Eva Bengtsson

Inhibition of Tumor Necrosis Factor-{alpha} Reduces Atherosclerosis in Apolipoprotein E Knockout Mice.

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-&agr; (TNF-&agr;), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-&agr; in atherogenesis. Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-&agr; were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-&agr; exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation of apoEo mice with apoEotnf-&agr;o bone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. In apoE knockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018). Conclusions—These findings demonstrate that TNF-&agr; is actively involved in the progression of atherosclerosis. Accordingly, TNF-&agr; represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.

Inhibition of Tumor Necrosis Factor-α Reduces Atherosclerosis in Apolipoprotein E Knockout Mice

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-&agr; (TNF-&agr;), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-&agr; in atherogenesis. Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-&agr; were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-&agr; exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation of apoEo mice with apoEotnf-&agr;o bone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. In apoE knockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018). Conclusions—These findings demonstrate that TNF-&agr; is actively involved in the progression of atherosclerosis. Accordingly, TNF-&agr; represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.

The Primary Structure of a Basic Leucine-rich Repeat Protein, PRELP, Found in Connective Tissues

We have determined the primary structure of a connective tissue matrix protein from the nucleotide sequence of a clone isolated from a human articular chondrocyte cDNA library. The major part of the amino acid sequence has also been determined by direct protein sequencing. The translated primary sequence corresponds to 382 amino acid residues, including a 20-residue signal peptide. The molecular mass of the mature protein is 41,646 Da. The main part of the protein consists of 10 leucine-rich repeats ranging in length from 20 to 26 residues, with asparagine at position 10 (B-type). The N-terminal part is unusual in that it is basic and rich in arginine and proline. There are four potential N-linked glycosylation sites present. In three of these sites, post-translational modifications are likely to be present since Asn was not found by direct protein sequencing. The amino- and carboxyl-terminal parts contain four and two cysteine residues, respectively, probably forming disulfide bonds by analogy with the other members of this family. The protein shows highest identity (36%) to fibromodulin and 33% to bovine lumican, two other leucine-rich repeat connective tissue proteins. Northern blot analysis showed the presence of an 3.8-kilobase mRNA in different types of bovine cartilage and cultured osteoblasts, whereas RNAs isolated from bovine kidney, skin, spleen, thymus, and trabecular bone and rat calvaria were negative. Human articular chondrocyte and rat chondrosarcoma cell RNAs contained an additional mRNA of 1.6 and 1.8 kilobases, respectively.

Lack of the Cysteine Protease Inhibitor Cystatin C Promotes Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Degradation of extracellular matrix plays an important role in growth and destabilization of atherosclerotic plaques. Cystatin C, inhibitor of the collagen- and elastin-degrading cysteine proteases of the cathepsin family, is produced by virtually all cell types. It is present in the normal artery wall but severely reduced in human atherosclerotic lesions. Methods and Results—To determine the functional role of cystatin C in atherosclerosis, we crossed cystatin C–deficient (cysC−/−) mice with apolipoprotein E–deficient (apoE−/−) mice. After 25 weeks of atherogenic diet, mice lacking apoE and cystatin C (cysC−/− apoE−/−) had larger subvalvular plaques compared with cysC+/+ apoE−/− mice (766 000±20 000 &mgr;m2 per section versus 662 000±19 000 &mgr;m2 per section; P=0.001), suggesting an atheroprotective role of cystatin C. The plaques from cysC−/− apoE−/− mice were characterized by increased total macrophage content. To determine which cellular source is important for the antiatherosclerotic effect of cystatin C, we performed bone marrow transplantations. ApoE−/− mice were transplanted with either cysC−/− apoE−/− or cysC+/+ apoE−/− bone marrow. No significant differences in plaque area, macrophage, collagen, or lipid content of subvalvular lesions between the 2 groups were detected. Conclusions—The result suggests that the protective role of cystatin C in atherosclerosis is dependent primarily on its expression in nonhematopoietic cell types.

Chondroitin sulfate perlecan enhances collagen fibril formation - Implications for perlecan chondrodysplasias

Inactivation of the perlecan gene leads to perinatal lethal chondrodysplasia. The similarity to the phenotypes of the Col2A1 knock-out and the disproportionate micromelia mutation suggests perlecan involvement in cartilage collagen matrix assembly. We now present a mechanism for the defect in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional to the content of the 4,6-disulfated disaccharide in the different cartilage extracts, with growth plate cartilage glycosaminoglycan being the most efficient enhancer. These findings demonstrate a role for perlecan chondroitin sulfate side chains in cartilage extracellular matrix assembly and provide an explanation for the perlecan-null chondrodysplasia.

Inhibition of Tumor Necrosis Factor- Reduces Atherosclerosis in Apolipoprotein E Knockout Mice

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-&agr; (TNF-&agr;), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-&agr; in atherogenesis. Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-&agr; were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-&agr; exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation of apoEo mice with apoEotnf-&agr;o bone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. In apoE knockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018). Conclusions—These findings demonstrate that TNF-&agr; is actively involved in the progression of atherosclerosis. Accordingly, TNF-&agr; represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.

Plasma fibronectin deficiency impedes atherosclerosis progression and fibrous cap formation

Atherosclerotic lesions are asymmetric focal thickenings of the intima of arteries that consist of lipids, various cell types and extracellular matrix (ECM). These lesions lead to vascular occlusion representing the most common cause of death in the Western world. The main cause of vascular occlusion is rupture of atheromatous lesions followed by thrombus formation. Fibronectin (FN) is one of the earliest ECM proteins deposited at atherosclerosis‐prone sites and was suggested to promote atherosclerotic lesion formation. Here, we report that atherosclerosis‐prone apolipoprotein E‐null mice lacking hepatocyte‐derived plasma FN (pFN) fed with a pro‐atherogenic diet display dramatically reduced FN depositions at atherosclerosis‐prone areas, which results in significantly smaller and fewer atherosclerotic plaques. However, the atherosclerotic lesions from pFN‐deficient mice lacked vascular smooth muscle cells and failed to develop a fibrous cap. Thus, our results demonstrate that while FN worsens the course of atherosclerosis by increasing the atherogenic plaque area, it promotes the formation of the protective fibrous cap, which in humans prevents plaques rupture and vascular occlusion.

Atheroprotective Effects of Alum Are Associated With Capture of Oxidized LDL Antigens and Activation of Regulatory T Cells

The immune system represents a promising novel target for prevention of atherosclerosis. Several pilot vaccines that reduce atherosclerosis in experimental animals have been developed. The aluminum hydroxide adjuvant Alum has been shown to have antiatherogenic properties in itself, suggesting that it may be a suitable adjuvant in possible future atherosclerosis vaccines. To characterize the immune pathways mediating this protection, we treated wild-type C57BL/6 and Apoe−/− mice with Alum or PBS. Analyses of splenocytes isolated from 12-week-old mice demonstrated that Alum increased the presence of CD4+CD25+FoxP3+ regulatory T cells and downregulated the expression of T cell activation markers CD28 and ICOS in Apoe−/− mice but not in C57BL/6 wild-type mice. A similar immunosuppressive phenotype was found also in 25-week-old Apoe−/− mice and was associated with reduced atherosclerosis. Alum precipitates recovered from the injection site of Apoe−/− mice contained antigens derived from oxidized LDL. These findings demonstrate that treatment of Apoe−/− mice with Alum results in an increase of regulatory T cells and suggest that these are activated by tolerogenic antigen-presenting cells presenting oxidized LDL antigens. Our findings provide improved mechanistic understanding of the atheroprotective properties of aluminum hydroxide adjuvants but also point to the importance of determining if hypercholesterolemia may compromise the efficacy of Alum-containing vaccines used clinically today.

Associations between autoantibodies against apolipoprotein B-100 peptides and vascular complications in patients with type 2 diabetes

Aims/hypothesisOxidation of LDL in the arterial extracellular matrix is a key event in the development of atherosclerosis and autoantibodies against oxidised LDL antigens reflect disease severity and the risk of developing acute cardiovascular events. Since type 2 diabetes is associated with increased oxidative stress, we tested the hypothesis that autoantibodies against oxidised LDL antigens are biomarkers for vascular complications in diabetes.MethodsWe studied 497 patients with type 2 diabetes without clinical signs of coronary heart disease. Oxidised LDL autoantibodies were determined by ELISA detecting IgG and IgM specific for native and malondialdehyde (MDA)-modified apolipoprotein B-100 peptides p45 and p210. The severity of coronary disease was assessed as the coronary artery calcium score.ResultsPatients affected by retinopathy had significantly higher levels of IgG against MDA-p45 and MDA-p210. In contrast, high levels of autoantibodies against the corresponding native peptides were associated with less coronary calcification and a lower risk of progression of coronary disease.Conclusions/interpretationOur observations suggest that LDL oxidation is involved in the pathogenesis of diabetic retinopathy and that autoantibodies against apolipoprotein B peptides may act as biomarkers for both micro- and macrovascular complications in diabetes.

Elevated Plasma Levels of MMP-12 Are Associated With Atherosclerotic Burden and Symptomatic Cardiovascular Disease in Subjects With Type 2 Diabetes

Objective— Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins and play important roles in development and tissue repair. They have also been shown to have both protective and pathogenic effects in atherosclerosis, and experimental studies have suggested that MMP-12 contributes to plaque growth and destabilization. The objective of this study was to investigate the associations between circulating MMPs, atherosclerosis burden, and incidence of cardiovascular disease with a particular focus on type 2 diabetes mellitus. Approach and Results— Plasma levels of MMP-1, -3, -7, -10, and -12 were analyzed by the Proximity Extension Assay technology in 1500 subjects participating in the SUMMIT (surrogate markers for micro- and macrovascular hard end points for innovative diabetes tools) study, 384 incident coronary cases, and 409 matched controls in the Malmö Diet and Cancer study and in 205 carotid endarterectomy patients. Plasma MMP-7 and -12 were higher in subjects with type 2 diabetes mellitus, increased with age and impaired renal function, and was independently associated with prevalent cardiovascular disease, atherosclerotic burden (as assessed by carotid intima-media thickness and ankle-brachial pressure index), arterial stiffness, and plaque inflammation. Baseline MMP-7 and -12 levels were increased in Malmö Diet and Cancer subjects who had a coronary event during follow-up. Conclusions— The plasma level of MMP-7 and -12 are elevated in type 2 diabetes mellitus, associated with more severe atherosclerosis and an increased incidence of coronary events. These observations provide clinical support to previous experimental studies, demonstrating a role for these MMPs in plaque development, and suggest that they are potential biomarkers of atherosclerosis burden and cardiovascular disease risk.