Effect of oral and intravenous heparin tetrasaccharide on allergic airway responses: Critical role of N-sulfation

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Abstract

We have shown that inhaled heparin (hep) oligosaccharides attenuate allergic airway responses in sheep and that this anti-allergic activity resides in a tetrasaccharide sequence. Here we determined: (a) the anti-allergic activity of oral and intravenous hep-tetrasaccharide on allergic airway responses in the sheep model of asthma; and (b) the role of N-sulfation in mediating this anti-allergic activity. Ascaris suum-induced early (EAR) and Late (LAR) airway responses and airway hyperresponsiveness (AHR) to carbachol were measured in allergic sheep without and after treatment with different doses of oral or intravenous hep-tetrasaccharide. At doses of 0.06 mg/kg, 0.125 mg/kg, and 0.25 mg/kg, oral hep-tetrasaccharide caused a dose-dependent inhibition of EAR and LAR. Post-antigen AHR was also inhibited dose dependently. The same doses of intravenous hep-tetrasaccharide yielded comparable inhibition of EAR, LAR and AHR, confirming that orally delivered hep-tetrasaccharide has good bioavailability. The protection by hep-tetrasaccharide on EAR and LAR was dependent on N-sulfation, as N-desulfated/N-acetylated tetrasaccharide had a markedly reduced effect. However, inhibition of the post-antigen AHR was independent of N-sulfation. These results demonstrate that orally administered hep-tetrasaccharide inhibits allergic airway responses in the sheep model of asthma. Hep-tetrasaccharide has good oral bioavailability and its anti-allergic activity is critically dependent on N-sulfation of the glucosamine ring.

Introduction

Heparin is a complex, highly sulfated glycosaminoglycan that exerts a variety of biochemical and biological effects, including its well known anticoagulant action [1]. The basic polymeric structure of heparin consists of repeating disaccharide units comprising of alternating sequence of l-iduronic acid and d-glucosamine [2]. Commercial heparin is derived from mast cells and has undergone a variety of chemical modifications during maturation, including C5 epimerization, C2 sulfation of l-iduronic acid, and C6 and N-sulfation of d-glucosamine [3], [4]. As a result of these modifications, mature heparin polymers contain disaccharide units that have varying degrees of sulfation, with a heterogeneous molecular organization [2], [3], [4].

The sugar sequence, the sulfation pattern and the structural variability play a unique role in determining the biological actions of heparin [2], [3], [5]. Recent structural studies of the binding domain to antithrombin III and basic fibroblast growth factor are the best known examples, showing the relationship between the fine structure of heparin-derived oligosaccharides and biological functions [6], [7]. The antithrombin III binding site requires a pentasaccharide sequence, while the binding domain of basic fibroblast growth factor requires a hexasaccharide sequence [6], [7]. Heparin is also known to regulate cell growth and angiogenesis [8], [9], [10], modulate various proteases [11], [12], and possess anti-inflammatory properties [13], [14]. Inhaled heparin has been shown to attenuate antigen-induced bronchoconstriction in allergic sheep [15], as well as to prevent the bronchoconstrictor responses to exercise and antigen in asthmatic subjects [16], [17]. The anti-allergic activity of heparin is molecular weight dependent [18], [19], and the tetrasaccharide fraction is the minimal effective chain length and the most potent heparin oligosaccharide inhibiting the antigen-induced airway responses in sheep [20], [21].

Because of its large size and anionic structure, unfractionated heparin is poorly absorbed when administered orally [22], [23]. Heparin depolymerizing activity has been detected in the rat and canine intestinal mucosa [24]; therefore, heparin is rapidly degraded in the gastrointestinal tract, requiring intravenous administration for its anti-coagulant action. Larsen et al. [25] have suggested that the gastrointestinal tract degrades heparin to smaller fragments that are absorbed into the circulation and may mediate the “non-anticoagulant” functions of heparin, including inhibition of angiogenesis [10], [26]. Thus, it is possible that smaller heparin oligosaccharides, when administered orally, may be absorbed into the circulation for mediation of “non-anticoagulant” actions. Therefore, in this study, we tested the hypothesis that oral heparin (hep-) tetrasaccharide possess anti-allergic activity. To do this, we used the sheep model of experimental asthma to determine: a) if orally administered hep-tetrasaccharide blocked allergen-induced airway responses; b) if the anti-allergic action of oral hep-tetrasaccharide was comparable to parentally administered hep-tetrasaccharide; and c) the role of N-sulfation in mediation of anti-allergic activity of hep-tetrasaccharide to further elucidate the structural determinants necessary for this action.

Section snippets

Animal preparation

All procedures used in this study were approved by the Mount Sinai Animal Research Committee, which is responsible for ensuring the humane care and use of experimental animals. Eight unsedated adult female sheep, with an average weight of 30 kg (26–35 kg), were suspended in an upright position in a specialized body harness in a modified shopping cart, with their heads secured as published previously [15], [27]. All sheep were allergic to Ascaris suum antigen and had previously been shown to

Effect of oral hep-tetrasaccharide

Oral administration of hep-tetrasaccharide inhibited the antigen-induced airway responses in a dose-dependent fashion (Fig. 1A, Table 1). In control studies, with antigen alone, mean ± SE peak early SRL increased by 339 ± 39%, while peak late SRL increased by 176 ± 24%. The corresponding mean ± SE Area Under the Curve for EAR (EAR-AUC0-4 h) and Area Under the Curve for LAR (LAR-AUC4-8 h) were 508 ± 53% and 359 ± 49%, respectively. Increasing doses of oral hep-tetrasaccharide produced

Discussion

Aerosol administration of unfractionated heparin and hep-oligosaccharides has been shown to inhibit allergic airway responses, and this anti-allergic activity resides in a tetrasaccharide sequence [20], [21]. Inhaled hep-tetrasaccharide, while lacking anti-coagulant activity, has unique biological properties and is a potent inhibitor of antigen-induced airway responses in sheep [21]. The results of this study extend our previous observations and demonstrate that: (a) orally administered

Conclusions

In summary, the present study has demonstrated that intravenous and orally administered hep-tetrasaccharide inhibits allergic airway responses in sheep. This activity is critically dependent on N-sulfation of the glucosamine ring. These findings extend and support our previous work in sheep and mice showing both anti allergic and anti-inflammatory activity of aerosolized hep-tetrasaccharide [21]. Collectively, these findings, in addition to our demonstration that hep-tetrasaccharide has good

References (47)

  • R.J. Linhardt et al.

    Heparin, heparinoids and heparin oligosaccharides: structure & biological activities

  • U. Lindahl

    Biosynthesis of heparin and related polysaccharides

  • M. Salmivirta et al.

    Heparan sulfate: a piece of information

    FASEB J

    (1996 Sep)
  • U. Lindahl et al.

    Evidence for a 3-O-sulfated D-glucosamine residue in the antithrombin-binding sequence of heparin

    Proc Natl Acad Sci U S A

    (1980 Nov)
  • J.J. Castellot et al.

    Effect of heparin on vascular smooth muscle cells. I. Cell metabolism

    J Cell Physiol

    (1985 Jul)
  • J. Folkman et al.

    Angiogenesis: regulatory role of heparin and related molecules

  • F. Redini et al.

    Inhibition of leucocyte elastase by heparin and its derivatives

    Biochem J

    (1988 Jun 1)
  • H. Hochart et al.

    Low-molecular weight and unfractionated heparins induce a downregulation of inflammation: decreased levels of proinflammatory cytokines and nuclear factor-kappaB in LPS-stimulated human monocytes

    Br J Haematol

    (2006 Apr)
  • T. Ahmed et al.

    Effects of inhaled heparin on immunologic and nonimmunologic bronchoconstrictor responses in sheep

    Am Rev Respir Dis

    (1992 Mar)
  • T. Ahmed et al.

    Preventing bronchoconstriction in exercise-induced asthma with inhaled heparin

    N Engl J Med

    (1993 Jul 8)
  • Z. Diamant et al.

    Effect of inhaled heparin on allergen-induced early and late asthmatic responses in patients with atopic asthma

    Am J Respir Crit Care Med

    (1996)
  • J. Martinez-Salas et al.

    Inhibition of allergic airway responses by inhaled low-molecular-weight heparins: molecular-weight dependence

    J Appl Physiol

    (1998)
  • C. Campo et al.

    Molecular-weight-dependent effects of nonanticoagulant heparins on allergic airway responses

    J Appl Physiol

    (1999 Feb)
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