Mikko P.S. Ares

HMG-CoA Reductase Inhibitors Regulate Inflammatory Transcription Factors in Human Endothelial and Vascular Smooth Muscle Cells

Objective—Pleiotropic atheroprotective effects of HMG-CoA reductase inhibitors may be mediated on the level of vascular gene transcription. The aim of this study was to characterize the effects of statins on the activation of transcription factors known to regulate inflammation and cell proliferation/differentiation. Methods and Results—Simvastatin, atorvastatin, and lovastatin (0.1 to 10 &mgr;mol/L) inhibited the binding of nuclear proteins to both the nuclear factor-kappa B (NF-&kgr;B) and activator protein-1 (AP-1) DNA consensus oligonucleotides in human endothelial and vascular smooth muscle cells as assessed by electrophoretic mobility shift assay (EMSA). The inhibitory effects of statins on NF-&kgr;B or AP-1–dependent transcriptional activity were examined by transient transfection studies. HMG-CoA reductase inhibitors upregulated I&kgr;B-&agr; protein levels in endothelial cells and decreased c-Jun mRNA expression in smooth muscle cells as analyzed by Western and Northern blotting, respectively. Furthermore, statins inhibited DNA binding of hypoxia-inducible factor-1&agr;. Downstream effects of statins included inhibition of plasminogen activator inhibitor-1 and vascular endothelial growth factor-A mRNA levels in endothelial cells. Conclusions—HMG-CoA reductase inhibitors downregulate the activation of transcription factors NF-&kgr;B, AP-1, and hypoxia-inducible factor-1&agr;. These findings support the concept that statins have antiinflammatory and antiproliferative effects that are relevant in the treatment of atherosclerotic diseases.

Recombinant human antibodies against aldehyde-modified apolipoprotein B-100 peptide sequences inhibit atherosclerosis

Background—Accumulation and oxidation of LDL are believed to be important initiating factors in atherosclerosis. Oxidized LDL is recognized by the immune system, and animal studies have suggested that these immune responses have a protective effect against atherosclerosis. Aldehyde-modified peptide sequences in apolipoprotein B-100 (apoB-100) are major targets for these immune responses. Methods and Results—Human IgG1 antibodies against 2 malondialdehyde (MDA)-modified apoB-100 peptide sequences were produced through screening of a single-chain antibody-fragment library and subsequent cloning into a pcDNA3 vector. Three weekly doses of these antibodies were injected into male apoE−/− mice. Phosphate-buffered saline and human IgG1 antibodies against fluorescein isothiocyanate were used as controls. One of the IgG1 antibodies significantly and dose-dependently reduced the extent of atherosclerosis as well as the plaque content of oxidized LDL epitopes and macrophages. In cell culture studies, human monocytes were incubated with native LDL or oxidized LDL, in the presence of antibodies. The same antibody induced an increase in monocyte binding and uptake of oxidized LDL. Conclusions—These findings suggest that antibodies are important mediators of atheroprotective immune responses directed to oxidized LDL. Thus, passive immunization against MDA-modified apoB-100 peptide sequences may represent a novel therapeutic approach for prevention and treatment of cardiovascular disease.

Very Low-Density Lipoprotein Activates Nuclear Factor-κB in Endothelial Cells

High plasma levels of VLDL are associated with increased risk for atherosclerosis. Here we show that VLDL (75 to 150 microg/mL) activates nuclear factor-kappaB (NF-kappaB), a transcription factor known to play a key role in regulation of inflammation. Oxidation of VLDL reduced its capacity to activate NF-kappaB in vitro, whereas free fatty acids such as linoleic and oleic acid activated NF-kappaB to the same extent as did VLDL. Intravenous injection of human VLDL (6 mg protein per kg) into rats resulted in arterial activation of NF-kappaB as assessed by electrophoretic mobility shift assay. Aortic endothelial cells showed positive nuclear staining for the activated RelA (p65) subunit of NF-kappaB at 6 to 24 hours after injection. There was also a parallel expression of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, as well as the cytokine tumor necrosis factor-alpha. Pretreatment of the rats with diet containing 1% of the antioxidant probucol for 8 weeks did not inhibit arterial activation of NF-kappaB in response to injection of VLDL. Moreover, injection of triglycerides (10% Intralipid, 5 mL/kg) activated arterial expression of NF-kappaB to the same extent as VLDL. Our results suggest that VLDL may promote the development of atherosclerotic lesions by activation of the proinflammatory transcription factor NF-kappaB. The effect appears to be mediated by a release of VLDL fatty acids but not to involve VLDL oxidation.

Human Monocytes/Macrophages Release TNF-α in Response to Ox-LDL

The uptake of oxidatively modified low density lipoprotein (Ox-LDL) by intimal macrophages is believed to play a key role in the development of atherosclerosis. The present study demonstrates that Ox-LDL in low concentrations activates monocyte/macrophage release of factors that stimulate smooth muscle cell growth, whereas higher concentrations are inhibitory. Exposure of monocytes/macrophages to 8 μg/mL Ox-LDL increased expression of tumor necrosis factor-α (TNF-α) mRNA but had no effect on interleukin-1β, platelet-derived growth factor B and heparin-binding epidermal growth factor–like mitogen mRNA levels. Ox-LDL also stimulated monocyte/macrophage release of TNF-α in a dose-dependent manner, with maximal effect at an LDL concentration of 8 μg/mL. Addition of TNF-α–blocking antibodies to conditioned medium from monocytes/macrophages already exposed to Ox-LDL reduced mitogenic activity by 44.7±8.4% (P<.005). Stimulation of TNF-α release by Ox-LDL was associated with activation of transcription factor AP-...

Identification of Immune Responses Against Aldehyde-Modified Peptide Sequences in ApoB Associated With Cardiovascular Disease

Atherosclerosis develops as a result of a chronic arterial inflammation and intimal fibrosis. The disease represents in many respects a vascular repair process activated in response to injury caused by toxic breakdown products of aggregated and oxidized lipoproteins. The initial response of the artery involves expression of adhesion molecules and recruitment of leukocytes. Degenerated lipoproteins are removed from the extracellular space by macrophages. If lipoproteins continue to accumulate, the inflammatory process becomes chronic and cytokines stimulate smooth muscle to migrate into the intima. These cells proliferate and form an atherosclerotic plaque. Plaque cell death and inflammation in response to oxidized lipids and other toxic factors may cause plaques to rupture.Objective—Atherosclerosis is associated with an immune response against oxidized LDL, which modulates the progression of the disease process. Methods and Results—Using a library of polypeptides covering the complete sequence of apoB-100, the only major protein of LDL, we have identified over 100 different human antibodies reacting against aldehyde-modified apoB-100 sequences. IgM antibody titer levels decreased with age and were associated with the intima-media thickness of the carotid artery in subjects younger than 60 years. There were also inverse associations between IgM levels and oxidized LDL in plasma. In prospective clinical studies, antibody levels against several aldehyde-modified apoB-100 sites were associated with cardiovascular disease in this age group. Whether this immune response is adaptive (protective) or maladaptive (causal) in atherosclerosis requires further investigation. Conclusions—We have characterized a large number of epitopes within the apoB-100 component of oxidized LDL that provoke an immune response in humans. These findings will make it possible to study the role of immune responses against specific sites in oxidized LDL and to determine whether these immune responses influence the risk for future cardiac events.

7β-Hydroxycholesterol induces Ca2+ oscillations, MAP kinase activation and apoptosis in human aortic smooth muscle cells

Abstract In the present study, we characterize the early cytotoxic effects of 7β-hydroxycholesterol, a major cytotoxin in oxidized LDL, in human aortic smooth muscle cells. Within a few minutes after addition, 7β-hydroxycholesterol induced Ca 2+ oscillations with a frequency of ≈0.3–0.4 min −1 . A few hours later, thapsigargin-sensitive Ca 2+ pools were depleted, indicating that 7β-hydroxycholesterol perturbs intracellular Ca 2+ homeostasis. The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (but not JNK) were activated within 5 min after addition of 7β-hydroxycholesterol. The side-chain hydroxylated oxysterols 25-hydroxycholesterol and 27-hydroxycholesterol were more potent in inducing apoptosis than 7β-hydroxycholesterol and cholesterol-5α,6α-epoxide, as determined by TUNEL staining. Addition of TNFα (10 ng/ml) and IFNγ (20 ng/ml) enhanced the cytotoxicity of oxysterols and potentiated apoptosis. The cytokines alone were not toxic to smooth muscle cells at these concentrations. 25-Hydroxycholesterol and 7β-hydroxycholesterol but not cholesterol inhibited protein synthesis at 4–8 h as determined by [ 35 S]methionine incorporation assay. Morphologically, oxysterol-induced cell death was characterized by disorganization of the ER and Golgi membranes. The Ca 2+ and ERK signals preceded the ultrastructural changes induced by 7β-hydroxycholesterol.

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.

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 ≥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.

Oxidized LDL and lysophosphatidylcholine stimulate plasminogen activator inhibitor-1 expression in vascular smooth muscle cells

Plasminogen activator inhibitor-1 (PAI-1) functions as an important regulator of fibrinolysis by inhibiting both tissue-type and urokinase-type plasminogen activator. PAI-1 is produced by smooth muscle cells (SMCs) in atherosclerotic arteries, but the mechanisms responsible for induction of PAI-1 in SMCs are less well understood. In cultured human aortic SMCs, PAI-1 mRNA expression and protein secretion were increased after incubation with oxidized low-density lipoprotein (LDL) and the lipid peroxidation product lysophosphatidylcholine, whereas the effects of native LDL on PAI-1 production and release were more variable and did not reach statistical significance. The effect of LDL on arterial expression of PAI-1 in vivo was also studied in an animal model. Intravenous injection of human LDL in Sprague-Dawley rats resulted in accumulation of apolipoprotein B in the aorta within 12 hours as assessed by immunohistochemical testing. Epitopes specific for oxidized LDL began to develop in the aorta 12 hours after injection of LDL and peaked at 24 hours; this peak was accompanied by intense expression of PAI-1 immunoreactivity in the media. Also, increased aortic expression of PAI-1 mRNA after LDL injection was detected by using in situ hybridization. The transcription factor activator protein-1, which is known to bind to the promoter of the PAI-1 gene, was activated in the aortic wall 24 hours after LDL injection as assessed by electrophoretic mobility shift assay. Pretreatment of LDL with the antioxidant probucol decreased expression of oxidized LDL and PAI-1 immunoreactivity and activator protein-1 induction in the aorta but did not affect expression of apolipoprotein B immunoreactivity. These findings demonstrate that LDL oxidation enhances secretion of PAI-1 from cultured SMCs and that a similar mechanism may be involved in vascular expression of PAI-1.

Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells

Oxidized low-density lipoprotein (oxLDL) is known to induce apoptosis in endothelial cells, and this is believed to contribute to the progression of atherosclerosis. In the present study we made the novel observation that oxLDL-induced death of HMEC-1 cells is accompanied by activation of calpain. The mu-calpain inhibitor PD 151746 decreased oxLDL-induced cytotoxicity, whereas the general caspase inhibitor BAF (t-butoxycarbonyl-Asp-methoxyfluoromethylketone) had no effect. Also, oxLDL provoked calpain-dependent proteolysis of cytoskeletal alpha-fodrin in the HMEC-1 cells. Our observation of an autoproteolytic cleavage of the 80 kDa subunit of mu-calpain provided further evidence for an oxLDL-induced stimulation of calpain activity. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B and caspases. Treating the HMEC-1 cells with oxLDL did not result in detectable activation of procaspase 3 or cleavage of PARP [poly(ADP-ribose) polymerase], but it did cause polyubiquitination of caspase 3, indicating inactivation and possible degradation of this protease. Despite the lack of caspase 3 activation, oxLDL treatment led to the formation of nucleosomal DNA fragments characteristic of apoptosis. These novel results show that oxLDL initiates a calpain-mediated death-signalling pathway in endothelial cells.