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Clinical Chemistry 49: 380-388, 2003; 10.1373/49.3.380
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(Clinical Chemistry. 2003;49:380-388.)
© 2003 American Association for Clinical Chemistry, Inc.

Anomalous Structure of Urinary Glycosaminoglycans in Patients with Pseudoxanthoma Elasticum

Francesca Maccari1, Dealba Gheduzzi2 and Nicola Volpia1

Departments of
1 Biologia Animale and
2 Scienze Biomediche, University of Modena and Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy

aAuthor for correspondence. Fax 39-59-2055548; e-mail volpi{at}unimo.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Pseudoxanthoma elasticum (PXE) is a hereditary connective tissue disease in which proteoglycans have altered properties. We investigated whether altered proteoglycan metabolism occurs in vivo and may be reflected in the urine of PXE individuals by analyzing the excreted polysaccharides.

Methods: We measured sulfated glycosaminoglycans in the urine of 10 PXE-affected patients, 12 healthy carriers, and 20 healthy controls by agarose gel electrophoresis. Chondroitin sulfate and heparan sulfate disaccharides were also quantified by treatment with specific lyases and separation of products by chromatography.

Results: Total polysaccharides were 34% lower in the urine of PXE-affected patients and 17% lower in healthy carriers than in the control group. Chondroitin sulfate was significantly (P <0.01) decreased, and heparan sulfate was significantly increased. The ratio of chondroitin sulfate to heparan sulfate was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for controls. In PXE-affected individuals and carriers, chondroitin sulfate contained more 4-sulfated disaccharide, less 6-sulfated disaccharide, and decreased nonsulfated disaccharide. Heparan sulfate from PXE-affected individuals and healthy carriers produced significantly less N-sulfated disaccharide and more disaccharide sulfated at the C-6 position with no significant abnormality of the nonsulfated disaccharide percentage and sulfates:disaccharide ratio.

Conclusions: The urinary data support the concept that the inherited defect of the ABCC6/MRP6 transporter in PXE alters metabolism of key polysaccharides. Structural analysis of urinary sulfated polyanions may be useful in the diagnosis of PXE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glycosaminoglycans [GAGs;1 hyaluronic acid, cheratan sulfate, chondroitin sulfate (CS), heparan sulfate (HS), and heparin] are linear, complex, polydisperse polysaccharides (1)(2). With the exception of cheratan sulfate and hyaluronic acid (hyaluronate), they are sulfated heteropolysaccharides of alternating copolymers of uronic acids and amino sugars, with different degrees of charge density attributable to sulfate groups that vary in amount and are linked in different positions. Polysaccharides are very heterogeneous in terms of relative molecular mass, charge density, physicochemical properties, and biological and pharmacologic activities (3) and are macromolecules of great importance in several pathologic processes. GAGs, generally bound to proteins forming proteoglycans (4), are found inside cells, at their surfaces, and in extracellular matrices, as well as in biological fluids such as plasma and urine.

Normal human urine contains mainly CS (~85–90% of the total), HS (~10–15%), and trace amounts of hyaluronic acid and dermatan sulfate (5)(6). Urinary GAGs have long been investigated for their possible quantitative and qualitative modifications in pathologic conditions, such as in cancer (5)(7), Werner syndrome (8), Weber Christian disease (9), Rothmund Thomson syndrome (10), epidermolysis bullosa (11), glomerular diseases (12), interstitial cystitis (13), osteopetrosis (14), and mucopolysaccharidoses (15). In some cases, results useful for diagnosis have been observed.

Pseudoxanthoma elasticum (PXE) is an inherited connective tissue disorder characterized by accumulation of ion precipitates within the elastic fibers of skin, eyes, and the whole cardiovascular system (16). Mineralization is progressive with time and leads to elastic fiber fragmentation and disruption (17). However, PXE is also characterized by collagen fibril abnormalities and by accumulation in the extracellular space of abnormal masses of materials containing proteoglycans and other matrix molecules (18)(19).

The PXE gene has recently been found to encode for ABCC6/MRP6, a protein that belongs to the ABC family of membrane transporters (20)(21)(22). There are indications that MRP6 in both rats and humans is expressed mainly on the plasma membrane of hepatocytes and tubular cells in the kidneys, similar to other members of the same family of transporters. However, MRP6 has also been found expressed in low amounts in several other cell types, where its function could be even more crucial and where its deficiency could lead to rather dramatic metabolic derangements.

Elastin is not primarily involved, and elastin mineralization as well as collagen and proteoglycan alterations represent secondary events of a cell metabolic disorder whose comprehension may help in understanding the pathogenesis of clinical manifestations. Abnormal amounts of proteoglycans are localized nearby and within mineralized elastin fibers (19)(23)(24), and abnormal amounts of GAGs, as well as alterations in their synthesis and deposition, have been detected in patients with PXE (25)(26). Moreover, cells from PXE-affected patients have been shown to produce proteoglycan species with altered properties, such as stronger polyanion properties, increased hydrodynamic size, abnormal hydrophobic interactions, and different content and distribution of HS (27)(28). It is well known that proteoglycans play an important role in assembly of the extracellular matrix (29), are involved in collagen fibrillogenesis and elastin fiber formation (30), and control gene expression, regulating cell survival, growth, differentiation, motility, and synthetic capability (31). As a consequence, proteoglycan abnormalities might be important factors in the phenotypic expression of PXE.

These findings led us to enquire whether the modification of proteoglycans and GAG metabolism might be reflected in the urine of patients with PXE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patient population and urine collection
We obtained 20 urine specimens from 20 different healthy human volunteers plus another 22 urine specimens, 10 from PXE-affected patients and 12 from healthy carriers (see Data Supplements available with the online version of this article at http://www.clinchem.org/content/vol49/issue3/). After obtaining written consent, we obtained a 50–100 mL urine sample from each volunteer or patient and stored the samples frozen at -20 °C for analytical investigation.

The majority of controls were chosen from among the 50 healthy individuals analyzed for ABCC6/MRP6 mutations and considered as "controls". They were all Italian and from the same regions as the patients. The majority of both controls and PXE patients were from Northern Italy. The age range for the controls and the majority of PXE patients was 20–50 years.

materials
We obtained high-purity agarose and barium acetate from Bio-Rad; 1,2-diaminopropane from Merck; cetylpyridinium chloride from Aldrich; cetyltrimethylammonium bromide from BDH; and toluidine blue from Sigma. Chondroitinase ABC from Proteus vulgaris (EC 4.2.2.4) and chondroitinase AC II from Arthrobacter aurescens (EC 4.2.2.5) were from Sigma. Unsaturated CS/dermatan sulfate disaccharides, heparinase from Flavobacterium heparinum (EC 4.2.2.7), and heparitinase I from F. heparinum (EC 4.2.2.8) were from Seikagaku Corporation.

CS from bovine trachea with a molecular mass of ~23 760 Da and a sulfate-to-carboxyl ratio of 0.93 was obtained from Institut Biochimique SA. HS with a molecular mass of 13 950 Da and a sulfate-to-carboxyl ratio of ~1.06 was prepared from bovine spleen (32). All other reagents were analytical grade.

extraction and purification of GAGS from human urine
We centrifuged 10 mL of urine from healthy volunteers, PXE-affected patients, and healthy carriers and brought the supernatant to pH 6.0 with 1 mol/L HCl. After the addition of 170 µL of 50 g/L cetyltrimethylammonium bromide, the urine was stored at 4 °C for 24 h. After centrifugation at 5000g, the pellet was washed with 2 mL of ethanol, stored at 4 °C for 24 h, centrifuged, and dried at 60 °C. After the addition of 1 mL of 2 mol/L NaCl and centrifugation at 5000g, 5 mL of ethanol was added to the supernatant, and the mixture was stored at -20 °C for 2 h. After centrifugation, the pellet was dried at 60 °C. After the addition of 500 µL of distilled water, the polysaccharides were lyophilized. The material was further dissolved in 500 µL of distilled water, and after 1 mL of acetone was added, the solution was stored at -20 °C for 24 h. After centrifugation at 5000g, the pellet was dried at 60 °C. The purified GAGs were dissolved in 40 µL of distilled water and further analyzed by agarose gel electrophoresis (5–20 µL) and HPLC (10 µL; see below).

agarose gel electrophoresis
The human urinary GAGs were identified with use of polysaccharide calibrators and quantified by agarose gel electrophoresis and densitometric scanning using specific calibration curves as reported elsewhere (33)(34) with minor modifications. GAGs were separated by agarose gel electrophoresis in barium acetate–1,2-diaminopropane. A Multiphor II (Pharmacia LKB Biotechnology) electrophoretic cell instrument was used. Agarose gel was prepared at a concentration of 0.5% in 0.04 mol/L barium acetate buffer, pH 5.8. Plates with a thickness of ~4–5 mm were prepared. We layered 5 µL of CS and HS calibrators (0.5–5.0 µg) with micropipets to construct calibration curves. The run was in 0.05 mol/L 1,2-diaminopropane (buffered at pH 9.0 with acetic acid) for 180 min at 50 mA. After migration, the plate was soaked in a solution containing 10 g/L cetyltrimethylammonium bromide for at least 6 h. The plates were dried and then stained with toluidine blue (2 g/L in ethanol–water–acetic acid, 50:49:1 by volume) for 30 min. After the gel was decolored with ethanol–water–acetic acid (50:49:1 by volume), we performed quantitative analysis of GAGs with a densitometer composed of a Macintosh IIsi computer interfaced with a Microtek Color Scanner from Microtek International. The plates were scanned in the red-green-blue (RGB) mode and saved in grayscale. The image processing and analysis program (Ver. 1.41) from the Jet Propulsion Laboratory, NASA, was used for densitometry.

qualitative and quantitative evaluation of unsaturated disaccharides from GAGS
The disaccharide products formed by the action of the chondroitin ABC lyase or chondroitin AC lyase on urinary CS were identified by strong-anion-exchange HPLC, as reported elsewhere (35)(36)(37). We incubated 10 µL of urinary extract with 25 mU of chondroitinase ABC in 50 mmol/L Tris-HCl buffer (pH 8.0) or 25 mU of chondroitinase AC in 50 mmol/L Tris-HCl buffer (pH 7.3). The reactions were stopped after incubation for 3 h at 37 °C by boiling for 1 min. Constituent disaccharides were determined by HPLC as reported, identified by use of unsaturated disaccharide calibrators, and quantified by specific calibration curves. As tested by HPLC, no oligosaccharides resistant to treatment with lyase were evident; the cleavage of CS with chondroitinase ABC or chondroitinase AC therefore produced 100% disaccharides.

The sulfate-to-carboxyl ratio (charge density) was determined by enzymatic degradation after HPLC separation of unsaturated disaccharides. The ratio was calculated taking into consideration the presence and the percentage of carboxyl and sulfate groups for each disaccharide.

HS from the urine of 20 healthy human volunteers, 10 PXE-affected patients, and 12 healthy carriers was also qualitatively and quantitatively analyzed after treatment with heparinase and heparitinase I prepared from F. heparinum (38). Heparitinase I digests HS isomers with relatively low-sulfated disaccharides, whereas heparinase degrades more highly sulfated compounds (38). Concomitant treatment with both enzymes generates >95% of the structural HS disaccharides. The nonsulfated and variously sulfated unsaturated disaccharides were then separated and quantified by HPLC, as reported above. We incubated 10 µL of urinary extract with 0.1 U of heparinase and 0.1 U of heparitinase I in 50 mmol/L acetate buffer (pH 7.3) in the presence of 25 mmoles of calcium acetate. The reactions were stopped after incubation for 6 h at 37 °C by boiling for 1 min.

light and electron microscopy
Skin biopsies were taken from the axilla of patients under local anesthesia and after signed informed consent. Fragments were immediately fixed in 100 mL/L formalin and embedded in paraffin or in 25 mL/L glutaraldehyde in Tyrode’s buffer (pH 7.2), postfixed in 10 g/L OsO4 in the same buffer, dehydrated in ethanol, and embedded in Spurr resin. Paraffin sections were stained for calcium by the Von Kossa method, and resin-embedded semi-thin sections were stained with toluidine blue or with the malachite green and basic fuchsin method; all were observed by light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Jeol 1200 EM electron microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The study participants are reported in the Data Supplements. All patients presented with skin and ocular lesions typical of PXE. Skin alterations were in the form of white-yellow papules, more or less coalescent, giving the skin a rough and redundant appearance. The diagnosis of PXE, made by ophthalmologists and/or dermatologists, was confirmed by the structural analysis of a skin biopsy that revealed the presence of Von Kossa-positive precipitates within elastic fibers and, by electron microscopy, the alterations typical of PXE, such as fragmentation and calcification of elastic fibers, collagen flowers, and aggregates of microfilaments in the reticular dermis (data not shown).

PXE is inherited as an autosomal recessive disease, whereas only a few cases of autosomal dominant forms have been reported. Except for those patients listed in the Data Supplements, no other members of the families analyzed in the present study were affected by PXE, nor were they in previous generations. Therefore, an autosomal recessive inheritance of the disorder may be supposed for all these families. If this is true, all children of the patients must be asymptomatic carriers of the disorder. In some of the families, this has been confirmed by ultrastructural analysis of a skin biopsy, which as reported previously (39), can reveal the presence of alterations typical of heterozygous carriers of PXE. In particular, in family 8, the ultrastructural analysis of the skin of clinically unaffected daughters of PXE patient A revealed the presence of small alterations in the reticular dermis, represented by polymorphisms and fragmentation of elastic fibers, small mineral deposits within some elastic fibers, collagen fibril alterations (collagen flowers), and active fibroblasts, already described in heterozygous carriers of PXE and confirmed by haplotyping (39). Similar alterations were also observed in the skin of patient B (family 7), indicating their state as heterozygous carriers for the genetic defect.

Urine samples from human healthy volunteers (20 males and females; age range, 20–50 years), 10 PXE-affected patients, and 12 healthy carriers (total for the latter two groups, 22 males and females; age range, 10–58 years) were analyzed. GAGs were extracted and purified by conventional methods and qualitatively and quantitatively analyzed by conventional agarose gel electrophoresis and densitometric scanning. Fig. 1 shows the electrophoretic pattern of the urinary sulfated GAGs from some of the patients compared with that obtained from the urine of healthy individuals. Densitometric analysis was performed by scanning single polysaccharide bands separated by electrophoresis and saved in grayscale vs specific calibration curves constructed with increasing absolute amounts (from 0.5 to 5.0 µg) of CS or HS (Fig. 1 ).



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  		Figure 1. Agarose gel electrophoresis of increasing amounts of CS, HS, and GAGs extracted and purified from normal human urine (10 mL) and urine from PXE-affected patients (10 mL).

CS: lane 1, 0.5 µg; lane 2, 3.0 µg; lane 3, 5.0 µg. HS: lane 1, 0.5 µg; lane 2, 3.0 µg; lane 3, 5.0 µg. A total of 5–20 µL was layered on the gel. Lane C1, volunteer 5; lane C2, volunteer 11; lane C3, volunteer 12; lane P1, PXE-affected patient B in family 1; lane P2, PXE-affected patient C in family 1; lane P3, PXE-affected patient A in family 6 (see Data Supplements). For quantitative purposes, samples of urine extracts were eventually diluted up to CS and HS concentrations of 0.5–5.0 µg.

The urinary concentrations of total GAGs, CS, and HS in the healthy group, PXE-affected patients, and healthy carriers are shown in Fig. 2 . The total amount of GAGs, mainly sulfated polysaccharides, CS, and HS, because hyaluronic acid is present only in a very low percentage (5)(6), was calculated to be 4.7 mg/L for the control group, 3.3 mg/L for the PXE-affected patients, and 3.9 mg/L for healthy carriers. This amount of urinary GAGs agrees with data reported by Dietrich et al. (5), who obtained urinary values of ~5.0 mg/L for healthy adults. The amount of total polysaccharides in the urine of PXE-affected patients was ~34% lower than in the control group, and this decrease was significant (P <0.05). Total GAGs in the urine of healthy carriers were not significantly modified (Fig. 2 ). Other studies have shown an increase of total polysaccharides in the urine of individuals affected by osteopetrosis (14), Rothmund Thomson syndrome (10), several kinds of cancer (7)(40)(41), and mucopolysaccharidosis (15); a decrease in patients affected by interstitial cystitis (13); or no significant differences, as for sulfated GAGs excreted by cancer patients and healthy individuals (5).



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  		Figure 2. Correlation between the total GAGs and the relative concentrations of CS and HS obtained from the urine of healthy volunteers, PXE-affected patients, and healthy carriers.

NS, not significant.

Normal urinary sulfated polysaccharides are composed of ~90% CS and 10% HS, as also reported by others (5)(42). GAGs measured in the urine of the 10 PXE-affected patients showed a significant decrease (P <0.01) in CS (approximately -44%) and a significant increase (P <0.05) in the percentage of HS (~125%). Urine from healthy carriers also showed a significant (P <0.01) decrease in CS (approximately -35%) and a significant (P <0.05) increase in HS (~200%; Fig. 2Up ). Thus, the average ratio of CS to HS was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for healthy controls.

The urinary CS from healthy and PXE-affected individuals was degraded by chondroitin ABC lyase, and the unsaturated disaccharides formed were analyzed by HPLC. The disaccharide products formed from the CS in the urine of a healthy control and of a PXE-affected patient by the action of chondroitinase ABC are shown, as an example, in Fig. 3 . It is clear that CS from PXE-affected individuals and healthy carriers produced relatively higher amounts of the 4-sulfated disaccharide ({Delta}Di-4s, 22%, P <0.05 for PXE; 18%, P <0.05 for carriers) and relatively smaller amounts of the disaccharide sulfated at the C-6 position of the galactosamine unit ({Delta}Di-6s, -7% for PXE and -6% for carriers) with a significant decrease of nonsulfated disaccharide [{Delta}Di-0s, -59% (P <0.01) for PXE; -47% (P <0.01) for carriers] compared with the disaccharide species formed from the CS of normal urine. This last polysaccharide is composed of ~10% {Delta}Di-0s, 50% {Delta}Di-6s, and 40% {Delta}Di-4s (Table 1 ). These changes in the percentages of urinary CS disaccharides are indicative of a different kind of polysaccharide in PXE-affected patients and healthy carriers compared with healthy individuals, in particular for a greater charge density, calculated as the amount of sulfate groups for disaccharide unit [5.5% (P <0.01) for PXE; 4.4% (P <0.01) for carriers] and an increase of {Delta}Di-4s (22.2% of the {Delta}Di-4s:{Delta}Di-6s ratio for PXE and 18.3% for carriers; P <0.05; Table 1 ).



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  		Figure 3. HPLC separation of unsaturated nonsulfated and variously sulfated disaccharides of CS after treatment with chondroitinase ABC.

HPLC chromatograms of unsaturated disaccharide calibrators (A), unsaturated disaccharides from CS extracted and purified from normal urine (B), and unsaturated disaccharides from CS extracted and purified from PXE-affected urine (C). {Delta}-di0S, {Delta}HexA-GalNAc; {Delta}-di4S, {Delta}HexA-GalNAc(4-OSO3); {Delta}-di6S, {Delta}HexA-GalNAc(6-OSO3); {Delta}-di2,6-diS, {Delta}HexA(2-OSO3)-GalNAc(6-OSO3); {Delta}-di2,4-diS, {Delta}HexA(2-OSO3)-GalNAc(4-OSO3); {Delta}-di4,6-diS, {Delta}HexA-GalNAc(4-OSO3)(6-OSO3); {Delta}-di2,4,6-triS, {Delta}HexA(2-OSO3)-GalNAc (4-OSO3)(6-OSO3); HexA, D-hexuronic acid; GalNAc, N-acetylgalactosamine.


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  		Table 1. Relative percentages of nonsulfated ({Delta}Di-0s), 6-sulfated ({Delta}Di-6s), and 4-sulfated ({Delta}Di-4s) disaccharides, charge density, and 4-sulfated/6-sulfated ratio for CS from PXE-affected patients and healthy carriers compared with healthy controls.

The amount of unsaturated disaccharides formed by chondroitinase ABC or chondroitinase AC on the urinary CS was essentially the same in PXE-affected patients, carriers, and volunteers (data not shown). This indicates that urinary CS contains glucuronic acid and not iduronic acid residues and that no modification of this characteristic was present in the CS of PXE-affected patients or carriers. Furthermore, the presence of dermatan sulfate was excluded.

The disaccharide products formed from the HS of the urine of a healthy control and a PXE-affected patient by the action of lyases are shown, as an example, in Fig. 4 . HS from PXE-affected patients produced significantly lower amounts of the N-sulfated disaccharide ({Delta}DiH-Ns; -64.2%; P <0.01) and relatively higher amounts of the disaccharide sulfated at the C-6 position of the glucosamine unit ({Delta}DiH-6s; 64.5%; P <0.01) with no significant variations in the percentage of nonsulfated disaccharide and the sulfates:disaccharide ratio compared with the HS of normal urine. According to previous studies (38), this last polysaccharide is composed of ~56% nonsulfated disaccharide, 20% N-sulfated and 12% 6-sulfated disaccharides, and ~7% disulfated and 4% trisulfated disaccharides (Table 2 ). These changes in the percentages of urinary HS disaccharides are indicative of a different kind of polysaccharide in PXE-affected patients compared with healthy individuals, in particular, a lower amount of the N-sulfate groups with a proportional increase in the N-acetyl groups and 6-O-sulfation. HS from healthy carriers also showed significantly lower amounts of {Delta}DiH-Ns (-32.8%; P <0.01) and relatively higher amounts of {Delta}DiH-6s (41.9%; P <0.01) with no significant variation in the nonsulfated disaccharide percentage and sulfates:disaccharide ratio compared with the HS of normal urine. The HS purified from the urine of healthy carriers contains, to a lesser extent than the polysaccharide from urine of the PXE-affected patients, decreased N-sulfate groups with an increase of the N-acetyl groups and 6-O-sulfation (Table 2 ).



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  		Figure 4. HPLC separation of unsaturated nonsulfated and variously sulfated disaccharides of HS after treatment with heparinase and heparitinase I.

HPLC chromatograms of unsaturated disaccharides from HS extracted and purified from normal (A) and PXE-affected (B) urine. 0s, {Delta}HexA-GlcNAc; Ns, {Delta}HexA-GlcSO3; 6s, {Delta}HexA-GlcNAc(6-OSO3); N,6dis, {Delta}HexA-GlcSO3(6-OSO3); 2,Ndis, {Delta}HexA(2-OSO3)-GlcSO3; 2,N,6tris, {Delta}HexA(2-OSO3)-GlcSO3(6-OSO3). HexA, D-hexuronic acid; GlcNAc, N-acetylglucosamine.


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  		Table 2. Relative percentages (CVs) for nonsulfated, monosulfated, disulfated, and trisulfated constituent disaccharides and charge density values of urine HS from 10 PXE-affected patients and 12 healthy carriers compared with 20 healthy controls.

The GAG concentrations were constant between the ages of 20 and 50 years in healthy individuals, and they were not sex-dependent, as already reported (43). No significant correlation was found between the age or sex of patients and carriers and the GAG content, such as the structure of CS and HS (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies showed that cultured PXE-affected fibroblasts from carriers and affected patients secrete anomalous families of proteoglycans, such as a proteoglycan population with stronger polyanion properties and a different content and distribution of HS proteoglycans, especially of those secreted into the growth medium (27)(28). These results indicate that PXE-affected fibroblasts in culture exhibit an abnormal proteoglycan metabolism, which could affect the normal assembly of the extracellular matrix and could account for the abnormal content and types of GAGs observed in vivo (25)(26)(44). As a consequence, the altered proteoglycan metabolism in vivo could lead to an abnormal concentration and structure of urinary GAGs. In the present study, the concentration of urinary GAGs was decreased in PXE patients with respect to controls, in contrast to data published by others, who found an increase of total urinary polysaccharides in PXE-affected individuals (45). The discrepancy could be dependent on the small number of cases studied, i.e., two PXE-affected patients. We also found a significant decrease in the amount of CS and a significant increase in HS. This could indicate an increase in HS proteoglycan production by fibroblast in patients affected with PXE and carriers, as already demonstrated with in vitro fibroblasts isolated from PXE skin (27).

The present study also shows that asymptomatic carriers of the PXE gene exhibit alterations in urinary GAGs, with values between those of patients and healthy controls. Data in the literature have already pointed out that heterozygous carriers of the mutated PXE gene show mild or limited phenotypic expression of the disease (46)(47) and that asymptomatic PXE carriers have dermal morphologic alterations similar to, although less dramatic than, those of homozygous patients (39). All these data support the hypothesis that cellular functions and extracellular matrix integrity are affected by mutations in a single allele of the PXE gene. PXE is a genetic disorder whose gene (ABCC6) encodes a transmembrane transporter called ABCC6/MRP6, with still unknown function(s) (20)(21)(22). Apart from clinical and ultrastructural alterations, PXE patients are characterized by fibroblasts with altered cell-cell and cell-matrix interactions, associated with a modified proliferation index and abnormal synthetic capabilities (48). These findings support the hypothesis that the ABCC6 gene might have a regulatory role in maintaining cellular and matrix homeostasis and that PXE may be regarded as an inherited metabolic disorder. The patients analyzed in the present study were all severely affected by the clinical manifestations of PXE. Their gene mutations are under investigation. Four of them were heterozygous for a stop codon mutation on one allele and a missense mutation on the other allele, one was heterozygous for a deletion on one allele and a nonsense mutation on the second allele, and five are still under investigation. The great majority of patients had the R1141X stop codon mutation in homozygosity or heterozygosity with another mutation. Among these same patients, five new mutations have been found, one of which is in intron 17 (I. Pasquali-Ronchetti, manuscript in preparation). From both the clinical manifestations and mutations identified to date, it can be reasonably concluded that ABCC6/MRP6 function is heavily impaired in these patients and that this may lead to the phenotypic alterations of in vitro cell behavior and metabolism already described (27)(48) and to the abnormal proteoglycan metabolism observed in the present investigation. These results and those of previous studies (19)(23)(24)(25)(26)(27)(28) clearly indicate that the gene responsible for PXE has to play a broad regulatory role on mesenchymal cell behavior and metabolism, leading to an unbalanced production of matrix components. Therefore, proteoglycans and the extracellular matrix in general would be dramatically involved only as a final event.

Urinary CS of PXE-affected patients and healthy carriers is structurally different from that of controls. It is a more highly sulfated polysaccharide containing a lower percentage of nonsulfated disaccharide units. Moreover, it has a lower number of sulfated groups in position 6 of the galactosamine and a higher degree of sulfation in position 4 of the same monosaccharide unit. These structural modifications produce a CS that is more sulfated in position 4 than in position 6 compared with controls. This abnormal urinary CS very likely reflects abnormalities of CS proteoglycans produced by PXE fibroblasts in vivo. The present data are in agreement with those by Longas and coworkers, who described abnormal GAGs isolated from lesional skin of PXE patients (26) and could also explain the observation that PXE-affected fibroblasts in vitro produce and secrete into the growing medium a CS proteoglycan population with polyanion properties stronger than those of control cells (27). Furthermore, HS from human skin fibroblasts is a polymer in which ~53% of disaccharides are N-acetylated, 47% are N-sulfated, and O-sulfate groups are ~26 per 100 disaccharide units (49). This polysaccharide is similar to that excreted and purified from human urine (38). These data strongly support the hypothesis that HS produced by pathologic cells can then be eliminated in urine.

The abnormal excretion of GAGs in urine and the modification of their structure might be the result of metabolic disorders of these polysaccharides in several pathologic conditions. The quantitative and qualitative tests for urinary GAGs are a tool to aid in the diagnosis of many connective tissue disease states (50), such as mucopolysaccharidosis (15)(51)(52), systemic scleroderma (53), and psoriasis (54). Furthermore, a direct correlation has been found between urinary GAG modification and the extent of skin affection in psoriasis (54). As a consequence, the three distinguishing differences of the urinary GAGs, i.e., the CS:HS ratio, the 4-sulfated:6-sulfated CS ratio, and the high degree of CS sulfation, as well as the increase in N-acetyl groups and 6-O-sulfation of HS could be useful for the diagnosis of PXE and/or for monitoring the progression of the disorder.


   Acknowledgments
 
This research was supported by "Ministero dell’Università e della Ricerca Scientifica e Tecnologica" (to N.V.) entitled "Proteoglycans and glycosaminoglycans metabolism in pseudoxanthoma elasticum (PXE)". The National Research Program entitled "Cellular and molecular studies on the pathogenesis of pseudoxanthoma elasticum" is the responsibility of Prof. Pasquali-Ronchetti I.


   Footnotes
 
1 Nonstandard abbreviations: GAG, glycosaminoglycan; CS, chondroitin sulfate; HS, heparan sulfate; PXE, pseudoxanthoma elasticum; {Delta}Di-4s, 4-sulfated disaccharide; {Delta}Di-6s, 6-sulfated disaccharide; {Delta}Di-0s, nonsulfated disaccharide; {Delta}DiH-Ns, HSN-sulfated disaccharide; and {Delta}DiH-6s, HS 6-sulfated disaccharide.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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