PAX3 in neuroblastoma: oncogenic potential, chemosensitivity and signalling pathways.

Transcription factor PAX3/Pax3 contributes to diverse cell lineages during embryonic development and is important in tumourigenesis. We found that PAX3 is re-expressed in neuroblastoma and malignant neuroblastic (N-type) neuroblastoma cells had significantly higher PAX3 protein expression than their benign substrate-adherent (S-type) counterparts. Knock-down of PAX3 expression by siRNA transfection resulted in persistent cell growth inhibition in both types of neuroblastoma cell, owing to G1 cell cycle arrest and progressive apoptosis. Inhibition of PAX3 expression significantly decreased the attachment of S-type SH-EP1 cells to extra-cellular matrix proteins, fibronectin, laminin and collagen IV. Migration and invasion of both neuroblastoma cell types were markedly reduced after PAX3 down-regulation. PAX3 knock-down significantly augmented the cytotoxic effect of chemotherapeutic agents, etoposide, vincristine and cisplatin, commonly used to treat neuroblastoma. Microarray analyses revealed that particularly signalling pathways involving cell cycle, apoptosis, cell adhesion, cytoskeletal remodelling and development were altered by PAX3 down-regulation. Changes in PAX3 downstream genes identified by microarray analyses were validated in 47 genes by quantitative PCR. These novel findings lead us to propose that PAX3 might contribute to oncogenic characteristics of neuroblastoma cells by regulating a variety of crucial signalling pathways.

Bioportide: an emergent concept of bioactive cell-penetrating peptides.

Cell-penetrating peptides (CPPs) have proven utility for the highly efficient intracellular delivery of bioactive cargoes that include peptides, proteins, and oligonucleotides. The many strategies developed to utilize CPPs solely as pharmacokinetic modifiers necessarily requires them to be relatively inert. Moreover, it is feasible to combine one or multiple CPPs with bioactive cargoes either by direct chemical conjugation or, more rarely, as non-covalent complexes. In terms of the message-address hypothesis, this combination of cargo (message) linked to a CPP (address) as a tandem construct conforms to the sychnological organization. More recently, we have introduced the term bioportide to describe monomeric CPPs that are intrinsically bioactive. Herein, we describe the design and biochemical properties of two rhegnylogically organized monometic CPPs that collectively modulate a variety of biological and pathophysiological phenomena. Thus, camptide, a cell-penetrant sequence located within the first intracellular loop of a human calcitonin receptor, regulates cAMP-dependent processes to modulate insulin secretion and viral infectivity. Nosangiotide, a bioportide derived from endothelial nitric oxide synthase, potently inhibits many aspects of the endothelial cell morphology and movement and displays potent anti-angiogenic activity in vivo. We conclude that, due to their capacity to translocate and target intracellular signaling events, bioportides represent an innovative generic class of bioactive agents.

Monomeric C-Reactive Protein - A Feature of Inflammatory Disease Associated With Cardiovascular Pathophysiological Complications?

Monomeric C-reactive protein (mCRP), the dissociated form of native C-reactive protein, is a critical molecule that causes and perpetuates inflammation in serious diseases. It has 'adhesive'-like properties causing aggregation of blood cells and platelets, and can stick permanently within arterial tissue where it can contribute to further complications including thrombosis, linking it potentially to atherosclerosis and subsequent acute coronary events. In this mini review, we discuss briefly the implications and the potential value of measuring and manipulating it for clinical diagnostics and therapeutic purposes.

CD105 (Endoglin): A Potential Anticancer Therapeutic Inhibits Mitogenesis and Map Kinase Pathway Activation.

BACKGROUND: CD105 is highly expressed on human activated endothelial cells (ECs), is an important component of the TGF-ß1 receptor complex and is essential for angiogenesis. CD105 expression is up-regulated in activated ECs and is an important potential marker for cancer prognosis. MATERIALS AND METHODS: In vitro rat myoblasts transfected with the L-CD105 and S-CD105 transfectants. The transfectants were treated with TGF-ß1 for the angiogenesis study. RESULTS: L-CD105 affects cell proliferation in the presence and absence of TGF-ß1, and inhibits p-ERK1/2, p-MEK1/2 and p-c-Jun in L-CD105 transfectants compared to controls. The induction of phospho-ERK1/2 following treatment with TGF-ß1 remained significantly lower in L-CD105 transfectants compared to controls. CONCLUSION: L-CD105 inhibits the phosphorylation of ERK1/2, MEK1/2, c-Jun1/2/3, and associated signalling intermediates. CD105 modulates cell growth and TGF-ß1 induced cell signalling through ERK-c-Jun expression.

Role of stromal fibroblasts in cancer: promoting or impeding?

The basement membrane, immune cells, capillaries, fibroblasts and extracellular matrix (ECM) constitute the tumour stroma, commonly referred to as the 'reactive stroma'. The fibroblasts from the initial stages of a tumour, as the main constituents of the reactive stroma, present a different phenotype from the normal fibroblasts and play a crucial role in tumour progression. This review presents the differences between normal and tumour stromal fibroblasts and analyzes the molecular mechanisms (which involve growth factors, ECM components, matrix metalloproteinases, integrins and cell adhesion molecules) in the complex interactions between stromal fibroblasts and tumour cells. To date, several examples of heterotypic interactions between tumour stromal fibroblasts and tumour cells have supported the hypothesis that the tumour stroma promotes the growth of the tumour mass, as well as invasion and metastasis. However, it remains possible that the stroma acts essentially as a local modulator to impede tumorigenesis at an early stage and that the desmoplastic response is a host defence reaction designed to confine the developing tumour. The latter hypothesis has largely been neglected. The review aims to give a broader view on the role of stromal fibroblasts in tumour growth, invasion and metastasis.

Modulation of endothelium and endothelial progenitor cell function by low-density lipoproteins: implication for vascular repair, angiogenesis and vasculogenesis.

The endothelium regulates vascular homeostasis and is responsible for angiogenesis, a process mediated by the sprouting of endothelial cells from pre-existing vessels. Several lines of evidence indicate that endothelial progenitor cells (EPCs) also play a role in adult neovascularization as well as in the maintenance of endothelial integrity and function. Hypercholesterolemia is associated with increased cardiovascular risk by inducing a cascade of events leading to endothelial dysfunction and injury. Growing evidence indicates that low-density lipoproteins (LDLs) impair endothelial reparative processes by inducing endothelial cell apoptosis but also by reducing the number and function of EPCs. The involvement of LDLs in mechanisms associated with vascular repair and neovascularization is also suggested by data from studies using lipid-lowering drugs (statins). This review is focused on the central role of the cholesterol pathway in the biology of the endothelium and EPCs.

Dynamin and perforin are associated with neovascularisation in advanced carotid plaques.

Intimal plaque neovascularization is associated with the development of symptomatic disease and thrombosis, with new 'leaky' fragile microvessels prone to haemorrhage. Perforin or pore forming protein is involved in vascular cell death by forming pores in target cells. Enzymes, in particular, granzyme B are secreted by immune infiltrates present in inflammatory plaque regions and have been shown to induce endothelial cell apoptosis. Similarly, dynamin-2 is a GTPase which mediates oxidised low density lipoprotein-induced apoptosis and is also required for granzyme B-mediated exocytosis and apoptosis. Our pilot studies identified increased expression of these proteins in complicated atherosclerotic plaques. Here we demonstrate by immunohistochemistry that both proteins are over-expressed in angiogenic regions of complicated carotid plaques. Dynamin-2 was extensively localised around microvessels and in immune infiltrating cells whilst perforin was localised in immune infiltrating cells, endothelial cells and smooth muscle cells. Over-expression of these proteins may contribute to plaque destabilisation by increasing cellular apoptosis in vulnerable atherosclerotic plaques.

The normal cellular prion protein and its possible role in angiogenesis.

Cellular Prion Protein (PrPc) is a ubiquitous glycoprotein present on the surface of endothelial cells. Resting vascular endothelial cells show minimum expression of PrPc and can constitutively release PrPc. PrPc participates in cell survival, differentiation and angiogenesis. During development, neonatal brain endothelial cells transiently express PrPc. Our group recently reported upregulation of PrPc in microvessels from ischemic brain regions in stroke patients. Ischemia/hypoxia induces PrPc expression through the activation of extracellular signal-regulated kinase (ERK). All these data suggest that PrPc plays an important role in angiogenic responses. In addition, PrPc participates in cellular function in the central nervous system, since PrPc is also highly expressed in neurons. PrPc binds copper, suggesting a role in copper metabolism. PrPc also protects cells against oxidative stress and it seems to be involved in neuroprotection. Several studies have demonstrated that PrPc prevents cells from apoptosis and subsequent tissue damage. Moreover, PrPc plays an important role in the immune response. Here, we review the multiple functions of PrPc with a special attention to its recently reported role in angiogenesis.

The role of endothelial progenitor cells in tumour vasculogenesis.

OBJECTIVE: To review the biological behaviour of endothelial progenitor cells and their role and significance in tumour vasculogenesis. DATA SOURCES: The data in this review were mainly from Medline and PubMed for the relevant articles in English published from March, 1997, to March, 2008. The search terms were 'endothelial progenitor cells' (EPCs) and 'neoplasm'. Articles about the biological behaviour of EPCs and their roles in tumour vasculogenesis were included. RESULTS: EPCs, whose characteristics are similar to those of endothelial cells (ECs) and stem cells, contribute to tumour vasculogenesis during tumour progression. The mobilisation, recruitment, homing and incorporation of EPCs into tumours are multi-step and multi-factor events during tumour vasculogenesis. This complex process requires the participation of many growth factors and cells, such as tumour cells, ECs, stromal cells and EPCs in the tumour microenvironment. However, there is still some debate about EPC distribution, contribution, origin and differentiation in tumour vasculogenesis. CONCLUSIONS: The characterisation of tumour-associated EPCs may provide valuable clues for more specific anti-angiogenesis therapy and/or tumour diagnosis. Many challenges remain in understanding definition, differentiation, mobilisation and recruitment of EPCs.

Expression of cyclin-dependent kinase 5 mRNA and protein in the human brain following acute ischemic stroke.

Neuronal cell death after brain ischemia may be regulated by activation of cyclin-dependent kinase 5 (Cdk5). In this study, expression of Cdk5 and its activator p35/p25 was examined in human post-mortem stroke tissue and in human cerebral cortical fetal neurons and human brain microvascular endothelial cells exposed to oxygen-glucose deficiency and reperfusion. The majority of patients demonstrated increased expression of Cdk5 and p-Cdk5 in stroke-affected tissue, with about a third showing increased p35 and p25 cleaved fragment as determined by Western blotting. An increase in Cdk5-, p-Cdk5- and p35-positive neurons and microvessels occurred in stroke-affected regions of patients. Staining of neurons became irregular and clumped in the cytoplasm, and nuclear translocation occurred, with colocalization of p35 and Cdk5. Association of Cdk5 with nuclear damage was demonstrated by coexpression of nuclear Cdk5 in TUNEL-positive neurons and microvessels in peri-infarcted regions. In vitro studies showed up-regulation and/or nuclear translocation of Cdk5, p-Cdk5 and p35 in neurons and endothelial cells subjected to oxygen-glucose deficiency, and strong staining was associated with propidium iodide positive nuclei, an indicator of cellular damage. These results provide new evidence for a role of Cdk5 in the events associated with response to ischemic injury in humans.

Hyaluronan-mediated angiogenesis in vascular disease: uncovering RHAMM and CD44 receptor signaling pathways.

The correct formation of new blood vessels from existing vasculature (angiogenesis) is essential for embryogenesis and the effective repair of damaged or wounded tissues. However, excessive and detrimental vascularization also occurs in neoplasia, promoting tumour growth and metastasis, as well as in proliferative diabetic retinopathy and atherosclerosis. Greater understanding of the mechanisms controlling the angiogenic process will allow optimization of wound healing, and provide mechanisms to inhibit vascularization in tumours and other diseases. Evidence supports a cascade of events in which the perturbation of one of the steps is sufficient to significantly inhibit neovascularization. The extracellular macromolecules, notably glycosaminoglycans (GAGs), are important mediators of angiogenesis. Hyaluronan (HA), a large, non-sulphated GAG, was first discovered in the vitreous of the eye [.], and is ubiquitously expressed in the extracellular matrix (ECM) of tissues. Native high molecular weight HA (n-HA) is anti-angiogenic, whereas HA degradation products (o-HA; 3-10 disaccharides) stimulate endothelial cell (EC) proliferation, migration and tube formation following activation of specific HA receptors in particular, CD44 and Receptor for HA-Mediated Motility (RHAMM, CD168). The involvement of HA in the regulation of angiogenesis makes it an attractive therapeutic target. We review the role of o-HA in modulation of angiogenesis during tissue injury, and vascular disease, focusing on receptor-mediated signal transduction pathways that have been evaluated.

A microarray study of gene and protein regulation in human and rat brain following middle cerebral artery occlusion.

BACKGROUND: Altered gene expression is an important feature of ischemic cerebral injury and affects proteins of many functional classes. We have used microarrays to investigate the changes in gene expression at various times after middle cerebral artery occlusion in human and rat brain. RESULTS: Our results demonstrated a significant difference in the number of genes affected and the time-course of expression between the two cases. The total number of deregulated genes in the rat was 335 versus 126 in the human, while, of 393 overlapping genes between the two array sets, 184 were changed only in the rat and 36 in the human with a total of 41 genes deregulated in both cases. Interestingly, the mean fold changes were much higher in the human. The expression of novel genes, including p21-activated kinase 1 (PAK1), matrix metalloproteinase 11 (MMP11) and integrase interactor 1, was further analyzed by RT-PCR, Western blotting and immunohistochemistry. Strong neuronal staining was seen for PAK1 and MMP11. CONCLUSION: Our findings confirmed previous studies reporting that gene expression screening can detect known and unknown transcriptional features of stroke and highlight the importance of research using human brain tissue in the search for novel therapeutic agents.

PAX3 and PAX3-FKHR promote rhabdomyosarcoma cell survival through downregulation of PTEN.

PAX3 or PAX3-FKHR expression is implicated in cell transformation and tumourigenesis. Here, C2C12 myoblasts were transfected with a sense Pax3 vector and a pTet-On system to induce Pax3 expression, whereas to downregulate PAX3-FKHR, Rh18 was transfected with an antisense Pax3 with a pTet-On system. The inhibition of PAX3-FKHR in Rh18 induced upregulation of PTEN. Decreased resistance to apoptosis and increased transformation ability were observed in the Rh18 cells with PAX3-FKHR downregulation. Conversely, Pax3 induction in C2C12 cells downregulated the expression of PTEN and p27(Kip1). These results indicate that the involvement of PAX3 and PAX3-FKHR in rhabdomyosarcoma tumourigenesis may be through downregulation of PTEN tumour suppressor gene, affecting the PTEN/AKT survival pathway.

Expression of signaling molecules associated with apoptosis in human ischemic stroke tissue.

There is growing evidence that, because of the highly significant differences in gene activation/protein expression between animal models of stroke and stroke patients, the current treatment strategies based on animal stroke models have been unsuccessful. Therefore, it is imperative that the pathobiology of human stroke be studied. As a first step here, Western blotting and immunohistochemistry were employed to examine expression and tissue localization of key apoptotic proteins in infarct and peri-infarcted (penumbra) from grey and white matter in human postmortem tissue of 18 patients who died between 2 and 37 d after stroke caused by large vessel disease. The contralateral hemisphere was used as a control. JNK1, JNK2, and p53 were upregulated in the majority of samples, whereas Bcl-2, caspase-3, active caspase-3, phosphorylated p53 (p-p53), phosphorylated JNK1 (p-JNK1), and phosphorylated JNK2 (p-JNK2) were upregulated in approximately half of the samples. JNK1 expression was positively correlated with JNK2 expression in grey and white matter infarct and penumbra, whereas active caspase-3 levels were positively correlated with p-JNK2 levels in grey and white matter infarct. Using indirect immunoperoxidase staining of paraffin-embedded sections, active caspase-3 was found in infarcted neurons that co-localized with TUNEL-positive cells. p-JNK localization in the nuclei of TUNELpositive cells with the morphological appearance of neurons from infarct and penumbra was also demonstrated. The use of Kaplan Meier survival data demonstrated that the presence of Bcl-2 in penumbra of grey matter correlated significantly with shorter survival (p = 0.006). In conclusion, the present study has identified significantly altered expression of apoptotic proteins in human stroke tissue and shown that the presence of Bcl-2 in penumbra of grey matter has prognostic value. It is tempting to suggest that further studies of apoptotic proteins in human stroke may lead to identification of novel targets for drug discovery.

Distinct roles of VEGF-A and VEGF-C in tumour metastasis of gastric carcinoma.

The expression of VEGF-A and -C was investigated immunohistochemically in 51 specimens of gastric carcinoma. The expression intensity was evaluated by means of a semi-quantitative scoring system and a quantitative analysis. The results showed that VEGF-A and -C were located mainly in the cytoplasm of tumour cells: 68.6% (34/51) and 62.8% (32/51) positive for VEGF-A and VEGF-C. VEGF-C expression was more frequent in tumours of the deceased patients [88.2% (15/17)] compared to that in the survivors [47.0% (16/34)]. Quantitative VEGF-C levels in the deceased patients were 0.304+/-0.023, which was significantly elevated compared to that in the survivors (0.131+/-0.018) (p<0.01). The frequency of VEGF-C expression was higher in patients with lymph node metastases (80.0%, 28/35) compared to those with negative nodes (25%, 4/16). Accordingly, its levels were markedly elevated in the former (0.230+/-0.017) than in the latter (0.089+/-0.011) (p<0.01). Furthermore, tumour cell invasion into the enlarged lymphatic vessels was also associated with VEGF-C expression. Its expression levels were 0.271+/-0.028 in lymph vessel invasion and 0.153+/-0.021 in the absence of invasion (p<0.01). VEGF-A expression, although correlated with survival, distant metastasis, ascites formation, and blood vessel invasion, was not associate with lymph node metastasis, lymphatic vessel invasion. The expression levels of the two factors were inversely correlated with overall survival rates. It can be conclude that VEGF-C expression in tumour tissues is indicative of lymphatic metastasis, whereas VEGF-A expression is more likely to be associated with haematogenous metastasis. Both VEGF-A and -C could serve as a prognostic biomarker in patients with gastric carcinoma.

Changes in hyaluronan production and metabolism following ischaemic stroke in man.

The extent of recovery from stroke is dependent on the survival of neurons, particularly in peri-infarcted regions. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion and better recovery. Hyaluronan (HA) is an important component of the brain extracellular matrix and a regulator of cellular differentiation, migration, proliferation and angiogenesis. We have found that the production of total HA and low molecular mass 3-10 disaccharides of HA (o-HA) was increased in post-mortem tissue and in the serum of patients 1, 3, 7 and 14 days (peaking at 7 days) after ischaemic stroke. Hyaluronidase activity was also increased in serum samples (peaking after 3 days), which might explain the subsequent increase in o-HA. Affinity-histochemical staining was performed using a HA-specific biotinylated binding protein, and it showed enhanced deposition of HA in blood vessels and intracellularly as well as in the nuclei of peri-infarcted neurons. Western blotting and immunohistochemistry demonstrated upregulation of HA synthases (HAS1 and 2) and hyaluronidases (HYAL1 and 2) in inflammatory cells from both stroke and peri-infarcted regions of the brain. HYAL1 was upregulated in microvesssels and intracellularly in neurons, whilst HAS2 became translocated into the nuclei of neurons in peri-infarcted areas. Receptor for HA-mediated motility was observed intracellularly and in the nuclei of neurons, in the tunica media of larger blood vessels and in the endothelial cells of microvessels in stroke-affected tissue, whilst expression of other receptors for HA, CD44 and tumour necrosis factor-stimulated gene 6 (TSG-6) were mainly increased in infiltrating mononuclear cells from inflammatory regions. The data presented here demonstrate that HA breakdown is a feature of the acute stage of stroke injury. Increased o-HA production soon after stroke may be detrimental through enhancement of the inflammatory response, whilst activation of HA and/or o-HA-induced cellular signalling pathways in neurons and microvessels may impact on the remodelling process by stimulating angiogenesis and revascularization, as well as the survival of susceptible neurons.

Vascular endothelial growth factor-C in patients with breast cancer.

Metastatic spread of tumours is the major cause of death in patients with breast cancer. Despite the importance of the lymphatic system in tumour metastasis, little is known about the role of lymphangiogenesis in tumour growth and metastasis. This study was undertaken to evaluate the potential usefulness of plasma levels of lymphanagiogenesis factor, vascular endothelial cell growth factor-C (VEGF-C) as a prognostic factor in 122 patients with breast cancer. There was no significant difference between plasma levels of VEGF-C in patients with early (n =81), advanced (n =32) or inflammatory breast cancer (n =9) and 64 age matched healthy controls. We found no significant correlation between VEGF-C with age, tumour size, tumour grade, or disease-free and over-all survival. Plasma VEGF-C levels did not significantly differ in patients with positive oestrogen, progesterone, and Her-2 neu compared to those who were negative for these parameters. In conclusion our study has failed to show any prognostic value for plasma VEGF-C level in patients with breast cancer.

Vascular endothelial growth factors and receptors in colorectal cancer: implications for anti-angiogenic therapy.

There are conflicting associations between growth factor expression and clinicopathological variables in colorectal cancer. This study aimed to define the expression of members of the VEGF family and the receptor, VEGFR2, in primary and metastatic sites of colorectal cancer and their relationship to metastatic potential. Thirty colorectal cancers, 12 lymph node metastases and 9 liver metastases were immunostained for VEGF-A, VEGF-C, VEGF-D and VEGFR2. VEGFR2 was expressed by endothelial cells and by the malignant epithelium. VEGF-C and VEGFR2 were co-expressed in the same territory and correlated throughout the primary tumour and in metastatic lymph nodes, but not in liver metastases. Their expression at the invasive tumour edge correlated with expression in metastatic nodes. The benefit of anti-VEGF antibodies might be increased by directing additional therapies against VEGF-C or against the kinase receptors to target redundancy in the system. A component of the therapeutic benefit might be due to a direct anti-tumour effect as well as an anti-angiogenic effect.

Comparative evaluation of microvessel density determined by CD34 or CD105 in benign and malignant gastric lesions.

Microvessel density (MVD) is regarded as a surrogate marker for angiogenesis and has been used for tumor prognosis. In this study, MVD was identified immunohistochemically by monoclonal antibodies against CD105 and CD34 in the tissues representing gastric carcinoma, chronic gastritis, and hyperplastic polyps, and the results were correlated with clinicopathologic features. The expression of CD105 in the microvessels within benign lesions was barely visible, and MVD was markedly lower than that determined by CD34. CD34 was strongly expressed in the microvessels within hyperplastic polyps and tissues with gastritis. In gastric carcinoma, CD105 expression in microvessels was as high as the MVD, compared with benign lesions. CD105 stained well-formed mature and newly formed immature vessels within the cancer mass. Correlation analysis showed that MVD determined by CD105 correlated with blood vessel invasion, distant metastasis, and formation of ascites. Survival analysis demonstrated an inverse correlation between MVD count and overall survival: patients with MVD counts of 32 or higher survived for a much shorter time than those with counts lower than 32. Multivariate analysis confirmed that MVD determined by CD105 was an independent prognostic factor for survival. Microvessel density determined by CD34 inversely correlated with overall survival, but it did not correlate with other clinicopathologic parameters except formation of ascites. In conclusion, CD34 was universally expressed in blood vessels within benign and malignant tissues, whereas CD105 expression was minimal in benign tissues but stronger in gastric carcinoma. These data suggest that both CD105 and CD34 could be used for quantification of angiogenesis, but preference should be given to CD105 in the evaluation of prognosis in gastric carcinoma.