extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28–29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes occupied almost 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%. Neural lobes incubated in either the high KCl solution, or in the high KCl-low NaCl solution, exhibited a significantly reduced extracellular space, to about 20% of the total area, or a reduction from controls of about one-third. The reduction was complemented by an increased area occupied by axons plus pituicytes. In the high KCl group, the contribution of the narrow spaces (22–24 nm) between apposed processes to the total extracellular space was greatly increased. The group exposed to low NaCl showed high variability in the size of extracellular space, and was thus not significantly different from any other group. No changes in any group were observed in the enclosure of axons by pituicytes, or in the relative amounts of axon and pituicyte apposition to the basal lamina.The subsequent buffering of K⁺ and other ions during periods of increased neuronal activity may be affected by changes in the extracellular space, thereby influencing stimulus-secretion coupling. A shrinkage of the extracellular space and the relative increase in the narrow spaces between processes initiated by elevated K⁺ could also alter the diffusion properties of the neural lobe.     

Gene expression profiling in an in vitro model of angiogenesis

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.     

Downregulation of GLTSCR2 expression is correlated with breast cancer progression

Glioma tumor-suppressor candidate region gene2 (GLTSCR2) is a recently identified nucleolus-localized protein participating in the regulation of cell cycle progression and apoptosis. Down-regulation of GLTSCR2 in several types of cancers and increased transforming activity in GLTSCR2-downregulated cancer cells indicated its tumor suppressive potential. The aim of this study was to evaluate GLTSCR2 expression in breast cancer and to investigate the question of whether reduced expression of GLTSCR2 may have any pathological significance in breast cancer development or progression. In this study, we performed quantitative RT-PCR and immunohistochemistry to evaluate the expression of GLTSCR2 and relevance with clinicopathological factors in the invasive ductal carcinoma (IDC). GLTSCR2 expression was reduced in 48% of IDC (n = 426) by a semi-quantitative scoring system using tissue microarray. GLTSCR2 mRNA was significantly reduced by 0.16 fold in 15 out of 17 (88%) cases of IDC. Reduction of GLTSCR2 was significantly correlated with increased histological grade (p < 0.005), increased tumor size (p < 0.001), axillary lymph node involvement (p < 0.001) and decreased disease free survival (p < 0.025). In addition, we show that upregulation of GLTSCR2 decreases the invasive potential of breast cancer cells. Taken together, our data suggest that GLTCR2 may play a role in the tumorigenesis, progression and biological behavior in breast cancer.     

Wnt expression is not correlated with β-catenin dysregulation in Dupuytren's Disease

Background  

Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in finger flexion contractures. Increased cellular β-catenin levels have been identified as characteristic of this disease. As Wnts are the most widely recognized upstream regulators of cellular β-catenin accumulation, we have examined Wnt gene expression in surgical specimens and in DD-derived primary cell cultures grown in two-dimensional monolayer culture or in three-dimensional FPCL collagen lattice cultures.     

Angiopoietins and angiopoietin-like proteins in angiogenesis.

Vascular network formation requires several endothelial cell growth factors. These factors have a potent angiogenic effect, and their precise coordination is essential for vascular development. Among them, angiopoietins function through the Tie2 receptor, whose signaling is critical to regulate vascular stabilization and remodeling. It has been reported that the angiopoietin/Tie2 signal is involved in survival and migration of endothelial cells and regulates vascular remodeling and maintenance of vascular integrity. More recent studies demonstrate that angiopoietin/Tie2 signaling is also required for lymphangiogenesis. The authors and several other groups have identified six angiopoietin-like proteins (Angptls) containing a coiled-coil domain and a fibrinogen-like domain, both of which are characteristic of angiopoietins. Interestingly, Angptls also function in angiogenesis through regulating survival and migration of endothelial cells, although Angptls do not bind the angiopoietin receptor Tie2. Currently, Angptls are orphan ligands, but they have been reported to have pleiotropic effects not only on vascular cells but also on metabolism and tumor biology. Here, the authors review current findings relating to the roles of angiopoietins and Angptls in vascular biology and discuss molecular mechanisms relevant to these factors and angiogenesis.     

Apoptosis in gestational trophoblastic disease is correlated with clinical outcome and Bcl-2 expression but not Bax expression.

Apoptosis has been found to play a crucial role in the pathogenesis and prognosis of many human diseases. The pathogenesis of gestational trophoblastic disease (GTD), which encompasses hydatidiform moles (HMs) and choriocarcinomas (CCAs), is not fully understood. Prognostic indicators of HM have also been scanty. In this study, we investigated apoptotic activity and the expression of two apoptosis regulatory genes, Bcl-2 and Bax, in an attempt to determine the role of apoptosis in GTD. Formalin-fixed paraffin-embedded tissue of 33 normal placentas, 14 spontaneous abortions, 14 partial moles, 34 complete moles, and eight CCAs were examined. Apoptotic activity was assessed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. Quantitative assessment of apoptotic index (AI) was calculated as a percentage of TUNEL-positive nuclei. Expression of Bcl-2 and Bax were assessed immunohistochemically. Extensive apoptosis was located in syncytiotrophoblasts, cytotrophoblasts, and villous stromal cells in all HM cases. Apoptosis was detected at a much lower level in spontaneous abortions and normal placentas. Moreover, in normal placentas, TUNEL positive nuclei were exclusively found in syncytiotrophoblasts. AIs were significantly different among various categories of trophoblastic lesions (P < .001) in an ascending order: normal placentas less than spontaneous abortions less than CCAs less than HMs. Furthermore, AIs of those cases that spontaneously regressed was statistically higher than those that developed persistent trophoblastic disease requiring chemotherapy. AIs of trophoblastic lesions in general inversely correlated with Bcl-2 expression (P < .001), but no significant correlation was found between AI and Bax expression (P > .5). We conclude that AI may be a useful prognostic marker for clinical progress of HMs. Bcl-2 expression is probably regulating apoptosis in normal placentas and GTD, whereas Bax expression is not. The difference in AI and Bcl-2 expression between non-molar placentas and HMs offers a potential adjunctive diagnostic tool to distinguish the two entities.