Sex-Specific Effects of Cholesteryl Ester Transfer Protein (CETP) on the Perivascular Adipose Tissue

Abstract

Cholesteryl ester transfer protein (CETP) increases the atherosclerosis risk by lowering HDL-cholesterol levels. It also exhibits tissue-specific effects independent of HDL. However, sexual dimorphism of CETP effects remains largely unexplored. Here, we hypothesized that CETP impacts the perivascular adipose tissue (PVAT) phenotype and function in a sex-specific manner. PVAT function, gene and protein expression, and morphology were examined in male and female transgenic mice expressing human or simian CETP and their non-transgenic counterparts (NTg). PVAT exerted its anticontractile effect in aortas from NTg males, NTg females, and CETP females, but not in CETP males. CETP male PVAT had reduced NO levels, decreased eNOS and phospho-eNOS levels, oxidative stress, increased NOX1 and 2, and decreased SOD2 and 3 expressions. In contrast, CETP-expressing female PVAT displayed increased NO and phospho-eNOS levels with unchanged NOX expression. NOX inhibition and the antioxidant tempol restored PVAT anticontractile function in CETP males. Ex vivo estrogen treatment also restored PVAT function in CETP males. Moreover, CETP males, but not female PVAT, show increased inflammatory markers. PVAT lipid content increased in CETP males but decreased in CETP females, while PVAT cholesterol content increased in CETP females. CETP male PVAT exhibited elevated leptin and reduced Prdm16 (brown adipocyte marker) expression. These findings highlight CETP sex-specific impact on PVAT. In males, CETP impaired PVAT anticontractile function, accompanied by oxidative stress, inflammation, and whitening. Conversely, in females, CETP expression increased NO levels, induced an anti-inflammatory phenotype, and preserved the anticontractile function. This study reveals sex-specific vascular dysfunction mediated by CETP.

Keywords: CETP; oxidative stress; perivascular adipose tissue.

Graphical Abstract

Figure 1.

CETP expression impairs the anticontractile effect of PVAT in male but not in female mice. Concentration-response curves to phenylephrine (PE) in aortas with (PVAT+, empty circles) or without PVAT (PVAT−, filled circles) from male NTg to hCETP mice (A) and from female NTg to hCETP mice (B). PE-induced contraction is expressed as millinewton (mN) per aorta length in millimeters (mm). Bar graphs represent maximum responses (Emax) to PE in males (A) and females (B). Data are mean ± SE; the number of animals in each group is in parenthesis. *P ≤ .05 and **P ≤ .01 by ANOVA two-way.

Figure 2.

Male and female hCETP-transgenic mice present similar phenotypes. Plasma CETP activity and hCETP mRNA expression in aorta and PVAT from males to females (A). Plasma lipoprotein profile and quantification in male and female NTg, male and female hCETP mice (B). Bars show mean ± SE of individual values. **P ≤ .01, ***P ≤ .001, and ****P ≤ .0001 by Student’s t-test. #P ≤ .05 and ##P ≤ .01 by Mann-Whitney.

Figure 3.

CETP expression modulates NO levels, eNOS expression, and phosphorylation in a sex-dependent way. NO production and eNOS mRNA expression in PVAT from male (A) to female (C) NTg and hCETP mice. Representative image and quantification of eNOS and phospho-eNOS protein expression in PVAT from male (B) to female (D) NTg and hCETP mice. Nuclei were stained with DAPI. Scale bar = 100 µm. Gene expression data are corrected by Rplp0 internal control and protein expression by β-actin protein levels. Data are mean ± SE, expressed relative to NTg controls (fold change). *P ≤ .05, **P ≤ .01 and ***P ≤ .001 by the Student’s t test.

Figure 4.

CETP expression induces oxidative stress in PVAT from males only, while NOX inhibition as well as SOD mimetic and 17β-estradiol restore the PVAT anticontractile function in CETP male mice. DHE fluorescence was evaluated in PVAT with or without the NOS inhibitor (L-NAME) in male (A) and female (C) NTg and hCETP mice. mRNA expression of NADPH oxidase subunits NOX1, NOX2 (Cybb), and NOX4, as well as superoxide dismutase (SOD) 1, 2, and 3, were evaluated in PVAT from male (B) and female (D) NTg and hCETP mice. Concentration-response curves to phenylephrine (PE) in aortas with PVAT+, with or without NOX nonspecific inhibitor DPI, NOX2 specific inhibitor GSK2795039, SOD mimetic TEMPOL, and 17β-Estradiol in male NTg (E) and male hCETP mice (F). Bar graphs represent maximum responses (Emax) to PE in NTg (E) and hCETP (F) males. Nuclei were stained with DAPI. Scale bar = 100 µm. Gene expression data are corrected by the Rplp0 internal control. Data are mean ± SE, expressed relative to NTg controls (fold change). A to D *P ≤ .05 and ***P ≤ .001 by Student’s t-test. E and F ***P ≤ .001 by one-way ANOVA.

Figure 5.

CETP expression induces sex-specific effects on the expression of pro- and anti-inflammatory markers in the PVAT of male and female mice. mRNA expression of markers of general macrophages (F4/80, CD68), pro-inflammatory markers (CD11c, CD80, TNF-α, and iNOS), and anti-inflammatory markers (CD163, CD206, IL-10, and ARG1) in male NTg and hCETP mice (A) and female NTg and hCETP mice (B). Gene expression was corrected by the Rplp0 internal control. Data are mean ± SE, expressed relative to NTg (fold change). *P ≤ .05 and **P ≤ .01 by the Student’s t-test.

Figure 6.

CETP expression induces sex-dependent alterations in PVAT morphology and adipose markers. PVAT adipocyte morphology by HE staining and quantification of fat area from male NTg to hCETP mice (A) and female NTg and hCETP mice (B). Gene expression of adipokines and its receptors and markers of brown (BAT) and white (WAT) adipocytes in PVAT from male NTg to hCETP mice (C) and female NTg to hCETP mice (D). Scale bar = 50 µm. Typical images obtained with a 40× objective. Gene expression is relative to Rplp0 control. Data are mean ± SE in % area or fold-change. *P ≤ .05 and **P ≤ .01 by the Student’s t-test. ##P ≤ .01 by Mann-Whitney. Abbreviations: Adipoq: adiponectin, Lep: leptin, Retn: resistin, Adipor1: adiponectin receptor 1, Ob-rb (Lepr): leptin receptor long form, Ucp-1: uncoupling Protein 1, Prdm16: PR/SET Domain 16, Cidea: cell death inducing DFFA like effector A, Nrip1: nuclear receptor interacting protein 1, Tmem26: transmembrane protein 26, Tbx1: tata-box transcription factor 1.