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Overexpression of perilipin1 protects against atheroma progression in apolipoprotein E knockout mice

Open AccessPublished:January 15, 2018DOI:https://doi.org/10.1016/j.atherosclerosis.2018.01.019

      Highlights

      • Overexpression of PLIN1 in macrophages protected against atheroma progression.
      • PLIN1's atheroprotective effect was independent of changes in major risk factors.
      • Overexpression of PLIN1 might induce macrophage polarity changes in plaques.

      Abstract

      Background and aims

      Perilipin1 (PLIN1), a lipid droplet-associated protein, plays an important role in the regulation of lipolysis and lipid storage in adipocytes. PLIN1 has recently been reported to be expressed in macrophages within atheroma plaques, suggesting PLIN1 may play a role in the accumulation of lipids at the arterial wall and in the development of atherosclerosis. To clarify the role of PLIN1 in the pathophysiology of atherosclerosis, we assessed the progression of atherosclerosis in PLIN1 transgenic mice (Plin1Tg).

      Methods

      Plin1Tg were crossed with apolipoprotein E knockout mice (ApoeKO). C57BL/6J mice, ApoeKO and Plin1Tg/ApoeKO received a normal chow diet for 20 weeks. Body weight, gonadal fat mass and plasma lipid concentrations were measured. Aortas were collected for quantification of atheroma lesions and histological analysis by Oil Red O staining.

      Results

      Body weight, gonadal adipose mass and plasma triglyceride concentrations were not significantly different among the three groups. In contrast, the atherosclerotic lesion area was significantly increased in ApoeKO (14.2 ± 3.2%; p < .01) compared with C57BL/6J mice (3.3 ± 1.2%) and Plin1Tg/ApoeKO (5.6 ± 1.9%).

      Conclusions

      Overexpressed PLIN1 in macrophages had a protected role against atheroma progression in ApoeKO in the absence of changes in gonadal fat mass or plasma lipid levels, presumably due to modification of the stability and/or inflammatory profile of macrophages.

      Keywords

      1. Introduction

      Perilipin1 (PLIN1, perilipin A) is one of the lipid droplet-associated proteins that was originally identified in adipocytes [
      • Greenberg A.S.
      • et al.
      Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets.
      ]. In adipocytes, PLIN1 coats the lipid droplets and regulates both lipid storage and lipolysis. In the basal state, PLIN1 associates with the protein CGI-58, a coactivator of adipose triglyceride lipase (ATGL), and regulates the access of hormone-sensitive lipase (HSL) to lipid droplets, thus down-regulating basal lipolysis [
      • Miyoshi H.
      • et al.
      Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes.
      ] [
      • Miyoshi H.
      • et al.
      Adipose triglyceride lipase regulates basal lipolysis and lipid droplet size in adipocytes.
      ]. Conversely, when adipocytes are stimulated by catecholamine, increased cAMP activates protein kinase A (PKA), resulting in the phosphorylation of HSL and PLIN1 in adipocytes [
      • Miyoshi H.
      • et al.
      Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosphorylation-dependent and -independent mechanisms.
      ]. Consequently, CGI-58 dissociates from phosphorylated PLIN1, and recruits phosphorylated HSL at the surface of the lipid droplets, thus activating lipolysis [
      • Greenberg A.S.
      • et al.
      The role of lipid droplets in metabolic disease in rodents and humans.
      ].
      Following the discovery of PLIN1, a series of additional lipid droplet-associated proteins were identified. The perilipin family comprised five proteins: PLIN1, PLIN2 (adipose differentiation-related protein, adipophilin) [
      • Gross D.N.
      • et al.
      Dynamics of lipid droplet-associated proteins during hormonally stimulated lipolysis in engineered adipocytes: stabilization and lipid droplet binding of adipocyte differentiation-related protein/adipophilin.
      ], PLIN3 (tail-interacting protein 47, TIP47) [
      • Kimmel A.R.
      • et al.
      Adoption of PERILIPIN as a unifying nomenclature for the mammalian PAT-family of intracellular lipid storage droplet proteins.
      ], PLIN4 (S3-12) [
      • Scherer P.E.
      • et al.
      Cloning of cell-specific secreted and surface proteins by subtractive antibody screening.
      ] [
      • Wolins N.E.
      • et al.
      Adipocyte protein S3-12 coats nascent lipid droplets.
      ], and PLIN5 (PAT1, LSPD5, OXPAT, MLDP) [
      • Dalen K.T.
      • et al.
      LSDP5 is a PAT protein specifically expressed in fatty acid oxidizing tissues.
      ] [
      • Wolins N.E.
      • et al.
      OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.
      ] [
      • Yamaguchi T.
      • et al.
      MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor alpha.
      ]. PLIN1 localizes generally in adipocytes and steroidogenic tissue. Under specific conditions, PLIN1 expression occurs also in macrophages, where it plays a crucial role in the progression of atherosclerosis [
      • Faber B.C.
      • et al.
      Identification of genes potentially involved in rupture of human atherosclerotic plaques.
      ]. However, the function of PLIN1 in macrophages is not yet known. Meanwhile, PLIN2 is the most abundant PAT protein in macrophage-derived foam cells. PLIN2 augments TNFα, MCP-1, and IL-6 expression in acetylated LDL-induced macrophages, suggesting that enhancing inflammation might be one role of PLIN2 in atherosclerosis [
      • Chen F.L.
      • et al.
      Adipophilin affects the expression of TNF-alpha, MCP-1, and IL-6 in THP-1 macrophages.
      ].
      Atherosclerosis is a chronic inflammatory disease that arises from an imbalance in lipid metabolism and a maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall [
      • Moore K.J.
      • Sheedy F.J.
      • Fisher E.A.
      Macrophages in atherosclerosis: a dynamic balance.
      ]. Macrophages play a principal role in atherosclerosis, having at least two different phenotypes: M1 and M2 macrophages, the former classically activated and the latter formed by alternative activation. A number of previous studies have demonstrated that M1 macrophages promote inflammation but M2 macrophages have anti-inflammatory properties [
      • Canton J.
      • Neculai D.
      • Grinstein S.
      Scavenger receptors in homeostasis and immunity.
      ].
      We [
      • Cho K.Y.
      • et al.
      The phenotype of infiltrating macrophages influences arteriosclerotic plaque vulnerability in the carotid artery.
      ] previously reported the polarity of macrophages in human carotid artery plaques obtained by carotid endarterectomy. In that study, predominant infiltration of M1 macrophages was shown in probably-unstable plaque specimen. Conversely, in stable plaques from asymptomatic patients, M2 macrophages were dominantly observed despite the fact of less infiltration of total macrophage number compared with that in unstable plaques.
      Following the observation above, we examined PLIN1 and PLIN2 expression in the plaques. In the stable plaques, PLIN1 expression was dominant compared with PLIN2 expression along with M2 macrophage ascendancy. In contrast, PLIN2 expression was prominent in unstable plaques in parallel with M1 macrophage infiltration. Therefore, we hypothesized that PLIN1/PLIN2 expression and macrophage polarity might be related to each other, consequently playing a role in the progression of atherosclerosis. We therefore conducted the present study to assess the effect of PLIN1 overexpression in macrophages on the progression of atherosclerosis in vivo using our PLIN1 transgenic mice (Plin1Tg) [
      • Miyoshi H.
      • et al.
      Perilipin overexpression in mice protects against diet-induced obesity.
      ].

      2. Materials and methods

      2.1 Antibodies

      Polyclonal anti-PLIN1 antibody and anti-PLIN2 antibody were generated as previously described [
      • Miyoshi H.
      • et al.
      Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes.
      ] [
      • Souza S.C.
      • et al.
      Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.
      ]. Anti-CD68 antibody and anti-CD206 antibody were purchased from Abcam (Cambridge, England) Anti-CD11c antibody was purchased from Bio-Rad (Hercules, United States).

      2.2 Animal experiments

      Plin1Tg were generated using the aP2 promotor on a C57BL/6J background as previously described. The PLIN1 expression level in white adipose tissue in Plin1Tg was shown to be two times higher than that in control mice [
      • Miyoshi H.
      • et al.
      Perilipin overexpression in mice protects against diet-induced obesity.
      ]. Apolipoprotein E knockout mice (ApoeKO) were purchased from the Jackson Laboratory (Bar Harbor, United States). C57BL/6J mice were purchased from Charles River Japan (Yokohama, Japan). Plin1Tg and ApoeKO were crossed to obtain Plin1Tg/ApoeKO mice. The mice were housed at the Graduate School of Medicine's Institute for Animal Experimentation at Hokkaido University in accordance with the institutional guidelines of Hokkaido University Graduate School of Medicine. All mice were housed at room temperature, maintained on a 12 h light/dark cycle, and given free access to water.
      All mice received a normal chow diet (MF from Oriental Yeast, Tokyo, Japan) for 20 weeks. Body weight and gonadal fat mass were measured. Blood was collected from inferior vena cava and plasma separated by centrifugation for enzymatic determination (Wako, Tokyo, Japan; R&D Systems, Minneapolis, United States) of lipid concentrations and proinflammatory cytokine levels. Aortic sinuses and whole aortas were collected for quantification of atheroma lesions and histology.

      2.3 Reverse transcription polymerase chain reaction (RT-PCR) analysis

      Thioglycollate-elicited macrophages were isolated from C57BL/6J mice and Plin1Tg by washing the peritoneal cavity with 3 ml of phosphate-buffered saline one day after the mice were intraperitoneally injected with 50 μl of 4% thioglycollate in phosphate-buffered saline. Individual cell suspensions were washed with red blood cell lysis buffer (eBioscience, San Diego, United States). Total RNA was isolated from the isolated macrophages using an RNeasy Mini kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's recommendations, and was used as the starting material for cDNA preparation. RT-PCR was performed using ReverTra-Plus (Toyobo, Osaka, Japan) in accordance with the manufacturer's protocols. Primer sequences are shown in Table 2 in Ref. [

      K. Yamamoto, et al, Atheroprotective Effect of Perilipin 1 Overexpression in Vivo (Data in Brief, Submitted).

      ].

      2.4 Histology

      Aortas were dissected from the aortic root to the iliac bifurcation, carefully separated from periarterial adipose tissue, and stained for lipid deposits with Oil Red O. The Oil Red O-positive areas were quantified and expressed as a percentage of total aorta area using a BZ-II Analyzer (Keyence). For histological studies, aortic sinuses were fixed in phosphate-buffered formalin and frozen before sectioning. Hematoxylin staining and immunocytology for whole macrophages (using anti-CD68 antibody), M1 macrophages (using anti-CD11c antibody), M2 macrophages (using anti-CD206 antibody), PLIN1 (using anti-PLIN1 antibody), and PLIN2 (using anti-PLIN2 antibody) were performed.

      2.5 Statistical analysis

      Results are expressed as means ± SD. The differences between the groups were assessed by un-paired t-test, analysis of variance, or Dunn's test. p < .05 was considered statistically significant. Data were analyzed using JMP Pro version 12.0.1.

      3. Results

      3.1 Biochemical markers did not differ between ApoeKO and Plin1Tg/ApoeKO

      Thioglycollate-elicited peritoneal macrophages were isolated from C57BL/6J mice or Plin1Tg. Total RNA was prepared and analyzed by RT-PCR. Expressions of the PLIN1 transgene and PLIN2 gene in the Plin1Tg macrophages was confirmed (Fig. 1). We next examined whether PLIN1 overexpression affected biochemical markers in ApoeKO. Body weight (C57BL/6J 26.6 ± 3.1 g; ApoeKO 29.0 ± 4.5 g; Plin1Tg/ApoeKO 27.5 ± 3.9 g) and gonadal fat mass (C57BL/6J 356 ± 78 mg; ApoeKO 332 ± 124 mg; Plin1Tg/ApoeKO 424 ± 190 mg) were comparable among C57BL/6J mice, ApoeKO and Plin1Tg/ApoeKO. Plasma total cholesterol levels were significantly higher in ApoeKO (395 ± 80 mg) and in PLIN1Tg/ApoeKO (471 ± 138 mg) than in C57BL/6J mice (72 ± 11 mg), but there was no significant difference between ApoeKO and Plin1Tg/ApoeKO. Plasma TNFa was undetectable in all groups. Plasma IL-6 levels (C57BL/6J 22.2 ± 14.1 pg/ml; ApoeKO 64.2 ± 37.4 pg/ml; Plin1Tg/ApoeKO 28.2 ± 31.1 pg/ml) tended to be high in ApoeKO, but did not differ significantly between ApoeKO and Plin1Tg/ApoeKO (p = .069) (Table 1).
      Fig. 1
      Fig. 1Peritoneal macrophages were isolated from C57BL/6J mice and PLIN1 transgenic mice (Plin1Tg), and mRNA was extracted.
      (Left three lanes) Macrophages from C57BL/6J mice; (right three lanes) macrophages from PLIN1Tg. tPLIN1 refers to the transgene induced in PLIN1 transgenic mice. ePLIN1 refers to the endogenous PLIN1 gene in macrophages.
      Table 1Biochemical markers.
      C57BL/6JApoeKOPlin1Tg/ApoeKO
      Body weight (g)26.6 ± 3.129.0 ± 4.527.5 ± 3.9
      Gonadal fat (mg)356 ± 78332 ± 124424 ± 190
      Triglyceride (mg/dl)69 ± 2599 ± 43120 ± 71
      Total cholesterol (mg/dl)72 ± 11395 ± 80*471 ± 138*
      IL-6 (pg/ml)22.2 ± 14.164.2 ± 37.428.2 ± 31.1
      *p < .01 vs. C57BL/6J.

      3.2 Overexpression of PLIN1 suppressed atherosclerosis development in ApoeKO

      We examined the size of the atherosclerotic lesions in the aortic sinus area (Fig. 2) and in the whole aorta using an en face method (Fig. 3A) with Oil Red O staining. The lesions were quantified as a percentage of total aorta area (Fig. 3B). Wild type mice had almost no atherosclerotic lesions (3.3 ± 1.0%), whereas a lack of apolipoprotein E resulted in an increase in lesion size (14.2 ± 0.9%). Interestingly, the atherosclerotic lesion size was significantly decreased in Plin1Tg/ApoeKO compared with ApoeKO (5.6 ± 0.7%, p < .01) despite the comparable body weight, visceral fat and plasma lipid levels in the two strains. Therefore, PLIN1 overexpression protects against atherosclerosis development in ApoeKO.
      Fig. 2
      Fig. 2Representative sections of atherosclerotic lesions stained with Oil Red O in aortic sinuses isolated from wild-type mice, apolipoprotein E knockout mice (ApoeKO) and Plin1Tg/ApoeKO.
      Fig. 3
      Fig. 3Atherosclerotic lesions and atheroma.
      (A) Representative sections of atherosclerotic lesions in aorta isolated from wild type mice, ApoeKO and Plin1Tg/ApoeKO and stained with Oil Red O. (B) Quantification of atheroma in aortas of wild-type mice, ApoeKO and Plin1Tg/ApoeKO. Atheroma was quantified via Oil Red O staining of lipid deposits (en face method). Results are shown as individual values and box-and-whisker plots. **p < .01 vs. wild-type. ‡p < .01 vs. Plin1Tg/ApoeKO.

      3.3 Immunohistological analysis

      We next assessed the polarities of the infiltrated macrophages and the expression of PLINs in the plaques (Fig. 4). In wild-type mice, there were almost no plaques. Neither macrophage infiltration nor expression of PLINs was evident. Conversely, both ApoeKO and Plin1Tg/ApoeKO exhibited plaques with massive macrophage infiltration. The expression level of CD11c, an M1 macrophage marker, showed higher intensity in ApoeKO than in Plin1Tg/ApoeKO, whereas no obvious difference in the expression level of the M2 macrophage marker CD206 was observed between the strains. PLIN1 expression was clearly identified in Plin1Tg/ApoeKO, but was not detected in ApoeKO. Conversely, PLIN2 expression was observed in both ApoeKO and Plin1Tg/ApoeKO, and its levels were obviously higher in ApoeKO than in Plin1Tg/ApoeKO.
      Fig. 4
      Fig. 4Macrophage infiltration and expression of lipid droplet-associated proteins in plaques.
      Frozen sections of aortic sinuses were stained with hematoxylin and anti-CD68 (macrophages), anti-CD11c (M1 macrophages), anti-CD206 (M2 macrophages), anti-PLIN1 (PLIN1) or anti-PLIN2 (PLIN2) antibody. Representative sections of atheroma plaques (ApoeKO and Plin1Tg/ApoeKO) or aortic valve base (wild-type mice) are shown.

      4. Discussion

      In this study, we explored the effect of PLIN1 overexpression on atherosclerosis progression. Our results support an atheroprotective role of PLIN1 by the observation that the extent of atheroma lesions was decreased in ApoE knockout mice overexpressing PLIN1 compared with in standard ApoE knockout mice. PLIN1 is expressed only in adipocytes and macrophages, and ordinary risk factors such as body weight, gonadal fat mass, plasma lipid levels, were not altered by PLIN1 overexpression at least in younger Plin1Tg. Accordingly, the atheroprotective property of PLIN1 was likely to be related with the effects of PLIN1 on macrophages. Similar information regarding atheroprotective role of Plin1 were recently published by Langlois et al. [
      • Langlois D.
      • et al.
      Increased atherosclerosis in mice deficient in perilipin1.
      ]. In their study, the extent of atheroma lesions was increased in LDL receptor knockout mice with PLIN1 ablated. However, we can see major limitations in the interpretation of their study. Body weight and fat mass were highly reduced by PLIN1 ablation, thus the effect of PLIN1 ablation on atheroma could include such “indirect effect” of PLIN1, apart from “direct” interaction of PLN1 with macrophages. Our study has, not only supported the atheroprotective role of PLIN1 proven by the previous studies, but also clarified the direct effect of PLIN1 on macrophages for the first time.
      In addition, we found that PLIN1 overexpression was correlated with a change in macrophage polarity in plaques; explaining in part the decrease in atherosclerosis. The balance of macrophages in plaques is dynamic and both macrophage numbers and the inflammatory phenotype influence plaque fate [
      • Moore K.J.
      • Sheedy F.J.
      • Fisher E.A.
      Macrophages in atherosclerosis: a dynamic balance.
      ]. PLIN1 has been detected in THP-1 cells and human monocyte-derived macrophages, but its role in regulating foam cell formation remains controversial [
      • Faber B.C.
      • et al.
      Identification of genes potentially involved in rupture of human atherosclerotic plaques.
      ] [
      • Hofnagel O.
      • et al.
      Expression of perilipin isoforms in cell types involved in atherogenesis.
      ] [
      • Larigauderie G.
      • et al.
      Perilipin, a potential substitute for adipophilin in triglyceride storage in human macrophages.
      ]. PLIN2 is the most abundant PAT protein in human and mouse macrophage-derived foam cells [
      • Buechler C.
      • et al.
      Adipophilin is a sensitive marker for lipid loading in human blood monocytes.
      ] [
      • Larigauderie G.
      • et al.
      Adipophilin enhances lipid accumulation and prevents lipid efflux from THP-1 macrophages: potential role in atherogenesis.
      ] [
      • Paul A.
      • et al.
      Deficiency of adipose differentiation-related protein impairs foam cell formation and protects against atherosclerosis.
      ], and augments tumor necrosis factor-alpha (TNFα), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6) induction in acetylated LDL-induced macrophages. Those observations have suggested that enhancing inflammation might be one role of PLIN2 in atherosclerosis [
      • Chen F.L.
      • et al.
      Adipophilin affects the expression of TNF-alpha, MCP-1, and IL-6 in THP-1 macrophages.
      ]. The hypothesis is supported by the study that PLIN2 gene inactivation in ApoeKO significantly reduced the number of lipid droplets in the foam cells in atherosclerotic lesions and protected mice against atherosclerosis [
      • Paul A.
      • et al.
      Deficiency of adipose differentiation-related protein impairs foam cell formation and protects against atherosclerosis.
      ].
      The close relationship between PLIN1 and PLIN2 in adipocytes has been well established. During normal cultured adipocyte differentiation, PLIN2 expression occurs first, then after several days of culture PLIN1 reciprocates PLIN2 expression. However, during PLIN1-null adipocyte differentiation, PLIN2 expression remains constant [
      • Miyoshi H.
      • et al.
      Perilipin promotes hormone-sensitive lipase-mediated adipocyte lipolysis via phosphorylation-dependent and -independent mechanisms.
      ]. In PLIN1 knockout mice, PLIN2, rather than PLIN1, coats the surface of lipid droplets in adipocytes [
      • Tansey J.T.
      • et al.
      Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity.
      ]. It is likely that the presence of PLIN2 protein reflects the lack of PLIN2 displacement by PLIN1 in the PLIN1 null animals.
      However, a different interaction between PLIN1 and PLIN2 in macrophages is possible. PLIN1 overexpression did not decrease the PLIN2 expression level noticeably in cultured macrophages derived from human monocytes (data not shown) or in peritoneal macrophages. There were no significant differences among the three groups in proinflammatory cytokine mRNA expression in adipose tissue (data not shown), and there was also no significant difference in plasma IL-6 concentration between ApoeKO and Plin1Tg/ApoeKO. Therefore, we believe that PLIN1 overexpression might induce the atheroprotective effect in Plin1Tg directly, rather than via changes in PLIN2 expression. However, to confirm that PLIN1 overexpression by itself influences atherosclerosis outcome, additional investigation should be performed in a PLIN2 knockout background.
      PLIN1 stably stores triglycerides in lipid droplets in adipocytes in the basal state. Compared with PLIN1, PLIN2 cannot store lipids effectively and induces continuous low-level lipolysis without PKA stimulation. Thus, increased lipolysis and diminished adipocyte size were observed in PLIN1 knockout mice [
      • Martinez-Botas J.
      • et al.
      Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice.
      ]. During macrophage-derived foam cell formation, PLIN2 is normally located on cytoplasmic lipid droplets [
      • Yuan Y.
      • Li P.
      • Ye J.
      Lipid homeostasis and the formation of macrophage-derived foam cells in atherosclerosis.
      ]. When an excess volume of lipids flows into a macrophage, some degree of lipolysis is sustained because the lipid storage ability of PLIN2 is lower than that of PLIN1, resulting in more fatty acid and cholesterol accumulating inside and possibly also around the macrophage. If PLIN1 is highly expressed compared with PLIN2 in macrophages, triglyceride and cholesterol ester would be stably stored in lipid droplets and fatty acid production via lipolysis would be suppressed, thus leading to the suppression of inflammation in plaques. Further study will clarify these mechanisms.
      In conclusion, overexpression of PLIN1 in macrophages protected against atheroma progression in the absence of changes in common risk factors. Changes in PLIN1 expression on lipid droplets in macrophages may play a role in the progression of atherosclerosis by modifying lipid droplet stability and subsequently influencing macrophage polarity and inflammation.

      Conflict of interest

      The authors declare they do not have anything to disclose regarding conflicts of interest with respect to this manuscript.

      Author contributions

      H Miyoshi designed the study. K Yamamoto performed the experiments and wrote the article. All authors participated in the analysis and interpretation of the data.

      Acknowledgements

      We thank N Fujimori and M Watanabe for technical assistance. HM thanks Dr. James W. Perfield II (University of Missouri, MO, USA) for his continued support and mentorship.

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