Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

Sophie M Thompson,Ruoyan Xu,Edwin C Jesudason,Jeremy E Turnbull,David G Fernig,Marilyn G Connell,Toin H Van Kuppevelt,Paul D Losty

doi:10.1186/1471-213x-11-38

Abstract

BackgroundHeparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.ResultsThe pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme.We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.ConclusionsThe observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.

Highlights

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome
HS chains are usually attached to core proteins to form HS proteoglycans (HSPGs), which are expressed by most mammalian cells and represent a major component of the cell surface and ECM
Seven HS ‘phage display antibodies were chosen on the basis that they have been raised against HS/heparin from a variety of tissue sources and display distinct binding specificities, which we have characterised in depth [24]

Summary

Introduction

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. The majority of the extracellular proteins involved in regulating embryonic development interact with heparin/heparan sulfate (HS), and, require HS for their cellular activities [1]. These include proteins required for lung morphogenesis [2,3]. Chains are variably modified by N-deacetylation/N-sulfation of N-acetyl glucosamines, O-sulfation at various positions and conversion of glucuronic acid to its C-5 epimer, iduronic acid These modifications do not occur at every potential site within a chain, resulting in a diverse range of HS chain structures displayed by a cell [1]. Characterising HS fine structure in vivo is, important as it equates to a view of HS function

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