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【摘要】
Objective- The dyslipidemia of insulin resistance, with high levels of albumin-bound fatty acids, is a strong cardiovascular disease risk. Human arterial smooth muscle cell (hASMC) matrix proteoglycans (PGs) contribute to the retention of apoB lipoproteins in the intima, a possible key step in atherogenesis. We investigated the effects of high NEFA levels on the PGs secreted by hASMCs and whether these effects might alter the PG affinity for low-density lipoprotein.
Methods and Results- hASMC exposed for 72 hours to high concentrations (800 µmol/L) of linoleate (LO) or palmitate upregulated the core protein mRNAs of the major PGs, as measured by quantitative PCR. Insulin (1 nmol/L) and the PPAR agonist rosiglitazone (10 µmol/L) blocked these effects. In addition, high LO increased the mRNA levels of enzymes required for glycosaminoglycan (GAG) synthesis. Exposure to NEFA increased the chondroitin sulfate:heparan sulfate ratio and the negative charge of the PGs. Because of these changes, the GAGs secreted by LO-treated cells had a higher affinity for human low-density lipoprotein than GAGs from control cells. Insulin and rosiglitazone inhibited this increase in affinity.
Conclusions- The response of hASMC to NEFA could induce extracellular matrix alterations favoring apoB lipoprotein deposition and atherogenesis.
We examined the effects of increased NEFA and insulin on the proteoglycans secreted by hASMC and whether these effects might affect LDL binding. The results indicate that increased fatty acids could induce qualitative and quantitative alterations of the intima extracellular matrix proteoglycans favoring LDL retention and possibly atherogenesis.
【关键词】 proteoglycans smooth muscle cells LDL fatty acids insulin
Introduction
Insulin resistance (IR) and type 2 diabetes (T2D) are associated with a 2- to 4-fold increase in atherosclerotic coronary artery disease. 1,2 Changes in circulating lipoproteins, chronic high levels of albumin-bound nonesterified fatty acids (NEFA, and hyperinsulinemia are important contributors to this association. 3-5 Atherogenesis involves a tissue response to deposition of apoB lipoproteins and insulin signaling defects that may affect vascular cells. 6 The accelerated intimal thickening of large arteries observed in IR and diabetes includes excessive production of matrix components, such as proteoglycans (PGs) and collagens by smooth muscle cells (SMCs). 7 Cell culture experiments suggest that increased endothelial cell permeability and SMC alterations of matrix production could be caused by exposure to excessive amounts of NEFA. 8-10
In IR, arterial cells are chronically exposed to increased levels of circulating NEFA. In addition, NEFA could be produced locally by lipolytic degradation of lipoproteins, additionally increasing their concentration in the arterial intima. 11,12 Retention of low-density lipoprotein (LDL) in the intima by chondroitin sulfate (CS)-rich PGs appears to be a key step in atherogenesis at sites of intima thickening. 13-15 We reported that the expression of genes for extracellular matrix proteins and PGs is increased when human arterial SMCs (hASMCs) are exposed to NEFA. Furthermore, the extracellular matrix produced by the NEFA-treated hASMC had a higher affinity for LDL. 9
In this study we describe how linoleate (LO) and palmitate (PA) increased the expression of genes encoding the core proteins of the main secreted PGs from hASMC and of the key enzymes in CS-glycosaminoglycan (GAG) biosynthesis. Exposure to LO also altered the GAG composition. Insulin, dose dependently, attenuated the effects of LO. The results explain why the changes in PG composition and structure increase the affinity of the secreted matrix for LDL and suggest a mechanism through which IR and its dyslipidemia can contribute to atherogenesis.
Methods
Please see http://atvb.ahajournals.org for a full-length version of this section.
Fatty Acid-Albumin Complex Preparation
Solutions containing sodium LO or PA bound to albumin were prepared from stock solutions of the fatty acid sodium salt and fatty acid-free BSA. Agarose gel electrophoresis of final solutions containing 10 5 cpm/µL of 14 C-labeled LO indicated that 99.9% of the radioactivity was bound to albumin.
Cell Culture
Primary cultures of hASMCs from inner media of uterine arteries were established by an explantation technique. 16 Cells were grown in medium 199 (M-199) with Earle?s salts, antibiotics, sodium pyruvate, and L-glutamine (M199+) containing 10% (v/v) human serum.
GAG Biolabeling
Cells were synchronized in M199+ containing 0.02% human serum, because the amount of PGs and the length and sulfation degree of their GAG chains may change with proliferation. 17 After 48 hours, the medium was replaced with sulfate-depleted minimum essential medium with 10% (v/v) human serum, antibiotics, sodium pyruvate, and L-glutamine. The next day, radioactive precursors ([ 35 S] Sulfate, D-[6- 3 H] glucosamine hydrochloride) and BSA, fatty acids, and/or insulin were added. In some experiments, the PPAR agonist rosiglitazone and the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) were added together with LO. Media were collected after 72 hours.
GAG Isolation and Analysis
GAGs were isolated by precipitation with 95% ethanol and characterized with agarose gel electrophoresis and chondroitinase treatment. 18 Evaluation of the chain length was performed by PAGE. 19
RT-PCR
Total RNA was isolated and reversed transcribed. Quantification of mRNA was performed using the ABI PRISM 7700 Sequence Detector (Applied Biosystems). 20 Primer and probe sequences were designed targeting PG core protein mRNAs. Primers and probes for versican were placed in a region common to all of the isoforms. 21 The mRNAs for enzymes involved in CS synthesis were also evaluated.
Extraction and Analysis of Lipids
Analyses of SMC lipid classes were performed by high-performance liquid chromatography and evaporative light-scattering mass detection. 22 Conventional Oil Red O staining was performed as described. 23
LDL Binding
LDL (d=1.019 to 1.063 g/mL) was isolated from fresh human plasma by differential ultracentrifugation. 24 Binding of LDL to 35 S/ 3 H-labeled GAGs was evaluated by electrophoretic band shift analysis. 25
Data Analysis
All of the densitometric evaluations were done with a Bio-Rad molecular imager system using the Quantity One software. Results are given as means±SE. Differences between 2 groups were identified with a Student t test. Multiple mean values were compared with a 1-way ANOVA and Dunnett?s post hoc test to identify differences versus fatty acid-treated cells. A value of P <0.05 was accepted as statistically significant. The SSPS software was used for statistical analysis.
Results
Effects of High NEFA and Insulin on mRNA of the PG Core Proteins
Both LO and PA upregulated the mRNA for the core protein of versican, a large CS-containing PG ( Figure 1 ). The mRNA of biglycan, a small leucine-rich PG carrying 2 chondroitin or dermatan sulfate (DS) chains, and that of perlecan, a membrane-bound PG with predominantly heparan sulfate (HS) chains, were also increased compared with controls but to a lesser extent ( Figure 1 ). Decorin expression was increased by LO, but significant differences were not reached. Insulin alone from 1 to 100 nmol/L had no effect on any of the analyzed mRNAs (Table I, available online at http://atvb.ahajournals.org). However, when insulin (10 nmol/L) was added together with the fatty acid, a reduction of the fatty acid-induced upregulation of versican, biglycan, and perlecan mRNAs (50%, 60%, and 74%, respectively) was observed. It should be noted that the basal concentration of insulin in the media, which contained 10% of human serum, was <10 pmol/L. We additionally explored the effect of increasing doses of insulin on the augmented expression of core protein genes for versican and biglycan as induced by LO and PA. The results for versican and biglycan mRNAs indicate that the EC 50 of insulin for this effect was &1 nmol/L (Table II, available online at http://atvb.ahajournals.org).
Figure 1. Expression of PG core proteins after exposure to fatty acids and insulin. HASMCs were exposed to 300 µmol/L BSA (control) and 800 µmol/L fatty acids (LO or PA) with and without 10 nmol/L insulin. Data are presented as percentage of expression in control cells corrected for ß-actin expression as means±SE (n=4). * P <0.05 vs corresponding fatty acid addition for each individual mRNA, as analyzed with ANOVA and Dunnett?s test.
We also found that the PPAR agonist rosiglitazone at 10 µmol/L inhibited the LO-induced changes in the mRNA for the core proteins of versican, biglycan, and perlecan. This effect was concentration dependent ( Figure 2 ).
Figure 2. Expression of PG core proteins after exposure to LO and a PPAR- agonist, rosiglitazone. HASMCs were cultured in 300 µmol/L BSA; 800 µmol/L LO with and without 1, 5, or 10 µmol/L rosiglitazone. Values are relative to ß-actin expression and presented as means±SE (n=4). * P <0.05 vs LO-treated cells.
Structural Changes of the PGs Induced by LO
Versican, a PG that can carry 12 CS chains with smaller amounts of DS GAGs, is the most abundant PG of those secreted by hASMC in culture. 26 Therefore, the relative content of CS chains secreted by the cells into the media should be augmented, if increased expression of mRNA for the core protein is accompanied by increased secretion of the versican isoforms with GAG attachment domains, v 0 or v 1. Three isoforms denoted V 1, V 2, and V 3 can be generated by alternative splicing from the larger transcript, V 0. These isoforms vary in their relative content of the 2 GAG attachment domains, that is, GAG- and GAG-ß, which are differentially omitted in the V 2 and V 1 isoforms and are entirely absent in the V 3 isoform. 21,27 Figure 3 shows the (CS+DS):HS ratios determined after releasing the media GAGs by proteolysis and electrophoretic separation under different conditions. Treatment with chondroitinases AC-I and ABC, and subsequent electrophoresis, indicated that CS constitutes 70% to 85% of the secreted GAGs. LO 800 µmol/L increased the (CS+DS):HS ratio 1.5 times the value for control cells. This effect was concentration dependent and observed already with additions of 50 to 100 µmol/L (Figure I, available online at http://atvb.ahajournals.org). Insulin (10 nmol/L) blunted the effect of LO, although it had no effect by itself on the (CS+DS):HS ratio. The changes in the (CS+DS):HS ratio caused by LO were also blocked by the addition of 10 µmol/L of the unspecific PKC inhibitor BIM I (Figure II, available online at http://atvb.ahajournals.org).
Figure 3. Effect of LO on glycosaminoglycan composition of secreted PGs. Secreted 35 S/ 3 H-GAGs were released with papain and separated by agarose gel electrophoresis. Autoradiographs of the gels were evaluated densitometrically, and the CS+DS:HS ratio was calculated. Values are means±SE (n=7). * P <0.05 vs LO-treated cells.
LO increased the mRNAs for the core proteins of CSDS-PGs, like versican and biglycan, but also that of perlecan, a HS-PG ( Figure 1 ). Considering the stoechiometry GAG chain to core protein of all examined PGs, we concluded that the increased (CS+DS):HS ratio caused by LO was determined by an increase in CS and DS PGs.
Changes in Enzymes Required for GAG synthesis
CS synthase and CS-6 and CS-4 sulfotransferases are key enzymes for the polymerization and sulfation of chondroitin chains. Therefore, we investigated whether the observed upregulation of core proteins for CS-containing PGs induced by LO was accompanied by changes in the mRNA for these enzymes. High LO increased the levels of mRNA corresponding with the 3 enzymes from 1.6 to 2 times the controls. Insulin at 10 nmol/L, when added together with LO, blunted the upregulation with no effect when added alone. Rosiglitazone at 10 µmol/L reduced the fatty acid-induced upregulation of CS-synthase, CS-6, and CS-4 sulfotransferases by 37%, 41%, and 22%, respectively ( Figure 4 ).
Figure 4. Expression of CS-synthase and CS-4 and CS-6 sulfotransferases. haSMCs were exposed to 300 µmol/L BSA, 800 µmol/L LO with and without 10 nmol/L insulin, and 800 µmol/L LO with 10 µmol/L rosiglitazone. Data are corrected for ß-actin mRNA content in the respective total RNA preparation. Values are means±SE (n=3). * P <0.05 vs LO-treated cells.
LO Increases the Affinity of LDL for Secreted GAGs
Metabolically labeled GAGs from the media were isolated and used for evaluation of binding parameters using increasing amounts of human LDL and electrophoretic band shift analysis. This method allows an estimation of interactions at physiological ionic conditions. 25 The GAGs isolated from LO-treated cells bound more efficiently to LDL than the GAGs from control cells. Insulin (10 nmol/L) and rosiglitazone (10 µmol/L) abrogated this fatty acid-dependent increase ( Figure 5 ). The results suggest that the LO-induced structural changes in GAGs caused a higher affinity for LDL and that these changes and the associated increased affinity were inhibited by insulin and the PPAR agonist.
Figure 5. LDL binding to secreted glycosaminoglycans. Labeled GAGs were incubated with human LDL at physiological ionic strength and pH. (a) The GAGs-LDL complexes were separated with agarose gel electrophoresis. On binding to LDL, the GAGs-LDL complexes are retained in the origin of the gel (arrow). (b) Quantification of retained GAGs by 2 LDL concentrations. Values are means±SE (n=4). * P <0.05 vs LO-treated cells.
LO Increases Sulfation of Secreted GAGs
The number of sulfate groups per disaccharide unit determines the negative charge density of the GAGs. Negative charge density and chain length of the GAGs are the main properties controlling the binding of PGs to many proteins, including LDL. 14,28 We found no differences in GAG chain length, as evaluated by PAGE. However, agarose gel electrophoresis indicated that the major CS-containing GAGs from LO-treated cells had a 9% to 10% ( P <0.05) higher anodic electrophoretic mobility relative to control cells (n=7), suggesting an increase in negative charge. Insulin (10 nmol/L) and rosiglitazone (10 µmol/L) returned the electrophoretic mobility of the CS-GAGs to control values.
Because we performed the metabolic labeling of GAGs with both [ 35 S] Sulfate and D-[6- 3 H] glucosamine, measurement of the 35 S: 3 H ratio in the isolated GAGs provided an additional measure of the relative amount of sulfate groups per GAG chain. Treatment with LO increased the relative sulfate content by 16% ( P <0.05) and, consequently, the negative charge density of secreted GAGs (n=5). This increase was eliminated by 10 nmol/L of insulin. Rosiglitazone showed a tendency to decrease the 35 S: 3 H ratio, but the differences did not reach statistical significance.
Changes in Intracellular Lipid Content
After incubation with 800 µmol/L of LO, the triacylglycerol 20-fold, from 1.22±0.20 to 26.48±0.70 µg/mg of cell protein ( P <0.05, n=3). Also, the content of diacylglycerol (DAG) increased significantly from 0.10±0.01 to 0.23±0.02 µg/mg of cell protein ( P <0.05, n=3). Insulin addition did not alter these values. No increases in other lipid classes were observed (Table III, available online at http://atvb.ahajournals.org). Oil Red O staining revealed lipid droplets in the cytoplasm of fatty acid-treated cells (Figure III, available online at http://atvb.ahajournals.org).
Discussion
IR, hyperinsulinemia, and hyperglycemia in T2D have been suggested as agents that could alter PGs of the vascular extracellular matrix. 29,30 Increased levels of albumin-bound NEFA, a component of the dyslipidemia of IR, have also been suggested to alter the GAGs of the basement membrane of endothelial cells increasing the permeability. 31 However, most of the arterial intima is made of extracellular matrix secreted by SMCs. This is the environment where key initial interactions contributing to atherogenesis take place. 12-15,32 Therefore, it is important to explore whether metabolic alterations associated with IR and T2D can be responsible for the atherogenic changes observed in the human extracellular intima. 7
The present experiments indicate that linoleic and palmitic acid increased the expression of genes for the core proteins of versican, the small leucine-rich PGs with either 1 (decorin) or 2 (biglycan) chondroitin/DS side chains, and perlecan, the main HS-containing PG of the basement membrane. These NEFA effects were accompanied by an increase of CS-rich PGs secreted into the media. In previous experiments, we found that the extracellular matrix synthesized by cells exposed to fatty acids had a higher affinity for LDL than that of unexposed cells. The present data using band shift analysis, conducted with the isolated secreted GAGs, indicate that this increase is most likely caused by CS-GAG chains contributed by versican and biglycan. Versican is the most abundant PG of the human intima and, when isolated from human arteries or SMC cultures in vitro, shows a high affinity for LDL. 26,33 Biglycan, on the other hand, has been shown to colocalize with apoB and apoE-containing lipoproteins in human coronary atherosclerotic intima and has been found to bind LDL. 34,35 Moreover, changes in the composition of arterial GAGs in human diabetes have been detected. 36,37
Our data suggest also that the increase in negative charge density of the GAGs caused by the NEFA contributed to the increased LDL binding. This increase in negative charge, indicated by the higher anodic mobility of the CS chains, appears to be a product of an increase of sulfate groups relative to hexosamine units, as indicated by the changes in the 35 S: 3 H ratio. The elevated expression of the genes for CS-4 and CS-6 sulfotransferases, the enzymes that attach negative sulfate groups to the polymerizing GAG chain, observed on LO exposure supports this conclusion. It is important to mention in this context that GAGs isolated from the intima of atherosclerosis-prone human arteries are enriched in highly sulfated CS. 38 These GAGs also show a higher affinity for LDL, a property that, as mentioned, could contribute to increased LDL entrapment in lesion-prone arteries. 14
Intramyocellular TAG levels correlate tightly with the severity of IR. 39 In addition to inducing TAG biosynthesis, NEFA also stimulates the synthesis of other less abundant metabolites, such as DAG and ceramides. 40 Both derivatives of fatty acyl CoA have been implicated as primary mediators of the antagonistic effects of NEFA in skeletal muscle. 41-43 DAG is hypothesized to activate a signaling cascade leading to the inhibition of IRS-1, 42 whereas ceramides have been shown to block activation of Akt/protein kinase B. 44 As a result of increased intracellular DAG, PKC is chronically activated in diabetes and nondiabetic IR. Also, high-glucose concentrations or diabetes cause increased activity of membrane-associated PKC and are associated with increased intracellular DAG concentrations in several tissues and in cultured aortic endothelial cells and vascular SMCs. 45-47 Furthermore, in skeletal muscle of PKC knockout mice, PKC activation during hyperlipidemia was necessary for the inhibition of skeletal muscle IRS-1 tyrosine phosphorylation and insulin-stimulated glucose uptake. 48 We found that incubation with LO increased the cellular content of TAG and DAG compared with control cells. In addition, BIM I, a nonspecific PKC inhibitor, blocked the effects of linoleic acid on the (CS+DS):HS ratio of secreted GAGs, suggesting that activation of PKC by fatty acids could be the cause of impairment of insulin signaling involved in PG and GAG biosynthesis, possibly by increasing the intracellular DAG content. 49 Thus, it is possible that in our experiments NEFA weakens insulin signaling by activation of PKC.
In addition, we found that the PPAR agonist rosiglitazone blocked the effects of LO on PGs, GAGs, and LDL binding. However, we did not find a consensus peroxisome proliferating response element in the upstream sequence spanning 6000 bases before the starting codon in any of the genes screened. Because PPAR is expressed in SMCs, we speculate that it may oppose the actions of NEFA by accelerating their conversion into stored TAGs. This could limit the concentration of DAG or other bioactive lipids that could modulate the insulin-signaling cascade. 50 It may also limit the actions of fatty acyl-CoA on glucose use and lower the contribution of the hexosamine pathway. The end products of this pathway, UDP-Glc-NAc and UDP-Gal-Nac, are the building blocks for the synthesis of the GAG chains of PGs. This action could explain why rosiglitazone inhibited the effects of NEFA in a dose-dependent manner.
Under our culture conditions, insulin on its own added on top of the basal level of <10 pmol/L had no effect on any of the PG or GAG properties. On the other hand, the changes caused by the fatty acids on genes encoding the PG core proteins, the substantial changes in GAG structure, and increased affinity 1 nmol/L. These results suggest that levels of insulin in the picomole range are sufficient to maintain a basal rate of PG synthesis and composition. However, under overexposure to NEFA, 1 to 5 nmol/L of insulin was required to restore the basal biosynthesis and structure of secreted PGs. In hASMCs, it has been reported previously that the insulin effects on 2-deoxy- D -glucose transport have an approximate K d of 25 nmol/L. 51 Arterial SMCs are responsive to both insulin and IGF-I, and their effects are additive at near physiological concentrations. 52,53 Both ligands can cross-react with the receptors of the other but with 100- to 1000-fold less potency than to their own receptors. 54 Insulin receptors are few in SMCs, whereas the IGF-I receptor is highly expressed in SMCs in intact arteries and in cultured SMCs. 54 Insulin and IGF-I receptors also show great similarities in their signaling pathways. Our results, indicating that 1 to 5 nmol/L of insulin is required to abrogate the fatty acid effects on secreted PGs, may indicate that the hormone is acting via both receptors pathways. It is important to stress that this is a direct possible beneficial effect of insulin on hASMC but that another important one present in vivo is the capacity of the hormone to reduce circulating NEFA levels. 3,55
In summary, the current study indicates that exposure of hASMC to high levels of fatty acids can cause structural alterations of secreted PGs that explain the increased binding of LDL. Such effects were blunted by insulin and PPAR activation. If similar effects are present in vivo in conditions of IR, overexposure of the arterial smooth muscle to fatty acids could induce extracellular matrix changes favoring apoB lipoprotein deposition and atherogenesis.
Acknowledgments
This work was supported by grants from the Heart and Lung Foundation and AstraZeneca. We thank Kristina Skålén, Lillemor Mattsson-Hultén, and Aira Lidell for technical advice.
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作者单位：Wallenberg Laboratory for Cardiovascular Research (M.R-L., G.Ö-L., G.B., J.M., G.C.), Sahlgrenska Academy at Göteborg University, Gothenburg, and AstraZeneca Discovery (B.W., G.C.), Mölndal, Sweden.