The angiogenesis inhibitor protease-activated kringles 1-5 reduces the severity of murine collagen-induced arthritis

During rheumatoid arthritis there is enlargement and increased cellularity of the synovial lining of joints, before invasion by the synovium of the underlying cartilage and bone. This increased tissue mass requires a network of blood vessels to supply nutrients and oxygen. Disruption of synovial angiogenesis is thus a desirable aim of antiarthritic therapies. Protease-activated kringles 1-5 (K1-5) is an angiogenesis inhibitor related to angiostatin. In common with angiostatin, K1-5 contains the first four kringle domains of plasminogen, but also encompasses the kringle 5 domain, which confers enhanced antiangiogenic activity when compared with angiostatin. The purpose of the present study was to assess the effect on murine arthritis of K1-5. Arthritis was induced in DBA/1 mice by a single injection of bovine collagen. Treatment with K1-5 was commenced on the day of arthritis onset and continued for 10 days, until the end of the experiment. Daily intraperitoneal administration of K1-5 (2 mg/kg body weight) significantly reduced both paw swelling and clinical score (a composite index of the number of arthritic limbs and the severity of disease). The clinical efficacy of this treatment was reflected by a reduction in joint inflammation and destruction, as assessed histologically. These data suggest that antiangiogenic therapies, which block formation of new blood vessels and hence reduce synovial expansion, might be effective in treating rheumatoid arthritis.     

Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition

The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention in RA.     

A window on disease pathogenesis and potential therapeutic strategies: molecular imaging for arthritis

Novel molecular imaging techniques are at the forefront of both preclinical and clinical imaging strategies. They have significant potential to offer visualisation and quantification of molecular and cellular changes in health and disease. This will help to shed light on pathobiology and underlying disease processes and provide further information about the mechanisms of action of novel therapeutic strategies. This review explores currently available molecular imaging techniques that are available for preclinical studies with a focus on optical imaging techniques and discusses how current and future advances will enable translation into the clinic for patients with arthritis.     

Notes on the rotifers of coal mine water in Eastern Poland

The species composition and quantitative structure of the rotifer fauna was investigated in a reservoir containing coal mine water. Only nine mainly planktonic species of rotifers, were found. Two of these were dominating: Brachionus angularis and B. rubens. They are typical indicators of eutrophic waters. Chlorides and sulphates may have an influence on the occurrence and quantitative structure of rotifer assemblages in the investigated reservoir.     

Novel splice variants derived from the receptor tyrosine kinase superfamily are potential therapeutics for rheumatoid arthritis

Introduction  

Despite the advent of biological therapies for the treatment of rheumatoid arthritis, there is a compelling need to develop alternative therapeutic targets for nonresponders to existing treatments. Soluble receptors occur naturally in vivo, such as the splice variant of the cell surface receptor for vascular endothelial growth factor (VEGF) – a key regulator of angiogenesis in rheumatoid arthritis. Bioinformatics analyses predict that the majority of human genes undergo alternative splicing, generating proteins – many of which may have regulatory functions. The objective of the present study was to identify alternative splice variants (ASV) from cell surface receptor genes, and to determine whether the novel proteins encoded exert therapeutic activity in an in vivo model of arthritis.     

Human epidermal growth factor receptor bispecific ligand trap RB200: abrogation of collagen-induced arthritis in combination with tumour necrosis factor blockade

Introduction  

Rheumatoid arthritis (RA) is a chronic disease associated with inflammation and destruction of bone and cartilage. Although inhibition of TNFα is widely used to treat RA, a significant number of patients do not respond to TNFα blockade, and therefore there is a compelling need to continue to identify alternative therapeutic strategies for treating chronic inflammatory diseases such as RA. The anti-epidermal growth factor (anti-EGF) receptor antibody trastuzumab has revolutionised the treatment of patients with EGF receptor-positive breast cancer. Expression of EGF ligands and receptors (known as HER) has also been documented in RA. The highly unique compound RB200 is a bispecific ligand trap that is composed of full-length extracellular domains of HER1 and HER3 EGF receptors. Because of its pan-HER specificity, RB200 inhibits responses mediated by HER1, HER2 and HER3 in vitro and in vivo. The objective of this study was to assess the effect of RB200 combined with TNF blockade in a murine collagen-induced arthritis (CIA) model of RA.     

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-NRP1^B 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.     

Contrasting effects of interferon gamma and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor alpha.

Tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelial cells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-alpha-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colony-stimulating factor (GM-CSF) from TNF-alpha-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-alpha-induced responses to those observed with endothelial cells of foetal origin. Additionally, we report here that TNF-alpha and interferon gamma (IFN-gamma) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-alpha-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-gamma plus TNF-alpha was markedly decreased when compared to the response induced by TNF-alpha alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.     

Proteolytic system of the sperm of Apis mellifera drones

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.     

From the cradle to the clinic: VEGF in developmental, physiological, and pathological angiogenesis

Formation of new blood vessels, which is fundamental in embryonic development, occurs through a combination of angiogenesis and vasculogenesis. Angiogenesis also plays a vital role postnatally, especially in reparative processes such as wound and fracture healing. Some of these events, especially in fracture healing, recapitulate processes observed in developmental angiogenesis. However, dysregulated angiogenesis is well documented to underlie a number of pathological disorders, including rheumatoid arthritis (RA). The vascular endothelial growth factor (VEGF)/VEGF receptor system is the best characterized regulator of angiogenesis. VEGF is expressed in a range of cells in response to soluble mediators (such as cytokines and growth factors), cell-bound stimuli (such as CD40 ligand), and environmental factors (such as hypoxia). As a consequence, this molecule is vital in the modulation of physiological and pathological angiogenesis. This review will focus in particular on the role played by VEGF in embryogenesis and skeletal growth, in fracture healing (in which increased angiogenesis is likely to be beneficial in promoting union), and in RA (in which excessive angiogenesis is thought to play a significant role in disease pathogenesis). In the not-too-distant future, targeting VEGF may prove to be of benefit in the treatment of diseases associated with excessive or aberrant angiogenesis, such as malignancies and RA.