Venous hypoxia: a poorly studied etiological factor of varicose veins.

Venous hypoxia has long been postulated as a potential cause of varicosity formation. This article aimed to review the development of this hypothesis, including evidence supporting and controversies surrounding it. Vein wall oxygenation is achieved by oxygen diffusing from luminal blood and vasa vasorum. The whole media of varicosities is oxygenated by vasa vasorum as compared to only the outer two-thirds of media of normal veins. There was no evidence that differences exist between oxygen content of blood from varicose and non-varicose veins, although the former demonstrated larger fluctuations with postural changes. Studies using cell culture and ex vivo explants demonstrated that hypoxia activated leucocytes and endothelium which released mediators regulating vein wall remodelling similar to those observed in varicosities. Venoactive drugs may improve venous oxygenation, and inhibit hypoxia activation of leucocytes and endothelium. The evidence for hypoxia as a causative factor in varicosities remains inconclusive, mainly due to heterogeneity and poor design of published in vivostudies. However, molecular studies have shown that hypoxia was able to cause inflammatory changes and vein wall remodelling similar to those observed in varicosities. Further studies are needed to improve our understanding of the role of hypoxia and help identify potential therapeutic targets.

What have we learnt from targeted anti-TNF therapy?

Anti-tumour necrosis factor (anti-TNF) therapy of patients with rheumatoid arthritis dates back to 1992, when the first proof-of-principle trials were performed in London by Maini and Feldmann. Considerable studies of the mechanism of action were performed, and insights into the way in which anti-TNF therapy delivers its benefit were obtained. In this brief review, certain aspects of knowledge acquired and the many gaps will be reviewed. Focus will be on the TNF-dependent cytokine cascade and what it means, and potential new approaches to treatment. Finally, an entertaining challenge: might many or even all unmet clinical needs be dealt with through cytokine analysis?

Inhibitory effect of TIMP influences the morphology of varicose veins.

OBJECTIVES: Imbalance of matrix metalloproteinase enzymes (MMP) and their inhibitors (TIMPs) may contribute to the development of varicose veins. We hypothesised that, histological changes in varicose vein wall correlate with alterations in expression of MMP/TIMP. METHODS: Varicose veins (n=26) were compared with great saphenous vein (GSV) segments (n=11) from arterial bypass, and with arm and neck veins from fistula and carotid operations (n=13). Varicose vein wall thickness was measured, enabling categorisation as atrophic and hypertrophic. MMP-2, MT1-MMP, TIMP-2, and TIMP-3 expression were quantitatively analysed by immunohistochemistry. RESULTS: There was significantly higher expression of TIMP-2 (immunopositive area 4.34% versus 0.26%), linked with connective tissue accumulation in the tunica media of varicose veins as compared with arm and neck vein controls. TIMP-2 and TIMP-3 expression was higher in hypertrophic than atrophic segments (3.2% versus 0.99% for TIMP-2, 1.7% versus 0.08% for TIMP-3). Similarly, TIMP-2 and TIMP-3 had elevated expression in the thicker proximal varicose vein segments compared to distal (4.3% versus 1.3% for TIMP-2 and 0.94% versus 0.41% for TIMP-3). CONCLUSIONS: This study linked morphological changes in varicose vein walls with MMP/TIMP balance. A higher TIMP expression favours deposition of connective tissue and thus thicker vein wall, reducing matrix turnover by suppression of protease activity.

In vitro target validation and in vivo efficacy of p38 MAP kinase inhibition in established chronic collagen-induced arthritis model: a pre-clinical study.

OBJECTIVES: The aim of the present study was to determine the in vivo efficacy of p38 mitogen-activated protein kinase (MAPK) inhibitors, namely GW856553X and GSK678361, in murine models of arthritis. METHODS: The effect of p38 MAPK inhibitors was tested in 2 variants of the collagen-induced arthritis model (CIA) in DBA/1 mice, acute arthritis induced by heterologous collagen and chronic relapsing arthritis induced by homologous collagen. Animals were treated after onset of arthritis. Furthermore, post-onset disease efficacy of GSK678361 was tested in the chronic model, so as to determine the effects on established arthritis. In vitro studies were carried out with GW856553X, using human umbilical vein endothelial cells, to determine potential effects of GW856553X on the vasculature. RESULTS: In both acute and chronic arthritis, GW856553X reduced signs and symptoms of disease, and protected joints from damage. The effect of GW856553X in chronic CIA was confirmed using an alternative compound, GSK678361. Importantly, treatment with GSK678361 from 14 days post-onset of chronic arthritis completely reversed signs of established disease and joint destruction. Mechanism of action studies demonstrated that GW856553X inhibited endothelial cell migration and angiogenesis in vitro, with reduced pro-inflammatory cytokine production. CONCLUSIONS: Suppression of murine CIA by the p38 MAPK inhibitors GW856553X and GSK678361 suggests that they may have therapeutic potential for future use in RA if safe clinical dosing achieves adequate compound exposure.

Adenovirus-mediated VEGF gene therapy enhances venous thrombus recanalization and resolution.

OBJECTIVE: Rapid thrombus recanalization reduces the incidence of post-thrombotic complications. This study aimed to discover whether adenovirus-mediated transfection of the vascular endothelial growth factor gene (ad.VEGF) enhanced thrombus recanalization and resolution. METHODS AND RESULTS: In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376+/-299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7+/-5.1 mm(2) to 22.0+/-4.0 mm(2), P=0.0003) and increased recanalization by >3-fold (3.9+/-0.69% to 13.6+/-4.1%, P=0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% (P=0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3+/-0.89 mm(2) to 8.7+/-1.4 mm(2), P=0.04) and enhanced vein lumen recanalization (3.39+/-0.34% to 5.07+/-0.57%, P=0.02). CONCLUSIONS: Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.

Human neutrophils secrete vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil-derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation.

The role of receptors for tumour necrosis factor-alpha in the induction of human polymorphonuclear neutrophil chemiluminescence.

Tumour necrosis factor-alpha (TNF-alpha) is a potent mediator of inflammation, which exerts profound effects on polymorphonuclear neutrophils (PMN). TNF-alpha binds to distinct cell surface receptors termed p55 and p75, expressed in approximately equal amounts on the PMN surface. We have studied the effects of TNF-alpha on the priming of F-Met-Leu-Phe (FMLP)-stimulated oxidative metabolism of PMN, using a luminol-enhanced chemiluminescence assay, and have examined the relative roles of PMN receptors for TNF-alpha in priming this oxidative metabolism, using antibodies with p55 and p75 receptor-specific agonistic and antagonistic activities. We have obtained the following results: (1) Antibody Htr-9 with agonistic activity at the p55 receptor mimicked the effect of TNF-alpha; however, a combination of Htr-9 and TNF-alpha did not results in any further increase in chemiluminescence relative to the response observed with TNF-alpha alone. The p75 agonistic antibody MR2-1 actually decreased basal and FMLP-enhanced chemiluminescence. Additionally, MR2-1 substantially inhibited the effects of both TNF-alpha itself and of the p55 agonist Htr-9. (2) Addition of antibodies with antagonistic activities at the p55 (antibody TBP-2) and p75 (antibody Utr-1) receptors resulted in a marked inhibition of the PMN response to TNF-alpha. A combination of both Utr-1 and TBP-2 was most effective at inhibiting the action of TNF. We have confirmed previously published observations that TNF-alpha alone effectively stimulates the oxidative metabolism of PMN in vitro, and that pre-incubation of PMN with TNF-alpha enhances subsequent generation of oxidative metabolites in response to FMLP. We conclude that both p55 and p75 receptors play a critical role in mediating the activation of PMN by TNF-alpha.

The role of the angiogenic molecule VEGF in the pathogenesis of rheumatoid arthritis.

The expansion of the synovial lining of joints in rheumatoid arthritis (RA), and the subsequent invasion by the pannus of underlying cartilage and bone, necessitates an increase in the vascular supply to the synovium, to cope with the increased requirement for oxygen and nutrients. New blood vessel formation - 'angiogenesis' - is now recognised as a key event in the formation and maintenance of the pannus in RA. Although many pro-angiogenic factors have been demonstrated to be expressed in RA synovium, the potent pro-angiogenic cytokine vascular endothelial growth factor (VEGF) has been demonstrated to have a central involvement in the angiogenic process in RA. The additional activity of VEGF as a vascular permeability factor may also increase oedema and hence joint swelling in RA. Several studies, including those from the Kennedy Institute of Rheumatology Division, have shown that targeting angiogenesis in animal models of arthritis ameliorates disease. Inhibition of angiogenesis, as an adjunct to existing therapy of RA, or even as a stand-alone treatment, would not only prevent delivery of nutrients to the synovium, but could also lead to vessel regression and possibly reversal of disease.

von Willebrand factor is an acute phase reactant in man.

Plasma von Willebrand factor antigen (vWfAg) concentrations in 19 patients with acute infectious illnesses of bacterial, viral or parasitic origin were significantly elevated with a mean greater than 3-fold above normal. In individual patients the elevation of vWf correlated strongly with the elevation of serum C-reactive protein (CRP). When patients were studied longitudinally, vWfAg and CRP concentrations both returned to normal values over 3-4 weeks. Similarly, in 14 volunteers infected with cold virus, vWfAg and CRP levels rose significantly and fell together during the course of infection. VWf is thus an acute phase reactant in man.

Neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression in endothelial cells.

Defensins, a family of small, cationic, antimicrobial peptides, are found in mammals, insects and plants. alpha-defensins are stored in granules of neutrophils and released upon activation by exocytosis. It was shown here that human neutrophil peptide (HNP), at concentrations of 10(-8) -10(-9) M, up-regulated the expression of TNF-alpha and IL-1 beta in monocytes activated with Staphylococcus aureus or PMA, while expression of IL-10 mRNA was down-regulated and production of IL-8 was not affected. HNP alone was unable to induce TNF-alpha or IL-1 beta expression in resting monocytes. At concentrations of 10(-4) -10(-5)M, HNP was cytotoxic for monocytes in serum-free medium. The cytotoxicity was abrogated in the presence of serum, while a cytokine-modulating effect of HNP was observed in the presence of serum and in whole blood, suggesting that this mechanism may function in vivo. Similarly, serum did not abrogate bactericidal activity of HNP. It was also demonstrated herein that HNP at 10 (-8) -10(-9) M, attenuated the inhibitory action of dexamethasone on TNF-alpha production. In parallel to monocyte studies, we have showed that HNP at concentrations ranging from 10(-9)M to 10(-6)M caused about 5-fold suppression of VCAM-1 expression in TNF-alpha-activated human umbilical vein endothelial cells, while the ICAM-1 expression was not affected. Our findings suggest that neutrophil defensins have the potential to modulate the inflammatory responses through regulation of cytokine production and adhesion molecule expression.

VEGF release is associated with reduced oxygen tensions in experimental inflammatory arthritis.

OBJECTIVE: The contribution of local VEGF production and subsequent angiogenesis within the synovial membrane to the propagation of arthritis is unclear. The relationship between synovial oxygenation and blood flow in the development of arthritic disease is unknown. We have therefore measured oxygen levels and perfusion rates in the synovial space in a murine model of arthritis. METHODS: Arthritis was induced in DBA/1 mice by immunisation with type II collagen. Oxygen and perfusion levels were measured polarographically using silver needle microelectrodes within the knee joints prior to and 10 days after the onset of arthritis. In addition, synovial cells were isolated from knee joints of naive, pre-arthritic and arthritic mice. RESULTS: Onset of arthritis was associated with a marked reduction in synovial oxygen tensions (pO2). The perfusion rates in naive and arthritic animals were not significantly different: in naive mice, the rate was 0.58 +/- 0.11 ml/min/g and in arthritic joints, 0.64 +/- 0.17 ml/min/g. Furthermore, synovial cells isolated from the knee joints of naive animals did not express mRNA for VEGF, but significant levels were detected in cells from non-arthritic mice immunised with collagen. The onset of arthritis was associated with expression of VEGF mRNA and protein, and correlated negatively with pO2 levels. CONCLUSION: These data demonstrate that decreases in intra-articular pO2 occur in established arthritic conditions and may be the stimulus for local VEGF production. However, perfusion was not increased in arthritic animals and vascular density was unaltered, suggesting that the neovascularisation associated with inflammatory arthritis, is insufficient to restore oxygen homeostasis in the joint.

Comparison of the functional activities of two different preparations of human recombinant tumor necrosis factor alpha.

The role of tumor necrosis factor (TNF-alpha) in physiological and pathological reactions has resulted in a progressive increase of expensive TNF-alpha consumption for laboratory and clinical purposes. Following this trend, the first chemical synthesis of the gene for rHuTNF-alpha gene in Poland and its subsequent successful expression in E. coli was recently reported. In the present paper, we verify the in vitro biological activities of this TNF-alpha preparation (CMMS/TNF-alpha) in comparison with a commercially available preparation of TNF-alpha. We demonstrate that our TNF-alpha possesses strong cytotoxic activity against WEHI 164 (clone 13) cells, binds the p55 and p75 TNF receptors on cell lines, induces intercellular adhesion molecule-1 (ICAM-1) expression, and interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) release from human umbilical vein endothelial cells (HUVEC). We demonstrate its usefulness for further investigations as an effective reagent for in vitro assays.

[Cellular mechanisms of regulation of the inflammatory response in atherosclerosis: the role of cell contact-mediated signaling]. / I meccanismi cellulari di regolazione ed amplificazione del processo infiammatorio nella placca aterosclerotica: le interazioni cellulari da contatto.

The regulatory mechanisms of the inflammatory process in the atherosclerotic plaque are still not clearly understood. Stimulated T cells may have a key role in enhancing and perpetuating inflammation at the atherosclerotic site. They activate endothelial cells, macrophages and smooth muscle cells in the atherosclerotic plaque, not only via the production of soluble mediators, but also through cell-cell contact-mediated interactions (via membrane receptors and their ligands). Cell/cell contact between stimulated T lymphocytes and monocytes/macrophages and endothelial cells induces the production of pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-6) and chemokines (interleukin-8, monocyte chemotactic factor-1). Thus, these interactions could play a relevant role in the disregulation of the inflammatory process in the atherosclerotic plaque, representing a novel mechanism of progression and complication of the atherosclerotic disease. Understanding the key ligands and receptors involved may permit the definition of new therapeutic targets.

Circulating endothelial progenitor cells as a link between synovial vascularity and cardiovascular mortality in rheumatoid arthritis.

Cardiovascular disease refers to the class of diseases that involve the heart and/or blood vessels (arteries and veins). Most Western countries face high and ever-increasing rates of cardiovascular disease. Each year, more Americans are killed by heart disease than by cancer. Diseases of the heart alone cause 30% of all deaths, with other diseases of the cardiovascular system causing substantial further deaths and disability. Indeed, cardiovascular disease is the major cause of death and disability in the USA and most European countries. The development of the vascular systems requires an intricate interplay of molecules such as vascular endothelial growth factor and endothelial progenitor cells. A defective vascular repair/regeneration is thought to be responsible for propagation of atherosclerosis, a key feature of cardiovascular disease. This is partly attributed to a reduction in the circulating endothelial progenitor cells in peripheral blood. Patients with rheumatoid arthritis (RA) have a higher than average incidence of cardiovascular disease in comparison with the general population, with an increased risk of stroke and myocardial infarction, and an increased risk of fatality following myocardial infarction. This review focuses on the current evidence linking the role played by endothelial progenitor cells to the development of cardiovascular disease and why this might relate to the increased risk observed in RA patients.

Target effector role of vascular endothelium in the inflammatory response: insights from the clinical trial of anti-TNF alpha antibody in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterised by chronic joint inflammation and infiltration by cells from the blood, especially activated T cells and macrophages, together with formation of new blood vessels. The overgrowth of the synovial lesion results eventually in destruction of cartilage and bone. Cytokines play a major role in RA, both in systemic inflammatory processes, such as induction of acute phase protein synthesis, and in the stimulation of new blood vessel development and recruitment of leucocytes to developing lesions. The focus for the interplay of many cytokines is the endothelium, the lining layer of the vasculature. This is the primary target for circulating mediators, and it controls the traffic of cells and molecules from the bloodstream into underlying tissues. Targeting the action of individual cytokines--for example, using antibody against tumour necrosis factor alpha (TNF alpha), has been shown to be very effective in the treatment of RA. Blockade of TNF alpha activity results in deactivation of the endothelium, manifested as reduced expression of adhesion molecules and chemoattractant cytokines, leading to diminished trafficking of inflammatory cells to synovial joints. In addition anti-TNF alpha decreases circulating levels of the potent angiogenic cytokine VEGF, suggesting that new blood vessel formation, and hence the supply of nutrients to the growing synovial lesion, is also affected. These observations lend further support to the hypothesis that interruption of a component of the cytokine network in RA may modulate disease progression, and point the way towards the development of new therapeutic strategies for the treatment of chronic inflammatory disease states.