Vimentin is required for normal accumulation of body fat.

Intermediate filaments (nanofilaments) have many functions, especially in response to cellular stress. Mice lacking vimentin (Vim-/-) display phenotypes reflecting reduced levels of cell activation and ability to counteract stress, for example, decreased reactivity of astrocytes after neurotrauma, decreased migration of astrocytes and fibroblasts, attenuated inflammation and fibrosis in lung injury, delayed wound healing, impaired vascular adaptation to nephrectomy, impaired transendothelial migration of lymphocytes and attenuated atherosclerosis. To address the role of vimentin in fat accumulation, we assessed the body weight and fat by dual-energy X-ray absorptiometry (DEXA) in Vim-/- and matched wildtype (WT) mice. While the weight of 1.5-month-old Vim-/- and WT mice was comparable, Vim-/- mice showed decreased body weight at 3.5, 5.5 and 8.5 months (males by 19-22%, females by 18-29%). At 8.5 months, Vim-/- males and females had less body fat compared to WT mice (a decrease by 24%, p < 0.05, and 33%, p < 0.0001, respectively). The body mass index in 8.5 months old Vim-/- mice was lower in males (6.8 vs. 7.8, p < 0.005) and females (6.0 vs. 7.7, p < 0.0001) despite the slightly lower body length of Vim-/- mice. Increased mortality was observed in adult Vim-/- males. We conclude that vimentin is required for the normal accumulation of body fat.

Effects of doxorubicin on myocardial expression of apolipoprotein-B.

OBJECTIVE: Doxorubicin (DOX) is an effective antitumour agent against a variety of human malignancies but is associated with deleterious side effects, including myocardial damage and heart failure. Myocardial apoB-containing lipoprotein (apoB) is upregulated post myocardial infarction and has been shown to be cardioprotective in this setting by unloading excessive lipid. The aim of this study was to investigate whether apoB expression is increased also in DOX-induced heart failure and whether apoB overexpression protects the heart in DOX-induced myocardial injury. DESIGN: Cardiac function and energy metabolism was studied in mice and rats 24 hours after intraperitoneally administered DOX. RESULTS: We found that the content of apoB was decreased in rat myocardium 24 hours after DOX injection. In contrast, apoB content was increased in the infarcted myocardium of rats 24 hours post ischemia-reperfusion. Moreover, transgenic mice overexpressing apoB had better cardiac function and lower intracellular lipid accumulation compared to wild type mice 24 hours post DOX. CONCLUSIONS: Our findings indicate that depression of the myocardial apoB system may contribute to DOX-induced cardiac injury and that overexpression of apoB is protective, not only in ischemically damaged myocardium, but also in DOX-induced heart failure.

Macrophages exposed to hypoxia secrete proteoglycans for which LDL has higher affinity.

Macrophages are prominent in hypoxic areas of atherosclerotic lesions. Their secreted proteoglycans (PG) can modulate the retention of lipoproteins as well as the activity of enzymes, cytokines, and growth factors involved in atherogenesis. Versican appears to be one of the main extracellular matrix components binding LDL in the arterial intima. We have recently shown that hypoxia increases versican and perlecan expression in macrophages, and that this increase was regulated by the hypoxia inducible factor (HIF). Here we report effects of hypoxia on human monocyte-derived macrophage (HMDM) secreted glycosaminoglycans (GAG), and its interaction with LDL. After 24 h exposure to 0.5% O2 (hypoxia), metabolically labeled GAG of secreted PG had higher affinity for LDL compared to 21% O2 (control cells). GAG secreted by HMDM in hypoxia were found to be more sulfated and longer which might be responsible for the increased affinity of LDL for these GAG chains. These results indicate that hypoxia induced changes in macrophage GAG biosynthesis have important consequences for the interaction with LDL. If present in vivo, an augmented association of GAG with LDL might contribute to the development of atherosclerosis in hypoxic intima.

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.

Overexpression of apolipoprotein-B improves cardiac function and increases survival in mice with myocardial infarction.

BACKGROUND: The heart produces apolipoprotein-B containing lipoproteins (apoB) whose function is not well understood. The aim of this study was to evaluate importance of myocardial apoB for cardiac function, structure and survival in myocardial infarction (MI) and heart failure (HF). METHODS AND RESULTS: MI was induced in mice (n=137) and myocardial apoB content was measured at 30 min, 3, 6, 24, 48, 120 h and 8 weeks post-MI. Transgenic mice overexpressing apoB (n=27) and genetically matched controls (n=27) were used to study the effects of myocardial apoB on cardiac function, remodeling, arrhythmias and survival after MI. Echocardiography was performed at rest and stress conditions at baseline, 2, 4 and 6 week post-MI and cumulative survival rate was registered. The myocardial apoB content increased both in the injured and the remote myocardium (p<0.05) in response to ischemic injury. ApoB mice had 2-fold higher survival rate (p<0.05) and better systolic function (p<0.05) post-MI. CONCLUSION: Overexpression of apoB in the heart increases survival and improves cardiac function after acute MI. Myocardial apoB may be an important cardioprotective system in settings such as myocardial ischemia and HF.

Hypoxia increases macrophage motility, possibly by decreasing the heparan sulfate proteoglycan biosynthesis.

Macrophages are recruited and retained in hypoxic sites in atherosclerotic lesions and tumors. Furthermore, macrophages are suggested to be a major source of HSPG synthesis in atherosclerotic lesions. HSPG are, among other things, known to regulate cell motility, cell adhesion, and receptor interaction. The aim of this study was to investigate the effect of hypoxia on HSPG expression and macrophage motility. We also explored the potential regulation of HSPG by the transcription factor HIF-1alpha. The nondirected cell motility was increased in HMDM after 24 h exposure to hypoxia (0.5% O(2)) compared with normal cell culture condition (21% O(2)). Enzymatic degradation of HS GAG further increased the motility of the HMDM in hypoxia, indicating a role of reduced cell-associated HSPG in the increased HMDM motility. HMDM exposed to 24 h of hypoxia had lower mRNA expressions of syndecan-1 and -4 compared with cells exposed to normal cell culture conditions. Protein levels of syndecan-1 were also decreased significantly in response to hypoxia, and cells subjected to hypoxia had lower mRNA expression for key enzymes involved in HS biosynthesis. In addition, hypoxia was found to reduce the relative content of HS GAG. Transfecting THP-1 cells with siHIF-1alpha indicated that this transcription factor was not involved in the hypoxia-induced modifications of HSPG expression. Given the documented multiple functions of HSPG in macrophage behavior, the hypoxia-induced modifications of HSPG may be of relevance for the development of atherosclerotic lesions and tumor progression.

Adipocyte differentiation-related protein promotes fatty acid storage in cytosolic triglycerides and inhibits secretion of very low-density lipoproteins.

OBJECTIVE: We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes. METHODS AND RESULTS: An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into beta-oxidation. CONCLUSIONS: ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to beta-oxidation and promotes the secretion of apoB-48 VLDL1.

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL.

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.

Relation of the size and intracellular sorting of apoB to the formation of VLDL 1 and VLDL 2.

In this study, we tested the hypothesis that two separate pathways, the two-step process and an apolipoprotein B (apoB) size-dependent lipidation process, give rise to different lipoproteins. Expression of apoB-100 and C-terminally truncated forms of apoB-100 in McA-RH7777 cells demonstrated that VLDL particles can be assembled by apoB size-dependent linear lipidation, resulting in particles whose density is inversely related to the size of apoB. This lipidation results in a LDL-VLDL 2 particle containing apoB-100. VLDL 1 is assembled by the two-step process by apoB-48 and larger forms of apoB but not to any significant amount by apoB-41. The major amount of intracellular apoB-80 and apoB-100 banded with a mean density of 1.10 g/ml. Its formation was dependent on the sequence between apoB-72 and apoB-90. This dense particle, which is retained in the cell, possibly by chaperones or association with the microsomal membrane, is a precursor of secreted VLDL 1. The intracellular LDL-VLDL 2 particles formed during size-dependent lipidation appear to be the precursors of intracellular VLDL 1. We propose that the dense apoB-100 intracellular particle is converted to LDL-VLDL 2 by size-dependent lipidation. LDL-VLDL 2 is secreted or converted to VLDL 1 by the uptake of the major amount of triglycerides.