Role of PVAT in obesity-related cardiovascular disease through the buffering activity of ATF3

Abstract

Thoracic aortic perivascular adipose tissue (PVAT) is an adipose organ exhibiting similarities to brown adipose tissue (BAT), including cellular morphology and thermogenic gene expression. However, whether the PVAT phenotype is indistinguishable from the BAT phenotype in physiological vasculature remains unclear. We demonstrated that PVAT is distinguishable from classical BAT, given its specific vessel-tone-controlling function. Activating transcription factor 3 (ATF3) is a key factor in hypertension. Compared with wild-type mice, ATF3-deficient (ATF3 ^-/- ) mice fed a high-fat diet exhibited elevated mean arterial pressure, increased monocyte chemoattractant protein-1 expression and hypertrophy, plus abnormal fatty tissue accumulation in the thoracic aortic PVAT, and enhanced vascular wall tension and vasoconstrictive responses of potassium chloride, U46619, and norepinephrine in isolated aortic rings, which were restored after administration of adeno-associated ATF3 vector. We suggest that PVAT, not BAT, modulates obesity-related vascular dysfunction. ATF3 within PVAT could provide new insights into the pathophysiology of obesity-related cardiovascular diseases.

Keywords: Cardiovascular medicine; Molecular biology.

Graphical abstract

Figure 1

PVAT possesses the potential for resting vasodilation and attenuating contractile responses induced by vasoconstrictors This strategy was performed using inguinal WAT (iWAT) (A), suprascapular BAT (B), and thoracic aortic PVAT (C) extracts collected from WT mice fed a high-fat diet (HFD) for 22 weeks, removing PVAT and endothelial cells of the aortic arteries of control WT mice and placing arterial rings into the organ bath to assess vascular function. Representative tracing showing only the VSMCs of the aorta isolated from WT mice fed a normal diet (ND; A–C), the vascular response at the resting tone assessment for strategy 1 (D–F), and phenylephrine-induced vasocontraction (PhE; 1 μM) for strategy 2 (G–I). Determination of iWAT (A, D, and G), suprascapular BAT (B, E, and H), and thoracic aortic PVAT extracts (C, F, and I) of WT mice fed an HFD for recording the vasodilation effects at 22 weeks or the end of the experiments. PVAT extract-induced time-dependent vasorelaxation is summarized in J. Data are presented as mean ± SEM. Statistical comparisons between two groups were performed using unpaired t test (G‒I), one-way ANOVA in more than two groups (D‒F), and two-way ANOVA (J). ∗p < 0.05, compared with the control solvent group in each experiment. ns: not significant. The dotted symbols in each column represent the number of mice examined. W: wash.

Figure 2

Analysis of ATF3 expression level among aorta, kidney, heart, and liver from mouse models of hypertension and human subjects in the NCBI GEO datasets (A–E) ATF3 expression levels in the aorta (A), kidney (B), heart (C), and liver (D) of normotensive and hypertensive mouse models. The expression of ATF3 in human peripheral blood cells was assessed, as shown in (E). The dotted symbol in each column represents the number of mice examined (A–D); the number of experiments in humans = 3. Statistical comparisons were performed using unpaired t test (A‒D). Data are mean ± SEM; ∗p < 0.05 compared with the normotension (control) group in mice and the healthy group in humans. ns: not significant.

Figure 3

Deletion of ATF3 aggravated high-fat diet (HFD)-induced body weight and blood pressure abnormality regulation in mice ATF3-gene-deleted mice (ATF3^−/−) and their wild-type (WT) littermates were fed a normal diet (ND) or high-fat diet (HFD) for 22 weeks. Body weight measurements were performed after 22 weeks of ND (A) or HFD (B) feeding in both groups. Determination of systolic pressure (C–D) and diastolic pressure (E–F) of WT and ATF3^−/− mice fed ND (C–E) or HFD (D–F) for 3 days of tail-cuff recording at 22 weeks or the end of the experiment. G shows the mean arterial pressure (MAP) and heart rate (HR) represented in H. The number of experiments in body weight measurement = 14 (A–B); the systolic and diastolic pressure represented the dot symbol in each column representing the number of mice examined (G–H). Statistical comparisons were performed using one-way ANOVA (G and H) and two-way ANOVA (A‒F). Data are presented as mean ± SEM; ∗p < 0.05 compared with the WT group in each experiment. ^#Indicates a statistically significant difference among the sets of curves by the generalized estimating equation (GEE) analysis (A and B). ns: not significant.

Figure 4

ATF3-deficient mice indicated PVAT pathological conversion after HFD feeding The characterization of thoracic aortic PVAT morphology and phenotype in wild-type (WT) and ATF3^−/− mice subjected to a normal diet (ND) and high-fat diet (HFD). To compare all the pathological changes caused by HFD induction, mice fed an ND (4% kcal from fat) were used as controls. Representative image illustrating blood vessels with PVAT structure isolated from the thoracic artery of mice (A). Lower magnification shows the entire vascular system with a standard scale, indicating that HFD-fed mice caused the PVAT morphology to be significant in irregular and fat hypertrophy in ATF3-deficient mice (A). Quantification of the thoracic aortic PVAT area (B), width (C), and tissue weight (D) was performed after 22 weeks of ND or HFD feeding for both groups. Representative hematoxylin and eosin Y staining of blood vessels with PVAT structure (E), and further measured the inner diameter (blue dotted line, F), media area (yellow dotted line to blue dotted line, G), and adventitia area (red dotted line to yellow dotted line, H) in both WT and ATF3^−/− mice. The gray box shows the zoomed-in capture location of the image (E). The effects of ND and HFD feeding strategies were assessed by the pathological examination of the cross-sections of mouse aortas, as shown in the enlarged picture in E, and further quantified the A to M (A/M) ratio as collagen deposition summarized in I. Lipid droplets were classified into three groups according to diameters of <20 μm, 20–40 μm, and >40 μm. Average proportions were calculated from 100 lipid droplets in each microscopic field. The data were obtained from six field/mouse in both WT and ATF3^−/− mice fed ND (J) and HFD (K). A, adventitia; L, lumen; M, media. Scale bar: 50 μm for hematoxylin and eosin Y staining. Statistical comparisons were performed using one-way ANOVA. Data are presented as mean ± SEM. ∗p < 0.05 compared with the WT group in each experiment. ns: not significant. The dotted symbols in each column represent the number of mice examined.

Figure 5

The vascular reactivity in VSMCs is hyperresponsive to vasoconstrictor stimuli in aortic rings isolated from ATF3 deficiencies in HFD feeding To determine vascular smooth muscle responses to vasoconstrictors, both PVAT and endothelium of the aortic rings were wholly removed before myography. Challenges in normal diet (ND) and high-fat diet (HFD) ingestion in wild-type (WT) and ATF3^−/− mice preserved only in vascular smooth muscle cells (VSMCs only; EC and PVAT were denudated) in response to vasoconstrictors potassium chloride (KCl; A‒F), U46619 (G‒L), and phenylephrine (PhE)-induced vasoconstriction (M). Representative tracing showing only the VSMCs of the aorta isolated from WT and ATF3^−/− mice fed normal chow (A) or HFD (D) was induced by a cumulative-dose KCl (7.5 mM–120 mM). In B and E, the percentage (%) of contraction was normalized to the changes observed in response to treatment with 120 mM KCl. The maximal contraction to KCl was analyzed and represented by the absolute tension (T) unit in grams (g) (C and F). Following similar procedures, representative tracing in G and J showed U46619-induced concentration-dependent curve responses in ND (H) and HFD (K)-fed mice. The maximal contraction induced by U46619 represented the absolute tension (T) unit as gram (g), shown in ND feeding (I) or HFD feeding (L). The α1-adrenergic receptor agonist phenylephrine (PhE) at 3 μM was administered to induce constriction for comparison between WT and ATF3^−/− mice in the HFD challenge (M). Compared with WT littermates, sodium nitroprusside (SNP) led to a significant reduction in PhE-induced contraction in the original tracing (N); however, there was no difference in either group (N and O). The dot symbol in each column represents the number of mice examined (C, F, I, L, and M). Number of experiments in SNP-induced relaxation = 5 (O). Statistical comparisons were performed using unpaired t test (C, F, I, L, and M). Figures B, E, H, K, and O were performed by two-way ANOVA. Data are mean ± SEM; ∗p < 0.05 compared with the WT group in each experiment and single concentration application of the concentration curve. ^#Indicates a statistically significant difference among the sets of curves by generalized estimating equation (GEE) analysis. ns: not significant. W: wash.

Figure 6

ATF3 deficiency in PVAT and VSMCs enhances norepinephrine-induced vascular contractility on HFD feeding Representative tracing showing concentration-dependent contractions of norepinephrine (NE) in thoracic aortic arteries with PVAT (A) and removal of PVAT (B) from wild-type (WT) littermates and ATF3-deficient (ATF3^−/−) mice 22 weeks after HFD-induced obesity (A and B). The concentration-response curve shows the logarithm of NE-induced vasoconstriction in the presence of PVAT (C) and the absence of PVAT is referred as VSMC only (D). (E) The quantitative analysis of the constriction tension (T) of isolated aortic rings in response to maximal concentration of NE (1 μM). +PVAT represents preserved with PVAT structure; −PVAT represents the removal of the PVAT structure shown in panel E. Statistical analysis of correction in thoracic aortic PVAT weights and NE-induced contractility in both groups subjected to the HFD (F). The response to treatment with 120 mM KCl was considered 100% contraction. The percentage (%) of NE-induced contractions was normalized to that of KCl. The number of experiments in the NE-induced contraction was six (C and D). Dotted symbols in each column represent the number of mice examined (E). A statistical comparison was performed using one-way ANOVA in E and two-way ANOVA in C and D. Data are mean ± SEM; ∗p < 0.05 compared with the WT group in each experiment and single concentration application of the concentration curve. ^#Indicates a statistically significant difference among the sets of curves by generalized estimating equation (GEE) analysis. SNP: sodium nitroprusside.

Figure 7

ATF3-deficient PVAT failed to elicit an anticontractile effect Representative trace in (A) showing concentration-response curves to serotonin (5-HT) in intact(+) thoracic aortic PVAT preparations and aortic rings without EC and PVAT (VSMC only). Quantitative analysis of constriction percentage (%) of aortic rings in response to 5-HT on vessel rings with PVAT (B) or without PVAT (C). Aortic rings with PVAT (+PVAT/VSMC) in (D) and only preserved vascular smooth muscle cells (VSMCs) in F from WT and ATF3-deficient (ATF3^−/−) mice were exposed to an increasing concentration of 5-HT. Each bar graph inset in (D and F) represents the percentage of contraction when each concentration of 5-HT was applied to the organ chamber. The total area under the curve (AUC) of the concentration-response curve is expressed as an arbitrary unit (AU) in E and G. Overlapping the whole area profiles in arteries with PVAT and preserved VSMC in both groups for comparison (H), summarized data from H are expressed as Δabc in I. The number of experiments on 5-HT-induced contractions was 10 (B and C). The dot symbol in each column represents the number of mice examined (E, G, and I). Statistical comparisons were performed using unpaired t test in E, G, and I and two-way ANOVA in B and C. Data are mean ± SEM; ∗p < 0.05 compared with the WT group in each experiment and single concentration application of the concentration curve. ^#Indicates a statistically significant difference among the sets of curves by generalized estimating equation (GEE) analysis. ns: not significant.

Figure 8

ATF3 influences vascular reactivity via obese PVAT-mediated ADCF MCP-1 release and downregulated adiponectin receptor expression in VSMCs Protein levels of adipokine and inflammation-related expression in wild-type (WT) and ATF3^−/− mice of thoracic aortic PVAT after HFD feeding for 22 weeks by adipokine assays (A), and the ImageJ analyzer software was used for quantification of densitometry of blots in B‒G. Protein levels of MCP-1 (H) and adiponectin (I) in PVAT tissue and adipoR1 in VSMC (J) were examined by immunoblot assay and summarized in a bar graph presented as the relative densities (%) for each dependent experiment in both groups. Actin was used as the internal control. Bar graphs in K and L quantify RT-PCR analysis of mRNA levels of MCP-1 (K) and adiponectin (L) in PVAT of WT and ATF3^−/− mice. Representative double-immunofluorescence (IF) images of MCP-1 (red IF) and adiponectin (green IF) in WT and ATF3^−/− mice (M). In N, images of adipoR1 (red IF) and SM22 (green IF) are shown. The yellow line indicates the Z-plot measurement of fluorescence intensity. The fluorescence intensity changes are shown in O for MCP-1, P for adiponectin, Q for adipoR1, and R for the SM22 VSMC marker for comparison. The fluorescence intensity was determined from six field/mouse (n = 3) in both WT and ATF3^−/− mice fed an HFD (O‒R). Scale bar for image: 100 μm; enlarged picture illustrates scale bar: 50 μm. Arrow indicates MCP-1 expression of PVAT in panel M and adipoR1 expression of VSMC in N. The dotted symbols in each column represent the number of mice examined. Statistical comparisons were performed using unpaired t test. Data are presented as mean ± SEM; ∗p < 0.05 compared with the WT group in each experiment. ns: not significant.