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Atheroprotective and atheroregressive potential of azapeptide derivatives of GHRP-6 as selective CD36 ligands in apolipoprotein E-deficient mice

  • Author Footnotes
    1 Joint first authors.
    Geneviève Frégeau
    Footnotes
    1 Joint first authors.
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Author Footnotes
    1 Joint first authors.
    ,
    Author Footnotes
    2 Present address: Department of Immunobiology, Center of Molecular Immunology, 216 St. & 15th Ave, Atabey, Playa, PO Box 16040, Havana 11,600, Cuba.
    Roger Sarduy
    Footnotes
    1 Joint first authors.
    2 Present address: Department of Immunobiology, Center of Molecular Immunology, 216 St. & 15th Ave, Atabey, Playa, PO Box 16040, Havana 11,600, Cuba.
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Hanan Elimam
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada

    Department of Biochemistry, Faculty of Pharmacy, University of Sadat City, Sadat City, Egypt
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  • Cloé L. Esposito
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Katia Mellal
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Liliane Ménard
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Silas D. Leitão da Graça
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Author Footnotes
    3 Present address: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27,695–8204, United States.
    Caroline Proulx
    Footnotes
    3 Present address: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27,695–8204, United States.
    Affiliations
    Department of Chemistry, Université de Montréal, Montréal, Québec, Canada
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  • Author Footnotes
    4 Present address: School of Pharmaceutical Sciences, Chongquing University, Chongquing 401,331, P. R. China.
    Jinqiang Zhang
    Footnotes
    4 Present address: School of Pharmaceutical Sciences, Chongquing University, Chongquing 401,331, P. R. China.
    Affiliations
    Department of Chemistry, Université de Montréal, Montréal, Québec, Canada
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  • Maria Febbraio
    Affiliations
    Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada
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  • Author Footnotes
    2 Present address: Department of Immunobiology, Center of Molecular Immunology, 216 St. & 15th Ave, Atabey, Playa, PO Box 16040, Havana 11,600, Cuba.
    Yosdel Soto
    Footnotes
    2 Present address: Department of Immunobiology, Center of Molecular Immunology, 216 St. & 15th Ave, Atabey, Playa, PO Box 16040, Havana 11,600, Cuba.
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • William D. Lubell
    Affiliations
    Department of Chemistry, Université de Montréal, Montréal, Québec, Canada
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  • Huy Ong
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Sylvie Marleau
    Correspondence
    Corresponding author. Faculty of Pharmacy, Université de Montréal, P.O. Box 6128, Station Centre-Ville, Montréal, Québec, H3C 3J7, Canada.
    Affiliations
    Faculty of Pharmacy, Université de Montréal, Montréal, Québec, Canada
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  • Author Footnotes
    1 Joint first authors.
    2 Present address: Department of Immunobiology, Center of Molecular Immunology, 216 St. & 15th Ave, Atabey, Playa, PO Box 16040, Havana 11,600, Cuba.
    3 Present address: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27,695–8204, United States.
    4 Present address: School of Pharmaceutical Sciences, Chongquing University, Chongquing 401,331, P. R. China.
Open AccessPublished:July 06, 2020DOI:https://doi.org/10.1016/j.atherosclerosis.2020.06.010

      Highlights

      • Azapeptide analogs of GHRP-6 were synthesized as selective CD36 ligands.
      • Azapeptides elicit lesion regression in aortic sinus in a CD36-dependent manner.
      • Azapeptides halted atherosclerotic lesion development in aorta.
      • Azapeptides induce macrophage skewing towards the anti-inflammatory phenotype.

      Abstract

      Background and aims

      Scavenger receptor class B member 3, also known as cluster of differentiation-36 (CD36) receptor, is involved in the uptake and accumulation of modified lipoprotein in macrophages, driving atherosclerosis progression. Azapeptide analogs of growth hormone-releasing peptide-6 (GHRP-6) have been developed as selective CD36 ligands and evaluated for their anti-atherosclerotic properties in apoe−/− mice.

      Methods

      From 4 to 19 weeks of age, male apoe−/− mice were fed a high fat high cholesterol (HFHC) diet, then switched to normal chow and treated daily with 300 nmol/kg of MPE-001 ([aza-Tyr4]-GHRP-6) or MPE-003 ([aza-(N,N-diallylaminobut-2-ynyl)Gly4]-GHRP-6) for 9 weeks. In another protocol, mice were fed a HFHC diet throughout the study.

      Results

      Azapeptides decreased lesion progression in the aortic arch and reduced aortic sinus lesion areas below pre-existing lesions levels in apoe−/− mice which were switched to chow diet. In mice fed a HFHC throughout the study, azapeptides reduced lesion progression in the aortic vessel and sinus. The anti-atherosclerotic effect of azapeptides was associated with a reduced ratio of iNOS+/CD206+ macrophages within lesions, and lowered plasma inflammatory cytokine levels. Monocytes from azapeptide-treated mice showed altered mitochondrial oxygen consumption rates, consistent with an M2-like phenotype. These effects were dependent on CD36, and not observed in apoe−/−cd36−/− mice.

      Conclusions

      Azapeptides MPE-001 and MPE-003 diminished aortic lesion progression and reduced, below pre-existing levels, lesions in the aortic sinus of atherosclerotic mice. A relative increase of M2-like macrophages was observed in lesions, associated with reduced systemic inflammation. Development of CD36-selective azapeptide ligands merits consideration for treating atherosclerotic disease.

      Graphical abstract

      Keywords

      1. Introduction

      A hallmark of atherosclerosis is scavenger receptor-mediated accumulation of lipid-laden cells into the intima [
      • Parthasarathy S.
      • Printz D.J.
      • Boyd D.
      • et al.
      Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor.
      ,
      • Rong J.X.
      • Shapiro M.
      • Trogan E.
      • et al.
      Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading.
      ]. Within the arterial wall, the class B scavenger cluster of differentiation-36 receptor (CD36, Scarb3) has been shown to play a prominent role in the extensive endocytosis of modified forms of low-density lipoproteins (LDL) by macrophages. Notably, CD36-mediated uptake of oxidized LDL (oxLDL) promotes an inflammatory and oxidative stress burden [
      • Endemann G.
      • Stanton L.W.
      • Madden K.S.
      • et al.
      CD36 is a receptor for oxidized low density lipoprotein.
      ,
      • Podrez E.A.
      • Febbraio M.
      • Sheibani N.
      • et al.
      Macrophage scavenger receptor CD36 is the major receptor for LDL modified by monocyte-generated reactive nitrogen species.
      ,
      • Moore K.J.
      • Sheedy F.J.
      • Fisher E.A.
      Macrophages in atherosclerosis: a dynamic balance.
      ].
      Synthetic peptide analogs of growth hormone-releasing peptide-6 (GHRP-6) (e.g. hexarelin and EP80317) have been previously shown to bind to the ectodomain of CD36 at a site overlapping that of oxLDL [
      • Demers A.
      • McNicoll N.
      • Febbraio M.
      • et al.
      Identification of the growth hormone- releasing peptide binding site in CD36: a photoaffinity cross-linking study.
      ,
      • Sabatino D.
      • Proulx C.
      • Pohankova P.
      • et al.
      Structure-activity relationships of GHRP-6 azapeptide ligands of the CD36 scavenger receptor by solid-phase submonomer azapeptide synthesis.
      ]. Administration of the synthetic peptide EP80317 in apoe-KO (apoe−/−) mice exerted both preventive and therapeutic effects on chronic atherosclerotic lesion progression [
      • Marleau S.
      • Harb D.
      • Bujold K.
      • et al.
      EP 80317, a ligand of the CD36 scavenger receptor, protects apolipoprotein E-deficient mice from developing atherosclerotic lesions.
      ] and fostered transintestinal cholesterol excretion [
      • Bujold K.
      • Mellal K.
      • Zoccal K.F.
      • et al.
      EP 80317, a CD36 selective ligand, promotes reverse cholesterol transport in apolipoprotein E-deficient mice.
      ]. The atheroprotection induced by EP80317 treatment was CD36-dependent; no effect was observed in apoe/cd36 double deficient (apoe−/− cd36−/−) mice [
      • Marleau S.
      • Harb D.
      • Bujold K.
      • et al.
      EP 80317, a ligand of the CD36 scavenger receptor, protects apolipoprotein E-deficient mice from developing atherosclerotic lesions.
      ]. In peritoneal macrophages, EP80317 elicited a modest (~20%) reduction of oxLDL internalization [
      • Marleau S.
      • Harb D.
      • Bujold K.
      • et al.
      EP 80317, a ligand of the CD36 scavenger receptor, protects apolipoprotein E-deficient mice from developing atherosclerotic lesions.
      ]. Moreover, EP80317 increased cholesterol efflux through the peroxisome proliferator-activated receptor gamma (PPARγ)-liver X receptor alpha (LXR-α)-ATP binding cassette A1/G1 (ABCA1/G1) pathway [
      • Bujold K.
      • Rhainds D.
      • Jossart C.
      • et al.
      CD36-mediated cholesterol efflux is associated with PPARgamma activation via a MAPK-dependent COX-2 pathway in macrophages.
      ]. PPARγ has been identified as a crucial transcriptional regulator of macrophage phenotypic polarization towards a non-inflammatory, M2 macrophage phenotype [
      • Chawla A.
      Control of macrophage activation and function by PPARs.
      ], thus playing an important role in the resolution of inflammation [
      • Jiang C.
      • Ting A.T.
      • Seed B.
      PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines.
      ]. Studies of GHRP-6 analogs have supported the development of more selective peptide modulators to target CD36 for the treatment of atherosclerosis. In this context, azapeptide derivatives of GHRP-6, in which the α-CH of one of the central residues was replaced by a nitrogen atom, have shown high receptor selectivity and low micromolar CD36 binding affinity [
      • Proulx C.
      • Picard E.
      • Boeglin D.
      • et al.
      Azapeptide analogues of the growth hormone releasing peptide 6 as cluster of differentiation 36 receptor ligands with reduced affinity for the growth hormone secretagogue receptor 1a.
      ]. Azapeptides have also been shown to exert anti-inflammatory activity in vitro, reducing stimulated nitric oxide production in macrophage cell lines [
      • Chignen Possi K.
      • Mulumba M.
      • Omri S.
      • et al.
      Influences of histidine-1 and azaphenylalanine-4 on the affinity, anti-inflammatory, and antiangiogenic activities of azapeptide cluster of differentiation 36 receptor modulators.
      ].
      Preclinical studies aiming to identify novel therapeutic avenues for the treatment of atherosclerosis have focused on preventing or reducing lesion progression in early phases of the pathology [
      • Baylis R.A.
      • Gomez D.
      • Owens G.K.
      Shifting the focus of preclinical, murine atherosclerosis studies from prevention to late-stage intervention.
      ]. Potential drug candidates have been mainly assessed for their ability to attenuate lesion development in early phases of atherosclerosis [
      • Charo I.F.
      • Taub R.
      Anti-inflammatory therapeutics for the treatment of atherosclerosis.
      ]; however, beneficial effects are also desirable at later phases of atherosclerosis, specifically the ability to induce plaque regression [
      • Kalanuria A.A.
      • Nyquist P.
      • Ling G.
      The prevention and regression of atherosclerotic plaques: emerging treatments.
      ,
      • Back M.
      • Yurdagul Jr., A.
      • Tabas I.
      • et al.
      Inflammation and its resolution in atherosclerosis: mediators and therapeutic opportunities.
      ,
      • Feig J.E.
      Regression of atherosclerosis: insights from animal and clinical studies.
      ].
      In addition to reduced apoB-containing lipoproteins, biomarkers of regressive mechanisms include enhancement of cholesterol and lipid efflux, reverse cholesterol transport and foam cell emigration out of lesions or clearance by efferocytosis [
      • Feig J.E.
      Regression of atherosclerosis: insights from animal and clinical studies.
      ,
      • Fisher E.A.
      Regression of atherosclerosis: the journey from the liver to the plaque and back.
      ]. Impaired efferocytosis is associated with a reduced number of phagocytic macrophages, and may perpetuate lesion inflammation and foam cell expansion, promoting plaque vulnerability [
      • Schrijvers D.M.
      • De Meyer G.R.
      • Kockx M.M.
      • et al.
      Phagocytosis of apoptotic cells by macrophages is impaired in atherosclerosis.
      ,
      • Thorp E.
      • Cui D.
      • Schrijvers D.M.
      • et al.
      Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accumulation and plaque necrosis in atherosclerotic lesions of apoe−/− mice.
      ]. CD36 expression has been observed to increase in the anti-inflammatory macrophage subtype [
      • Korns D.
      • Frasch S.C.
      • Fernandez-Boyanapalli R.
      • et al.
      Modulation of macrophage efferocytosis in inflammation.
      ]. The roles of CD36 in dysfunctional efferocytosis during atherogenesis progression and regression require additional clarification.
      In the present study, the atheroprotective potential of CD36-selective aza-GHRP-6 analogs has been further evaluated. The therapeutic effect of two novel azapeptides, MPE-001 and MPE-003, have been studied in high fat high cholesterol (HFHC)-fed apoe−/− mice. The effects of azapeptides were assessed on both atherosclerotic lesions progression and regression. Moreover, a mechanism has been delineated to explain, in part, the observed vasculoprotection afforded by the CD36-selective azapeptides.

      2. Materials and methods

      2.1 Experimental protocols

      In a first experimental setting (Fig. 1A), 4-week-old apoe−/− male mice were randomly assigned to one of four experimental groups: basal group (n = 6), which were euthanized at week 19, MPE- 001 (300 nmol/kg), MPE-003 (300 nmol/kg) or 0.9% NaCl (n = 11 per group) which were euthanized at week 28. Mice were fed a HFHC, cholate free diet (D12108c, Research Diets Inc., New Brunswick, NJ, USA) containing 20 kcal% protein, 40 kcal% fat and 1.25% cholesterol (wt/wt), until 19 weeks of age. The diet was then changed to normal chow and daily subcutaneous (s.c.) treatments with MPE-001, MPE-003 or 0.9% NaCl were initiated and pursued for 9 more weeks. Additional studies were performed in apoe−/−cd36−/− mice subjected to the same diet/treatment conditions (Fig. 3B ) (n = 8–14 mice per group).
      Fig. 1
      Fig. 1Azapeptides MPE-001 and MPE-003 induced regression of atherosclerotic lesions in apoe−/− mice.
      (A) Study design. (B) Representative photomicrographs of Oil red-O/hematoxylin-
      eosin-stained aortic sinuses (scale bar: 200 μm). (C) Percentage aortic sinus lesion areas expressed as dot plots and mean ± SEM (n = 4 per group, 2–4 sections per mice). Each symbol denotes the sections from a single mouse. (D) Representative photomicrographs of aortic arches stained en face with Oil red-O. (E) Dot plots and mean ± SEM of percentage aortic arch lesion areas (n = 6, week 19 and n = 11, mice per group). (F) Dot plots and mean ± SEM of total plasma cholesterol levels (n = 11 per group). Dotted line represents the mean basal value. (G) Mean ± SEM of IL-1β (n = 10–11), IL-6 (n = 5–7) and TNFα (n = 11) plasma levels at sacrifice. Statistical analysis was performed by one-way ANOVA followed by Tukey's Multiple Comparison post hoc test for all figures except (C) aortic sinus lesion areas (nested one-way ANOVA followed by Tukey); *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
      Fig. 2
      Fig. 2Azapeptides reduced the ratio of pro-inflammatory to anti-inflammatory macrophages in 28-week-old mice fed a HFHC diet until 19 weeks of age and then switched to chow.
      (A) Representative serial cross-sections of immunostained BCA
      sections at weeks 19 and 28, for F4/80, iNOS and CD206 antibodies at 10×
      magnification (scale bar: 100 μm). Enlarged areas (100X) are displayed from the inset (scale bar: 100 μm). (B) BCA lesion areas. (C) Absolute F4/80+ cell counts in BCA lesion areas. (D) Relative percentage of iNOS+ and CD206+ stained cells versus 0.9% vehicle. (E) Ratio of iNOS to CD206 positive cells in BCA lesions. Results are expressed as mean ± SEM of 4 (0.9% NaCl), 5 (MPE-001) and 4 (MPE-003) mice, n = 2–4 sections per mice. Dotted line represents the mean basal value. *p < 0.05, **p < 0.01, nested one-way ANOVA followed by Tukey's Multiple Comparison post hoc test.
      Fig. 3
      Fig. 3Double immunofluorescence staining of macrophages in BCA lesions of apoe−/− mice showed the colocalization of F4/80 and iNOS.
      (A) Representative staining of macrophages showing F4/80 (green), iNOS (red) and their colocalization (yellow) in BCA
      sections at 10×
      magnification (scale bar: 100 μm). Enlarged areas of the merging (100X) are displayed from the inset (scale bar: 100 μm). (B) Study design for apoe−/−cd36−/−mice. (C) Representative en face Oil red-O-stained aortic arches from apoe−/−cd36−/−. (D) Dot plots and mean ± SEM of aortic arch lesion areas. Dotted line represents the mean basal value. One-way ANOVA followed by Tukey's Multiple Comparison post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
      In another study, 4-week-old apoe−/− mice were fed a HFHC diet throughout the protocol (Fig. 4A) (n = 9–13 mice per group). A fifth group was added, in which mice were treated daily with 300 nmol/kg s.c. of [aza-Lys6]-GHRP-6 as a negative control [
      • Turcotte S.
      • Mellal K.
      • Chingle R.
      • et al.
      Azasulfurylpeptide modulation of CD36- mediated inflammation without effect on neovascularization.
      ]. At week 12, the basal group was euthanized and treatments were initiated in the other groups for 8 more weeks. Similar studies were also performed in apoe−/−cd36−/− mice (Supplemental Fig. 9A).
      Fig. 4
      Fig. 4MPE-001 and MPE-003 reduced the progression of atherosclerotic lesions in apoe−/−mice.
      (A) Study design. (B) Representative photomicrographs of aortas stained with Oil red-O. (C) Dot plots and mean ± SEM of aorta lesion areas of 11 (0.9% NaCl, MPE-001), 9 (MPE-003) and 13 ([aza-Lys6]-GHRP-6) 20-week-old mice. (D) Total plasma cholesterol expressed as dot plots. (E) Representative photomicrographs of aortic sinuses after staining with Oil red-O and hematoxylin-eosin (scale bar: 200 μm). (F) Percentage aortic sinus lesion areas showed as dot plots of 4 mice per group, 2–4 cross-sections per mice. Each symbol denotes the sections from a single mouse. Dotted line represents the mean basal value. (G) Plasma levels of CRP (n = 11), IL-6 (n = 10–11), CCL-2 (n = 11), TNFα (n = 10–11) and IL-1β (n = 9–11) at week 20. Results are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA followed by Tukey's Multiple Comparison post hoc test for all figures except (F) aortic sinus lesion areas (nested one-way ANOVA followed by Tukey) and (G) Il-1β (unpaired t-test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
      After fasting for 12 h, blood samples were drawn from the submandibular vein and plasma (heparin sodium, 10 U/mL) or serum was collected. Mice were then euthanized under isoflurane anesthesia (Fresenius Kabi, Toronto, ON, Canada), followed by exsanguination via an intracardiac puncture and perfusion with PBS.

      2.2 Azapeptides

      Azapeptide analogs of GHRP-6, MPE-001, MPE-003 and [aza-Lys6]-GHRP-6 (Supplemental Fig. 1A–C) were respectively synthetized and characterized as described previously [
      • Proulx C.
      • Picard E.
      • Boeglin D.
      • et al.
      Azapeptide analogues of the growth hormone releasing peptide 6 as cluster of differentiation 36 receptor ligands with reduced affinity for the growth hormone secretagogue receptor 1a.
      ,
      • Doan N.D.
      • Zhang J.
      • Traore M.
      • et al.
      Solid-phase synthesis of C-terminal azapeptides.
      ,
      • Zhang J.
      • Proulx C.
      • Tomberg A.
      • et al.
      Multicomponent diversity-oriented synthesis of aza-lysine-peptide mimics.
      ], and reconstituted in sterile 0.9% NaCl before injection.
      See Supplemental Materials for more detailed methods.

      3. Results

      3.1 Azapeptides reduce atherosclerotic lesions below the baseline level in aortic sinuses of apoe−/− mice after switching diet from HFHC to normal chow

      In a first setting, apoe−/− mice were fed a HFHC diet from 4 to 19 weeks of age. A group was then euthanized (to determine baseline values), whereas vehicle- and azapeptide-treated mice received their treatment and were switched to normal chow diet until week 28 (Fig. 1A).
      Both aortic sinuses and aortic crosses were evaluated for lesion areas. Representative photomicrographs of aortic sinuses from mice at week 19 (baseline) and week 28 (vehicle, MPE- 001 and MPE-003) are shown in Fig. 1B and lesion areas, expressed as a percentage of the total area, are illustrated in Fig. 1C. After reversion to normal chow for 9 weeks, the vehicle-treated group (week 28) displayed no significant difference in the mean lesion size from that of the basal group (week 19), suggesting no lesion progression after the change of diet. In contrast, mice treated with azapeptides featured mean lesion areas which were reduced by ~20% (p < 0.01) compared to pre-existing lesion areas at week 19. Treatment with azapeptides was associated with a ~30% (p < 0.01) reduction in necrotic areas within aortic sinus lesions (Supplemental Fig. 2).
      Contrary to aortic sinus lesion areas, lesions in aortic arches continued to grow from weeks 19–28 in vehicle-treated apoe−/− mice, as shown by the 63% (p < 0.01) increase in lesion areas compared to the basal group (Fig. 1D and E). Lesion areas expanded despite a halving of plasma cholesterol levels (Fig. 1F), which reached the values previously observed in apoe−/− mice fed a normal chow diet (~13 mM) [
      • Marleau S.
      • Harb D.
      • Bujold K.
      • et al.
      EP 80317, a ligand of the CD36 scavenger receptor, protects apolipoprotein E-deficient mice from developing atherosclerotic lesions.
      ]. Azapeptides MPE-001 and MPE-003 attenuated the progression of aortic arch lesion areas by 26 and 30% (p < 0.05), respectively, compared to vehicle-treated mice (Fig. 1E). Compared to vehicle treatment, azapeptides did not modulate plasma cholesterol (Fig. 1F) nor triglyceride levels (Supplemental Fig. 3). Azapeptide MPE-001, but not MPE-003, reduced plasma levels of the pro-inflammatory cytokine Il-1β by 24% (p < 0.05) (Fig. 1G). Neither body weight nor food intake were modulated by treatment with azapeptides (Supplemental Fig. 4). Furthermore, total and differential leukocytes blood counts were not altered by azapeptide treatment (Supplemental Fig. 5).

      3.2 Azapeptides reduced macrophage content and the relative numbers of pro-inflammatory macrophages in the brachiocephalic artery of apoe−/− mice after switching from HFHC to normal chow diet

      Immunostainings were performed on sequential, 4 μm cross-sections of brachiocephalic artery (BCA) from five randomly selected mice, using universal-macrophage marker EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80, EMR1), the M1-macrophage marker nitric oxide synthase 2 (iNOS, NOS2), the M2-macrophage marker mannose receptor c type 1 (CD206, MCR-1) and Lillie's trichrome to detect collagen deposition (representative images shown in Fig. 2A and Supplemental Fig. 7B). Isotype control antibodies were used to detect nonspecific binding (Supplemental Fig. 6). In a manner similar to that observed in the aortic arch, BCA lesions from apoe−/− mice increased in size from week 19 (dotted line, basal group) to week 28 by ~2-fold (p < 0.001) versus vehicle despite a change to chow diet (Fig. 2B). Compared to vehicle-treated mice, MPE-001 and MPE-003-treated mice tended to have reduced lesion areas (Fig. 2B), while MPE-003 diminished the absolute number of F4/80+ macrophages by 40% (p < 0.01) (Fig. 2C).
      However, azapeptides did not alter F4/80+ cellularity (cell count per mm2 of lesions) (Supplemental Fig. 7D). In addition, MPE-003 elicited a change towards a non-inflammatory macrophage phenotype, expressed as a relatively lower expression of iNOS+ versus CD206+ cells within plaques (Fig. 2D). In agreement, the ratio of iNOS+/CD206+ was reduced in favor of the M2-like macrophage phenotype (Fig. 2E). The shift towards the M2-macrophage phenotype associated with MPE-003 (p < 0.01) was further validated using chitinase 3-like (YM1, Chil3) as a second M2 marker (Supplemental Fig. 7E). No differences in collagen deposition were associated with azapeptide treatment (Supplemental Fig 7C). Immunofluorescent F4/80 and iNOS double staining confirmed colocalization of both markers in BCA from apoe−/− mice treated respectively, with vehicle and azapeptides (Fig. 3A).
      Treatment with MPE-001 and MPE-003 caused, respectively, 30 and 26% (p < 0.05) reductions of abdominal aortic mRNA expression of adhesion g protein-coupled receptor e1 (Adgre1), the F4/80 gene (Supplemental Fig. 8). In addition, mRNA of matrix metalloproteinase 14 (Mmp14), which is highly expressed in macrophages [
      • Ray B.K.
      • Shakya A.
      • Turk J.R.
      • et al.
      Induction of the MMP-14 gene in macrophages of the atherosclerotic plaque: role of SAF-1 in the induction process.
      ], was decreased in the MPE-003-treated group (p < 0.05) (Supplemental Fig. 8). Azapeptide treatments did not significantly modulate the mRNA levels of CD68, ABCA1, matrix metalloproteinase 2 (Mmp2) and caspase-1 (Casp1) (Supplemental Fig. 8).

      3.3 In apoe−/−cd36−/− mice, no anti-atherosclerotic effects of MPE-001 and MPE-003 were observed

      The anti-atherosclerotic effects induced by the azapeptides were shown to be CD36-dependent using apoe−/−cd36−/− mice. After switching from a HFHC to a normal chow diet and initiating treatment with azapeptide (MPE-001 or MPE-003) or vehicle (0.9% NaCl) (Fig. 3B), apoe−/−cd36−/− mice exhibited atherosclerotic lesions that tended to increase from week 19–28, as illustrated in photomicrographs (Fig. 3C), but at a lower level than those observed for apoe−/− mice. In the aortic arches of apoe−/−cd36−/− mice, neither MPE-001 nor MPE-003 were able to attenuate atherosclerosis progression relative to vehicle-treated mice (Fig. 3D). Similar results were obtained after treatment of apoe−/−cd36−/− mice maintained on a HFHC diet throughout the study (Supplemental Fig. 9).

      3.4 Azapeptides MPE-001 and MPE-003 reduced atherosclerotic lesions progression in apoe−/− mice fed a HFHC diet throughout the study

      In another setting, the atheroprotective effects of MPE-001 and MPE-003 were evaluated in male apoe−/− mice in which atherosclerotic lesions were induced by feeding a HFHC diet from 4 to 20 weeks of age. Mice were treated with a daily dose of 300 nmol/kg of azapeptides, s.c., from weeks 12–20. Azapeptide [aza-Lys6]-GHRP-6 and 0.9% NaCl were respectively used as a negative control and vehicle (Fig. 4A). Nearly 3- and 2.6-fold increases in aortic lesion areas were observed in the vehicle- and [aza-Lys6]-GHRP-6-treated groups, respectively, compared to the basal group (12 weeks of age) (Fig. 4B and C). After 8 weeks of treatment with MPE-001 and MPE-003, the extent of atherosclerotic lesion areas at the aortic level was reduced by more than 24% (p < 0.01) and 29% (p < 0.001) (Fig. 4C), respectively, relative to those of vehicle-treated mice maintained on the HFHC diet. The azapeptide negative control ([aza-Lys6]-GHRP-6) was unable to arrest atherosclerosis progression compared to vehicle. Atherosclerosis progression was inhibited under hypercholesterolemic conditions by azapeptides MPE-001 and MPE-003, which did not modulate plasma cholesterol (Fig. 4D), nor triglyceride levels (Supplemental Fig. 10). Oil red-O staining of aortic sinus sections (Fig. 4E) and morphometric quantification demonstrated an increase in lesion areas of 37% (p < 0.0001) in the vehicle group, compared to the basal group (Fig. 4F). Azapeptides MPE-001 and MPE-003 reduced aortic sinus lesion areas by 24% (p < 0.0001) and 29% (p < 0.0001), respectively, compared with those from vehicle-treated mice. In the MPE-003-treated group, the anti-atherosclerotic effect was associated with a significant reduction in circulating pro-inflammatory cytokines, including C-reactive protein (CRP) (25%, p < 0.05), IL-6 (52%, p < 0.01) and CCL-2 (40%, p < 0.05) (Fig. 4G). In the MPE-001-treated group, IL-1β was significantly reduced (60%, p < 0.05). Treatment with azapeptide had no observable effect on body weight gain nor food intake (Supplemental Fig. 11). Representative BCA cross-sections illustrated a significant reduction of lesion areas by azapeptide MPE-003 (p < 0.0001) compared to vehicle (Supplemental Fig. 12B and C); however, the absolute number of F4/80+ cells (Supplemental Fig. 12D) and F4/80+ cellularity (Supplemental Fig. 13B) were unchanged. Furthermore, MPE-003 reduced the relative expression of iNOS+ versus CD206+ cells (Supplemental Fig. 12E). In agreement, MPE-003 decreased the iNOS+/CD206+ cell ratio by 65% (p < 0.001) (Supplemental Fig. 12F). A similar trend was observed using YM1 as an additional marker of M2 macrophages (Supplemental Fig. 13C). No difference in collagen deposition was found in the BCA after azapeptide treatment (Supplemental Fig. 12G).

      3.5 Azapeptides promoted aerobic metabolic shift in bone marrow-derived monocytes

      The predominance of M2-like macrophages in BCA lesions was investigated in bone marrow-derived monocytes (BMM) from apoe−/− and apoe−/−cd36−/− mice after 12 weeks of treatment with azapeptides (Fig. 5A). The BMM from azapeptide-treated apoe−/− mice exhibited increased basal levels of oxygen consumption rates (OCR) compared to those of vehicle-treated animals (Fig. 5B). After the addition of the potent uncoupler of mitochondrial oxidative phosphorylation carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the observed increase in OCR was more than 2-fold in azapeptide-treated compared to vehicle-treated mice (Fig. 5B). On the contrary, BMM from apoe−/cd36−/− mice treated with vehicle and azapeptide, respectively, exhibited no differences in OCR levels indicating once more the CD36-dependant effect of the latter (Fig. 5C).
      Fig. 5
      Fig. 5MPE-001 and MPE-003 increased oxygen consumption rate of BMM from azapeptide-treated-mice.
      (A) Experimental protocol of the study. (B and C) Time-course of OCR in BMMs of apoe−/− and apoe−/−cd36−/− mice, respectively. Data are representative of 3 independent experiments. (D and E) Bar graphs of basal and maximal (FCCP) OCR in BMM from apoe−/− and apoe−/−cd36−/− mice. Each independent experiment was performed with four replicates per treatment. ****p < 0.0001, Kruskal-Wallis test followed by Dunn's Multiple Comparisons test.
      Azapeptides also reduced pro-inflammatory cytokine TNFα and chemokine CCL-2 secretion by activated macrophages (Supplemental Fig. 14), as reported previously [
      • Mellal K.
      • Omri S.
      • Mulumba M.
      • et al.
      Immunometabolic modulation of retinal inflammation by CD36 ligand.
      ].

      4. Discussion

      Pharmacological targeting of the CD36 receptor in male apoe−/− mice was examined under two diet regimens; HFHC diet and HFHC diet followed by normal chow. Azapeptides MPE-001 and MPE- 003 reduced lesion progression in mice fed a HFHC diet throughout the study and elicited regression at the aortic sinus level, where lesion progression was halted by the switch of diet regimen from HFHC to normal chow. The CD36-selective ligands, MPE-001 and MPE-003, reduced lesion areas below pre-existing levels in the aortic sinus and greatly attenuated lesion progression in the aortic arch of apoe−/− mice fed a HFHC diet for 15 weeks then switched to chow and treated with azapeptide for 9 weeks. Under these conditions, characterization of macrophages within the BCA lesions of treated mice revealed reduced absolute numbers and a notable change of relative cell phenotype with a reduction in pro-inflammatory (M1) markers and an increase in anti-inflammatory (M2) cell markers. Neither total plasma cholesterol nor triglyceride level was modified by azapeptide treatment. The anti-atherosclerotic effects of the azapeptides were dependent on CD36 expression, as they were not observed in apoe−/−cd36−/− mice. The azapeptides exhibited similar anti-atherosclerotic effects in mice fed a HFHC diet throughout treatment. In contrast to the striking increase in lesion areas exhibited by apoe−/− mice fed a HFHC diet between 12 (basal group) and 20 weeks, marked reductions in lesion areas were observed at the aortic arch and sinus levels of mice treated with MPE-001 and MPE-003, but not to the extent of decreasing to baseline levels. Consistently, the azapeptides caused a change in the relative amounts of macrophage pro- and anti-inflammatory markers, indicative of a reduced M1 phenotype, which coincides with the observed diminishment of inflammatory mediators in circulation.
      The roles of CD36 in atherosclerosis have been linked to Toll-like receptor (TLR)-dependent and independent pathways in early lesion formation [
      • Febbraio M.
      • Podrez E.A.
      • Smith J.D.
      • et al.
      Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice.
      ,
      • Baranova I.N.
      • Kurlander R.
      • Bocharov A.V.
      • et al.
      Role of human CD36 in bacterial recognition, phagocytosis, and pathogen-induced JNK-mediated signaling.
      ] and in plaque resolution through macrophage efferocytosis [
      • Parks B.W.
      • Black L.L.
      • Zimmerman K.A.
      • et al.
      CD36, but not G2A, modulates efferocytosis, inflammation, and fibrosis following bleomycin-induced lung injury.
      ]. In the pathogenesis of atherosclerosis, CD36 may be involved more extensively than initially anticipated. Previously, the GHRP-6-derived CD36 ligand EP80317 exhibited preventive and curative effects in atherosclerotic lesions [
      • Marleau S.
      • Harb D.
      • Bujold K.
      • et al.
      EP 80317, a ligand of the CD36 scavenger receptor, protects apolipoprotein E-deficient mice from developing atherosclerotic lesions.
      ] associated with impact on macrophage cholesterol metabolism and efflux, vascular inflammation and regulation of mononuclear cell trafficking [
      • Harb D.
      • Bujold K.
      • Febbraio M.
      • et al.
      The role of the scavenger receptor CD36 in regulating mononuclear phagocyte trafficking to atherosclerotic lesions and vascular inflammation.
      ]. Azapeptide analogs of GHRP-6 were later conceived as potent and selective CD36 ligands with CD36 binding affinities in the micromolar range [
      • Proulx C.
      • Sabatino D.
      • Hopewell R.
      • et al.
      Azapeptides and their therapeutic potential.
      ]. In particular, the series of GHRP-6 peptides possessing aza-Tyr4 and aza-Phe4 residues exhibited promising cardiovascular properties in vitro. For example, [Aza-Tyr4]-GHRP-6 (MPE-001) exerted anti-angiogenic activity [
      • Chingle R.
      • Proulx C.
      • Lubell W.D.
      Azapeptide synthesis methods for expanding side-chain diversity for biomedical applications.
      ] and also dampened the retinal inflammation induced by photo-oxidative stress under blue light exposure, which was associated with a reduced accumulation of M1-activated macrophages at the subretinal level [
      • Mellal K.
      • Omri S.
      • Mulumba M.
      • et al.
      Immunometabolic modulation of retinal inflammation by CD36 ligand.
      ]. The activity of MPE-001 correlated with the inhibition of certain pro-inflammatory pathways, such as NF-κB activation of the NLRP3 inflammasome, but initiation of others, like the anti-inflammatory PPARγ-PGC-1α pathways. The aza-Phe4 analog, CP-3 (iv), previously exhibited cardioprotective effects against myocardial ischemia-reperfusion by decreasing the generation of left ventricular reactive oxygen species and apoptosis [
      • Huynh D.N.
      • Bessi V.L.
      • Menard L.
      • et al.
      Adiponectin has a pivotal role in the cardioprotective effect of CP-3(iv), a selective CD36 azapeptide ligand, after transient coronary artery occlusion in mice.
      ]. The effects of CP-3 (iv) were associated with an increase in plasma adiponectin and a transient reduction in circulating free fatty acid levels. The observed activity of CP-3 (iv) was consistent with reduced lipolysis and dampened total fatty acid uptake in the hearts of mice treated with other CD36 ligands in the myocardial ischemia-reperfusion model [
      • Bessi V.L.
      • Labbe S.M.
      • Huynh D.N.
      • et al.
      EP 80317, a selective CD36 ligand, shows cardioprotective effects against post-ischaemic myocardial damage in mice.
      ]. In the present study, CD36-selective azapeptide analogs of GHRP-6 are now shown to reduce atherosclerosis progression and to initiate the regression of lesions at the aortic sinus level in apoe−/− mice, which were switched from a HFHC to normal diet. Despite some controversy, regression of atherosclerotic lesions has been experimentally documented and reported to be associated with reduced foam-cell accumulation [
      • Feig J.E.
      Regression of atherosclerosis: insights from animal and clinical studies.
      ] without significant change in plasma cholesterol [
      • Raffai R.L.
      • Loeb S.M.
      • Weisgraber K.H.
      Apolipoprotein E promotes the regression of atherosclerosis independently of lowering plasma cholesterol levels.
      ,
      • Posthuma J.J.
      • Posma J.J.N.
      • van Oerle R.
      • et al.
      Targeting coagulation factor xa promotes regression of advanced atherosclerosis in apolipoprotein-E deficient mice.
      ]. Regression of atherosclerotic lesions in the azapeptides-treated mice was consistent with the latter findings. Even though patients benefit from therapies lowering high LDL, some are still at risk of residual inflammatory cardiovascular events [
      • Aday A.W.
      • Ridker P.M.
      Antiinflammatory therapy in clinical care: the CANTOS trial and beyond.
      ]. Along that line, azapeptide treatment reduced systemic inflammation and diminished inflammatory macrophage numbers within lesions. These results coincide with other characteristics of lesion regression, including a reduction in foam cell accumulation and necrotic core areas within lesions [
      • Rahman K.
      • Fisher E.A.
      Insights from pre-clinical and clinical studies on the role of innate inflammation in atherosclerosis regression.
      ].
      In spite of a reduction in plasma cholesterol levels after switching apoe−/− mice from HFHC to chow, mice remained hypercholesterolemic. Therefore, lesions progressed between weeks 19 and 28, except at the level of the aortic sinus. The ability to observe regression in the aortic sinus of azapeptide-treated mice may be due, in part, to the absence of increased lesion area compared to baseline levels. In contrast, despite the change of diet, both the aortic arch and BCA displayed an increase in atherosclerotic lesion areas in vehicle-treated mice which may have impeded detection of azapeptide effects on lesion regression in these regions. Consequently, evidence of lesion regression was not observed. However, azapeptide-treated mice exhibited significant increases in markers characteristic of M2 macrophages and reduced inflammation [
      • Rahman K.
      • Fisher E.A.
      Insights from pre-clinical and clinical studies on the role of innate inflammation in atherosclerosis regression.
      ]. Moreover, mRNA levels of Adgre-1, encoding the F4/80 antigen, and of Mmp14 were both reduced, consistent with reduced macrophage accumulation at lesion sites of apoe−/− mice treated with the first generation CD36 ligand, EP80317 [
      • Harb D.
      • Bujold K.
      • Febbraio M.
      • et al.
      The role of the scavenger receptor CD36 in regulating mononuclear phagocyte trafficking to atherosclerotic lesions and vascular inflammation.
      ].
      Notwithstanding the complexity of the different phenotypic states of macrophages overtime during lesion development, regression of pre-established lesions is correlated with M2 markers [
      • Rahman K.
      • Fisher E.A.
      Insights from pre-clinical and clinical studies on the role of innate inflammation in atherosclerosis regression.
      ,
      • Stoger J.L.
      • Gijbels M.J.
      • van der Velden S.
      • et al.
      Distribution of macrophage polarization markers in human atherosclerosis.
      ]. Favoring tissue remodeling, repair and plaque stability, M2 macrophages clear lesions of dying cells and debris, and secrete anti-inflammatory mediators and cytokines [
      • Rahman K.
      • Fisher E.A.
      Insights from pre-clinical and clinical studies on the role of innate inflammation in atherosclerosis regression.
      ]. In the M2 macrophage subtype, CD36 and PPARγ expression are correlated [
      • Bouhlel M.A.
      • Derudas B.
      • Rigamonti E.
      • et al.
      PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties.
      ]. Moreover, the CD36 ligand EP80317 stimulated the PPARγ-LXRα-ABCA1/G1 transporter pathway and cholesterol efflux in macrophages [
      • Bujold K.
      • Rhainds D.
      • Jossart C.
      • et al.
      CD36-mediated cholesterol efflux is associated with PPARgamma activation via a MAPK-dependent COX-2 pathway in macrophages.
      ] and reversed cholesterol transport in apoe−/− mice [
      • Bujold K.
      • Mellal K.
      • Zoccal K.F.
      • et al.
      EP 80317, a CD36 selective ligand, promotes reverse cholesterol transport in apolipoprotein E-deficient mice.
      ]. Both PPARγ and LXRα were reported to contribute to dampening inflammation, which leads to enhanced M2-like macrophages and reduced M1 phenotype [
      • Moore K.J.
      • Sheedy F.J.
      • Fisher E.A.
      Macrophages in atherosclerosis: a dynamic balance.
      ,
      • Bouhlel M.A.
      • Derudas B.
      • Rigamonti E.
      • et al.
      PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties.
      ,
      • Moore K.J.
      • Tabas I.
      Macrophages in the pathogenesis of atherosclerosis.
      ,
      • Bensinger S.J.
      • Bradley M.N.
      • Joseph S.B.
      • et al.
      LXR signaling couples sterol metabolism to proliferation in the acquired immune response.
      ]. These observations are in agreement with the PPARγ-dependent cytoprotective effects of CD36-selective ligand MPE-001 [
      • Mellal K.
      • Omri S.
      • Mulumba M.
      • et al.
      Immunometabolic modulation of retinal inflammation by CD36 ligand.
      ] and coincide with the reduction of iNOS+/CD206+ ratio in azapeptides-treated mice in the current study.
      Furthermore, BMM isolated from apoe−/− mice fed a HFHC diet showed an increase in mitochondrial activity following treatment with azapeptides. Oxygen consumption rates were higher at basal levels, but also upon addition of the protonophore FCCP in BMM from azapeptide-treated mice. This indicates a shift to aerobic metabolism that is consistent with previous observations in bone-marrow-derived macrophages [
      • Mellal K.
      • Omri S.
      • Mulumba M.
      • et al.
      Immunometabolic modulation of retinal inflammation by CD36 ligand.
      ]. Such intracellular metabolic shift coincides with an anti-inflammatory M2 macrophage subtype, which is characterized by enhanced rates of oxidative phosphorylation and fatty acid oxidation [
      • Vats D.
      • Mukundan L.
      • Odegaard J.I.
      • et al.
      Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation.
      ] and a role for CD36 in facilitating fatty acid uptake and oxidation [
      • Wang F.
      • Zhang S.
      • Vuckovic I.
      • et al.
      Glycolytic stimulation is not a requirement for M2 macrophage differentiation.
      ]. In addition, the anti-inflammatory effect of azapeptides was shown in isolated macrophages by a reduction in cytokines secretion in a CD36-dependent manner (Supplemental Fig. 14).
      These observations and those of our previous studies [
      • Bujold K.
      • Rhainds D.
      • Jossart C.
      • et al.
      CD36-mediated cholesterol efflux is associated with PPARgamma activation via a MAPK-dependent COX-2 pathway in macrophages.
      ,
      • Harb D.
      • Bujold K.
      • Febbraio M.
      • et al.
      The role of the scavenger receptor CD36 in regulating mononuclear phagocyte trafficking to atherosclerotic lesions and vascular inflammation.
      ] are consistent with atherosclerotic plaque resolution [
      • Back M.
      • Yurdagul Jr., A.
      • Tabas I.
      • et al.
      Inflammation and its resolution in atherosclerosis: mediators and therapeutic opportunities.
      ], as they show reduced aortic lesion areas, reduced macrophage accumulation, reduced endothelial and systemic inflammation at lesion sites, as well as increased cholesterol efflux and reverse cholesterol transport in EP80317-treated mice. Moreover, the azapeptide effects were associated with polarization of macrophages to an M2-like subtype. Reduced lesion areas and absolute F4/80+ macrophage counts were observed in the BCA, a site known to develop advanced lesions in apoe−/− mice, although detection of azapeptide-induced regression in this region may have likely been impeded by diet-induced lesion progression.On the other hand, at the aortic sinus level, azapeptides reduced necrotic areas which have notably been implicated in plaque instability [
      • Rahman K.
      • Fisher E.A.
      Insights from pre-clinical and clinical studies on the role of innate inflammation in atherosclerosis regression.
      ].
      During plaque regression, little evidence exists for significant proliferation of macrophages [
      • Rahman K.
      • Vengrenyuk Y.
      • Ramsey S.A.
      • et al.
      Inflammatory Ly6Chi monocytes and their conversion to M2 macrophages drive atherosclerosis regression.
      ]. An increase expression of M2-like cell markers in the artery wall has been proposed to be attributed to the conversion of M1 macrophages to an M2-like state upon changes in the plaque environment, but could also be due to recruitment of new monocytes that become M2-like [
      • Fisher E.A.
      Regression of atherosclerosis: the journey from the liver to the plaque and back.
      ]. Although the origin of macrophages subpopulations within the different lesion sites and the immunomodulatory mechanism by which azapeptides alter macrophage numbers requires further study, ability to favor the M2-like state has probably significant beneficial consequences. The therapeutic effect of CD36-selective azapeptides on monocytes and macrophages are likely to be extended to other cell types and tissues expressing the scavenger receptor, including endothelium and vascular smooth muscle cells.
      The azapeptides also exhibited antiatherogenic effects in apoe−/− mice which were fed a HFHC diet throughout the experiment. Pivotal cytokines associated with the proatherogenic function of M1 macrophages, such as IL-1β, IL-6 and CCL-2, were decreased in circulation [
      • Ramji D.P.
      • Davies T.S.
      Cytokines in atherosclerosis: key players in all stages of disease and promising therapeutic targets.
      ,
      • Yadav A.
      • Saini V.
      • Arora S.
      MCP-1: chemoattractant with a role beyond immunity: a review.
      ]. A significant reduction in CRP plasma levels was also detected in the azapeptide-treated mice, probably as a consequence of decreasing the upstream regulator IL-6. A well-recognized indicator of inflammation, CRP expression often correlates with cardiovascular disease [
      • Koenig W.
      High-sensitivity C-reactive protein and atherosclerotic disease: from improved risk prediction to risk-guided therapy.
      ]. The results of the present study on the anti-atherosclerotic effects of azapeptides MPE-001 and MPE-003 encourage further examination. Additional studies aimed to ascertain cellular and molecular events implicated in their influence on monocyte trafficking at lesions sites, plaque macrophage apoptosis and CD36-mediated efferocytosis, remain to be done [
      • Korns D.
      • Frasch S.C.
      • Fernandez-Boyanapalli R.
      • et al.
      Modulation of macrophage efferocytosis in inflammation.
      ].

      4.1 Conclusion

      Azapeptide analogs of GHRP-6 have been shown to be potent and selective CD36 ligands. Treatment of apoe−/− mice fed a HFHC diet with such azapeptides reduced atherosclerosis progression, and elicited regression of aortic sinus lesions. These effects of azapeptides MPE-001 and MPE-003 were associated with a relative increase in M2-like macrophages within lesions. Azapeptide ligands of CD36 merit further development as a novel therapeutic avenue for treating atherosclerosis.

      Financial support

      This work was supported by the Heart and Stroke Foundation of Canada ( G-18-0022167 ), an educational grant from Mperia Therapeutics Inc., Natural Sciences and Engineering Research Council of Canada Discovery Grants (# 04079 and # 06647 ), and the Fonds de Recherche du Québec - Nature et Technologies from the Centre in Green Chemistry and Catalysis (FRQNT- 2020-RS4-265155-CCVC ).

      Declaration of competing interests

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

      CRediT authorship contribution statement

      Geneviève Frégeau: Conceptualization, Methodology, Validation, Investigation, Visualization, Project administration, Formal analysis, Writing - original draft, Writing - review & editing. Roger Sarduy: Conceptualization, Methodology, Validation, Investigation, Visualization, Formal analysis, Writing - original draft, Writing - review & editing. Hanan Elimam: Investigation, Validation, Formal analysis, Writing - original draft, Writing - review & editing. Cloé L. Esposito: Validation, Visualization, Writing - review & editing. Katia Mellal: Investigation, Visualization, Formal analysis, Writing - original draft, Writing - review & editing. Liliane Ménard: Methodology, Investigation, Visualization, Formal analysis, Writing - review & editing. Silas D. Leitão da Graça: Validation, Writing - review & editing. Caroline Proulx: Investigation. Jinqiang Zhang: Investigation. Maria Febbraio: Conceptualization, Methodology, Resources, Writing - review & editing. Yosdel Soto: Conceptualization, Writing - review & editing. William D. Lubell: Investigation, Resources, Funding acquisition, Resources, Writing - review & editing. Huy Ong: Conceptualization, Funding acquisition, Supervision, Resources, Writing - original draft, Writing - review & editing. Sylvie Marleau: Conceptualization, Methodology, Validation, Funding acquisition, Project administration, Supervision, Resources, Writing - original draft, Writing - review & editing.

      Acknowledgements

      RS received scholarship from the Ministère des relations internationales et de la francophonie du Québec and from the Emerging leaders in the America program. HE is a recipient of a grant from the Direction des affaires internationales, Université de Montréal. CP is grateful to NSERC and Boehringer Ingelheim for graduate student fellowships.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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