Activator protein-1 in carotid plaques is related to cerebrovascular symptoms and cholesteryl ester content.

INTRODUCTION: Transcription factor activator protein-1 regulates genes involved in inflammation and repair. The aim of this study was to determine whether transcription factor activator protein-1 activity in carotid plaques is related to symptoms, lipid accumulation, or extracellular matrix composition. METHODS: Twenty-eight atherosclerotic carotid plaques were removed by endarterectomy and divided into two groups based on the presence or absence of ipsilateral symptoms (<1 month ago). Activator protein-1 DNA binding activity was assessed, and subunit (c-Jun, JunD, JunB, c-Fos, FosB, Fra-1, Fra-2) protein levels analyzed by immunoblotting. Distribution of c-Jun in plaques was analyzed by immunohistochemistry. RESULTS: Plaques associated with symptoms had increased activator protein-1 activity and increased expression of c-Jun and JunD, as compared to asymptomatic plaques. Fra-1 and Fra-2 were present in equal amounts in both groups, whereas JunB, FosB, and c-Fos were undetectable. Activator protein-1 activity correlated with cholesteryl ester and elastin in plaques and decreased with age. Activator protein-1 activity did not correlate with collagen, calcified tissue, or proteoglycan content. CONCLUSIONS: Activator protein-1 is increased in plaques associated with symptoms. The correlation between activator protein-1 and cholesteryl esters suggests that high activator protein-1 is a marker of plaque vulnerability. Activator protein-1 expression can also reflect the activation of repair processes.

Hypoxic regulation of secreted proteoglycans in macrophages.

Macrophages are prominent in hypoxic areas of atherosclerotic lesions, and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia-inducible factor (HIF-1alpha and HIF-2alpha). We found that versican co-localized with HIF-1alpha in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O(2) (hypoxia) compared with 21% O(2) (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1alpha and HIF-2alpha in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1alpha and HIF-2alpha but expression of perlecan was influenced only by HIF-1alpha and not by HIF-2alpha knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.

Elastin- and collagen-rich human carotid plaques have increased levels of the cysteine protease inhibitor cystatin C.

BACKGROUND: Cystatin C is a major inhibitor of the elastin- and collagen-degrading cysteine proteases and may therefore have an important role in preserving atherosclerotic plaque stability. In this study we analyzed the associations between human carotid plaque cystatin C expression and the plaque content of collagen and elastin. METHODS: Thirty-one plaques were removed by endarterectomy and homogenized. Cystatin C levels were analyzed by densitometry of Western blots and elastin and collagen levels were determined colorimetrically. RESULTS: The plaque content of cystatin C correlated with total elastin (r = 0.58, p = 0.001) and collagen (r = 0.50, p = 0.004), as well as with cross-linked forms of elastin (r = 0.42, p = 0.022) and collagen (r = 0.52, p = 0.003). Immunohistochemical analysis demonstrated that cystatin C colocalized with elastin and collagen. No correlation was seen between cystatin C and the amount of degraded elastin or collagen in plaques. CONCLUSION: The positive correlation between cystatin C levels and collagen and elastin levels in plaques supports the notion that cystatin C plays an important role in maintaining atherosclerotic plaque stability.

IFNgamma regulates PDGF-receptor alpha expression in macrophages, THP-1 cells, and arterial smooth muscle cells.

The recruitment of monocyte-derived macrophages (MDMs) and arterial smooth muscle cells (ASMCs) contributes to inflammation and development of intimal hyperplasia during atherosclerosis. Platelet-derived growth factor (PDGF) is a potent mitogen for SMC, signalling through PDGF-receptor subunits alpha (Ralpha) and beta (Rbeta). We have previously found that interferon gamma (IFNgamma) upregulates PDGF-Ralpha mRNA expression in human MDM (hMDM) which causes an increased migration towards PDGF. In the present study, we found that IFNgamma mediated an upregulation of PDGF-Ralpha mRNA also in THP-1 cells. The induction of PDGF-Ralpha in both hMDM and THP-1 cells was caused by STAT1 binding to the PDGF-Ralpha promoter. In human ASMCs, IFNgamma again stimulated a transient STAT1-binding to the PDGF-Ralpha promoter. However, this was not followed by an upregulation of PDGF-Ralpha mRNA. IFNgamma-stimulation resulted in augmented expression of PDGF-Ralpha protein in differentiated hMDM. Early hMDM only expressed an immature and not fully glycosylated form of the PDGF-Ralpha protein. In contrast, THP-1 cells did not synthesize PDGF-Ralpha protein, implying further posttranscriptional inhibition. Our results contribute to a better understanding of the complex regulation of PDGF-Ralpha expression and how proinflammatory factors may contribute to PDGF-related hyperplasia in vascular diseases.

Fatty acids cause alterations of human arterial smooth muscle cell proteoglycans that increase the affinity for low-density lipoprotein.

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 micromol/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 PPARgamma agonist rosiglitazone (10 micromol/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.

Low density lipoprotein stimulation of human macrophage proteoglycan secretion.

Lipoprotein trapping in arterial intima increases the risk for lipoprotein oxidation, foam cell formation, and inflammatory response in surrounding cells. Modified lipoproteins increase smooth muscle cell production of proteoglycans likely to retain lipoproteins in intimal extracellular matrix. We hypothesized that macrophage proteoglycan production is regulated in a similar manner, and characterized glycosaminoglycan side chains of secreted proteoglycans. Incubation with native low density lipoproteins (LDL) strongly stimulates total proteoglycan secretion in a time and concentration dependent manner. The main secretion product is small-sized (120 kDa) with unusually long galactosaminoglycan chains, predominantly chondroitin-6-O-sulfated. The effect appears specific for native LDL; oxidized LDL, very low density lipoproteins or phospholipid liposomes have only minor effects compared to control. These observations suggest that native LDL stimulate macrophages to secrete a chondroitin sulfate-rich proteoglycan moiety likely to have high capacity for vascular extracellular trapping of apolipoprotein B-containing lipoproteins.

Primary human glomerular endothelial cells produce proteoglycans, and puromycin affects their posttranslational modification.

This article describes the possible role of the endothelial cell-surface coat, containing proteoglycans (PGs) with connected glycosaminoglycans (GAGs), in maintaining glomerular permselectivity. Primary human glomerular endothelial cells (HGEC) in culture were treated with the nephrosis-inducing agent puromycin aminonucleoside (PAN). Analysis was made by TaqMan real-time PCR, Western blot analysis, and by metabolic labeling with [(35)S]sulfate. The HGECs express several PGs: syndecan, versican, glypican, perlecan, decorin, and biglycan, which may contribute to the glomerular charge barrier. PAN treatment downregulated both the protein expression (by 25%) and the mRNA expression (by 37 +/- 6%, P < 0.001, n = 8) of versican compared with control. Transferases important for chondroitin and heparan sulfate biosynthesis were also significantly downregulated by PAN, resulting in less sulfate groups, shorter GAG chains, and reduced PG net-negative charge. Moreover, analysis of the cell media after PAN treatment revealed a reduced content of [(35)S]sulfate-labeled PGs (40% of control). We conclude that PAN may cause proteinuria by affecting the endothelial cell-surface layer and not only by disrupting the foot process arrangement of the podocytes. Thus the endothelium may be a more important component of the glomerular barrier than hitherto acknowledged.

Elastin and calcium rather than collagen or lipid content are associated with echogenicity of human carotid plaques.

BACKGROUND AND PURPOSE: Echolucent carotid plaques have been associated with increased risk for stroke. Histological studies suggested that echolucent plaques are hemorrhage- and lipid-rich, whereas echogenic plaques are characterized by fibrosis and calcification. This is the first study to relate echogenicity to plaque composition analyzed biochemically. METHODS: Echogenicity of human carotid plaques was analyzed by standardized high-definition ultrasound and classified into echolucent, with gray-scale median (GSM) <32 and echogenic with GSM > or =32. The biochemical composition of the plaques was assessed by fast-performance liquid chromotography and high-performance thin-layer chromotography. RESULTS: As assessed biochemically (milligrams per gram [mg/g]), echolucent plaques contained less hydroxyapatite (43.8 [SD 41.2] mg/g versus 121.6 [SD 106.2] mg/g; P=0.018), more total elastin (1.7 [SD 0.4] mg/g versus 1.2 [SD 0.4] mg/g; P=0.008), and more intermediate-size elastin forms (1.2 [SD 0.3] mg/g versus 0.8 [SD 0.4] mg/g; P=0.018). There was no difference in collagen amount between echogenic and echolucent plaques, neither biochemically (15.3 [SD 3.7] mg/g versus 14.4 [SD 3.4] mg/g) nor histologically (13.4 [SD 4.9] % versus 13.0 [SD 5.6] %). Cholesterol esters, unesterified cholesterol, and triglycerides were increased in plaques associated with symptoms (22.5 [SD 23.3] mg/g versus 13.3 [SD 3.2]; P=0.04), but no differences were detected between echolucent and echogenic plaques (13.5 [SD 4.0] versus 20.2 [SD 21.5] mg/g). Similar results were obtained by Oil Red O staining (symptomatic 7.6 [SD 4.7] % versus asymptomatic 4.2 [SD 3.6] %; P=0.03; echolucent 5.9 [SD 4.1] % versus echogenic 5.0 [SD 4.0] % of area). CONCLUSIONS: Echogenicity of carotid plaques is mainly determined by their elastin and calcium but not collagen or lipid content. In addition, echolucency is associated to higher elastin content.

Changes related to age and cerebrovascular symptoms in the extracellular matrix of human carotid plaques.

BACKGROUND AND PURPOSE: Many processes involved in the pathogenesis of atherosclerosis result in modifications of the extracellular matrix. These changes not only determine the mechanical stability of atherosclerotic lesions but can directly or indirectly influence further development of the lesions. The purpose of the present study was to compare the matrix composition of human carotid plaques from symptomatic patients with those obtained from patients without symptoms. Furthermore, matrix changes related to age were studied. METHODS: Thirty atherosclerotic carotid plaques were removed by endarterectomy from 27 patients and divided into 2 groups on the basis of the presence of ipsilateral symptoms. The plaques were homogenized, and the total levels of the major components of the extracellular matrix were determined. RESULTS: Plaques associated with symptoms were characterized by increased levels of elastin (1.58+/-0.46 versus 1.24+/-0.40 mg/g wet wt; P=0.03) and decreased levels of hydroxyapatite (45.1+/-46.3 versus 131.4+/-111.7 mg/g wet wt; P=0.02) compared with asymptomatic plaques. The increase in elastin in plaques from symptomatic patients was due to elevated levels of an intermediate-size fraction, as determined by liquid chromatography. Collagen and sulfated glycosaminoglycans were present in equal amounts in both groups. Elastin content in carotid plaques decreased with age. CONCLUSIONS: Carotid plaques from symptomatic patients have lower levels of hydroxyapatite than those from asymptomatic patients. The present study also raises the possibility that non-cross-linked forms of elastin, increased in plaques associated with symptoms, could be a marker of plaque vulnerability and/or directly induce harmful cellular activities or increase lipoprotein retention in the vascular wall.