Changes in glycosaminoglycan expression in the rat developing intestine

Synthesis of glycosaminoglycan (GAG) chains was studied in the developing rat intestine. Intestinal segments, taken at various developmental stages, were exposed to 3H-glucosamine and 35S-sulfate for 6 hours. The amounts of 3H-GAGs (total GAGs) and of 35S-GAGs (sulfated GAGs) showed a clear age-dependence, with a broad maximum in the fetal period when dramatic growth and morphogenesis occur. Characterization of individual GAG species indicated that hyaluronic acid (HA), heparan and chondroitin sulfate (HS and CS) synthesis was modified quantitatively or qualitatively during development: decrease of HA with age; production of undersulfated HS molecules during embryonic life; shift towards a lower hydrodynamic form of HA and HS molecules after birth. We postulate that these alterations are crucial in the elaboration of an age-related specific extracellular microenvironment allowing intestinal growth and differentiation.     

Effect of the gonadal status and the gender on glycosaminoglycans profile in the lower urinary tract of dogs

Glycosaminoglycans (GAGs) form a functional component of connective tissues that affect the structural and functional integrity of the lower urinary tract (LUT). The specific GAGs of physiological relevance are both nonsulfated (hyaluronan) and sulfated GAGs (chondroitin sulphate [CS], dermatan sulphate [DS], keratan sulphate [KS], and heparan sulphate [HS]). As GAG composition in the LUT is hormonally regulated, we postulated that gonadectomy-induced endocrine imbalance alters the profile of GAGs in the canine LUT. Four regions of the LUT (body and neck of the bladder as well as the proximal and distal urethra) from 20 clinically healthy dogs (5 intact males, 5 intact anoestrus females, 4 castrated males, and 6 spayed females) were collected, wax-embedded and sectioned. Alcian blue staining at critical electrolyte concentrations was performed on the sections to determine total GAGs, hyaluronan, total sulfated GAGs, combined components of CS and DS, as well as KS and HS. The amount of staining was evaluated in 3 tissue layers, i.e., epithelium, subepithelial stroma and muscle within a region. Overall, hyaluronan (67.1%) was the predominant GAG in the LUT. Among sulfated GAGs, a combined component of KS and HS was found to be 61.8% and 38.2% for CS and DS. Gonadal status significantly affected GAG profiles in the LUT (P < 0.01). All GAG components were lower (P < 0.05) in body of the bladder of gonadectomized dogs. Total sulfated GAGs and a combined component of KS and HS were lower (P < 0.05) in all 4 regions of gonadectomized dogs. Except for a combined component of CS and DS, decreases in all GAGs were found more consistently in the muscle compared to other tissue layers. Differences between genders became obvious only when considered along with the effect of gonadal status. In gonadectomized dogs, changes in GAG components in the LUT were more consistent in females compared to males; this may partly explain different levels of risk in the development of urinary incontinence between genders. Quantitative differences in GAG profiles found between intact and gonadectomized dogs indicate a potential role of gonadectomy-induced endocrine imbalance in modifying GAG composition in the canine LUT. Profound alteration in the pattern of GAGs in gonadectomized dogs may compromise structural and functional integrity of the LUT and is possibly involved in the underlying mechanism of urinary incontinence post neutering.     

Dermatan sulfate exerts an enhanced growth factor response on skeletal muscle satellite cell proliferation and migration

Skeletal muscle regeneration is a complex process in which many agents are involved. When skeletal muscle suffers an injury, quiescent resident myoblasts called satellite cells are activated to proliferate, migrate, and finally differentiate. This whole process occurs in the presence of growth factors, the extracellular matrix (ECM), and infiltrating macrophages. We have shown previously that different proteoglycans, either present at the plasma membrane or the ECM, are involved in the differentiation process by regulating growth factor activity. In this article, we evaluated the role of glycosaminoglycans (GAGs) in myoblast proliferation and migration, using C2C12, a satellite cell-derived cell line. A synergic stimulatory effect on myoblast proliferation was observed with hepatocyte growth factor (HGF) and fibroblast growth factor type 2 (FGF-2), which was dependent on cell sulfation. The GAG dermatan sulfate (DS) enhanced HGF/FGF-2-dependent proliferation at 1-10 ng/ml. However, decorin, a proteoglycan containing DS, was unable to reproduce this enhanced proliferative effect. On the other hand, HGF strongly increased myoblast migration. The HGF-dependent migratory process required the presence of sulfated proteoglycans/GAGs present on the myoblast surface, as inhibition of both cell sulfation, and heparitinase (Hase) and chondroitinase ABC (Ch(abc)) treatment of myoblasts, resulted in a very strong inhibition of cell migration. Among the GAGs analyzed, DS most increased HGF-dependent myoblast migration. Taken together, these findings showed that DS is an enhancer of growth factor-dependent proliferation and migration, two critical processes involved in skeletal muscle formation.     

Heparin--a unique stimulator of human colon cancer cells' growth

The cancer microenvironment and the interactions between cancer and surrounding tissue cells are thought to play a pivotal role in tumor development and progression. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are major constituents of the extracellular matrix, the composition of which may affect various cellular functions. In the present study, the effects of GAGs on the proliferation of HT29, SW1116, and HCT116 human colon cancer cell lines were examined using exogenously added GAGs, an inhibitor of endogenous GAG sulfation and specific glycosidase digestions. Our results demonstrate that colon cancer cell growth was exclusively stimulated by exogenously added heparin and insensitive to endogenous GAGs/PGs production, in a sulfation pattern-related manner. Treatment of the tested cell lines with the FGF-2 neutralizing antibody showed that the stimulatory effect of heparin on the cells' growth was not FGF-2-dependent. Responsiveness of colon cancer cell lines to exogenous heparin/heparan sulfate may play a role in their growth and metastasis.     

Wild blueberry (Vaccinium angustifolium) consumption affects the composition and structure of glycosaminoglycans in Sprague-Dawley rat aorta

It has been documented that increased intake of polyphenols may provide protection against coronary heart disease and stroke. Blueberries (Vaccinium angustifolium) are one of the richest sources of antioxidants among fruits and vegetables. Phenolic compounds from berry extracts inhibit human low density lipoprotein and liposome oxidation. Glycosaminoglycans (GAGs) and proteoglycans (PGs) are structural components of aortas with great structural diversity. Their interaction with compounds such as enzymes, cytokines, growth factors, proteins and lipoproteins and their subsequent role in degenerative diseases has been documented. We investigated the effects of a diet rich in blueberries on the content and structure of GAGs. Sprague-Dawley rats were fed either a control (C) or a blueberry (B) diet for 13 weeks. Aortic tissue GAGs were isolated with papain digestion, alkaline borohydride treatment and anion-exchange chromatography. Cellulose acetate electrophoresis and treatment of the fractions with specific lyases revealed the presence of three GAG populations, i.e. hyaluronan (HA), heparan sulfate (HS) and galactosaminoglycans (GalAGs). Disaccharide composition was determined by high-performance capillary electrophoresis following enzymatic degradation. A 13% higher amount of total GAGs in aortas of B-fed rats was attributed to a higher content of GalAGs (67%). Determination of the sulfated disaccharides showed an overall lower concentration of oversulfated disaccharides in both HS and GalAG populations in the aortas of the B group. Our results demonstrate for the first time that a diet rich in blueberries results in structural alterations in rat aortic tissue GAGs. These changes may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.     

Exogenous addition of a C-xylopyranoside derivative stimulates keratinocyte dermatan sulfate synthesis and promotes migration

As C-Xyloside has been suggested to be an initiator of glycosaminoglycan (GAG) synthesis, and GAGs such as Dermatan sulfate (DS) are potent enhancers of fibroblast growth factor (FGF)--10 action, we investigated if a C-Xylopyranoside derivative, (C-β-D-xylopyranoside-2-hydroxy-propane, C-Xyloside), could promote DS production by cultured normal human keratinocytes, how this occurs and if C-Xyloside could also stimulate FGF-dependent cell migration and proliferation. C-Xyloside-treated keratinocytes greatly increased secretion of total sulfated GAGs. Majority of the induced GAG was chondroitin sulfate/dermatan sulfate (CS/DS) of which the major secreted GAG was DS. Cells lacking xylosyltransferase enzymatic activity demonstrated that C-Xyloside was able to stimulate GAG synthesis without addition to core proteins. Consistent with the observed increase in DS, keratinocytes treated with C-Xyloside showed enhanced migration in response to FGF-10 and secreted into their culture media GAGs that promoted FGF-10-dependent cellular proliferation. These results indicate that C-Xyloside may enhance epithelial repair by serving as an initiator of DS synthesis.     

Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions: IMPLICATIONS FOR BINDING SPECIFICITY*

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K_D values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m⁻¹ s⁻¹ and FGF-9, 130,000 m⁻¹ s⁻¹). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.     

Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.     

Inducible expression of the cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan) on human activated macrophages can regulate fibroblast growth factor action.

Monocyte/macrophages play important roles in regulating tissue growth and angiogenesis through the controlled release of heparin-binding growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor, and heparin binding epidermal growth factor. The action of these potent growth mediators is known to be regulated by adsorption to heparan sulfate proteoglycans (HSPGs) on the surface and within the extracellular matrix of other neighboring cells, which respectively promote or restrict interactions with their signal-transducing receptors on target cells. Here we report on the nature of HSPGs inducibly expressed on the surface of macrophages that confer these cells with the capacity to regulate endogenous growth factor activity. We reveal that activated human macrophages express only a single major 48-kDa cell surface HSPG, syndecan-2 (fibroglycan) as the result of de novo RNA and protein synthesis. In addition, we demonstrate this macrophage HSPG selectively binds the macrophage-derived growth factors FGF-2, vascular endothelial growth factor and heparin binding EGF and can present FGF-2 in a form that transactivates receptor-bearing BaF32 cells. These results define a novel and unique proteoglycan profile for macrophages and imply a key role for syndecan-2 in the delivery of sequestered growth factors by inflammatory macrophages for productive binding to their appropriate target cells in vivo.     

Identification of common and specific growth factor binding sites in heparan sulfate proteoglycans

The structural complexity within heparan sulfate has suggested that it contains multiple protein-specific binding sites. To evaluate the selectivity of growth factor binding to heparan sulfate, we conducted a detailed study of the intercompetition of fibroblast growth factor-2 (FGF-2) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) binding to heparan sulfate (HS) on bovine aortic smooth muscle cells. Radioligand binding assays were conducted, and an analytical method was developed for determining the apparent binding constants and numbers of specific and shared binding sites within HS. These studies revealed the presence of two general classes of HS-binding sites for FGF-2 and HB-EGF. The major class (approximately 10(6) sites per cell) was able to bind to either growth factor with relatively low affinity (K(d) = 12 and 44 nM for FGF-2 and HB-EGF, respectively) and was termed "common" binding sites. However, both FGF-2 and HB-EGF also showed specific high affinity (0.6 and 6.1 nM for FGF-2 and HB-EGF, respectively) binding to a minor subset (118,000 and 28,000 sites per cell for FGF-2 and HB-EGF, respectively) of "unique" binding sites, which were unable to bind the other growth factor. These studies indicate that growth factor binding to HS involves multiple binding sites of variable affinity, density, and selectivity. The approach outlined in this study could be applied to aid in the evaluation of the relative biological roles of these selective and nonselective growth factor binding domains within HS.     

Heparan sulphate inhibition of cell proliferation induced by TGFbeta and PDGF

The effect of glycosaminoglycans (GAGs) on the proliferation of smooth muscle cells (SMC) and fibroblasts was assessed by culturing cells with or without GAGs. Porcine heparan sulphate (HS) inhibited proliferation in a dose dependent manner. At 167 mug/ml of HS this reached 88% and 72% inhibition of SMC and fibroblast growth, respectively. Pig and beef mucosal heparins also blocked proliferation, but to a lesser extent. In contrast, beef lung heparin, chondroitin sulphate, and dermatan sulphate failed to block growth factor induced proliferation. Continuous presence of HS was not required, suggesting that the inhibitory effects resulted from a direct effect on the cell rather than an interaction of the GAG with growth factors. The mechanism by which GAGs inhibit proliferation will be addressed in future studies.     

Growth-coupled changes in glucosaminoglycans (heparan sulfate and hyaluronic acid) in normal and transformed human fibroblasts

Changes in glycosaminoglycans (GAGs) were investigated in relation to cell density, growth and transformation of human fibroblasts. Relative amounts (percentages of the total GAGs) of heparan sulfate (HS) increased and those of hyaluronic acid (HA) decreased in growth-reduced (serum-starved, exogenous HS-treated and dense) cultures of normal (WI-38) cells. In contrast, transformed (WI-38 CT-1) cells exerted such GAG changes only in serum-starved cultures, but not in HS-treated or dense cultures. These results indicate that the changes in glucosaminoglycans (G1cAGs) (HS and HA) is coupled exclusively with cell growth.     

Sulfatases are determinants of alveolar formation

Alveolar formation or alveolarization is orchestrated by a finely regulated and complex interaction between growth factors and extracellular matrix proteins. The lung parenchyma contains various extracellular matrix proteins including proteoglycans, which are composed of glycosaminoglycans (GAGs) linked to a protein core. Although GAGs are known to regulate growth factor distribution and activity according to their degree of sulfation the role of sulfated GAG in the respiratory system is not well understood. The degree of sulfation of GAGs is regulated in part, by sulfatases that remove sulfate groups. In vertebrates, the enzyme Sulfatase-Modifying Factor 1 (Sumf1) activates all sulfatases. Here we utilized mice lacking Sumf1(-/-) to study the importance of proteoglycan desulfation in lung development. The Sumf1(-/-) mice have normal lungs up until the onset of alveolarization at post-natal day 5 (P5). We detected increased deposition of sulfated GAG throughout the lung parenchyma and a decrease in alveolar septa formation. Moreover, stereological analysis showed that the alveolar volume is 20% larger in Sumf1(-/-) as compared to wild type (WT) mice at P10 and P30. Additionally, pulmonary function test was consistent with increased alveolar volume. Genetic experiments demonstrate that in Sumf1(-/-) mice arrest of alveolarization is independent of fibroblast growth factor signaling. In turn, the Sumf1(-/-) mice have increased transforming growth factor β (TGFβ) signaling and in vivo injection of TGFβ neutralizing antibody leads to normalization of alveolarization. Thus, absence of sulfatase activity increases sulfated GAG deposition in the lungs causing deregulation of TGFβ signaling and arrest of alveolarization.     

Interaction of platelet factor 4 with fibroblast growth factor 2 is stabilised by heparan sulphate

The CXC chemokine platelet factor 4 (PF4) appears to inhibit tumour growth through its modulation of the activity of angiogenic growth factors. We investigated the heparan sulphate-dependent mechanism of PF4 inhibition of fibroblast growth factor 2 (FGF-2). The ability of PF4 to bind simultaneously to both FGF-2 and HS was assessed using affinity gel chromatography. Thirty-three to forty-two percent more HS bound to the FGF-2 affinity gel in the presence of PF4 than with HS alone. Protection assays showed that PF4 and FGF-2 bound to adjacent or overlapping sites together covering a 12 kDa stretch of HS. This study suggests that the three components may form a ternary complex. PF4 released at sites of angiogenesis may bind to angiogenic growth factors attached to endothelial cell surface HS to disrupt or prevent them from interacting with their signalling receptors. Manipulation of this mechanism may prove useful for clinical intervention of angiogenesis.     

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.     

Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes.

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.     

Increased deposition of glycosaminoglycans and altered structure of heparan sulfate in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant deposition of extracellular matrix (ECM) constituents, including glycosaminoglycans (GAGs), that may play a role in remodelling processes by influencing critical mediators such as growth factors. We hypothesize that GAGs may be altered in IPF and that this contribute to create a pro-fibrotic environment. The aim of this study was therefore to examine the fine structure of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) in lung samples from IPF patients and from control subjects. GAGs in lung samples from severe IPF patients and donor lungs were analyzed with HPLC. HS was assessed by immunohistochemistry and collagen was quantified as hydroxyproline content. The total amount of HS, CS/DS and HA was increased in IPF lungs but there was no significant difference in the total collagen content. We found a relative increase in total sulfation of HS due to increment of 2-O, 6-O and N-sulfation and a higher proportion of sulfation in CS/DS. Highly sulfated HS was located in the border zone between denser areas and more normal looking alveolar parenchyma in basement membranes of blood vessels and airways, that were immuno-positive for perlecan, as well as on the cell surface of spindle-shaped cells in the alveolar interstitium. These findings show for the first time that both the amount and structure of glycosaminoglycans are altered in IPF. These changes may contribute to the tissue remodelling in IPF by altering growth factor retention and activity, creating a pro-fibrotic ECM landscape.