Rebecca Lever

Interference with Heparin Binding and Oligomerization Creates a Novel Anti-Inflammatory Strategy Targeting the Chemokine System

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.

The effects of heparin and related molecules upon the adhesion of human polymorphonuclear leucocytes to vascular endothelium in vitro

The effects of an unfractionated heparin preparation (Multiparin), a low molecular weight heparin preparation (Fragmin) and a selectively O‐desulphated derivative of heparin lacking anticoagulant activity, have been investigated for their effects on the adhesion of human polymorphonuclear leucocytes (PMNs) to cultured human umbilical vein endothelial cells (HUVECs) in vitro. The effect of poly‐L‐glutamic acid, a large, polyanionic molecule was also studied. Unfractionated heparin (50–1000 U ml−1), the O‐desulphated derivative (0.3–6 mg ml−1) and the low molecular weight heparin (50 U–1000 U ml−1) all inhibited significantly the adhesion of 51Cr labelled PMNs to HUVECs stimulated with interleukin‐1β (IL‐1β; 10 U ml−1), bacterial lipopolysaccharide (LPS; 2.5 μg ml−1) or tumour necrosis factor‐α (TNF‐α; 125 U ml−1) for 6 h, whereas poly‐L‐glutamic acid had no effect. In addition, the three heparin preparations in the same concentration range inhibited significantly the adhesion of f‐met‐leu‐phe‐stimulated PMNs to resting HUVECs. The effects of unfractionated heparin upon the expression of adhesion molecules intercellular adhesion molecule‐1 (ICAM‐1) and E‐selection were also investigated, as were the effects of unfractionated heparin upon adhesion of human PMNs to previously stimulated HUVECs. Heparin had little effect upon levels of expression of these adhesion molecules on stimulated HUVECs. However, a profound effect upon PMN adhesion to previously stimulated HUVECs was demonstrated using the same preparation, suggesting that inhibition of adhesion molecule expression is not a major component of the described inhibitory effects of heparin. Pre‐incubation of PMNs with heparin followed by washing inhibited their adhesion to HUVECs, under different conditions of cellular activation, implying that heparin can bind to these cells and exert its anti‐adhesive effects even when not directly present in the system. These observations would suggest that both heparin and a low molecular weight heparin are capable of inhibiting adhesion of human PMNs to endothelial cells, an effect not dependent solely upon the polyanionic nature of these molecules, nor dependent upon their ability to act as anticoagulants.

Effects of heparin and related molecules upon neutrophil aggregation and elastase release in vitro.

Neutrophil‐derived elastase is an enzyme implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Heparin inhibits the enzymatic activity of elastase and here we provide evidence for the first time that heparin can inhibit the release of elastase from human neutrophils. Unfractionated and low molecular weight heparins (UH and LMWH, 0.01–1000 U ml−1) and corresponding concentrations (0.06–6000 μg ml−1) of nonanticoagulant O‐desulphated heparin (ODH), dextran sulphate (DS) and nonsulphated poly‐L‐glutamic acid (PGA) were compared for their effects on both elastase release from and aggregation of neutrophils. UH, ODH and LMWH inhibited (P<0.05) the homotypic aggregation of neutrophils, in response to both N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP, 10−6 M) and platelet‐activating factor (PAF, 10−6 M), as well as elastase release in response to these stimuli, in the absence and presence of the priming agent tumour necrosis factor‐alpha (TNF‐α, 100 U ml−1). DS inhibited elastase release under all the conditions of cellular activation tested (P<0.05) but had no effect on aggregation. PGA lacked efficacy in either assay, suggesting general sulphation to be important in both effects of heparin on neutrophil function and specific patterns of sulphation to be required for inhibition of aggregation. Further investigation of the structural requirements for inhibition of elastase release confirmed the nonsulphated GAG hyaluronic acid and neutral dextran, respectively, to be without effect, whereas the IP3 receptor antagonist 2‐aminoethoxydiphenylborate (2‐APB) mimicked the effects of heparin, itself an established IP3 receptor antagonist, suggesting this to be a possible mechanism of action.

Pharmacology of Heparin and Related Drugs

Heparin has been recognized as a valuable anticoagulant and antithrombotic for several decades and is still widely used in clinical practice for a variety of indications. The anticoagulant activity of heparin is mainly attributable to the action of a specific pentasaccharide sequence that acts in concert with antithrombin, a plasma coagulation factor inhibitor. This observation has led to the development of synthetic heparin mimetics for clinical use. However, it is increasingly recognized that heparin has many other pharmacological properties, including but not limited to antiviral, anti-inflammatory, and antimetastatic actions. Many of these activities are independent of its anticoagulant activity, although the mechanisms of these other activities are currently less well defined. Nonetheless, heparin is being exploited for clinical uses beyond anticoagulation and developed for a wide range of clinical disorders. This article provides a “state of the art” review of our current understanding of the pharmacology of heparin and related drugs and an overview of the status of development of such drugs.

Heparin : a century of progress

Part 1. Introduction.- Part 2. Unfractionated and Low Molecular Weight Heparins.- Part 3. Clinical Use of Heparin and LMWH.- Part 4. Non Anticoagulant Indictions for Heparin and Related Compounds.- Part 5. Heparin-like Entities.

Non-anticoagulant Effects of Heparin: An Overview

Heparin has long been known to possess biological effects that are unrelated to its anticoagulant activity. In particular, much emphasis has been placed upon heparin, or novel agents based upon the heparin template, as potential anti-inflammatory agents. Moreover, heparin has been reported to possess clinical benefit in humans, including in chronic inflammatory diseases and cancer, that are over and above the expected effects on blood coagulation and which in many cases are entirely separable from this role. This chapter aims to provide an overview of the non-anticoagulant effects that have been ascribed to heparin, from those involving the binding and inhibition of specific mediators involved in the inflammatory process to effects in whole system models of disease, with reference to the effects of heparin that have been reported to date in human diseases.

Activation of corticotropin-releasing factor receptor-2 causes bronchorelaxation and inhibits pulmonary inflammation in mice

Urocortins are members of the corticotropin‐releasing factor (CRF) family of peptides that bind to two receptors, CRF1 and CRF2. While CRF1 is a high‐affinity receptor for CRF, urocortin III binds with much greater affinity to CRF2. In the present study we investigated the effect of CRF2 receptor activation with urocortin III on airway smooth muscle tone in vitro and in an acute model of airway inflammation in mice. Urocortin III caused relaxation of methacholine‐pre‐contracted mouse tracheal segments. CRF caused similar relaxation, but with reduced potency compared to urocortin III, consistent with the CRF2 receptor subtype. Relaxation induced by urocortin III was concen‐tration‐dependently inhibited by the CRF2 antagonist, astressin 2B, with an IC50 in the nanomolar range. These relaxations were potentiated by inhibition of phosphodiesterases but unaffected by inhibition of cyclooxygenase and NO or by removal of the epithelium. Finally, the number of neutrophils retrieved by bronchoalveolar lavage after administration of bacterial LPS (LPS) was reduced by prior intraperitoneal (i.p.) injection of urocortin III. This effect was also suppressed by astressin 2B, implicating CRF2 receptors. Therefore, CRF2 agonists appear to have both bronchorelaxant and anti‐inflammatory activities and might represent an interesting therapeutic approach to the treatment of inflammatory lung diseases.—Moffatt, J. D., Lever, R., Page, C. P. Activation of corticotropin‐releasing factor receptor‐2 (CRF2) causes bronchorelaxation and inhibits pulmonary inflammation in mice. FASEB J. 20, E1181–E1187 (2006)

Locally available heparin modulates inflammatory cell recruitment in a manner independent of anticoagulant activity

Heparin is known to possess anti-inflammatory properties that in many cases appear to be separable from its anticoagulant activity. Mast cells, located in tissue, are the sole source of endogenous heparin, which may be involved in control of the inflammatory response. The majority of studies of the effects of heparin on the inflammatory response, carried out to date, have involved systemic administration and the potential influence of heparin in the site of inflammation has been less clear. In the present study, the effects of locally administered heparin and a non-anticoagulant derivative were investigated on leucocyte accumulation in the inflamed peritoneal cavity and leucocyte-endothelial interactions in the mesenteric microcirculation of the rat. Heparin and non-anticoagulant heparin inhibited the influx of neutrophils to the site of inflammation, as well as leucocyte rolling and adhesion in post-capillary venules. These effects were apparent only while heparin was present in the peritoneal cavity and plasma and may reflect, in part, an action on resident peritoneal cells, as the heparins tested were found also to inhibit the expression of endothelial adhesion molecules in response to products of activated mononuclear cells, in vitro. Our data show that locally administered heparin has anti-inflammatory effects in an in vivo model of peritoneal inflammation whilst present at the site of inflammation. These non-anticoagulant properties may involve interaction with cells in the site of inflammation, in addition to inhibiting cell adhesion at the level of the microcirculation.

The effects of heparin on the adhesion of human peripheral blood mononuclear cells to human stimulated umbilical vein endothelial cells.

The effects of unfractionated heparin (UH) and a selectively O‐desulphated derivative of heparin (ODSH), lacking anticoagulant activity, on the adhesion of human peripheral blood mononuclear cells (HPBMNC) to human stimulated umbilical vein endothelial cells (HUVECs), were investigated. For comparison, the effects of poly‐L‐glutamic acid (PGA), a large polyanionic molecule without sulphate groups and two different molecular weight sulphated dextrans (DS 5 k and DS 10 k) were studied. UH (50 – 1000 u ml−1) significantly (P<0.05) inhibited the adhesion of HPBMNC to HUVECs, stimulated with IL‐1β (100 u ml−1), TNF‐α (1000 u ml−1) or LPS (100 μg ml−1), when the drugs were added together with stimuli to HUVECs and coincubated for 6 h. Such effects on adhesion occurred with limited influence on expression of relevant endothelial adhesion molecules (ICAM‐1 and VCAM‐1). UH (100 – 1000 u ml−1), when added to prestimulated HUVECs, significantly (P<0.05) increased adhesion of mononuclear cells to endothelium at the higher concentrations tested, without any effect on adhesion molecule expression. In contrast, the opposite effect was observed when human polymorphonuclear leucocyte adhesion was examined, under the same experimental conditions, suggesting that the observed potentiation of HPBMNC adhesion is cell specific. The effects of UH on HPBMNC adhesion were shared by the non‐anticoagulant ODSH (600 – 6000 μg ml−1) but not by sulphated dextrans or PGA (300 – 6000 μg ml−1). Heparin affects the adhesion of HPBMNC to stimulated endothelium, in both an inhibitory and potentiating manner, effects which are unrelated to its anticoagulant activity and not solely dependent on molecular charge characteristics.

Size-fractionated heparins have differential effects on human neutrophil function in vitro.

Heparin is known to possess a range of activities, other than effects on blood coagulation, many of which are anti‐inflammatory. Effects with potential anti‐inflammatory applications include the inhibition of elastase release from neutrophils, as well as the adhesion of these cells to vascular endothelium. In the present study we aimed to investigate whether fractionation of heparin may yield molecules with enhanced or specific effects on human neutrophil function.