Oxidized LDL bind to nonproteoglycan components of smooth muscle extracellular matrices.

Arterial wall lipid retention is believed to be due primarily to ionic interactions between lipoproteins and proteoglycans. Thus, oxidized low density lipoproteins (LDL), with decreased positive charge relative to native LDL, should have decreased interaction with negatively charged proteoglycans. However, oxidized LDL does accumulate within arterial lesions. Therefore, this study investigated the binding of native and oxidized LDL to a complex smooth muscle extracellular matrix and the role of ionic charge interactions in their binding. LDL was modified with 2,2-azo-bis(2-amidinopropane)-2HCl, hypochlorite, soybean lipoxygenase, and phospholipase or copper sulfate. The extracellular matrix had 15- to 45-fold greater binding capacity for the different forms of oxidized LDL than for native LDL. However, the affinity of binding for all forms of oxidized LDL was high (K(a) = approximately 10(-9) M) and was similar to that for native LDL. Preincubation of the lipoproteins with chondroitin sulfate decreased the binding of native LDL, but had no effect on the binding of oxidized LDL. Digestion of matrices with chondroitin ABC lyase and heparinase decreased the binding of native LDL, but increased the binding of oxidized LDL; matrix digestion with pronase or trypsin markedly reduced the binding of both native and oxidized LDL.Thus, the binding of native LDL involves matrix proteoglycans, whereas the binding of oxidized LDL involves a nonproteoglycan component(s) of the matrix. The markedly enhanced retention of oxidized LDL compared with native LDL may play an important role in the progression of atherosclerosis.

Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells.

In the mechanically active environment of the artery, cells sense mechanical stimuli and regulate extracellular matrix structure. In this study, we explored the changes in synthesis of proteoglycans by vascular smooth muscle cells in response to precisely controlled mechanical strains. Strain increased mRNA for versican (3.2-fold), biglycan (2.0-fold), and perlecan (2.0-fold), whereas decorin mRNA levels decreased to a third of control levels. Strain also increased versican, biglycan, and perlecan core proteins, with a concomitant decrease in decorin core protein. Deformation did not alter the hydrodynamic size of proteoglycans as evidenced by molecular sieve chromatography but increased sulfate incorporation in both chondroitin/dermatan sulfate proteoglycans and heparan sulfate proteoglycans (p < 0.05 for both). Using DNA microarrays, we also identified the gene for the hyaluronan-linking protein TSG6 as mechanically induced in smooth muscle cells. Northern analysis confirmed a 4.0-fold increase in steady state mRNA for TSG6 following deformation. Size exclusion chromatography under associative conditions showed that versican-hyaluronan aggregation was enhanced following deformation. These data demonstrate that mechanical deformation increases specific vascular smooth muscle cell proteoglycan synthesis and aggregation, indicating a highly coordinated extracellular matrix response to biomechanical stimulation.

Biglycan, a vascular proteoglycan, binds differently to HDL2 and HDL3: role of apoE.

Lipoprotein retention by vascular extracellular matrix proteoglycans is important in atherogenesis. Proteoglycans bind apolipoprotein (apo)B- and apoE-containing lipoproteins. However, the colocalization of apoA-I and apoE with biglycan in atherosclerotic lesions suggests that vascular proteoglycans also may trap high density lipoproteins (HDLs). Because the major HDL subclasses may be atheroprotective to different degrees, we investigated the role of apoE in mediating HDL(2) and HDL(3) binding to the extracellular vascular proteoglycan, biglycan. ApoE-free HDL(2) and HDL(3) did not bind to purified [(35)S]SO(4)-biglycan, whereas apoE-containing HDL(2) and HDL(3) (HDL+E) did. The extent of binding correlated positively with the apoE content for both HDL(2) and HDL(3), although HDL(2)+E had a 3.5-fold higher affinity than did HDL(3)+E. ApoE on HDL(3) was cleaved into 22- and 12-kDa fragments, whereas apoE on HDL(2) remained intact. These results suggest that the cleaved apoE on HDL(3) results in diminished biglycan binding of HDL(3)+E relative to HDL(2)+E. Reducing positive charges on lysine and arginine residues on HDL+E eliminated biglycan binding, suggesting an ionic interaction. Thus, apoE is an important determinant of HDL binding to extracellular vascular proteoglycans and may play a role in HDL retention in the artery wall.

Oxidized low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced native low density lipoprotein binding properties.

Oxidized low density lipoproteins (Ox-LDL) affect several biological processes involved in atherogenesis. However, it is not known whether Ox-LDL can regulate proteoglycan expression and thus affect arterial wall lipoprotein retention. This study evaluated whether Ox-LDL, as compared with native LDL, regulates proteoglycan expression by monkey arterial smooth muscle cells in vitro and whether proteoglycans synthesized in the presence of Ox-LDL exhibit altered lipoprotein binding properties. Ox-LDL stimulated glycosaminoglycan synthesis, as measured by (35)SO(4) incorporation, by 30-50% over that of native LDL. The effect was maximal after 72 h of exposure to 5 microg/ml of Ox-LDL. The molecular sizes of versican, biglycan, and decorin increased in response to Ox-LDL, as indicated by size exclusion chromatography and SDS-polyacrylamide gel electrophoresis. These effects could be mimicked by the lipid extract of Ox-LDL. These size increases were largely due to chain elongation and not to alterations in the ratio of (35)SO(4) to [(3)H]glucosamine incorporation. Affinity chromatography indicated that Ox-LDL stimulated the synthesis of proteoglycans with high affinity for native LDL. Ox-LDL also specifically stimulated mRNA expression for biglycan (but not versican or decorin), which was correlated with increased expression of secreted biglycan. Thus, Ox-LDL may influence lipoprotein retention by regulating synthesis of biglycan and also by altering glycosaminoglycan synthesis of vascular proteoglycans so as to enhance lipoprotein binding properties.

Lipoprotein lipase enhances the binding of native and oxidized low density lipoproteins to versican and biglycan synthesized by cultured arterial smooth muscle cells.

Retention of low density lipoproteins (LDL) by vascular proteoglycans and their subsequent oxidation are important in atherogenesis. Lipoprotein lipase (LPL) can bind LDL and proteoglycans, although the effect of different proteoglycans to influence the ability of LPL to act as a bridge in the formation of LDL-proteoglycan complexes is unknown. Using an electrophoretic gel mobility shift assay, [(35)S]SO(4)-labeled versican and biglycan, two extracellular proteoglycans secreted by vascular cells, bound native LDL in a saturable fashion. The addition of bovine milk LPL dose-dependently increased the binding of native LDL to both versican and biglycan, approaching saturation at 30-40 microgram/ml LPL for versican and 20 microgram/ml LPL for biglycan. LDL was oxidized by several methods, including copper, 2, 2-azo-bis(2-amidinopropane)-2HCl and hypochlorite. Extensively copper- and hypochlorite-oxidized LDL bound poorly to versican and biglycan. Proteoglycan binding to LDL was correlated inversely with the extent of LDL; however, the addition of LPL to oxidized LDL together with biglycan or versican allowed the oxidized LDL to bind the proteoglycans in an LPL dose-dependent manner. Addition of LPL had a greater relative effect on the binding of extensively oxidized LDL to proteoglycans compared with native LDL. LPL had a slightly greater effect on increasing the binding of native and oxidized LDL to biglycan than versican. Thus, LPL in the artery wall might increase the atherogenicity of oxidized LDL, since it enables its binding to vascular biglycan and versican.

Ozone alters the expression of tenascin-C in cultured primate nasal epithelial cells.

Tenascin-C is an extracellular matrix component which is transiently expressed in association with epithelial cell detachment, proliferation, and migration. This molecule has been identified in respiratory tissue, but little is known about the cellular source of tenascin-C or the factors that regulate its production. Since air pollutants are known to disrupt epithelial integrity, we investigated the regulation of tenascin-C in response to 0.3 ppm ozone in differentiated primate nasal epithelial cells in culture at an air-medium interface. The expression of tenascin-C was upregulated in response to ozone, as determined by Northern blot analysis, Western blotting, and immunofluorescent staining. In contrast, there was no change in the mRNA levels for versican, biglycan, perlecan, or collagen type I. Reduced cellular attachment to the substrate was evident in ozone-treated cultures in association with tenascin-C deposition at the interfaces between cells and basal surfaces. The presence of tenascin-C on denuded areas of the matrix suggests that tenascin-C may have been instrumental in the loss of patches of cells. The modulation of tenascin-C synthesis and distribution may play a significant role in the response of respiratory epithelial cells to ozone exposure.

Heparin causes the accumulation of heparan sulfate in cultures of arterial smooth muscle cells.

Heparin infusion in an experimental animal model of arterial injury causes a significant increase in the proteoglycan (PG) component of the extracellular matrix in the injured arteries (Snow, A. D., Bolender, R. P., Wight, T. N., and Clowes, A. W. (1990) Am. J. Pathol. 137, 313-330). The mechanisms responsible for this heparin-induced increase in arterial PGs are not understood. To address this question, we have examined the effect of heparin on PG synthesis and accumulation by aortic smooth muscle cells in culture. Heparin causes a dose-dependent increase in the accumulation of PGs while the greatest percentage change among the different types of PGs is a doubling of the amount of heparan sulfate (HS) accumulated in the cell layer. There is also a selective enrichment of HS in the trypsin-resistant component of the cell layer indicating a specific modification in intracellular PGs. This change is due to the accumulation of large HS glycosaminoglycans (GAGs) which elute at K(av) approximately 0.3 on Sepharose CL-6B (Mr approximately 50,000) under dissociative conditions and which are absent from the controls. These GAGs are located inside the cell as is indicated by their retention after heparitinase or trypsin treatment of the cell layer. Furthermore, the increased accumulation of labeled HS in steady-state heparin-treated cells above controls does not appear for several hours after the addition of [35S]sulfate, indicating that HS accumulation is due to decreased degradation of the HS and not new synthesis. This possibility is further supported by the fact that degradation of the intracellular large HS in trypsinized cells is delayed for 2 h by incubation with heparin. These results suggest that heparin may reduce the intracellular degradation of HS through competitive inhibition of endogenous heparitinase.

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

Distinct rat aortic smooth muscle cells differ in versican/PG-M expression.

Smooth muscle cells (SMCs) with distinct phenotypes are present in blood vessels, and distinct culture types appear when SMCs are maintained in vitro. For example, cultured SMCs from rat adult media grow as bipolar cells, which differ in gene expression from the predominantly cobblestone-shaped SMCs from rat pup aortas and rat neointimas that we call pi SMCs. Since proteoglycans are present at different concentrations in the normal intima and media and are elevated in atherosclerotic plaque, we sought to determine whether pi and adult medial SMC types synthesize different or unique proteoglycans that are characteristic of each phenotype. [35S]sulfate-labeled proteoglycans were purified by ion-exchange chromatography. An adult medial SMC line synthesized a large proteoglycan (0.2 Kav on Sepharose CL-2B) that was not detectable in a pi SMC line. Digestion of this proteoglycan with chondroitin ABC lyase revealed three core glycoproteins of 330, 370, and 450 kD. By Western blot analysis, the two smallest of these reacted with two antibodies to the human fibroblast proteoglycan versican. RNAs hybridizing to versican probes were found only in adult medial-type SMCs, including an adult medial type clone from pup aorta, by Northern blot analysis. Both SMC types synthesize RNAs that hybridize to probes for other proteoglycans, such as perlecan, biglycan, and decorin. We conclude that rat pi SMC cultures, unlike monkey, human, and rat adult medial SMC cultures, express little or no versican. This difference in expression may be responsible for the different morphologies and growth properties of the two cell types.

Characterization of proteoglycans synthesized by murine embryonal carcinoma cells (P19) reveals increased expression of perlecan (heparan sulfate proteoglycan) during neuronal differentiation.

Proteoglycans (PGs) incorporated into cell layer and secreted into media were characterized during retinoic acid-induced neuronal differentiation of cultured P19 murine embryonal carcinoma cells. Heparan sulfate significantly increased (P < 0.01) in cell layer following neuronal differentiation of P19 cells by 3.9-fold. CL-4B gel chromatography revealed the major PGs present in cell layer of stem cells eluted as a broad peak with a Kav = 0.65, and was susceptible to chondroitin ABC lyase. The chondroitin ABC lyase resistant material eluted as a broad peak between Kav = 0.40 and Kav = 0.60, and was only partially digested with heparitinase/heparinase (with resistant material eluting at Kav = 0.70). Therefore, the cell layer of stem cells contained primarily chondroitin sulfate/dermatan sulfate (CS/DS) PGs, with lesser amounts of heparan sulfate proteoglycans (HSPGs). This was confirmed by SDS-PAGE. The CS/DS PGs in the cell layer of stem cells had an apparent M(r) of approximately > 200 kDa, and the HSPGs had an apparent M(r) of approximately 140-230 kDa. In contrast, the major PGs in the cell layer of neurons consisted primarily of HSPGs, with only a minor proportion of CS/DS PGs. Furthermore, both gel filtration chromatography and SDS-PAGE analysis revealed a larger HSPG in the cell layer of neurons (Kav = 0.3-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 170 kDa- > 400 kDa on SDS-PAGE) in comparison to stem cells (Kav = 0.4-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 140-230 kDa on SDS-PAGE). Likewise, the major PGs secreted into media of stem cells consisted almost exclusively of CS/DS PGs, with lesser amounts of HSPGs, whereas an increase in HSPGs in the media of neurons was apparent. Western, Northern, and immunocytochemical analysis demonstrated that mRNA transcript and protein levels for a specific HSPG (i.e., perlecan) markedly increased in cell layer following P19 neuronal differentiation. Perlecan core protein was identified by Western blot analysis using specific monoclonal and polyclonal antibodies, as a large HSPG with a core protein of apparent M(r) approximately 370-400 kDa, and was observed primarily in extracts from neurons. Northern blot analysis with a cDNA to perlecan revealed a significant (P < 0.01) 12.7-fold increase in expression of perlecan in neurons (day 9) in comparison to stem cells. The increase in perlecan message during P19 neuronal differentiation was concomitant with a significant (P < 0.01) 26.3-fold increase in message for beta-amyloid precursor protein (beta PP).(ABSTRACT TRUNCATED AT 400 WORDS)

Partial characterization of proteoglycans synthesized by human gingival epithelial cells in culture.

Proteoglycans (PGs) were extracted from the [35S]-sulfate labelled medium and cell layer of proliferating human gingival epithelial cells and analyzed by ion exchange and molecular sieve chromatography, and by SDS-PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sepharose CL-6B contained two species--a quantitatively major peak at K(av) = 0.30 (M(r) congruent to 235,000 on SDS-PAGE) and a quantitatively minor peak at K(av) = 0.39. The major peak was sensitive to alkaline borohydride, shifting to K(av) = 0.45, and nitrous acid degradation, indicating the presence of heparan sulfate PG with glycosaminoglycan chains with M(r) congruent to 26,000. The minor peak is chondroitin/dermatan sulfate PG with glycosaminoglycan chains of M(r) = 22,200 as indicated by sensitivity to alkaline borohydride (shifting to K(av) = 0.48) and chondroitin ABC lyase digestion. The [35S]-sulfate labelled material from the cell layer eluted in a broad peak between 0-0.50 M NaCl from DEAE Sephacel. Chromatography of this material on Sepharose CL-6B revealed the presence of three peaks at K(av) = 0.20, 0.31, and 0.75. The largest peak (K(av) = 0.20 and M(r) congruent to 245,000 on SDS-PAGE) shifted elution position to K(av) = 0.50 after alkaline borohydride treatment and was completely sensitive to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M(r) congruent to 20,000. Two peaks containing [35S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K(av)S = 0.42 (M(r) congruent to 30,500) and 0.75 (M(r) congruent to 5300). The larger peak was predominantly chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase while the chains at K(av) = 0.75 were predominantly heparan sulfate with 83% susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.

Altered proteoglycan synthesis via the false acceptor pathway can be dissociated from beta-D-xyloside inhibition of proliferation.

beta-D-Xylosides have been used to perturb proteoglycan (PG) synthesis to elucidate the function of PGs in a number of cellular processes, including proliferation, migration, and differentiation. This study was designed to examine whether specific xylosides affect the proliferation of several different cell types and, if so, whether this effect is dependent on altered PG synthesis via the false acceptor pathway. Both methylumbelliferyl beta-D-xylopyranoside and p-nitrophenyl beta-D-xylopyranoside (PNP beta-xyloside) inhibit cell proliferation and modulate PG synthesis; however, the alpha form of PNP xyloside which does not perturb PG synthesis inhibits the proliferation of cultured cells on a molar basis equally as well as the beta form. Conversely, beta-methyl xylopyranoside stimulates the synthesis of free glycosaminoglycan chains equally as well as PNP beta-xyloside and yet has no measurable effect on cell proliferation at comparable doses, indicating that cells can grow normally while experiencing disruption of their proteoglycan metabolism. At doses ranging from 0.5 to 5 mM, PNP beta-xyloside arrests cells in the G1 phase of the cell cycle at the same time point as serum starvation. It also delays the exist of cycling cells from the S phase. This treatment is not cytotoxic and is rapidly reversed by the replacement of PNP beta-xyloside containing medium with control medium. Dimethyl sulfoxide, the most commonly used solvent for beta-xyloside in proteoglycan studies, potentiates the inhibitory effect of PNP beta-xyloside on cell proliferation. These results indicate that the perturbation of PG synthesis via the false acceptor pathway can be uncoupled from control of cell proliferation.

Proteoglycans and vascular cell proliferation.

The relationships that exist between proliferative states and proteoglycan (PG) synthesis have been examined in monkey aortic smooth muscle cells in culture. These cells were made quiescent in medium containing low serum (0.1%) and stimulated to divide by addition of either nanogram quantities or platelet-derived growth factor (PDGF) or medium containing 5% serum. Incorporation of [35S]sulfate into PG was increased during the first 24 h of growth stimulation, and this increase appeared to be principally in the large chondroitin sulfate proteoglycan (CSPG). Furthermore, addition of p-nitrophenyl beta-D-xyloside, which perturbs PG metabolism, inhibits cells from proliferating, suggesting that PG may be involved in facilitating cell division. Inhibition of cell proliferation by heparin and/or TGF-beta also causes elevated levels of 35S-sulfate incorporation into PG by these cells. These studies indicate that proteoglycan metabolism is modulated as a function of the growth of arterial smooth muscle cells; however, it is still uncertain whether PG play a direct or indirect role in the control of cell growth.

Vascular cell proteoglycans: evidence for metabolic modulation.

Proteoglycans accumulate in the intimal layer of blood vessels during the early stages of atherosclerosis and predispose the vessel wall to further complications of this disease. Arterial endothelial and smooth muscle cell cultures have been used to study the metabolism of vessel wall proteoglycans in an attempt to determine whether cellular events associated with the genesis of this disease, such as cellular proliferation, ageing, migration and interaction with components of the extracellular matrix, influence the metabolism of arterial proteoglycans. Proteoglycan analyses of vascular cells reveal that endothelial cells synthesize multiple species of heparan sulphate proteoglycan while smooth muscle cells synthesize little heparan sulphate proteoglycan but significant quantities of chondroitin and dermatan sulphate proteoglycan. Each family of proteoglycans synthesized by each cell type differs with regard to charge density, hydrodynamic size, glycosaminoglycan type and size, oligosaccharide content and ability to form high molecular weight aggregates. A monoclonal antibody has been generated against the chondroitin sulphate proteoglycan and used to immunolocalize this antigen to the interstitial matrix of normal and diseased blood vessels. Experiments are presented to indicate that proteoglycan metabolism is modulated when cultured arterial cells are stimulated to proliferate and migrate. Other factors shown to influence proteoglycan metabolism include the age of the cell and the nature of the substratum upon which the cells are grown. These culture systems provide useful models with which to study the factors involved in the regulation of proteoglycan synthesis by vascular cells.

Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells.

Using an improved method to quantify the level of phosphorylation of the S6 ribosomal protein, we have analyzed the effect of growth stimuli on S6 phosphorylation in quiescent murine Swiss/3T3 cells to see if it can be dissociated from the later increase in DNA synthesis. Saturating concentrations of epidermal growth factor (EGF), insulin and serum each stimulate phosphorylation of the S6 ribosomal protein to the same maximal level; this is not so for DNA synthesis. Subsaturating concentrations of EGF and insulin act synergistically to stimulate both S6 phosphorylation and DNA synthesis, but qualitatively the two synergistic interactions are expressed differently. Insulin increases the maximal response of DNA synthesis to EGF, whereas it decreases the concentration of EGF required for half-maximal stimulation of S6 phosphorylation. We conclude that S6 phosphorylation is not a principal regulator of DNA synthesis, and that insulin and EGF regulate both S6 phosphorylation and DNA synthesis through different, but interacting, pathways of action.