Low-density lipoprotein stimulates mesangial cell proteoglycan and hyaluronan synthesis

Ravinder S. Chana*, David C. Wheeler*, Gareth J. Thomas, John D. Williams and Malcolm Davies

University of Wales College of Medicine, Institute of Nephrology, Heath Park, Cardiff, UK

Correspondence and offprint requests to: Professor Malcolm Davies, University of Wales College of Medicine, Institute of Nephrology, Heath Park, Cardiff CF11 4XN, UK. E-mail: daviesm6{at}cardiff.ac.uk.



   Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Hyperlipidaemia leads to glomerulosclerosis in small mammals and may contribute to progressive renal disease in man. One prominent feature of lipid-induced glomerular injury in animal models is the accumulation of mesangial matrix. These studies were designed to investigate whether low-density lipoprotein (LDL) enhanced mesangial cell (MC) matrix deposition by modulating the production of proteoglycans (PG) and hyaluronan (HA).

Methods. Growth arrested human MC were metabolically labelled with either 50 µCi/ml Na2[35S]sulphate or 25 µCi/ml [3H]glucosamine and stimulated with LDL (10–100 µg/ml). The radiolabelled PG and HA extracted from the cell layer and the culture medium were isolated, quantified and characterized. Comparison of the PG core proteins synthesized by MC was carried out using Western blot analysis.

Results. LDL stimulation led to a dose- and time-dependent increase in [35S]sulphate incorporation into PG in the culture medium and to a lesser extent in the cell layer. Analysis of the glycosaminoglycan (GAG) chains showed no difference in either their size or charge. Enzyme digestion studies demonstrated that the synthesis of both chondroitin sulphate PG (CSPG) and heparan sulphate PG (HSPG) was enhanced as was the production of the core proteins of versican (a large CSPG), perlecan (a basement membrane HSPG) and to a lesser extent decorin (a small dermatan sulphate PG (DSPG)). An increase in HA synthesis was also demonstrated in [3H]glucosamine labelled cells following LDL stimulation.

Conclusion. LDL selectively enhances the synthesis of specific PG and HA by mesangial cells. Such effects may contribute to the expansion of the mesangial matrix and modify cell-matrix interactions in lipid-induced renal damage.

Keywords: glomerulosclerosis; hyaluronan; lipid; low-density lipoprotein; mesangial cell; proteoglycans



   Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Although many different disease processes initiate kidney damage, all may lead eventually to a final common pathway characterized histologically by glomerulosclerosis and tubulointerstitial fibrosis [1]. Glomerular lesions similar to those seen in the end-stage kidney can be induced in small mammals by feeding high cholesterol diets whilst in several animal models of kidney disease, correcting the lipid abnormalities that complicate uraemia or heavy proteinuria slows the progression of renal injury [2]. Such observations suggest that abnormalities of lipid metabolism may exacerbate glomerular damage. Histological studies have demonstrated lipoprotein deposition in the glomerular mesangium during the early stages of glomerulosclerosis whilst accumulation of extracellular matrix is a critical event in the development of scarring [3]. Similar changes are observed in the arterial intima in atherosclerosis and it has been suggested that both glomerulosclerosis and atherosclerosis share common pathogenic mechanisms. This analogy is further strengthened by the fact that the cells playing a key role in these scarring processes, namely glomerular MC and vascular smooth muscle cells respectively, are closely related in terms of morphological and functional characteristics. In vitro studies have demonstrated that incubation of vascular smooth muscle cells with low-density lipoprotein (LDL) stimulates production of matrix components thus providing a possible link between lipid deposition and matrix accumulation in the arterial wall [4].

Glomerular mesangial matrix comprises a complex structure of interacting macromolecules which aggregate to form polymers, providing support for cells and influencing their behaviour through cell surface integrin receptors. Analysis of the lesions which characterize glomerulosclerosis in humans reveals that scarred areas contain an excess of the same molecules present in normal matrix, namely collagen, fibronectin, laminin, glycoproteins and PG [5]. Proteoglycans comprise one or more glycosaminoglycan chains covalently linked to a core protein. These macromolecules accumulate in the early stages of glomerular scarring and are found in abundance in sclerotic lesions. Hyaluronan is a high-molecular weight polysaccharide composed of repeated units of ß-1,4 glucuronate and ß1-3-N-acetylglucosamine. Within matrix, it has a number of important physicochemical functions, acting as a support for cell adhesion and locomotion and mediating cell-matrix interactions through specific binding to CD-44 and the large CSPG versican [6]. Human MC in culture have been shown to synthesize a number of different proteoglycans including versican, biglycan, decorin, and perlecan [7]. Furthermore, these cells secrete HA, which forms large aggregates with MC versican [8].

Previous studies have demonstrated that exposure of MC to LDL leads to activation, enhanced proliferation and the synthesis of eicosanoids, chemotactic cytokines, collagen type IV and fibronectin [9]. Oxidation of lipoprotein by the cells modifies its effects and may result in cytotoxic injury [10]. The following experiments are based on the hypothesis that hyperlipidaemia exacerbates renal injury by modifying MC matrix synthesis thereby causing glomerular scarring. In these experiments, the effect of LDL on the synthesis and secretion of PG and HA were investigated. The results demonstrate that activation of cells by lipoprotein stimulation leads to increase production of these matrix components with selective up-regulation of the synthesis of certain PG species.



   Methods
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 Methods
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Mesangial cell culture and metabolic labelling
Mesangial cell cultures were established from specimens of normal human kidney as previously reported by us [7]. Briefly, human MC were grown in 75 cm2 culture flasks (Falcon, Cowley, UK) in RPMI supplemented with 20% foetal calf serum, penicillin (100 i.u./ml), streptomycin (100 µg/ml), insulin (5 µg/ml), sodium selenite (5 ng/ml) and transferrin (5 µg/ml) (RPMI medium) and used between passages 3–9. For labelling experiments the cells were detached with Trypsin/EDTA, washed with medium by centrifugation at 1000 g for 5 min and plated at a density of 2x104 cells/well (24-microwell plates; Falcon). After 96 h the cells were growth arrested in serum-free RPMI for 48 h, then metabolically labelled with 50 µCi/ml carrier-free Na2[35S]sulphate (Amersham, Little Chalfont, UK) in sulphate-low medium in the absence or presence of LDL (0–100 µg/ml) [11]. After labelling, the culture medium was removed, the cells washed twice with PBS and the washes added to the culture medium together with a cocktail of proteinase inhibitors [11]. The cell layer was extracted with 4% CHAPS, 4 M guanidine HCl in 50 mM sodium acetate, pH 6.0, containing 0.05% sodium azide and proteinase inhibitors at -20°C overnight. The culture medium and the cell layer were stored at -20°C until used.

For all experiments, parallel cultures were set up in the absence of radiolabel, for the determination of cell numbers. At the termination of each experiment cultures were trypsinized and the detached cells counted using a haemocytometer.

Measurement and characterization of radiolabelled material
The incorporation of [35S]sulphate into macromolecules, the isolation of 35S-labelled PGs and 35S-labelled-GAGs and their subsequent analysis by chromatography on a dissociative Sepharose CL 4B column (0.006x1.5 m) before and after digestion with chondroitin ABC lyase or heparitinase have been described by us in detail [7,8,12,13].

The measurement of hyaluronan synthesis was undertaken on cells metabolically labelled with 25 µCi/ml D-[6-3H]glucosamine (specific activity 20 Ci/mMol, Amersham) as previously described [14]. In some experiments cycloheximide (10 ng/ml) or actinomycin (25 ng/ml) was included in the culture medium.

Western blot analysis of human MC PG
Confluent human MC were maintained in serum free RPMI medium in 75 cm2 flasks with and without LDL (100 µg/ml). Conditioned medium was harvested after 24 h and unlabelled PG concentrated by DEAE and Mono Q ion exchange chromatography [7]. PG were determined using the dye binding assay of Farndale [15]. Aliquots of the PG (100 µg) were incubated with buffer alone, with 50 mU of proteinase free chondroitin ABC lyase (ICN, Thane, UK) or with a mixture of heparitinase I, II and III [13]. SDS– polyacrylamide gel electrophoresis was carried out using 3–12% gradient gels following the method of Laemmli [16]. The proteins were then transferred to nitrocellulose, incubated with primary antibody and developed using enhanced chemiluminescence (ECL) (Amersham). The antibodies used were rabbit anti-bovine versican (the kind gift of Dr D. Heinegard, University of Lund, Lund, Sweden), rabbit anti-human decorin (LF-30) and anti-biglycan (LF-15) (the kind gift of Dr L. Fisher National Institute of Dental Health, Bethesda, MA, USA), and rabbit anti-human perlecan (the kind gift of Dr J. Hassell, Schreiner Hospital for Sick Children, Tampa, Fl, USA).

Isolation of LDL
LDL (density range 1.019–1.063 g/ml) was isolated by sequential ultracentrifugation of human plasma collected from healthy volunteers and stored under nitrogen at 4°C for up to 4 weeks [10,17]. The preparation was free from endotoxin as determined by the Limulus reaction and remained in an un-oxidized form under these storage conditions. For use in tissue culture the lipoprotein preparations were dialysed against 0.15 M NaCl, pH 7.4, containing 0.3 mM EDTA and sterilised by passage through a 0.22 µm filter (Millipore, Harrow, UK).

Statistical analysis
Statistical analysis was performed using a Mann–Whitney unpaired single-tailed test or by analysis of variance (ANOVA). Results are expressed as mean±1 standard deviation. P<0.05 was taken as significant.



   Results
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 Methods
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Effect of lipoproteins on the incorporation of [35S]sulphate into glycosaminoglycans
To investigate the effect of lipoproteins on the synthesis of PG by human MC, confluent quiescent cells were metabolically labelled with [35S]sulphate in the presence of native LDL (range 0–100 µg/ml). Over the concentration and time periods studied in these experiments there were no changes in cell number. The results showed a dose-dependent increase in the amount of 35S-labelled PG extracted from the cell layer and culture medium (Figure 1aGo). As lipoprotein concentration increased, a relatively greater proportion of labelled proteoglycan was secreted into the conditioned medium as opposed to being retained in the cell layer. Thus when compared to control cells grown in RPMI alone (100%), exposure to 50 µg/ml and 100 µg/ml LDL for 24 h led to a 238.0% (±44.4%, P<0.001; n=5) and 426.5% (±28.5%, P<0.001; n=5) increase in secreted 35S-labelled PG respectively. In contrast, at the same concentrations, LDL increased cell-associated 35S-labelled PG by only 140.6% (±25.1%, P<0.001; n=5) and 197.9% (±20.4%, P<0.001; n=5). Exposure of cells to 50 µg/ml LDL from 6 to 48 h led to a linear increase in total (secreted plus cell associated) 35S-labelled PG (Figure 1bGo). At all time-points, more [35S]sulphate was incorporated into macromolecules synthesized by LDL-stimulated cells than by control cells labelled in medium alone (Figure 1bGo).



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Fig. 1. Incorporation of [35S]sulphate into PG synthesised by MC exposed to LDL. (a) The effect of LDL concentration; MC were metabolically labelled with [35S]sulphate for 24 h either in the absence or presence of increasing concentrations of LDL. The culture medium ({blacksquare}) and cell layer ({square}) were obtained as outlined in Methods and the incorporation of radiolabel into PG determined. The results represent the mean±1 SD of 35S incorporation into PG corrected for cell number (n=5) (*P<0.05). (b) The effect of LDL with time; MC were labelled with [35S]sulphate for up to 48 h in the absence ({circ}) or presence (•) of 50 µg/ml LDL. The results represent the mean±1 SD (*P<0.05, **P<0.005).

 
Analysis of the 35S-labelled glycosaminoglycans and PGs
Analysis of the 35S-labelled glycosaminoglycans by gel chromatography and anion-exchange chromatography indicated that the enhanced levels of 35S-labelled PG extracted from treated cells was not accounted for by either elongation or increased sulphation of the GAG chains. Selective enzyme digestion studies, however, showed that with respect to the culture medium, there was a 5-fold increase in both the large CSPG (versican) (Kav 0.16) and small CSPG (decorin and biglycan) (Kav 0.5) above control levels (Figure 2Go panel a and Table 1Go). In addition a single HSPG species (Kav 0.36) was increased 3-fold (Figure 2Go panel b). With regards to the CL, of particular note was a 5-fold increase in a large CSPG (Figure 2Go panel c). In addition there was a 2-fold increase in HSPG (Kav 0.3) when cells were stimulated with lipoprotein (Figure 2Go panel d). LDL had no major effect on the material eluting between Kav 0.6 to 0.9, which has previously been shown to contain single GAG chains and their degraded product [7].



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Fig. 2. Sepharose CL-4B chromatography of MC PG, MC were metabolically labelled with [35S]sulphate for 24 h either in the absence ({circ}) or presence (•) of 100 µg/ml LDL. Aliquots of the 35S-labelled PG in the culture medium (panels a & b) and cell layer (panels c & d) were incubated with either heparitinase (a & c) or chondroitin ABC lyase (b & d) and the remaining CSPG and HSPG respectively chromatographed on a dissociative Sepharose CL 4B column. The excluded (Vo) and total volumes (Vt) of the columns are indicated with arrows.

 

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Table 1. The effect LDL on the distribution of proteoglycans
 
Western blot analysis of the proteoglycan core proteins
To confirm the selective synthesis of versican, biglycan, decorin and possibly perlecan, Western blotting analysis was performed on the culture medium using antibodies that recognize the core proteins of these PG. Anti-versican antibody recognized 2 core proteins (~Mr 380 and 400x103) which were increased three-fold in the presence of 100 µg/ml LDL (Figure 3aGo compare lanes 2 and 4). Using anti-perlecan antibody the two 2 core proteins (~Mr 260 and 300x103) identified were also increased (3.5-fold) in the presence of the LDL (Figure 3bGo lanes 2 and 4). The core protein of decorin (Figure 3cGo) was also increased (2-fold) but there was no apparent change in biglycan (Figure 3dGo). These findings suggest that LDL selectively increased the synthesis of specific proteoglycans by mesangial cells.



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Fig. 3. Western blot analysis of the PG core proteins. MC was cultured in the absence or presence of 100 µg/ml LDL and PG isolated from culture medium. Aliquots of the PG from cultures with medium alone (lanes 1 and 2) or stimulated with LDL (lanes 3 and 4) were incubated with buffer alone (lanes 1 and 3) or with either chondroitin ABC lyase (panels a, c & d) or heparitinase (panel b) (lanes 2 and 4) and subjected to gradient SDS PAGE. Western blots were generated with anti-sera raised to (a) versican, (b) perlecan, (c) decorin and (d) biglycan. The 64x103 Mr band in panel a (lanes 1–4) represents BSA present in the medium and immunoidentified by the anti-bovine versican antisera.

 
Effect of LDL on HA synthesis by MC
A further series of experiments was conducted to determine whether LDL also enhanced the synthesis of hyaluronan. At a concentration of 100 µg/ml and after 24 h incubation, LDL increased the incorporation of [3H]glucosamine into HA by 202.6% (±67.6%, P<0.05; n=5) of control. Approximately 60% of the newly synthesized hyaluronan was secreted and stimulation of MC with lipoproteins did not alter the ratio of secreted to cell-associated molecules. The inclusion of cycloheximide (10 ng/ml) or actinomycin (25 ng/ml) in the culture medium did not alter the basal synthesis of [3H]hyluronan, however the increased synthesis caused by the inclusion of LDL was inhibited by 80% and 60% respectively.



   Discussion
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 Methods
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These studies demonstrate that stimulation of human MC with LDL enhances proteoglycan production in the absence of cell proliferation. This was shown to be the result of increased de novo synthesis of versican, perlecan and decorin but not biglycan. LDL also enhanced hyaluronan production by a mechanism that probably involved increased synthetic enzyme activity.

LDL stimulation did not alter the hydrodynamic size of secreted or cell-associated PG or that of their constituent glycosaminoglycan chains. Furthermore, all GAGs had similar charge densities to those produced by the control cells. These results strongly suggest that the increased incorporation of radiolabelled sulphate into secreted and cell-associated macromolecules was the result of enhanced de novo synthesis. Selective digestion of labelled macromolecules followed by Western blotting studies confirmed these findings by demonstrating increased production of the core proteins of versican (a large CSPG) and perlecan (a basement membrane HSPG). A less marked effect on DSPG was noted with a small increase in decorin but not in biglycan. Our data therefore suggest that the various proteoglycans synthesized by MC may be differentially regulated. This conclusion is in agreement with other studies that show TGF-ß uniquely effects the metabolism of decorin and biglycan, while IGF-1 appears to selectively increase CSPGs [18,19]. In addition recent reports show that high glucose culture conditions mainly decrease heparan sulphate rather than dermatan/chondroitin sulphate [20].

The roles of different PG synthesized by MC are poorly understood making it difficult to predict the pathophysiological consequences of LDL stimulation. One possibility is that increased proteoglycan production will promote entrapment of lipid within the glomerular mesangium. CSPG bind apo-B-containing lipoproteins in the presence of calcium to form insoluble complexes, a process that may contribute to the development of atherosclerotic lesions [21]. It is possible that similar interactions occur in the glomerulus. Previous studies have shown that versican and decorin/biglycan extracted from human MC conditioned medium form insoluble complexes with LDL in the presence of calcium [9]. The binding, optimal at 30 mM Ca2+, was abolished by pre-treatment of PG fractions with chondroitin ABC lyase demonstrating the critical importance of GAG side chains. Thus, increased production of PG by MC following exposure to LDL may promote further lipid accumulation in the glomerular mesangium, thereby setting up a cycle of progressive scarring.

Matrix accumulation resulting from LDL stimulation may also assist cytokine accumulation within the mesangium. For example, decorin binds TGF-ß, which in turn has been implicated as a mediator of increased proteoglycan production, and perlecan immobilizes basic fibroblast growth factor [18,22]. Such interactions may modulate exposure of MC to the biological effects of these mediators. Additional mechanical effects of increased proteoglycan accumulation include changes in matrix charge, which may modulate filtration of plasma proteins and disruption of the normal interactions between various matrix components and cells [23].

LDL also enhanced hyaluronan production by MC. More than 60% of this glycosaminoglycan was secreted into the medium and lipoprotein stimulation did not alter the ratio of secreted to cell-associated molecules. Furthermore, inclusion of transcriptional and protein synthesis inhibitors blocked the effects of LDL suggesting that the lipoprotein modulates enzymes involved in hyaluronan synthesis. Apart from playing a critical role in the organization and structure of extracellular matrix, hyaluronan also regulates cell migration, adhesion and proliferation [6]. Although the role of hyaluronan in the mesangium has not been fully investigated, recent studies suggest that this glycosaminoglycan interacts with glomerular cells via specific receptors and binds to large versican PG to form aggregates [8]. These interactions may play an important role in glomerular cell migration and proliferation. Thus by enhancing HA synthesis, lipoproteins might modulate the interaction between matrix components and MC.

It is well established that the precise nature of extra cellular matrix exerts a stringent control on the phenotype of normal MC, our results also suggest that LDL may indirectly effect MC growth. Unmodified LDL has a biphasic effect on human MC growth [10]. At low concentrations (less than 50 µg/ml) it enhances cellular proliferation as determined by [3H]thymidine incorporation, whereas at 100 µg/ml it has no effect and if anything inhibits cell growth. The present results that are based on cell number therefore confirm our earlier finding that at 100 µg/ml LDL is not a mitogen for human MC. They also indicate that LDL induced synthesis of versican and hyaluronan is independent of cell proliferation. This conclusion is difficult to reconcile with preliminary in vitro data, which demonstrated that rat MC proliferation induced by serum is accompanied by increased production of CSPG [24]. Vascular smooth muscle cells stimulated to divide by PDGF also show increased PG synthesis, which is mainly accounted for by a large CSPG [25]. At the present, however, it is uncertain whether CSPG such as versican play a direct or indirect role in the control of cell growth.

In conclusion, this study demonstrates that LDL stimulates the de novo synthesis of chondroitin sulphate, dermatan sulphate, heparan sulphate PG and hyaluronan by cultured human MC. This increase in PG synthesis was due to enhanced production of versican, perlecan and decorin, but not to biglycan. Thus, upregulation in proteoglycan and hyaluronan synthesis resulting from lipoprotein stimulation is likely to contribute to the extracellular matrix expansion within the glomerulus. Furthermore, selective increases in certain proteoglycan species may lead to an imbalance of matrix components and adversely modify normal cell-matrix interactions.



   Acknowledgments
 
This study was supported by the Baxter Healthcare Extramural Grant Program and by the Kidney Research Foundation for Wales.



   Notes
 
* Present address: Department of Nephrology, University Hospital (Birmingham) NHS Trust, Queen Elizabeth Hospital, Birmingham B15 2TH, UK. Back



   References
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 Abstract
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 Methods
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 Discussion
 References
 

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Received for publication: 25. 5.99
Accepted in revised form: 31. 8.99