Dick Wågsäter

Adipose tissue inflammation and increased ceramide content characterize subjects with high liver fat content independent of obesity.

OBJECTIVE— We sought to determine whether adipose tissue is inflamed in individuals with increased liver fat (LFAT) independently of obesity. RESEARCH DESIGN AND METHODS— A total of 20 nondiabetic, healthy, obese women were divided into normal and high LFAT groups based on their median LFAT level (2.3 ± 0.3 vs. 14.4 ± 2.9%). Surgical subcutaneous adipose tissue biopsies were studied using quantitative PCR, immunohistochemistry, and a lipidomics approach to search for putative mediators of insulin resistance and inflammation. The groups were matched for age and BMI. The high LFAT group had increased insulin (P = 0.0025) and lower HDL cholesterol (P = 0.02) concentrations. RESULTS— Expression levels of the macrophage marker CD68, the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α, and plasminogen activator inhibitor-1 were significantly increased, and those of peroxisome proliferator–activated receptor-γ and adiponectin decreased in the high LFAT group. CD68 expression correlated with the number of macrophages and crown-like structures (multiple macrophages fused around dead adipocytes). Concentrations of 154 lipid species in adipose tissue revealed several differences between the groups, with the most striking being increased concentrations of triacylglycerols, particularly long chain, and ceramides, specifically Cer(d18:1/24:1) (P = 0.01), in the high LFAT group. Expression of sphingomyelinases SMPD1 and SMPD3 were also significantly increased in the high compared with normal LFAT group. CONCLUSIONS— Adipose tissue is infiltrated with macrophages, and its content of long-chain triacylglycerols and ceramides is increased in subjects with increased LFAT compared with equally obese subjects with normal LFAT content. Ceramides or their metabolites could contribute to adverse effects of long-chain fatty acids on insulin resistance and inflammation.

CXCL16/SR-PSOX is an interferon-gamma-regulated chemokine and scavenger receptor expressed in atherosclerotic lesions

Objective—Atherosclerosis is an inflammatory disease. Several chemokines are important for monocyte/macrophage and T-cell recruitment to the lesion. CXCL16 is a recently discovered chemokine that is expressed in soluble and transmembrane forms, ligates CXCR6 chemokine receptor, and guides migration of activated Th1 and Tc1 cells. It is identical to scavenger receptor SR-PSOX, which mediates uptake of oxidized low-density lipoprotein. We investigated whether CXCL16 expression is controlled by interferon-&ggr; (IFN-&ggr;)-cytokine abundant in atherosclerotic lesions. Methods and Results—CXCL16 and CXCR6 expression was identified by polymerase chain reaction and histochemistry in atherosclerotic lesions from humans and apolipoprotein-E–deficient mice. In vitro IFN-&ggr; induced CXCL16 in human monocytic THP-1 cells and primary human monocytes, which led to increased uptake of oxidized low-density lipoprotein in THP-1 cells, which could be blocked by peptide antibodies against CXCL16. In vivo IFN-&ggr; induced CXCL16 expression in murine atherosclerotic lesions. Conclusions—We demonstrate a novel role of IFN-&ggr; in foam cell formation through upregulation of CXCL16/SR-PSOX. CXCR6 expression in the plaque confirms the presence of cells able to respond to CXCL16. Therefore, this chemokine/scavenger receptor could serve as a molecular link between lipid metabolism and immune activity in the atherosclerotic lesion.

CD137 Is Expressed in Human Atherosclerosis and Promotes Development of Plaque Inflammation in Hypercholesterolemic Mice

Background— Atherosclerosis is a multifactorial disease in which inflammatory processes play an important role. Inflammation underlies lesion evolution at all stages, from establishment to plaque rupture and thrombosis. Costimulatory molecules of the tumor necrosis factor superfamily such as CD40/CD40L and OX40/OX40L have been implicated in atherosclerosis. Methods and Results— This study shows that the tumor necrosis factor superfamily members CD137 and CD137 ligand (CD137L), which play a major role in several autoimmune diseases, may constitute a pathogenic pair in atherogenesis. We detected CD137 protein in human atherosclerotic lesions not only on T cells but also on endothelial cells and showed that CD137 in cultured endothelial cells and smooth muscle cells was induced by proinflammatory cytokines implicated in atherosclerosis. Activation of CD137 by CD137L induced adhesion molecule expression on endothelial cells and reduced smooth muscle cell proliferation. In addition, treatment of atherosclerosis-prone apolipoprotein E–deficient mice with a CD137 agonist caused increased inflammation. T-cell infiltration, mainly of CD8+ cells, and expression of the murine major histocompatibility complex class II molecule I-Ab increased significantly in atherosclerotic lesions, as did the aortic expression of proinflammatory cytokines. Conclusions— Taken together, these observations suggest that CD137-CD137L interactions in the vasculature may contribute to the progression of atherosclerosis via augmented leukocyte recruitment, increased inflammation, and development of a more disease-prone phenotype.

Endurance exercise activates matrix metalloproteinases in human skeletal muscle

In the present study, the effect of exercise training on the expression and activity of matrix metalloproteinases (MMPs) in the human skeletal muscle was investigated. Ten subjects exercised one leg for 45 min with restricted blood flow and then exercised the other leg at the same absolute workload with unrestricted blood flow. The exercises were conducted four times per week for 5 wk. Biopsies were taken from the vastus lateralis muscles of both legs at rest before the training period, after 10 days and 5 wk of training, and 2 h after the first exercise bout for analysis of MMP and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA, enzyme activity, and protein expression. Levels of MMP-2, MMP-14, and TIMP-1 mRNA in muscle tissue increased after 10 days of training regardless of blood flow condition. MMP-2 mRNA level in laser-dissected myofibers and MMP-2 activity in whole muscle increased with training. The level of MMP-9 mRNA and activity increased after the first bout of exercise. Although MMP-9 mRNA levels appeared to be very low, the activity of MMP-9 after a single bout of exercise was similar to that of MMP-2 after 10 days of exercise. MMP-2 and MMP-9 protein was both present throughout the extracellular matrix of the muscle, both around fibers and capillaries, but MMP-2 was also present within the skeletal muscle fibers. These results show that MMPs are activated in skeletal muscle in nonpathological conditions such as voluntary exercise. The expression and time pattern indicate differences between the MMPs in regards of production sites as well as in the regulating mechanism.

Effects of PDGF-C and PDGF-D on monocyte migration and MMP-2 and MMP-9 expression

BACKGROUND AND AIMS Atherosclerosis is a chronic inflammatory process involving the activity of several cytokines and growth factors. Platelet-derived growth factor-A (PDGF-A) and PDGF-B are important mitogens and chemoattractants for monocytes as well as smooth muscle cells. We sought to identify the role of PDGF-C and PDGF-D, two new members of the PDGF family, in monocyte migration and differentiation. We also assessed their effects in regulating matrix metalloproteinase-2 (MMP-2) and MMP-9, which are important for cell migration. METHODS AND RESULTS PDGF-C and PDGF-D were expressed in macrophages, smooth muscle cells, and endothelial cells in human atherosclerotic plaques, as shown by immunohistochemical analysis. PDGF-C and PDGF-D mRNA and protein expression was induced after differentiation of THP-1 monocytes to macrophages, and both PDGF-C and PDGF-D induced MMP-9 mRNA expression in a concentration-dependent manner. Treatment of cells with PDGF-C or PDGF-D enhanced the secretion of MMP-2 and MMP-9 in a cell-dependent manner. In a migration assay using a Boyden chamber with 8 microm pore size, PDGF-C and PDGF-D attracted THP-1 monocytes in a concentration-dependent manner. CONCLUSIONS Our data suggest that PDGF-C and PDGF-D, like PDGF-A and PDGF-B, play important roles in atherosclerosis by stimulating MMP activity and influencing monocyte migration.

Unraveling divergent gene expression profiles in bicuspid and tricuspid aortic valve patients with thoracic aortic dilatation: the ASAP study.

Thoracic aortic aneurysm (TAA) is a common complication in patients with a bicuspid aortic valve (BAV), the most frequent congenital heart disorder. For unknown reasons TAA occurs at a younger age, with a higher frequency in BAV patients than in patients with a tricuspid aortic valve (TAV), resulting in an increased risk for aortic dissection and rupture. To investigate the increased TAA incidence in BAV patients, we obtained tissue biopsy samples from nondilated and dilated aortas of 131 BAV and TAV patients. Global gene expression profiles were analyzed from controls and from aortic intima-media and adventitia of patients (in total 345 samples). Of the genes found to be differentially expressed with dilation, only a few (<4%) were differentially expressed in both BAV and TAV patients. With the use of gene set enrichment analysis, the cell adhesion and extracellular region gene ontology sets were identified as common features of TAA in both BAV and TAV patients. Immune response genes were observed to be particularly overexpressed in the aortic media of dilated TAV samples. The divergent gene expression profiles indicate that there are fundamental differences in TAA etiology in BAV and TAV patients. Immune response activation solely in the aortic media of TAV patients suggests that inflammation is involved in TAA formation in TAV but not in BAV patients. Conversely, genes were identified that were only differentially expressed with dilation in BAV patients. The result has bearing on future clinical studies in which separate analysis of BAV and TAV patients is recommended.

Increased expression of leukotriene C4 synthase and predominant formation of cysteinyl-leukotrienes in human abdominal aortic aneurysm

Leukotrienes (LTs) are arachidonic acid-derived lipid mediators involved in the pathogenesis and progression of diverse inflammatory disorders. The cysteinyl-leukotrienes LTC4, LTD4, and LTE4 are important mediators of asthma, and LTB4 has recently been implicated in atherosclerosis. Here we report that mRNA levels for the three key enzymes/proteins in the biosynthesis of cysteinyl-leukotrienes, 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and LTC4 synthase (LTC4S), are significantly increased in the wall of human abdominal aortic aneurysms (AAAs). In contrast, mRNA levels of LTA4 hydrolase, the enzyme responsible for the biosynthesis of LTB4, are not increased. Immunohistochemical staining of AAA wall revealed focal expression of 5-LO, FLAP, and LTC4S proteins in the media and adventitia, localized in areas rich in inflammatory cells, including macrophages, neutrophils, and mast cells. Human AAA wall tissue converts arachidonic acid and the unstable epoxide LTA4 into significant amounts of cysteinyl-leukotrienes and to a lesser extent LTB4. Furthermore, challenge of AAA wall tissue with exogenous LTD4 increases the release of matrix metalloproteinase (MMP) 2 and 9, and selective inhibition of the CysLT1 receptor by montelukast blocks this effect. The increased expression of LTC4S, together with the predominant formation of cysteinyl-leukotrienes and effects on MMPs production, suggests a mechanism by which LTs may promote matrix degradation in the AAA wall and identify the components of the cysteinyl-leukotriene pathway as potential targets for prevention and treatment of AAA.

MMP-2 and MMP-9 are prominent matrix metalloproteinases during atherosclerosis development in the Ldlr-/-Apob100/100 mouse

Matrix-degrading proteases capable of degrading components of the extracellular matrix may play an important role in development and progression of atherosclerotic lesions. In the present study, we used the Ldlr(-/-)Apob(100/100) mouse model, which has a plasma lipoprotein profile similar to that of humans with atherosclerosis, to study the expression of matrix metalloproteinases (MMPs) during early stages of atherosclerosis development. We analyzed the expression of 11 proteases and three protease inhibitors in 5- to 40-week-old Ldlr(-/-)Apob(100/100) mice. Expression and activity of MMP-2 and MMP-9 was increased in advanced atherosclerotic lesions followed by macrophage infiltration as shown by real-time PCR, gel-based and in situ zymography and immunohistochemistry. Expression of other investigated MMPs did not increase during disease progression. However, the mRNA expression of MMP-8 and MMP-13 was down-regulated, which could explain the relatively high amount of collagen observed in the vessels in this model. In conclusion, low proteolytic expression at early stages of atherogenesis and a limited repertoire of proteolytic enzymes were associated with the progression of atherosclerosis in Ldlr(-/-)Apob(100/100) mice. The study suggests that MMP-2 and MMP-9 are the main proteases involved in atherogenesis in this mouse model.

The Antiviral Cytomegalovirus Inducible Gene 5/Viperin Is Expressed in Atherosclerosis and Regulated by Proinflammatory Agents

Objective—Inflammatory processes play an important role in atherosclerosis, and increasing evidence implies that microbial pathogens and proinflammatory cytokines are involved in the development and activation of atherosclerotic lesions. To find new inflammatory genes, we explored the vascular transcriptional response to an activator of innate immunity bacterial lipopolysaccharides (LPSs). Methods and Results—Gene arrays identified the cytomegalovirus-inducible gene 5 (cig5)/viperin among the genes most potently induced by LPS in human vascular biopsies. Viperin was expressed by endothelial cells in atherosclerotic arteries and significantly elevated in atherosclerotic compared with normal arteries. In culture, cytomegalovirus infection, interferon-γ, and LPS induced viperin expression. Conclusion—Viperin is expressed in atherosclerosis and induced in vascular cells by inflammatory stimuli and cytomegalovirus infection. The putative functions of viperin in atherosclerosis may relate to disease-associated microbes.

Gene Expression and Immunohistochemistry in Adipose Tissue of HIV Type 1–Infected Patients with Nucleoside Analogue Reverse-Transcriptase Inhibitor–Associated Lipoatrophy

BACKGROUND Long-term use of both zidovudine (AZT) and stavudine (d4T) is associated with lipoatrophy, but it occurs possibly through different mechanisms. METHODS Surgical biopsy specimens of subcutaneous adipose tissue were obtained from 18 human immunodeficiency virus type 1 (HIV-1)-infected lipoatrophic patients (the LA+ group) who were treated with either zidovudine (the AZT+LA+ group; n = 10) or stavudine (the d4T+LA+ group; n = 8) and from 10 nonlipoatrophic HIV-1-infected patients (the LA- group) who received antiretroviral therapy. Mitochondrial DNA (mtDNA) copy numbers, gene expression, and immunohistochemistry data were analyzed. RESULTS mtDNA copy numbers were significantly reduced in the LA+ group, compared with the LA- group, and in the d4T+LA+ group, compared with the AZT+LA+ group. The ratio of mtDNA-encoded cytochrome COX3 to nuclear DNA-encoded COX4 expression was significantly lower in the LA+ group than in the LA- group. Compared with the LA- group, the LA+ group had significantly lower expression of genes involved in adipogenesis (SREBP1c and CEBPB), lipid (fatty acid synthase), and glucose (GLUT4) metabolism. Expression of genes involved in mitochondrial biogenesis (PGC1B), apoptosis (FAS), inflammation (IL1B), oxidative stress (PCNA and SOD1), and lamin B was significantly higher in the LA+ group than in the LA- group. The d4T+LA+ group had significantly lower expression of genes involved in mitochondrial biogenesis (POLG1), energy metabolism (the COX3/COX4 ratio), adipogenesis (SREBP1c and CEBPA), perilipin, and hexokinase than did the AZT+LA+ group. There were 7-fold more macrophages in adipose tissue specimens obtained from patients in the LA+ group, compared with the LA- group. CONCLUSIONS Lipoatrophy is characterized by mtDNA depletion, inflammation, and signs of apoptosis. Changes were more profound in the d4T+LA+ group than in the AZT+LA+ group.