Scholarly article on topic 'Hematopoietic arginase 1 deficiency results in decreased leukocytosis and increased foam cell formation but does not affect atherosclerosis'

Hematopoietic arginase 1 deficiency results in decreased leukocytosis and increased foam cell formation but does not affect atherosclerosis Academic research paper on "Biological sciences"

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{"Nitric oxide" / "Bone marrow transplantation" / B-cells / Inos / Tie2Cre / oxLDL}

Abstract of research paper on Biological sciences, author of scientific article — Baoyan Ren, Erik Van Kampen, Theo J.C. Van Berkel, Sheena M. Cruickshank, Miranda Van Eck

Abstract Background and aims Arginase1 (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. Methods Ldlr KO mice were transplanted with Arg1 flox/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. Results After 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. Conclusions Deletion 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.

Academic research paper on topic "Hematopoietic arginase 1 deficiency results in decreased leukocytosis and increased foam cell formation but does not affect atherosclerosis"

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

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Baoyan Ren a' ■Erik Van Kampen a'Theo J.C. Van Berkel a, Sheena M. Cruickshank b, Miranda Van Eck a

a Division of Biopharmaceutics, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands b Manchester Immunology Group, Faculty of Life Sciences, The University of Manchester, Manchester, UK

ARTICLE INFO

Article history:

Received 3 May 2016

Received in revised form

4 November 2016

Accepted 15 November 2016

Available online 16 November 2016

Keywords: Nitric oxide

Bone marrow transplantation

B-cells

Tie2Cre

ABSTRACT

Background and aims: Arginasel (Argl), 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. Argl 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, Argl 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 Argl in atherosclerosis is largely unknown. The aim of this study is to specifically investigate the effect of Argl deletion in hematopoietic cells on atherosclerosis susceptibility.

Methods: Ldlr KO mice were transplanted with Arg1flox/flox;Tie2-Cre (Argl 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 l0 weeks to induce atherosclerosis.

Results: After l0-week WTD challenge, blood leukocyte counts were decreased by 25% (p < 0.00l), and spleen leukocytes were decreased by 35% (p = 0.05) in Ldlr KO mice transplanted with Argl 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 Argl KO BM compared to WT BM transplanted controls, whereas oxLDL-specific IgM was not affected. On the other hand, peritoneal foam cells in Argl KO BM recipients were increased 3-fold (p < 0.00l) 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 Argl function does not reduce plaque stability. Conclusions: Deletion of Argl in hematopoietic cells adversely affects blood leukocyte counts and increases foam cell formation. However, no effects on atherosclerosis could be demonstrated, indicating that hematopoietic Argl function is not a decisive factor in atherosclerotic plaque formation. © 20l6 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-

ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Inhibition of the activity of the enzyme arginase l (Argl) is considered a promising novel therapeutic strategy for the treatment of cardiovascular disease [l]. In line, arginase inhibition by

* Corresponding author. Division of Biopharmaceutics, Leiden Academic Centre for Drug Research, Gorlaeus Laboratories EE1.13, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

E-mail address: b.ren@lacdr.leidenuniv.nl (B. Ren). 1 These authors contributed equally to this work.

N(omega)-hydroxy-nor-/-arginine (nor-NOHA) improves endothelial function in familial hypercholesterolemia patients and reduces atherosclerotic lesion development in carotid arteries of apolipo-protein E (Apoe) knockout mice exposed to low shear stress [2—4]. Argl influences a number of processes implicated in the pathogenesis of atherosclerosis [5—8]. It is expressed in endothelial cells, vascular smooth muscle cells (VSMCs) and macrophages, which are all important cellular components of the atherosclerotic plaque [1]. Depending on the cell type it is expressed in, Arg1 function is expected to exert different effects on atherosclerotic plaque formation.

http://dx.doi.org/10.1016/j.atherosclerosis.2016.11.018

0021-9150/© 2016 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The primary function of Arg1 is production of urea and L-orni-thine from L-arginine [9]. L-arginine, however, is also used as a substrate by the enzymes inducible- and endothelial nitric oxide synthase (Inos and Enos) for the production of the endothelial-protective signalling molecule nitric oxide (NO) [2,10]. By competition for the common substrate L-arginine, Arg1 can thus indirectly inhibit the synthesis of NO [11,12]. In line, endothelial Arg1 contributes to endothelial activation and vascular stiffness by reducing the L-arginine pool, leading to Enos uncoupling and reduced NO production [10,13]. This results in endothelial activation and increased recruitment of immune cells to the plaque [10,13]. However, atheroprotective effects have also been described for Arg1 in macrophages and VSMCs. By producing L-ornithine, Arg1 contributes to the synthesis of L-proline by the enzyme Ornithine Amino Transferase (OAT), which is a precursor for collagen biosynthesis. Ornithine can also be metabolised into polyamines, which leads to increased VSMC differentiation and decreased inflammation [14-16]. In agreement, lentiviral-mediated upregu-lation of Arg1 in a balloon-injury rabbit model inhibited plaque inflammation and augmented VSMC proliferation. Plaque size was, however, not affected [8].

In macrophages, Arg1 is found in the alternatively activated M2 cells, a macrophage subtype with an anti-inflammatory and wound healing function [6]. Downregulation of Arg1 expression and inhibition of Arg1 activity in Raw264.7 macrophages resulted in augmented LPS-induced Tnf-a and IL-6 secretion [8]. On the other hand, Arg1 in macrophages suppresses Th2 dependent inflammation by dampening the production of anti-inflammatory cytokines by CD4+ T cells and suppressing T-cell proliferation in mice infected with the trematode Schistosoma mansoni [17]. Differential gene expression analysis in macrophages of atherosclerosis-susceptible and -resistant rabbits suggested that high macrophage Arg1 expression was associated with low atherosclerosis susceptibility [18]. Furthermore, M2 macrophages are found predominantly in carotid plaques of asymptomatic patients that have more stable plaques [19], indicating the positive association between macrophages of the M2 phenotype and atherosclerotic plaque stabilization. However, the functional role of macrophage Arg1 in atherosclerotic plaque development is currently still unknown.

In this study we specifically assessed the contribution of he-matopoietic Arg1 to the development of atherosclerosis by transplanting bone marrow from Arg1flox/flox;Tie2Cre mice into atherosclerosis-susceptible Ldl receptor knockout (Ldlr KO) mice.

2. Materials and methods

2.1. Animals

Ldlr KO mice and WT C57Bl/6 were obtained from the Jackson Laboratory and expanded at the Faculty of Science, Leiden University. Arg1flox/flox;Tie2Cre (Arg1 KO) mice [20] were bred at the Faculty of Life Sciences, University of Manchester. All animal studies in the Netherlands were approved by the regulatory authority of Leiden University and carried out in compliance with the Dutch government guidelines. All animal work in the United Kingdom was performed in accordance with Home Office regulations.

2.2. mRNA expression analysis by real time PCR

Thioglycollate-elicited peritoneal macrophages (PMs) from 12-week old male C57Bl/6 mice was obtained after injection of 1 mL of 3% thioglycollate solution 5 days prior to the experiment. After adherence and washing, the macrophages were incubated with/ without 10 mg/mL oxidized low density lipoprotein (oxLDL, prepared as described previously [21]) for 24 h. After that, cells were

collected for total RNA isolation [22]. Subsequently, RevertAid M-MuLV enzyme (Fermentas, Burlington, Canada) was used to transcribe RNA to cDNA. Quantitative PCR (qPCR, ABI PRISM 7500 system, Foster City, CA) was used to access the mRNA expression levels of genes interested using SYBR Green reagents (Applied Biosystems). Rpl27 and 36B4 were used as housekeeping genes.

2.3. Microarray analysis

Twelve-week old female Ldlr KO mice were first fed Western-type Diet (WTD; Special Diet Services) that contains 15% cacao butter and 0.25% cholesterol for a run-in period of 2 weeks before bilateral perivascular collar placement in the carotid arteries. Then the mice were challenged with WTD for another 2 weeks to induce early atherosclerotic lesion development. The carotid arteries were isolated directly after the run-in WTD period (baseline group) or 2 weeks after collar-placement (atherosclerotic plaque group) for microarray analysis as previously described [23].

2.4. Bone marrow transplantation

Bone marrow from male C57Bl/6 WT controls and Arg1 KO mice (around 12 weeks old) was prepared for bone marrow transplantation (BMT) to 12 weeks old female Ldlr KO recipient mice. In brief, lethally irradiated recipients received 5 x 106 bone marrow cells via tail vein injection. The mice were allowed to recover for 8 weeks on chow diet (RM3; Special Diet Services), after which they were fed WTD to induce atherosclerosis. After the 10-week WTD challenge, the mice were anaesthetized by a lethal dose anesthetic mixture that contains rompun, ketamine and atropine. Mice were bled and perfused with PBS, after which organs were isolated. The hematologic chimerism was confirmed in genomic DNA of recipient bone marrow using the PCR method (Supplementary Fig. 1A and B).

2.5. Generation of bone marrow-derived macrophages (BMDMs)

Bone marrow from Ldlr KO recipients transplanted with WT BM or Arg1 KO BM was isolated at sacrifice for the in vitro experiments. Bone marrow-derived macrophages were obtained as described previously [24]. Macrophages were cultured for 24 h with or without 100 mg/mL acetylated-low density lipoprotein (acLDL). The preparation of acLDL is described previously [25]. Subsequently the cells were analysed by an automated veterinary haematology analyzer (Sysmex Corporation, XT-2000iV, Japan) for foam cell formation as described previously [26,27]. Briefly, the Sysmex XT-2000iV analyzer applies a similar principle for cell differential analysis as patented fluorescent flow cytometric analysis [28]. Laser side scatter and side fluorescence lights were used for separating cell clusters. Lipid-rich macrophages (foam cells) are larger and contain more abundant granules compared to the non-foam cells [26,27]. Thus in a differential scattergram, the lipid-rich macrophage population shifts to a larger scale on the side scatter axis and side fluorescent light axis, enabling gaiting of a separate, shifted population of macrophage foam cells.

2.6. Flow cytometry analysis and WBC differential analysis

Blood samples, anti-coagulated with EDTA, as well as single splenic cell suspensions, were obtained using a 70 mm cell strainer (734-0003, VWR), were used for FACS analysis. Erythrocyte lysis buffer (0.15 M NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA, pH = 7.3) was used to lyse red blood cells in the blood samples and splenocyte preparations. Consecutively, the cells were analysed on a FACS Canto II (BD Biosciences, Mountain View, CA) using the relevant FACS antibodies (all obtained from eBioscience).

An automated Haematology analyzer (XT-2000iV, Sysmex Corporation, Japan) was used to analyse leukocyte counts in spleen and blood samples. Furthermore, peritoneal leukocytes collected at sacrifice from the bone marrow transplanted animals were analysed for quantification of macrophage foam cells formation.

2.7. Serum cholesterol level determination

Total and free cholesterol concentrations in serum were determined using an enzymatic colorimetric method as described previously [29]. Absorbance was read at 490 nm.

2.8. ELISA assay for anti-oxLDL antibodies

Copper-oxLDL was prepared as previously described [21]. The mouse immunoglobulin isotyping ELISA kit was obtained from BD Biosciences (Catalog No. 550487). HRP labelled polyclonal rabbit anti-Rat immunoglobulins (Ig) were obtained from DAKO (Product No. P045001). Total Ig and IgM antibodies against oxLDL were measured by a modified ELISA. In brief, oxLDL was coated in an ELISA plate (Corning, NY, 14831) in a concentration of 10 mg/mL overnight at 4 °C in coating buffer (0.42% w/v NaHCO3, 0.53% Na2CO3, pH of 9.6). After washing with 0.05% tween-20-PBS, wells were blocked for 30 min in 1% BSA blocking buffer. After washing, 4 mL plasma was added and plates were incubated at room temperature for an hour, then washed. For determination of total Ig, the HRP-labelled rat anti-mouse Ig antibody included in the BD ELISA kit was added. After incubation for 1 h at room temperature and subsequent washing, the plate is ready for colour development. For the IgM determination, after incubation with IgM-specific rat anti-mouse antibody for 1 h, wells were incubated with HRP-labelled rabbit anti-rat Ig for another hour before colour development following the manufacturer's instruction. Optical density (OD) were obtained by reading the plate in a plate reader (Biotek, poo-werWave 340) at 450 nm and 570 nm. Wavelength correction was performed by subtracting the values obtained at 570 nm from the values at 450 nm.

2.9. Histological analysis of the aortic root

After 10-week WTD feeding, mice were sacrificed. Seven-mm serial sections of the aortic root were cut using a Leica cryostat. Oil red-O staining, MoMa2 staining and Masson Trichrome kit were used for visualization of plaque area, macrophage positive area and collagen content respectively as described previously [29]. Quantification was performed using the Leica image analysis system (Leica Ltd, Cambridge, UK) [29].

2.10. Statistical analysis

Student's t-test or two-way ANOVA were used to determine the statistically significant differences (Graphpad Prism software). A Welch correction was applied to the t-test in the case of unequal variances in the dataset. The statistical significance was set at 0.05. Results are shown as the mean ± SEM.

3. Results

3.1. Argl expression is induced in macrophages by oxLDL loading and in carotid arteries upon induction of collar-induced atherosclerosis

The early stages of atherosclerotic lesion development are characterized by the accumulation of lipid-laden macrophages. Therefore, first the effects of incubation of macrophages with oxLDL

and induction of collar-induced atherosclerosis in the carotid artery on the expression of Arg1 was determined. In agreement with a previous study from Gallardo-Soler et al. [30], Arg1 mRNA expression was increased 4.3-fold in thioglycollate-elicited peritoneal macrophages after 24 h oxLDL (10 mg/mL) loading (p < 0.001; Fig. 1A) after a qPCR analysis. Oil-red O staining confirmed that after oxLDL incubation, foam cells were successfully induced (Fig. 1B). Moreover, microarray analysis showed that Arg1 expression was significantly increased in collar-induced early atherosclerotic lesions in the carotid artery of Ldlr KO mice (4.5-fold, p < 0.01; Fig. 1C).

3.2. Arg1 deletion in bone marrow-derived cells of Ldlr KO mice increases foam cell accumulation in the peritoneum in absence of effects on serum total cholesterol levels

To generate a mouse model that specifically lacks Arg1 in bone marrow-derived cells, bone marrow (BM) from Arg1flox/flox ; Tie2Cre (Arg1 KO) mice and WT controls was transplanted into Ldlr KO recipients. In Arg1floxflox;Tie2Cre mice Arg1 has been deleted in cells of the hematopoietic lineages and in endothelial cells [19,31].

Niese et al. previously showed that Arg1 deficiency does not affect bone marrow engraftment [32]. In agreement PCR analysis on genomic DNA isolated from bone marrow after 10 weeks of WTD feeding confirmed successful disruption of Arg1 functionality in the bone marrow and peritoneal cells of the Ldlr KO recipient mice (Supplementary Fig. A-C).

Differential haematology analysis was performed on peritoneal cells harvested from Ldlr KO mice transplanted with Arg1 KO or WT bone marrow after 10 weeks of WTD feeding. No difference in total number of peritoneal leukocytes (data not shown) or the percentage of macrophages within the peritoneal leukocyte population were found between Arg1 KO BM recipients and WT BM recipients (p > 0.05, Fig. 2A). Interestingly, a 3-fold larger fraction of foam cells in the peritoneum of Arg1 KO BM recipients was found when compared to the WT BM recipients (p < 0.001, Fig. 2B). This increased foam cell formation could not be attributed to increased serum cholesterol levels, as no difference in serum free cholesterol (data not shown) or total cholesterol was found (p > 0.05; Fig. 2C).

3.3. Arg1 KO BMDMs show increased foam cell formation and tend to differentiate to an M2 macrophage phenotype upon acLDL stimulation

BMDMs from WT and Arg1 KO mice were treated with 100 mg/ mL acLDL for 24 h to gain mechanistic insight into the observed increased foam cell formation in vivo. AcLDL-induced lipid loading led to foam cell formation in both WT BMDMs and Arg1 KO BMDMs (4.7 ± 0.3% to 10.4 ± 1.1% for WT BMDMs, p < 0.01; and 6.5 ± 0.66% to 15.1 ± 1.8% for Arg1 KO BMDMs, p < 0.001, respectively; Fig. 2D). Interestingly, both before and after lipid loading Arg1 KO BMDMs displayed a 50% increase in foam cell formation compared to WT BMDMs (p < 0.05, Fig. 2D). The mRNA expression of the genes related to foam cell formation were also assessed by qPCR analysis in BMDMs incubated with/without 100 mg/mL acetylated LDL for 24 h. Upon acLDL loading, the expression of Sr-b1, a receptor for native and modified lipoproteins, was effectively downregulated in WT BMDMs (p < 0.05 as compared to non-loaded cells; Fig. 3A), whereas Arg1 KO BMDMs failed to downregulate Sr-b1 upon acLDL loading (p > 0.05, Fig. 3A). No differences were observed in Ldlr expression between the 2 genotypes either with or without acLDL loading (p > 0.05; Fig. 3B). Foam cell formation is determined by the balance between cholesterol uptake and synthesis on the one hand and cholesterol efflux on the other hand. Although the expression of Abca1, the primary cholesterol efflux transporter, was increased

Fig. 1. Argl expression was induced by oxLDL loading in wild-type (WT) thioglycollate-elicited peritoneal macrophages (PMs) or Western-type diet (WTD) and collar-induced atherosclerosis in the carotid artery. (A) Thioglycollated-elicited PMs were incubated with/without l0 mg/mL oxLDL for 24 h, and then qPCR was used for analysis of Argl gene expression. (n = 6). (B) Oil-red O staining of PMs after incubation with/without l0 mg/mL oxLDL for 24 h. (C) Ldlr KO mice were challenged with WTD 2 weeks before the collar placement (baseline). The experimental group was fed WTD for another 2 weeks after collar placement (atherosclerotic plaque). Then the collar-induced atherosclerotic plaques in carotid arteries of Ldlr KO mice were used for microarray analysis of which the effects on Argl expression are shown (n = 6). Results are expressed as mean ± SEM, significance was assessed by Student t-test. **p < 0.0l; ***p < 0.00l.

in the BMDMs loaded with acLDL (WT, 15.7-fold, p < 0.001; Argl KO, 3.7-fold, p < 0.01; Fig. 3C), no difference in Abcal expression was found upon comparison of the Arg1 KO and the WT BMDMs (p > 0.05, Fig. 3C). Notably, expression of Srebpl was 2.45-fold higher in Arg1 KO BMDMs compare to WT BMDMS in response to acLDL loading, whereas no difference was found under control non-loaded conditions (Control, p > 0.05; +acLDL, p < 0.05; Fig. 3D). The difference in Srebpl expression between acLDL loaded Arg1 KO and WT macrophages is explained by a failure of the Arg1 KO macrophages to downregulate Srebp-l in response to acLDL loading (WT, p < 0.01; Arg1 KO, p > 0.05; Fig. 3D). Furthermore, the M1 and M2 markers Inos and Fizz-l were determined in both genotypes of BMDMs before and after acLDL loading. Under control non-loaded conditions, no differences were found in the expression of Inos or Fizz-l. However, in response to acLDL loading, Arg1 deficiency in BMDMs led to an M2-like phenotype, as evidenced by significantly downregulated Inos expression (67% decrease, p < 0.05; Fig. 3E) and extremely upregulated Fizz-l expression (19fold, p < 0.001; Fig. 3F), whereas no such changes were observed in WT BMDMs.

Furthermore, NO production was determined in the culture medium of WT and Arg1 KO BMDMs, both under control conditions and after acLDL lipid loading. Two-way ANOVA showed an

increased NO production in Arg1 KO BMDMs compared to the WT BMDMs (p=<0.05, Fig. 3G). NO production was significantly increased (1.6-fold, p < 0.05, Fig. 3G) in Arg1 KO BMDMs, but not in WT BMDMs in response to acLDL loading, leading to 2.2-fold (p < 0.01, Fig. 3G) higher NO concentrations in the supernatant of Arg1 KO BMDMs under these conditions.

3.4. Bone marrow Argl deficiency affects neither atherosclerotic plaque size nor plaque composition in Ldlr KO mice

After 10 weeks WTD feeding, the aortic root was sectioned and stained with oil red-O to analyse atherosclerotic lesion development. Despite the observed increase in macrophage foam cell formation upon deletion of Arg1, no difference in plaque size was found between the two experimental groups (647 ± 29 *103 mm2 for WT BM recipients vs. 634 ± 26 *103 mm2 for Arg1 KO BM recipients, Fig. 4A). Macrophages in the plaque were visualized by MOMA-2 staining. No difference in plaque macrophage content as a fraction of total plaque size was observed (0.171 ± 0.011 WT BM vs. 0.169 ± 0.016 Arg1 KO BM, Fig. 4B). Collagen in the plaque was stained using a Masson Trichrome method and Picosirius Red staining. A trend towards a reduction in the collagen content of plaques of Arg1 KO BM recipients was found in Masson Trichrome-

Fig. 2. Increased in vivo peritoneal foam cell formation in Ldlr KO mice transplanted with Arg1 KO BM in absence of effects on serum cholesterol and augmented in vitro foam cell formation in bone marrow-derived macrophages (BMDM) lacking Arg1. Serum and peritoneal leukocytes were collected at sacrifice after 10 weeks of high-fat, high-cholesterol Western-type diet feeding. (A) Macrophage percentage of total peritoneal leukocytes in BMT recipients (n = 9—14). (B) Foam cell percentage of total peritoneal leukocytes in the BMT recipients (n = 9—14). (C) Total cholesterol level in BMT recipients (n = 9—14). (D) In vitro, BMDMs from WT BM or Arg1 KO BM recipients were incubated 24 h in the presence or absence of 100 mg/mL acLDL. Foam cell percentage was detected using a haematology analyzer (n = 6—10). Results are expressed as mean ± SEM, significance was assessed by Student t-test or 2-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

stained sections (0.102 ± 0.009 WT BM vs. 0.084 ± 0.004 Arg1 KO BM, p = 0.06, Fig. 4C). However, analysis of Picosirius Red staining did not indicate any difference between the groups (0.124 ± 0.013 WT BM vs. 0.105 ± 0.012 Arg1 KO BM, Fig. 4D).

3.5. Transplantation of Argl KO bone marrow into Ldlr KO recipients results in reduced splenocyte and blood leukocyte counts

Flow cytometry was used to assess whether loss of Arg1 functionality in bone marrow-derived cells in the transplanted Ldlr KO mice affected leukocyte numbers in the circulation or the spleen. On chow diet, no difference in total blood leukocyte numbers was detected (p > 0.05, Fig. 5A) between the recipient mice with WT BM and the mice with Arg1 KO BM. WTD feeding for 10 weeks increased total leukocyte counts from in blood of Ldlr KO mice transplanted with WT BM (chow diet: 11410 ± 708 cells/ml; WTD: 15672 ± 689 cells/ml; p < 0.01; Fig. 5A). Mice transplanted with Arg1 KO bone marrow, however, failed to show an increase in blood leukocyte counts upon WTD feeding (chow diet: 12092 ± 906 cells/ ml; WTD: 11573 ± 491 cells/ml; p > 0.05; Fig. 5A). Therefore, it led to lower total leukocyte counts in the blood of Arg1 KO BM recipients as compared WT BM recipients after 10 weeks of WTD feeding (p < 0.001, Fig. 5A). Comparison of the subtypes of WBC, showed that, in contrast to WT BM recipients in which lymphocytes were 1.6-fold increases (p < 0.001) after 10 weeks WTD feeding,

lymphocytes did not increase in Ldlr KO mice transplanted with Argl KO BM (Fig. 5B). No differences were found in neutrophils, monocytes, and eosinophils (data not shown). Next, flow cyto-metric analysis was used to determine the effects on the different cellular subsets in blood at sacrifice. No differences were found in the absolute amounts of CDllb+ cells, CDllb+/Ly6Chi inflammatory, CDllb+/Ly6Clow+med patrolling monocytes and CDllb+/Ly6G+ neutrophils, nor the amounts of CD4+ T helper cells, CD25+/CD4+ activated T helper cells and CD8+ cytotoxic T-cells (Fig. 5C—H). Unexpectedly, the decrease in total blood leukocytes appeared to be driven by a 2-fold decrease in circulating CDl9+ B cells (p < 0.0l, Fig. 5I).

At the time of sacrifice spleens were taken and weighed. Organ weight was normalized for total body weight. A small but significant l0% decrease in spleen weight was found in Ldlr KO mice transplanted with Argl KO BM (p < 0.05, Fig. 6A), while there were no differences in total body weight (data not shown). Correspondingly, spleens from the Argl KO BM recipients contained 35% less splenocytes (p = 0.052, Fig. 6B). Next, splenocyte composition was assessed by flow cytometry. The absolute numbers of leukocyte subtypes, including CDllb+ cells, CDllb+/Ly6Chi pro-inflammatory monocytes, CDllb+/Ly6Clow+med patrolling cells, CDllb+/Ly6G+ neutrophils, CDl9+ B cells and CD8+ T cells in the spleen of Arg l KO recipients showed a significant decrease or a trend towards a decrease, suggesting that the decrease in splenocyte number was

Fig. 3. Gene expression and nitric oxide production in WT and Arg1 KO BMDMs after incubation with or without 100 mg/mL acLDL for 24 h. Relative mRNA expression of (A) Sr-b1, (B) Ldlr, (C) Abcal, (D) Srebpl, (E) Inos, and (F) Fizz-1. (Control group, n = 9-12; +acLDL group, n = 3-4). (G) Nitric oxide concentrations in the supernatant of BMDMs incubated with or without 100 mg/mL acLDL 24 h (Control group, n = 3; +acLDL group, n = 4). Results are expressed as mean ± SEM, significance was assessed by Student t-test or 2-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4. Deletion of Arg1 in bone marrow-derived cells does not influence atherosclerotic lesion development after 10-week WTD feeding. (A) Plaques were stained with oil-red O and plaque area was quantified. (p > 0.5; WT BMT, n = 16; Arg1 KO BMT, n = 15). (B) Plaque area stained positively with MOMA-2 antibody was measured and normalized for total lesion size. (p > 0.5; WT BMT, n = 14; Arg1 KO BMT, n = 16). (C) Plaque collagen was visualized using Masson's Trichrome Staining and plaque collagen content was analysed. (p = 0.064; WT BMT, n = 15; Arg1 KO BMT, n = 15). (D) Sirius Red staining was performed to examine plaque collagen content further. Collagen content was analysed (p > 0.5; WT BMT, n = 11; Arg1 KO BMT, n = 12). (E) Representative images of the staining described above. Results are expressed as mean ± SEM, significance was assessed by Student t-test.

Fig. 5. Decreased leukocytes in blood of Ldlr KO mice transplanted with Arg1 KO BM after 10 weeks of feeding a high-fat, high-cholesterol Western-type diet (WTD).

After BMT, recipient mice were fed chow diet for 8 weeks (shown as "chow diet" in figures) before the switch to WTD for an additional 10 weeks (shown as "WTD" in figures) to induce atherosclerosis development. Circulating leukocytes (A) and lymphocytes (B) on chow diet at 8 weeks after transplantation and after 10 weeks on WTD at 18 weeks after transplantation. (n = 15-19). (B) Circulating lymphocytes at 8 weeks after transplantation on chow diet and 10 weeks on WTD. (n = 15-19). (C-1) Circulating leukocytes were analysed by flow cytometry for expression of several immune cell markers. (n = 5). (F) Total CD19+ cells in circulation (n = 5). (J) Representative flow cytometry plots. Results are expressed as mean ± SEM, significance was assessed by Student t-test or 2-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.

not attributable to one specific cell type (p < 0.05, Fig. 6C and D). However, fractional analysis showed a 23% increase in CD4+ and CD25+/CD4+ Tcells in the spleen of Ldlr KO mice that received Arg1 KO BM compared to the recipients that received WT BM (*p < 0.05; $p < 0.05, respectively. Fig. 6E).

3.6. Argl deficiency in the bone marrow of Ldlr KO mice induces circulating oxLDL-specific antibody levels

The total level of ox-LDL specific immunoglobulins (Ig) and its isotype IgM were determined in plasma of the Ldlr KO mice

Fig. 6. Decreased spleen weight in Ldlr KO mice transplanted with Arg1 KO BM. Spleens were isolated at 10 weeks after feeding a high-fat, high-cholesterol Western-type diet. (A) A reduction in relative spleen weight was measured in the Arg1 KO BM recipients. The readout was corrected for the total body weight of mice (*p < 0.05; WT BMT, n = 11; Arg1 KO BMT, n = 14). (B) Reduction in total amount of splenocytes in the Arg1 KO BM recipients (p = 0.05; WT BMT, n = 5; Arg1 KO BMT, n = 5). (C) Flow cytometric analysis of splenocytes showing that the amount of CD11b+, CD11b+/Ly6Chi, CD11b+/Ly6Clow+med monocytes and CD11b+/Ly6G+ neutrophils was significantly decreased or trended to a decrease in spleens of Arg1 KO recipients (*p < 0.05; WT BMT, n = 5; Arg1 KO BMT, n = 5). (D and E) A trend to a decrease in the amount of CD19+ and CD8+ splenocytes. The fraction of CD4+ and CD4+CD25+ cells in the spleen was increased (*p < 0.05; $p < 0.05, respectively; WT BMT, n = 5; Arg1 KO BMT, n = 5), though the total amount of CD4+ and CD4+CD25+ cells in the spleen was unchanged (p > 0.05; WT BMT, n = 5; Arg1 KO BMT, n = 5). (F) Representative flow cytometry plots. Results are expressed as mean ± SEM, significance was assessed by Student t-test. *p < 0.05; $p < 0.05.

transplanted with WT or Arg1 KO BM after 10 weeks WTD challenge. As shown in Fig. 7, deletion of Arg1 in the bone marrow of Ldlr KO mice led to increased total oxLDL-specific Ig levels (1.2-fold, p < 0.05 compared to the WT BM Ldlr KO recipients after 10 weeks WTD challenge), which was not driven by IgM.

4. Discussion

In the current study for the first time the effects of Arg1 deletion in bone marrow-derived cells on macrophage foam cell formation and atherosclerosis susceptibility was determined. We show that Argl expression is increased in elicited peritoneal macrophages upon oxLDL-induced lipid loading and during the development of early murine atherosclerotic lesions in the carotid-artery. In

agreement, Gallardo-Soler, Alejandro, et al., previously demonstrated that Argl expression is highly induced in bone marrow-derived macrophage foam cells upon oxLDL or acLDL stimulation [30]. Inhibition of Ppar-g/5 suppresses the lipid-induced increase in macrophage Argl expression [30], indicating that Argl expression is increased during macrophage foam cell formation probably due to Ppar-g/5 activation. Combined, these results indicate that Argl might participate in macrophage foam cell formation, thereby influencing atherosclerosis development. However, so far there had been no reports on the effects of Argl function on macrophage foam cell formation. Interestingly, Argl KO BMDMs showed increased foam cell formation both under control conditions and after acLDL-induced lipid-loading compared to the WT BMDMs. Genes involved in foam cell formation were investigated to gain

Fig. 7. Increased oxLDL-specific antibody levels in Ldlr KO mice transplanted with Arg1 KO BM after 10 weeks of WTD feeding. Total oxLDL-speciflc antibody and isotype IgM levels were assessed using ELISA. Results are expressed as mean ± SEM, significance was determined by Student t-test. *p < 0.05 as compared between Ldlr KO recipient with WT BM and recipients with Argl KO BM in the level of total oxLDL-Specific antibody (Ig).

insight into the mechanism behind the enhanced foam cell formation in absence of Argl. No difference was found in the expression of Abcal, the primary cholesterol export transporter, between WT and Argl KO macrophages. In response to acLDL loading, Argl deficient macrophages however did display an impaired suppression of Srebpl and Sr-bl compared to WT macrophages. Srebpl is an important regulator of cellular lipid biosynthesis [33], while Sr-bl facilitates the uptake of native and modified lipoproteins [34]. Enhanced foam cell formation of macrophages lacking Argl might thus be explained by defective suppression of cellular lipid biosynthesis and modified LDL uptake. Moreover, upon lipid loading, macrophages lacking Argl seem more likely to skew towards the M2 phenotype, as evidenced by the significantly downregulated Ml marker Inos and strongly upregulated M2 marker Fizz-l expression in Argl KO macrophages. M2 macrophages exhibit increased cholesterol loading by oxLDL compared to Ml macrophages, providing one possible mechanism for the increased foam cell formation in our model [35,36]. Collectively, these findings suggest a link between Argl expression and macrophage foam cell formation. As Argl and Inos compete for the common substrate L-arginine, macrophage Argl deficiency indirectly leads to enhanced synthesis of NO upon lipid loading. The observed downregulation of Inos in the Argl deficient macrophages could thus be due to negative feedback by NO [l0,37—4l]. Notably, in agreement with our findings, excess NO has recently been shown to induce oxLDL-induced macrophage foam cell formation by inhibition of the LXR-Abcal-cholesterol efflux axis [42].

Next we investigated whether the observed increased foam cell formation in vitro also translated into increased macrophage foam cell formation in vivo and augmented atherosclerosis susceptibility. Hereto, bone marrow from ArglfloXflox;Tie2Cre mice was transplanted into Ldlr KO mice, a commonly used model to study atherosclerosis. We decided to use Arglfloxflox;Tie2Cre mice as donors as the Tie2-cre deleter has previously been shown to lead to a complete ablation of Argl activity in macrophages, while in ArglfloX flox;LysMCre only ~80% reduction in macrophage Argl activity was achieved [20]. In line with the observed increase in lipid accumulation in Argl KO macrophages in vitro, foam cell formation in the peritoneal cavity of Ldlr KO mice transplanted with Argl KO bone marrow was increased after l0 weeks WTD feeding.

Foam cell formation in the peritoneal cavity is a marker for atherosclerotic plaque development [43,44]. However, no differences in plaque size or plaque macrophage content were detected between the Ldlr KO mice transplanted with Argl KO or WT bone marrow. Increased expression of Argl in balloon-injured rabbits

results in augmented plaque stability as a consequence of enhanced VSMC proliferation [8]. Moreover, Arg1flox/flox;Tie2Cre (Argl KO) mice exhibit impaired cutaneous wound healing, to some extent due to impaired collagen deposition [43]. The reduced collagen content in wounds of Arg1flox/flox;Tie2Cre (Argl KO) mice was attributed to increased collagen degradation by neutrophils and macrophages lacking Argl [45]. In atherosclerosis, collagen deposition provides stability to the atherosclerotic plaque. However, no significant effect on the collagen-content of atherosclerotic plaques of Ldlr KO mice transplanted with Argl KO bone marrow was found. In the aortic root lesions, smooth muscle cells, the main producers of collagen deposited in atherosclerotic lesions [46], were not affected by deletion of Argl in bone marrow-derived cells [47]. Conclusively, deletion of Argl in bone marrow-derived cells does not affect atherosclerosis susceptibility despite a clear increase in macrophage foam cell formation, indicating other compensatory mechanisms.

Tie2 is expressed in all hematopoietic lineage cells [20]. Hence, in our bone marrow transplantation model using the Arg1floxflox;Tie2Cre mice as donors Argl is not only deleted in macrophages, but in all hematopoietic cells [48]. For long it has been thought that Argl is only expressed in the myeloid lineage and not in the lymphoid lineages [49]. Indeed Argl is expressed primarily in anti-inflammatory alternatively activated macrophages, however it can also be detected in neutrophils [50,5l], and innate lymphoid cells II (1LC2) [52].

To investigate whether bone marrow Argl deficiency had any atheroprotective effects to counteract the observed increase in foam cell formation, the leukocytes in blood and spleen, the major hematopoietic organ and an important reservoir for monocytes, were analysed in the BM transplanted mice. In agreement with previous studies by Niese et al. [32], deletion of Argl in bone marrow-derived cells did not affect circulating leukocytes on regular chow diet. Hypercholesterolemia is commonly known to induce leukocytosis in animal models [53,54] and humans [55,56]. Upon challenge with a high fat/high cholesterol WTD, Ldlr KO mice transplanted with Argl KO bone marrow failed to increase leukocyte counts in the circulation. The inflammatory and pro-atherogenic CDllb+/Ly6Chl subset of monocytes gives rise to classically activated Ml macrophages in the atherosclerotic plaque [57,58]. However, no difference was found in the numbers of CDllb+/Ly6Chi monocytes in blood, nor in the amount of circulating neutrophils between the 2 groups of bone marrow recipients after l0 weeks WTD challenge. Hypercholesterolemia-induced monocytosis and neutrophilia was thus not affected in the blood by deletion of Argl in bone marrow. 1n addition to myeloid cells mobilization from bone marrow, high fat diet-induced inflammatory conditions like atherosclerosis could also induce hematopoi-etic stem and progenitor cells (HSPCs) settlement in the spleen and leading to local production of monocytes and neutrophils [59,60]. Spleen-derived monocytes and neutrophils eventually infiltrate into the growing atherosclerotic lesion, giving rise to foam cell formation and pro-inflammatory cytokine production [60]. Notably, 30% of the total number of aortic monocytes were spleen-derived Ly-6Chi monocytes, which are also reported to contribute to foam cell formation in the lesions [60]. Argl depletion in bone marrow leads to a significant reduction of the CDllb+ cells, CDllb+/ly6Clow+med monocytes and neutrophils in the spleen of Ldlr KO mice and a tendency towards a decrease in proinflammatory CDllb+/Ly6Chi monocytes. This might provide an atheroprotective mechanism counteracting the increased foam cell in Ldlr KO mice transplanted with Argl KO BM.

As L-arginine is required for CD4+ T-cell function and maturation, Argl-mediated depletion of L-arginine by leukocytes results in decreased T cell proliferation [l7,6l]. T cell counts and activation

status were therefore also investigated. However, there was no difference in the amount of CD4+ cells and CD4+/CD25+ in blood and spleen, although a modest fractional increase in both subtypes was found in the spleen. This indicates that leukocyte Argl is not a strong regulating factor of T-cell proliferation in the spleen. Notably, strikingly lower amounts of CDl9+ B cells were found in both the circulation and the spleen of Argl KO transplanted Ldlr KO mice as compared to WT transplanted animals after l0 weeks WTD challenge, explaining the decrease in total blood leukocyte counts. L-arginine is an essential amino acid for B cell maturation in the bone marrow and arginase-mediated L-arginine depletion leads to reduced B cell emigration from the bone marrow and reduced B cell numbers in the spleen and lymph nodes [62]. If anything, leukocyte Argl deletion is thus anticipated to enhance B cell emigration from bone marrow, which clearly cannot explain the reduced B cell numbers in blood. B cells, as the antibodies producing cell of the immune system, play an important role in atherosclerosis. Anti-oxLDL antibodies, especially IgM anti-oxLDL, are inversely related to atherosclerotic plaque size in experimental studies [63]. Therefore, oxLDL antibody levels were determined in plasma obtained from recipient mice after l0 weeks WTD feeding. To our surprise, despite the reduced numbers of B cells in the Ldlr KO recipients transplanted with Argl KO BM, total oxLDL-specific antibody levels were increased. OxLDL-specific IgM that is suggested to be athe-roprotective [63], however, was not changed. Although in clinical cardiovascular disease diverging results have been described on the association between oxLDL-specific antibodies and atherosclerosis, animal studies consistently suggest an atheroprotective role for oxLDL antibodies [64]. Whatever the mechanism behind the reduction in B cells and increased circulating levels of oxLDL-specific antibodies, it might counteract the pro-atherogenic effects of enhanced foam cell formation in absence of Argl.

We conclude that despite leading to an increase in foam cell formation and a decrease in circulating B cells, Argl deficiency in bone marrow-derived cells does not significantly affect atherosclerotic plaque development.

Conflict of interest

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

BR contributed to the execution of experiments the acquisition, analysis, and interpretation of data for the work, and wrote the manuscript.

EVK contributed to the execution of experiments the acquisition, analysis, and interpretation of data for the work and wrote the manuscript.

TJCVB contributed significantly to conception of the study and made critical revisions to the manuscript for intellectual content.

SMC contributed significantly to conception of the study and made critical revisions to the manuscript for intellectual content.

MVE contributed significantly to conception of the study, experimental design, and made critical revisions to the manuscript for intellectual content.

Acknowledgements

This study was supported by 'the Netherlands Cardiovascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organisation for Health Research and Development, and the Royal Netherlands Academy of Sciences' for the GENIUS project 'Generating the best

evidence-based pharmaceutical targets for atherosclerosis' (CVON2011), the Netherlands Organisation for Scientific Research (VICI Grant 91813603 (M.V.E)), and the Medical Research Council (grant G.14000449 (SMC)). M.V.E. is an Established Investigator of the Netherlands Heart Foundation (Grant 2007T056). E.V.K was supported by The Netherlands Heart Foundation (Grant 2009B075 and B. R. by a grant from the China Scholarship Council.

We thank Martine Bot for sharing the Microarray data. We thank Ronald van der Sluis, Marco van der Stoep, Gijs van Puijvelde and Amanda C. Foks for their expert help with animal sacrifice.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2016.11.018.

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