Regulation of the angiopoietin-Tie ligand-receptor system with a novel splice variant of Tie1 reduces the severity of murine arthritis.

OBJECTIVES: To determine the function of the angiopoietin (Ang)-Tie ligand-receptor system, and to assess the effect of Tie1-751, a naturally occurring extracellular domain of the Tie1 receptor derived by alternative splicing, in an in vivo model of RA. METHODS: In the murine CIA model, expression of endogenous Ang1, Ang2, Tie1 and Tie2 in whole paws was analysed by quantitative RT-PCR. To assess the effect of inhibition of the Ang-Tie axis, Tie1-751 was expressed and fused to the Fc fragment of human IgG1. The effect of Tie1-751-Fc on human umbilical vein endothelial cell (HUVEC) cytoprotection and migration in response to Ang1, either alone or in combination with VEGF, was investigated. Furthermore, an in vitro angiogenesis assay was used to determine the effect of Tie1-751-Fc on Ang1-mediated angiogenesis. Finally, Tie1-751-Fc was administered in CIA, and the effects on clinical disease and joint architecture of hind foot specimens were determined. RESULTS: Gene expression levels of Ang1, Ang2, and receptors Tie1 and Tie2 in whole paws were significantly increased during the progression of arthritis. Tie1-751-Fc significantly inhibited HUVEC cytoprotection and migration in response to Ang1 alone, or Ang1 in combination with VEGF. Tie1-751-Fc also significantly inhibited angiogenesis induced by a combination of Ang1 plus VEGF. Finally, Tie1-751-Fc, when administered intra-peritoneally to arthritic mice, reduced clinical signs of arthritis, damage to joint architecture and infiltration of blood vessels into the synovium. CONCLUSIONS: Our data demonstrate that the Ang-Tie ligand-receptor system is dysregulated in CIA. Tie1-751, a novel splice variant of the Tie1 receptor, inhibits Ang1/VEGF signalling, suggesting that Ang inhibition may be of therapeutic benefit in inflammatory arthritis.

Chk1 and Wee1 control genotoxic-stress induced G2-M arrest in melanoma cells.

In the present report, the role of ATR-Chk1-Wee1 and ATM-Chk2-p53-p21 pathways in stress-induced cell cycle control is analysed in melanoma cells. Treatment of p53 wild-type melanoma cells with the genotoxic agent doxorubicin induces G2-M arrest, inhibitory phosphorylation of cell cycle kinase Cdc2 (CDK1) and enhanced expression of p53/p21. Wee1 inhibition under doxorubicin pulse-treatment reduces G2-M arrest and induces apoptosis. Inhibition of upstream kinase Chk1 under doxorubicin treatment almost completely abolishes stress-induced G2-M arrest and induces enhanced apoptosis. Interestingly, Chk1 inhibition alone even further increases apoptosis. While Chk1 inhibition alone almost completely abolishes G0-G1 arrest, combined treatment with doxorubicin re-establishes G0-G1 arrest. Moreover, Chk1 inhibition alone induces only a slight p53/p21 induction, while a strong induction of both proteins is observed by the combination with doxorubicin. These findings are suggestive for a particular role of p53/p21 in G0-G1, and Chk1 in G0-G1 and G2-M arrest. In line with this, the p53-mutant SK-Mel-28 melanoma cells do not mount a significant G0-G1 arrest under combined doxorubicin and Chk1 inhibitor treatment but rather show extensive apoptosis. Moreover, knockdown of p21 dramatically reduces stress-induced G0-G1 arrest under doxorubicin and Chk1 inhibitor treatment accompanied by massive DNA damage and apoptosis induction. Treatment of melanoma cells with an inhibitor of Chk2 upstream kinase ATM and doxorubicin almost completely abolishes G0-G1 arrest. Taken together, both Chk1 and Wee1 are mediators of G2-M arrest, while p53, p21 and Chk1 are mediators of G0-G1 arrest in melanoma cells. Combined treatment with chemotherapeutic agents such as doxorubicin and Chk1 inhibitors may help to overcome apoptosis resistance of p53-proficient melanoma cells. But treatment with Chk1 inhibitor alone may even be more efficient.

miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy.

The present study identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions of melanomas compared with primary melanomas. miR-638 enhanced the tumorigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling identified new candidate genes including TP53INP2 as miR-638 targets, the majority of which are involved in p53 signalling. Overexpression of TP53INP2 severely attenuated proliferative and invasive capacity of melanoma cells which was reversed by miR-638. Depletion of miR-638 stimulated expression of p53 and p53 downstream target genes and induced apoptosis and autophagy. miR-638 promoter analysis identified the miR-638 target transcription factor associated protein 2α (TFAP2A/AP-2α) as a direct negative regulator of miR-638, suggestive for a double-negative regulatory feedback loop. Taken together, miR-638 supports melanoma progression and suppresses p53-mediated apoptosis pathways, autophagy and expression of the transcriptional repressor TFAP2A/AP-2α.

Gene expression profiling and functional analysis of angiogenic markers in murine collagen-induced arthritis.

INTRODUCTION: Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA. METHODS: CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1. RESULTS: Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1B antibody significantly reduced disease severity and progression in CIA mice. CONCLUSIONS: Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.

Dynamical effects of epigenetic silencing of 14-3-3sigma expression.

The development and progression of malignant tumours are often due to deregulated cell cycle control involving a plethora of different molecules. Among these, tumour suppressor proteins like p53 play a crucial role. p53 induces 14-3-3sigma, a multifunctional protein kinase inhibitor, centrally involved in cell cycle control and DNA damage repair after genotoxic stress. Recently, it has been shown that 14-3-3sigma is epigenetically silenced in a variety of tumours, which might contribute to tumour development and progression via impaired cell cycle control. In addition, p53, its inhibitor MDM2 and 14-3-3sigma form a signalling module in which 14-3-3sigma positively regulates the activity of p53 through feedback regulation. Here we present a mathematical model integrating the effects of 14-3-3sigma gene silencing, the dynamics of 14-3-3sigma induction and compartmentalisation by genotoxic stress and the role of interacting molecules p53 and MDM2. In vitro experiments with different melanoma cell lines were performed and our mathematical model was subjected to computer simulations to analyse different scenarios of activation depending on gene methylation status and DNA damage levels. Our analysis indicates that 14-3-3sigma expression is silenced by high gene methylation, but also that strong stimulation is necessary to induce 14-3-3sigma expression in cases of intermediate levels of gene methylation. More intriguingly, the model suggests that epigenetic silencing of 14-3-3sigma affects p53 dynamics in a synergistic way, such that the accumulative effect of partial downregulation of p53 expression and reduction of its nuclear fraction could affect drastically the activity of p53 as a transcription factor.

Angiogenesis as a therapeutic target in arthritis: learning the lessons of the colorectal cancer experience.

The idea of a therapeutic modality aimed at 'starving' a tissue of blood vessels, and consequentially of oxygen and nutrients, was born from the concept that blood vessel formation (angiogenesis) is central to the progression and maintenance of diseases which involve tissue expansion/invasion. In the first instance, solid malignancies were the target for anti-angiogenic treatments, with colorectal cancer being the first disease for which an angiogenesis inhibitor--anti-vascular endothelial growth factor antibody bevacizumab--was approved in 2004. Our understanding of the pathogenesis of rheumatoid arthritis (RA) has lead to many parallels being drawn between this chronic inflammatory disease and solid tumours, in that both involve tissue expansion, invasion, expression of cytokines and growth factors and areas of hypoxia/hypoperfusion. As a result, angiogenesis blockade has been touted as a possible treatment for RA. The lessons learnt during the progression of eventually successful therapies such as bevacizumab should undoubtedly guide us in the future development of comparable treatments for RA.