Peritoneal Foam Cells Research Articles

Recombinant human proteoglycan 4 lowers inflammation and atherosclerosis susceptibility in female low-density lipoprotein receptor knockout mice.

Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin-like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti-atherogenic potential of chronic rhPRG4 treatment. Female low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet for 6 weeks and injected three times per week intraperitoneally with 0.5mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma-free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4-treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103μm2 compared with 339 ± 15 × 103μm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti-inflammatory M2-like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro-inflammatory/pro-atherogenic cytokine TNF-alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti-inflammatory agent. KEY POINTS: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility. In this study we investigated the anti-atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low-density lipoprotein receptor knockout mice. We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro-inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF-alpha levels and a lowered atherosclerosis susceptibility. Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions.

Low human and murine Mcl-1 expression leads to a pro-apoptotic plaque phenotype enriched in giant-cells

The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1fl/fl (Mcl-1−/−) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1−/− compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1−/− peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1−/− mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque.

Myeloid Kdm6b deficiency results in advanced atherosclerosis

Background and aimsAtherosclerosis is a lipid-driven chronic inflammatory disorder of the arteries, and monocytes and macrophages play a central role in this process. Within the atherosclerotic lesion, macrophages can scavenge modified lipids and become the so-called foam cells. We previously reported that the epigenetic enzyme Kdm6b (also known as Jmjd3) controls the pro-fibrotic transcriptional profile of peritoneal foam cells. Given the importance of these cells in atherosclerosis, we now studied the effect of myeloid Kdm6b on disease progression. MethodsBone marrow of myeloid Kdm6b deficient (Kdm6bdel) mice or wild type littermates (Kdm6bwt) was transplanted to lethally irradiated Ldlr−/− mice fed a high fat diet for 9 weeks to induce atherosclerosis. ResultsLesion size was similar in Kdm6bwt and Kdm6bdel transplanted mice. However, lesions of Kdm6bdel mice contained more collagen and were more necrotic. Pathway analysis on peritoneal foam cells showed that the pathway involved in leukocyte chemotaxis was most significantly upregulated. Although macrophage and neutrophil content was similar after 9 weeks of high fat diet feeding, the relative increase in collagen content and necrosis revealed that atherosclerotic lesions in Kdm6bdel mice progress faster. ConclusionMyeloid Kdm6b deficiency results in more advanced atherosclerosis.

Abstract 57: The Epigenetic Enzyme Kdm6b Controls the Pro-Fibrotic Transcriptome Signature of Foam Cells

Aim: Foam cells are a key hallmark of atherosclerotic lesion formation. Within the atherosclerotic lesion macrophages scavenge modified lipoproteins and thereby acquire their foam cell characteristics. Besides their foam cell phenotype, macrophages can have specific inflammation regulatory functions in atherosclerotic lesions. Epigenetic pathways are crucial for monocyte to macrophage differentiation and activation. The H3K27 demethylase Kdm6b (also known as Jmjd3) is regulated in response to various triggers and regulates several modes of macrophage activation. Given the crucial role of macrophage foam cells in atherosclerosis, we here studied Kdm6b in peritoneal foam cells in order to identify regulated pathways. Material and Methods: A myeloid deficient Kdm6b mice (LysMCre-Kdm6b fl/fl ) was generated and bone marrow of Kdm6b wt or Kdm6b del mice was transplanted to irradiated Ldlr -/- mice which were fed a high fat diet for 9 weeks to induce foam cell formation. Peritoneal foam cells from Kdm6b del or Kdm6b wt mice were isolated and used for RNA-sequencing analysis. Results: Among the list of downregulated genes many genes involving fibrosis were affected in Kdm6b deficient foam cells including Collagen genes ( Col1a1 , Col1a2 ), Alpha smooth muscle actin ( Acta2 ) and Fibronectin-1 ( Fn1 ). Pathway analysis on downregulated genes ( P -value < 0.05) indicated that pathways involved in epithelial to mesenchymaltransition (EMT) ( q- value=10 -13 ) and extracellular matrix organization ( q- value=10 -4 ) were significantly downregulated. Pro-fibrotic pathways were thus strongly suppressed in Kdm6b deleted foam cells. Analysis of published datasets of foam cells showed that foam cell formation induces these pro-fibrotic characteristics. Overlay of both data sets indicated that fibrotic genes which are induced upon foam cell formation, are reduced in the absence of Kdm6b. These data suggest that foam cell formation induces a pro-fibrotic gene signature in a Kdm6b-dependent manner. Conclusion: We identified Kdm6b as a novel regulator of the pro-fibrotic signature of peritoneal foam cells.

Macrophage Kdm6b controls the pro-fibrotic transcriptome signature of foam cells.

In order to identify regulators of foam cells, we studied the H3K27 demethylase Kdm6b (also known as Jmjd3), a known regulator of macrophages, in controlling the transcriptional profile of foam cells. Foam cells from Kdm6b-deleted or Kdm6b wild-type mice were isolated and used for RNA-sequencing analysis. Pathway analysis revealed that pro-fibrotic pathways were strongly suppressed in Kdm6b-deleted foam cells. Analysis of published datasets showed that foam cell formation induces these pro-fibrotic characteristics. Overlay of both datasets indicated that fibrotic genes which are induced upon foam cell formation, are reduced in the absence of Kdm6b. These data suggest that foam cell formation induces a pro-fibrotic gene signature in a Kdm6b-dependent manner. We identified Kdm6b as a novel regulator of the pro-fibrotic signature of peritoneal foam cells.

Hematopoietic arginase 1 deficiency results in decreased leukocytosis and increased foam cell formation but does not affect atherosclerosis

Background and aimsArginase1 (Arg1), an M2 macrophage marker, plays a critical role in a number of immunological functions in macrophages, which are the main cell type facilitating atherosclerotic lesion development. Arg1 uses the substrate l-arginine to create l-ornithine, a precursor molecule required for collagen formation and vascular smooth muscle cell differentiation. By reducing l-arginine availability, Arg1 limits the production of nitric oxide (NO), a pro-atherogenic factor in macrophages. In endothelial cells, conversely, NO is strongly anti-atherogenic. However, until now, the role of Arg1 in atherosclerosis is largely unknown. The aim of this study is to specifically investigate the effect of Arg1 deletion in hematopoietic cells on atherosclerosis susceptibility. MethodsLdlr KO mice were transplanted with Arg1flox/flox;Tie2-Cre (Arg1 KO) bone marrow (BM) or wildtype (WT) BM. After 8 weeks of recovery on chow diet, recipients mice were fed a Western-Type Diet (WTD) for 10 weeks to induce atherosclerosis. ResultsAfter 10-week WTD challenge, blood leukocyte counts were decreased by 25% (p < 0.001), and spleen leukocytes were decreased by 35% (p = 0.05) in Ldlr KO mice transplanted with Arg1 KO BM compared to mice transplanted with WT BM. The decrease in leukocytes was due to lower B lymphocyte counts. However, oxLDL-specific antibodies were increased in plasma of Ldlr KO mice transplanted with Arg1 KO BM compared to WT BM transplanted controls, whereas oxLDL-specific IgM was not affected. On the other hand, peritoneal foam cells in Arg1 KO BM recipients were increased 3-fold (p < 0.001) compared to WT BM recipients. No change in blood cholesterol was found. Despite changes in leukocyte counts and macrophage foam cell formation, we did not observe differences in atherosclerotic plaque size or plaque macrophage content in the aortic root. Surprisingly, there was also no difference in plaque collagen content, indicating that absence of macrophage Arg1 function does not reduce plaque stability. ConclusionsDeletion of Arg1 in hematopoietic cells adversely affects blood leukocyte counts and increases foam cell formation. However, no effects on atherosclerosis could be demonstrated, indicating that hematopoietic Arg1 function is not a decisive factor in atherosclerotic plaque formation.

Liver X receptor activation promotes polyunsaturated fatty acid synthesis in macrophages: relevance in the context of atherosclerosis.

Liver X receptors (LXRs) modulate cholesterol and fatty acid homeostasis as well as inflammation. This study aims to decipher the role of LXRs in the regulation of polyunsaturated fatty acid (PUFA) synthesis in macrophages in the context of atherosclerosis. Transcriptomic analysis in human monocytes and macrophages was used to identify putative LXR target genes among enzymes involved in PUFA biosynthesis. In parallel, the consequences of LXR activation or LXR invalidation on PUFA synthesis and distribution were determined. Finally, we investigated the impact of LXR activation on PUFA metabolism in vivo in apolipoprotein E-deficient mice. mRNA levels of acyl-CoA synthase long-chain family member 3, fatty acid desaturases 1 and 2, and fatty acid elongase 5 were significantly increased in human macrophages after LXR agonist treatment, involving both direct and sterol responsive element binding protein-1-dependent mechanisms. Subsequently, pharmacological LXR agonist increased long chain PUFA synthesis and enhanced arachidonic acid content in the phospholipids of human macrophages. Increased fatty acid desaturases 1 and 2 and acyl-CoA synthase long-chain family member 3 mRNA levels as well as increased arachidonic acid to linoleic acid and docosahexaenoic acid to eicosapentaenoic acid ratios were also found in atheroma plaque and peritoneal foam cells from LXR agonist-treated mice. By contrast, murine LXR-deficient macrophages displayed reduced expression of fatty acid elongase 5, acyl-CoA synthase long-chain family member 3 and fatty acid desaturases 1, as well as decreased cellular levels of docosahexaenoic acid and arachidonic acid. Our results indicate that LXR activation triggers PUFA synthesis in macrophages, which results in significant alterations in the macrophage lipid composition. Moreover, we demonstrate here that LXR agonist treatment modulates PUFA metabolism in atherosclerotic arteries.

(Pro)renin receptor blocker improves survival of rats with sepsis

BackgroundThe renin-angiotensin system (RAS) affects inflammatory responses during sepsis. Nonproteolytic activation of prorenin by the (pro)renin receptor has recently been shown to stimulate the tissue RAS. In the present study, the effect of (pro)renin receptor blocker (PRRB) pretreatment on sepsis in a rat cecal ligation and puncture (CLP) model was investigated. Materials and methodsMale Sprague-Dawley rats underwent CLP and were randomly divided into two groups: PRRB-treated group and control peptide–treated group. Survival was analyzed for 7 d after CLP. The serum concentrations of cytokines and high-mobility group box chromosomal protein 1 (HMGB1) were measured at three time points (0, 3, and 6 h after CLP). Hematoxylin-eosin staining and immunohistochemical staining for nonproteolytically activated prorenin and HMGB1 were performed on the cecum to assess pathologic changes found 6 h after CLP. ResultsTreatment with PRRB improved the survival rate of the post-CLP septic rats (P = 0.023). PRRB also significantly reduced serum tumor necrosis factor-α, interleukin-1β, and HMGB1 levels 6 h after CLP. In CLP rats that were treated with control peptide, the expression of activated prorenin was elevated in peritoneal foam cells. Moreover, expression of HMGB1 was increased in peritoneal inflammatory cells. In contrast, both were markedly suppressed in CLP rats that were treated with PRRB. ConclusionsPRRB significantly improved the survival rate of rats with clinically relevant sepsis, possibly by attenuating a sepsis-induced systemic inflammatory response. We propose that overactivation of the RAS by activation of prorenin in foam cells may be a significant contributor to sepsis.

11β-Hydroxysteroid Dehydrogenase Type 1 Gene Knockout Attenuates Atherosclerosis and In Vivo Foam Cell Formation in Hyperlipidemic apoE−/− Mice

BackgroundChronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11βHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated.Methodology/Principal FindingsTo examine the role of 11βHSD1 in atherogenesis, 11βHSD1 knockout mice were created on the pro-atherogenic apoE−/− background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11βHSD1−/−/apoE−/− mice vs. 11βHSD1+/+/apoE−/− mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11βHSD1−/−/apoE−/− mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11βHSD1−/−/apoE−/− mice. Bone marrow transplantation from 11βHSD1−/−/apoE−/− mice into apoE−/− recipients reduced plaque area 39–46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11βHSD1+/+/apoE−/− and 11βHSD1−/−/apoE−/− mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11βHSD1−/−/apoE−/− mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11βHSD1−/−/apoE−/− mice including TLR 1, 3 and 4. Cytokine release from 11βHSD1−/−/apoE−/−-derived peritoneal foam cells was attenuated following challenge with oxidized LDL.ConclusionsThese findings suggest that 11βHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11βHSD1 in modulating binding of pro-atherogenic TLR ligands.

Abstract 411: An Anion-Exchange Chromatography Isolated Subfraction of Mouse Apolipoprotein A-I Is Unable to Activate Cellular Cholesterol Release from Mouse Peritoneal Macrophage Foam Cells

Aim: Human and mouse plasma apoA-I separate on anion exchange chromatography (6M urea) in distinct subfractions. To investigate the nature of this diversity, we analyzed the protein subfractions by IEF and Urea-PAGE, and compared their ability to generate HDL in vitro. Methods: Human and mouse plasma HDLs were delipidated then applied to an DE52 ion-exchange column and eluted by 30-60mM Tris-HCl, pH7.80 containing 6M urea. Mouse, human, and recombinant apoA-Is were analyzed by IEF (Ettan IPGphor3) followed by CBB protein staining. Urea-PAGE analysis was performed with 14% acrylamide gel in 6M urea buffer. ApoA-Is abilities to efflux cholesterol from mouse peritoneal foam cells and to shift the size of the ACAT-accessible cholesterol pool were determined. Results: Human plasma and recombinant human apoA-I showed two apoA-I subtypes by IEF and Urea-PAGE analysis; whereas HDL from C57BL/6 and 129SV mice contained three apoA-Is at pI=5.37, 5.31, 5.27 (mP3-1, mP2, mP3-3), and pI=5.44, 5.37, 5.32, respectively. Subfractions mP2 and mP3-3 were isolated on a DE52 column and, despite their difference in pI, were able to mediate cellular cholesterol efflux at levels similar to human plasma apoA-Is. Subfraction mP3-1, with the highest pI, eluted between mP2 and mP3-3 on DE-52 column. Interestingly, for both mouse strains, mP3-1 showed very poor cholesterol efflux and the incorporation of 14C-oleic acid to cellular CE was significantly higher when compared to mP2 and mP3-3. Conclusion: We found that differences in the pI of anion-exchange chromatography apoA-I subfractions did not directly affect HDL generation except for mouse subtype mP3-1. mP3-1 binds strongly to the anionic DE52 resin, probably by hydrophobic interaction, and is unable to mediate cellular cholesterol efflux. Further investigation of the nature of the differences in these apoA-I subtypes will provide insights on factors that may be implicated in the biogenesis of HDL in vivo.