Cholesterol accumulation regulates expression of macrophage proteins implicated in proteolysis and complement activation.

OBJECTIVE: Cholesterol accumulation by macrophages plays a key role in atherogenesis. To begin to develop a global picture of this process, we used proteomics and transcriptomics to analyze foam cells generated with acetyl-low-density lipoprotein, a classic ligand for scavenger receptors. METHODS AND RESULTS: Tandem mass spectrometry and stringent statistical analysis revealed that foam cells differentially expressed 15 of 542 proteins (2.8%) detected in macrophage-conditioned medium. Apolipoprotein E was one of the most upregulated proteins, confirming that proteins involved in lipid metabolism are important targets for regulation by sterol accumulation. However, levels of proteins linked to complement activation and lysosomal proteolysis also changed markedly. Transcriptional analysis demonstrated that 698 of 19,700 genes (3.5%) were regulated in foam cells, including many genes important in sterol metabolism. We also found that cholesterol accumulation regulated genes implicated in complement activation but failed to affect genes linked to proteolysis and macrophage polarization. Changes in protein levels in macrophage-conditioned medium were largely independent of changes in mRNA levels. CONCLUSIONS: Loading sterol into macrophages regulates levels of complement proteins and lysosomal proteases-key players in the immune system and plaque rupture. Posttranscriptional mechanisms are likely important for controlling levels of most of the proteins detected in macrophage medium.

High-density lipoprotein suppresses the type I interferon response, a family of potent antiviral immunoregulators, in macrophages challenged with lipopolysaccharide.

BACKGROUND: High-density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation. METHODS AND RESULTS: To identify potential antiinflammatory mechanisms, we challenged macrophages with lipopolysaccharide, an inflammatory microbial ligand for Toll-like receptor 4. HDL inhibited the expression of 30 (277 of 911) of the genes normally induced by lipopolysaccharide, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by Toll-like receptor 4 and the TRAM/TRIF signaling pathway. Unexpectedly, the ability of HDL to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by Toll-like receptor 4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein A-I, the major protein of HDL. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses lipopolysaccharide, apolipoprotein A-I-deficient mice had 6-fold higher plasma levels of interferon-ß, a key regulator of the type I interferon response, than did wild-type mice. CONCLUSIONS: HDL inhibits a subset of lipopolysaccharide-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.

A hierarchical network controls protein translation during murine embryonic stem cell self-renewal and differentiation.

Stem cell differentiation involves changes in transcription, but little is known about translational control during differentiation. We comprehensively profiled gene expression during differentiation of murine embryonic stem cells (ESCs) into embryoid bodies by integrating transcriptome analysis with global assessment of ribosome loading. While protein synthesis was parsimonious during self-renewal, differentiation induced an anabolic switch, with global increases in transcript abundance, polysome content, protein synthesis, and protein content. Furthermore, 78% of transcripts showed increased ribosome loading, thereby enhancing translational efficiency. Transcripts under exclusive translational control included the transcription factor ATF5, the tumor suppressor DCC, and the beta-catenin agonist Wnt1. We show that a hierarchy of translational regulators, including mTOR, 4EBP1, and the RNA-binding proteins DAZL and GRSF1, control global and selective protein synthesis during ESC differentiation. Parsimonious translation in pluripotent state and hierarchical translational regulation during differentiation may be important quality controls for self-renewal and choice of fate in ESCs.

Combined analysis of monocyte and lymphocyte messenger RNA expression with serum protein profiles in patients with scleroderma.

OBJECTIVE: We attempted to elucidate possible pathogenetic mechanisms in scleroderma by analysis of gene expression patterns of purified monocytes and lymphocytes, as well as protein profiles of cytokines and growth factors. METHODS: Expression analysis was performed on messenger RNA (mRNA) from cells that had been purified with magnetic beads. Plasma samples from the same patients were used for multiplex cytokine analysis. Potential sources of proteins were also examined by in situ hybridization of skin specimens. RESULTS: A total of 1,800 genes from monocytes and 863 genes from CD4+ T cells were differentially expressed in scleroderma patients. As observed by other investigators using unfractionated peripheral blood cells from patients with autoimmune connective tissue diseases, the cell type-specific analyses of our scleroderma samples showed expression of genes suggesting the presence of interferon-alpha (IFNalpha), despite the apparent absence of this cytokine in plasma. IFNalpha RNA was, however, expressed at enhanced levels in vascular and perivascular cells in scleroderma skin samples. While levels of interleukin-1alpha (IL-1alpha) and IL-16 were among 10 proteins found to be significantly elevated in scleroderma patients, none of the large panel of plasma cytokines we analyzed correlated with the expression levels of putative IFN response genes. CONCLUSION: The pattern of up-regulation of mRNA in both the monocytes and CD4 lymphocytes of scleroderma patients, together with the detection of IFNalpha RNA in the microvasculature, suggests that leukocytes respond to this cytokine locally in the vessels. Detection of high levels of IL-1alpha and IL-16 in plasma and the independence of these protein levels from the IFN signature, implicates an independent contribution of other cytokines to immune activation and/or inflammation in scleroderma.

Capillary regeneration in scleroderma: stem cell therapy reverses phenotype?

BACKGROUND: Scleroderma is an autoimmune disease with a characteristic vascular pathology. The vasculopathy associated with scleroderma is one of the major contributors to the clinical manifestations of the disease. METHODOLOGY/PRINCIPAL FINDINGS: We used immunohistochemical and mRNA in situ hybridization techniques to characterize this vasculopathy and showed with morphometry that scleroderma has true capillary rarefaction. We compared skin biopsies from 23 scleroderma patients and 24 normal controls and 7 scleroderma patients who had undergone high dose immunosuppressive therapy followed by autologous hematopoietic cell transplant. Along with the loss of capillaries there was a dramatic change in endothelial phenotype in the residual vessels. The molecules defining this phenotype are: vascular endothelial cadherin, a supposedly universal endothelial marker required for tube formation (lost in the scleroderma tissue), antiangiogenic interferon alpha (overexpressed in the scleroderma dermis) and RGS5, a signaling molecule whose expression coincides with the end of branching morphogenesis during development and tumor angiogenesis (also overexpressed in scleroderma skin. Following high dose immunosuppressive therapy, patients experienced clinical improvement and 5 of the 7 patients with scleroderma had increased capillary counts. It was also observed in the same 5 patients, that the interferon alpha and vascular endothelial cadherin had returned to normal as other clinical signs in the skin regressed, and in all 7 patients, RGS5 had returned to normal. CONCLUSION/SIGNIFICANCE: These data provide the first objective evidence for loss of vessels in scleroderma and show that this phenomenon is reversible. Coordinate changes in expression of three molecules already implicated in angiogenesis or anti-angiogenesis suggest that control of expression of these three molecules may be the underlying mechanism for at least the vascular component of this disease. Since rarefaction has been little studied, these data may have implications for other diseases characterized by loss of capillaries including hypertension, congestive heart failure and scar formation.

cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages.

We report the systematic use of large-scale cDNA microarrays to study the gene expression profiles of primary human peripheral blood monocytes (MONO) in comparison with in vitro-differentiated, M-CSF-induced MONO-derived macrophages (MAC) and primary human alveolar MAC (AM), obtained by bronchoalveolar lavage from the lungs of normal volunteers. These studies revealed large-scale differences in the gene expression profile between both MAC types (MAC and AM) and MONO. In addition, large differences were observed in the gene expression profiles of the two MAC types. Specifically, 21% of genes on the array (2904 out of 13,582) were differentially expressed between AM and MONO, and 2229 out of 13,583 probes were differentially expressed between MAC and AM. Our expression data show remarkable differences in gene expression between different MAC subpopulations and emphasize the heterogeneity of different MAC populations. This study underscores the need to scrutinize models of MAC biology for relevance to specific disease processes.

TagSNP evaluation for the association of 42 inflammation loci and vascular disease: evidence of IL6, FGB, ALOX5, NFKBIA, and IL4R loci effects.

Inflammatory markers have consistently been associated with vascular disease. Evidence of genetic polymorphisms in inflammatory loci that predict severe carotid artery disease (CAAD) would suggest that this relationship is not secondary to other correlated factors, but related to inflammation itself. We examined the full common genetic variation in 42 inflammatory loci for prediction of severe CAAD versus ultrasound proven controls using a tagSNP approach. For selected loci, monocyte RNA levels were contrasted in subjects with and without CAAD. We confirm the association of IL6(-174), FGB (-455), and ALOX5 with CAAD and show that multiple ALOX5 SNPs independently predict CAAD. We provide evidence for previously unreported associations of SNPs in IL4R, NFKBIA, and PLG with CAAD, and weaker evidence for associations with CSF3, IL10RA, and VCAM1. The NFKBIA and IL10RA expression levels significantly differed between subjects with CAAD and controls. These results support a role for genetic variation related to inflammation in CAAD and a causal role for specific gene products.

Myeloperoxidase generates 5-chlorouracil in human atherosclerotic tissue: a potential pathway for somatic mutagenesis by macrophages.

Somatic mutations induced by oxidative damage of DNA might play important roles in atherogenesis. However, the underlying mechanisms remain poorly understood. Myeloperoxidase, a heme protein expressed by select populations of artery wall macrophages, initiates one potentially mutagenic pathway by generating hypochlorous acid. This potent chlorinating agent reacts rapidly with primary amines to yield long-lived, selectively reactive N-chloramines. In the current studies, we demonstrate that myeloperoxidase produced by human macrophages differentiated in the presence of granulocyte macrophage colony-stimulating factor generates 5-chlorouracil, a mutagenic thymine analog. The primary amine taurine fails to block the reaction, suggesting that N-haloamines produced by macrophages might oxidize uracil. Model system studies demonstrated that N-chloramines convert uracil to 5-chlorouracil. Interestingly, the tertiary amine nicotine dramatically enhances uracil chlorination, suggesting that cigarette smoke might promote nucleobase oxidation by N-chloramines. To look for evidence that myeloperoxidase promotes uracil oxidation in vivo, we measured 5-chlorouracil levels in human aortic tissue, using isotope dilution gas chromatography-mass spectrometry. The level of 5-chlorouracil was 10-fold higher in atherosclerotic aortic tissue obtained during vascular surgery than in normal aortic tissue, suggesting that halogenated nucleobases produced by macrophages might contribute to atherogenesis. Because 5-chlorouracil can be incorporated into nuclear DNA, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis, phenotypic modulation, and cytotoxicity during atherogenesis.