Beiging of perivascular adipose tissue regulates its inflammation and vascular remodeling

Adachi, Yusuke; Ueda, Kazutaka; Nomura, Seitaro; Ito, Kaoru; Katoh, Manami; Katagiri, Mikako; Yamada, Shintaro; Hashimoto, Masaki; Zhai, Bowen; Numata, Genri; Otani, Akira; Hinata, Munetoshi; Hiraike, Yuta; Waki, Hironori; Takeda, Norifumi; Morita, Hiroyuki; Ushiku, Tetsuo; Yamauchi, Toshimasa; Takimoto, Eiki; Komuro, Issei

doi:10.1038/s41467-022-32658-6

Download PDF

Article
Open Access
Published: 07 September 2022

Beiging of perivascular adipose tissue regulates its inflammation and vascular remodeling

Nature Communications volume 13, Article number: 5117 (2022) Cite this article

8177 Accesses
13 Citations
15 Altmetric
Metrics details

Subjects

Abstract

Although inflammation plays critical roles in the development of atherosclerosis, its regulatory mechanisms remain incompletely understood. Perivascular adipose tissue (PVAT) has been reported to undergo inflammatory changes in response to vascular injury. Here, we show that vascular injury induces the beiging (brown adipose tissue-like phenotype change) of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism. In a mouse model of endovascular injury, macrophages accumulate in PVAT, causing beiging phenotype change. Inhibition of PVAT beiging by genetically silencing PRDM16, a key regulator to beiging, exacerbates inflammation and vascular remodeling following injury. Conversely, activation of PVAT beiging attenuates inflammation and pathological vascular remodeling. Single-cell RNA sequencing reveals that beige adipocytes abundantly express neuregulin 4 (Nrg4) which critically regulate alternative macrophage activation. Importantly, significant beiging is observed in the diseased aortic PVAT in patients with acute aortic dissection. Taken together, vascular injury induces the beiging of adjacent PVAT with macrophage accumulation, where NRG4 secreted from the beige PVAT facilitates alternative activation of macrophages, leading to the resolution of vascular inflammation. Our study demonstrates the pivotal roles of PVAT in vascular inflammation and remodeling and will open a new avenue for treating atherosclerosis.

Introduction

Inflammation is deeply involved in the development of cardiovascular diseases, particularly in atherosclerosis as responses to vascular injury^1,2,3. Temporal inflammation and its resolution are required for wound healing and repair after vascular injury, and prolonged excessive inflammation causes pathological vascular remodeling that manifests as neointimal hyperplasia^4,5. It is widely accepted that the pathogenesis of vascular remodeling starts from the impaired endothelial cell function, leading to recruitment of circulating inflammatory cells to the intima–medial layer^3,6,7. The effects of potent vascular protective agents such as estrogen, statins and microRNAs have been mostly attributed to their beneficial functions on endothelial cells^8,9,10,11,12. There is also growing evidence suggesting that perivascular adipose tissue (PVAT), which accounts for the majority of the outer matrix surrounding systemic blood vessels, contributes to the pathophysiological vascular response in an ‘outside-in’ manner¹³. Adipose tissues are classified into two distinct phenotypes; white adipose tissue (WAT), which stores energy as triglycerides, and brown adipose tissue (BAT), which dissipates energy as heat¹⁴. A brown adipocyte-like phenotype has been reported to emerge within WAT in response to various stimuli such as cold exposure and β3-adrenergic receptor (β3AR) agonist^14,15. This unique phenomenon, called ‘browning’ or ‘beiging’, is controlled by transcriptional regulators, such as PRDM16, NFIA and EBF2^{16,17,18,19,20,21,22}, and observed in humans as well as in rodents, contributing to energy homeostasis^14,15. Recent basic and clinical studies reported that human PVAT surrounding coronary arteries show phenotypic changes in response to vascular inflammation, which can be detected by newly innovated computerized tomography-based methods^23,24. Although these findings suggest an association between PVAT phenotypic changes and vascular diseases, the causality and mechanisms involved remain unclear.

In this work, we examined the roles of PVAT in vascular inflammation and subsequent pathological remodeling. We show that vascular injury induces the beiging of PVAT, which fine-tunes inflammatory response and thus vascular remodeling as a protective mechanism.

Results

Endovascular injury induces PVAT beiging

We generated endovascular injury by inserting a wire into the femoral arteries (FAs) and examined inflammatory response in PVAT. Given the considerable effects of estrogen on the vasculature^25,26, male or ovariectomized female mice were used in the study. Genes of various types of immune cells were upregulated in PVAT at 24 h after vascular injury, including the monocyte-macrophage marker genes, Cd11b, Cd11c, Mrc1, and Adgre1 (F4/80) (Fig. 1a). Immunohistochemical staining revealed that F4/80⁺ macrophages accumulated predominantly in the outer tissues surrounding the vasculature, which were mainly comprised of PVAT, rather than in the vascular tissues, at the early phase (3 days) after injury (Fig. 1b, c). These F4/80⁺ macrophages then spread into the vascular tissues at the late phase (14 days) after injury (Fig. 1b, c). Concurrently, the expressions of inflammatory cytokine genes such as Tnf (TNF-α), Serpine1 (PAI-1), Il1a and Il1b, were significantly upregulated in the surrounding tissues after vascular injury (Fig. 1d).

**Fig. 1: Endovascular injury-induced macrophage accumulation and inflammation in PVAT followed by upregulation of BAT markers.**

Transcriptome analysis of PVAT surrounding injured or sham-operated FAs showed that in addition to the increase in expression levels of several genes encoding immune cell markers and inflammatory cytokines, vascular injury induced a significant increase in expression levels of BAT marker genes including Ucp1, Cidea, Cox8b, Ppargc1a, Elovl3 and Dio2 in the PVAT (Supplementary Fig. 1a), which was confirmed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis (Fig. 1e). Upregulation of UCP1 in PVAT after vascular injury was confirmed by in situ hybridization analysis (Fig. 1f) and immunohistochemical analysis (Fig. 1g, h). Morphologically, adipocytes in PVAT after vascular injury possessed the beige/BAT characteristics including smaller cell size and multilocular lipid droplets compared with those after sham operation (Fig. 1g, i). Western blot analysis also showed the increase in UCP1 exclusively in the outer tissues denuded from vessels rather than the remained arteries (Fig. 1j). Then, we examined the role of infiltrated macrophages in the upregulation of UCP1. Reduction of macrophages by the administration of clodronate liposome significantly attenuated upregulation of UCP1 after vascular injury (Fig. 1k, l). The effects of ovariectomy on the expression levels of BAT markers, such as Ucp1 and Elovl3, were marginal, while the upregulation of these markers after vascular injury was more prominent in ovariectomized mice than in controls (Supplementary Fig. 1b). A significant upregulation of UCP1 and the morphological shrinkage of adipocyte cell size in PVAT after vascular injury were observed in intact gonadal male mice (Supplementary Fig. 1c, d). Taken together, these results suggest that in male and ovariectomized female mice‚ vascular injury elicits the infiltration of macrophages in PVAT, which induces the change of PVAT into BAT-like phenotypes, resembling the “beiging”-phenomenon that is observed in subcutaneous and visceral WAT following exposure to cold or β3AR agonists.

PVAT beiging alters vascular inflammatory response

We next investigated the pathophysiological roles of the PVAT beiging in the development of vascular remodeling after injury using mice with an adipocyte-specific deletion of Prdm16 gene, an essential regulator of beiging^16,17,18, using Adipoq (Adiponectin)–Cre^+/−;Prdm16^fl°^x/fl°^x mice, referred to as Adipo^Cre+;Prdm16. Prdm16 mRNA expression was >90% lower in adipose tissues such as thoracic aorta PVAT and intra-scapular BAT compared with Adipo^Cre−;Prdm16 control littermates, despite comparable expression in other tissues such as lung and intestine (Supplementary Fig. 2a). Vascular injury induced more prominent remodeling accompanied by marked inflammation and suppressed beiging in the PVAT in both males and ovariectomized females of Adipo^Cre+;Prdm16 mice compared with Adipo^Cre−;Prdm16 mice at 14 days after injury (Fig. 2a, b and Supplementary Fig. 2b, c). To rule out the effects of Prdm16 deletion in adipose tissues of the whole body, we locally repressed Prdm16 by applying pluronic gel containing small interfering RNA (siRNA) against Prdm16 around FAs^27,28. A significant reduction of Prdm16 mRNA expression was confirmed in the outer tissues surrounding FA of wild type mice after siRNA treatment, whereas no difference was observed in contralateral untreated FA (Supplementary Fig. 2d). The Prdm16 siRNA treatment abolished PVAT beiging after vascular injury (Supplementary Fig. 2e) and exacerbated pathological intimal thickening 14 days after vascular injury (Fig. 2c).

Macrophages change their phenotypical and functional characteristics, termed macrophage polarization, in response to pro- or anti-inflammatory stimuli²⁹. Although the accumulation of F4/80⁺ cells was comparable between locally beiging-suppressed FA and control FA (Fig. 2c), the ratio of inducible nitric oxide synthase (iNOS)⁺ activated inflammatory macrophages to CD206⁺ alternatively activated anti-inflammatory macrophages was significantly increased in the beiging-suppressed FA PVAT as compared with that of control FA PVAT 14 days after vascular injury (Fig. 2c). These significant differences were not observed during the early phase (3 days) after injury (Supplementary Fig. 3a, b), suggesting that inhibition of PVAT beiging exacerbates pathological remodeling after vascular injury by prolonged activation of pro-inflammatory macrophages.

The effects of systemic activation of WAT beiging by exposure to cold or β3AR agonists on vascular pathogenesis in vivo remain controversial and depend on the experimental models studied^30,31. These conflicting results are considered to come from direct and/or indirect effects of systemic activation of beiging on vascular remodeling, such as brown/beige fat-mediated lipolysis, release of anti-inflammatory adipokines and glucose homeostasis^30,31,32. Therefore, we locally activated beiging of the FA PVAT using pluronic gel containing the specific β3AR agonist, CL316243 and evaluated the specific effects of PVAT beiging on the phenotypic transition of macrophage and vascular remodeling. Local β3AR activation significantly induced PVAT beiging in the outer surrounding tissue of FA, but not in the contralateral untreated FA, visceral WAT or BAT (Supplementary Fig. 3c), and significantly attenuated vascular intimal thickening 14 days after injury with a shift of macrophage polarization to anti-inflammatory phenotype in FA PVAT (Fig. 2d and Supplementary Fig. 3d). These significant differences were not observed during the early phase after injury (Supplementary Fig. 3a, b). In contrast, the beiging in PVAT elicited by vascular injury was attenuated by a β3AR antagonist (SR59230A) with pluronic gel (Supplementary Fig. 3e). These data suggest that pharmacological beiging stimulation exerts additional protection against pathological remodeling after vascular injury by suppression of prolonged activation of pro-inflammatory macrophages, and that the beiging in PVAT after the injury is mediated by the β3AR signaling.

Then, we examined the interactions between PVAT-derived beige adipocytes and macrophages. When RAW 264.7 macrophage cells or bone marrow-derived macrophages (BMDMs) were cultured in medium conditioned by beige adipocytes differentiated from the stromal vascular fraction of PVAT, expression levels of Mrc1 (CD206) were increased and those of inflammatory cytokines such as Tnf, Serpine1, and Il1b were decreased (Fig. 3a-d). These changes were abrogated by inhibition of PVAT beiging by siRNA knockdown of Prdm16 (Fig. 3e, f and Supplementary Fig. 3f). We further examined the effects of PVAT beiging on cell growth of macrophages. Culture medium conditioned by PVAT-derived beige adipocytes significantly decreased the number of RAW 264.7 macrophages activated by interferon-gamma (IFNγ) and lipopolysaccharide (LPS), but not that of macrophages alternatively activated by interleukin (IL) 4 (Fig. 3g and Supplementary Fig. 3g). Culture medium conditioned by PVAT-derived adipocytes with knockdown of Prdm16 did not decrease the number of classically activated macrophages induced by IFNγ and LPS (Fig. 3g). Taken together, these results suggest that factors secreted from PVAT-derived beige adipocytes selectively inhibit the growth of classically activated macrophages and shift macrophage phenotypes into an alternatively activated state, resulting in anti-inflammatory effects.

**Fig. 3: Beige-differentiated PVAT shifts macrophage phenotypes to alternatively activated state.**

NRG4 as an anti-inflammatory factor

Some studies have shown the contributions of UCP1 to atherogenesis^{30,32,33,34,35}. Therefore, we first examined UCP1 as a possible regulator of vascular remodeling after injury. Unexpectedly, vascular wall thickening 14 days after injury was comparable between Ucp1^−/− and littermate wild type (Ucp1^+/+) mice (Fig. 3h), suggesting that UCP1 is not involved in the beneficial effects of PVAT beiging on vascular remodeling.

To identify the key factors that control endovascular injury-induced inflammation in PVAT, we performed transcriptional RNA profiling using single-cell RNA sequencing (scRNA-seq) and subsequent computational analysis using publicly available scRNA-seq datasets of murine inguinal WAT (iWAT) treated with CL316243 or control (GSE 133486)³⁶. Cluster analysis of uniform manifold approximation and projection (UMAP) dimensionality reduction after integration with Seurat revealed that the cells of iWAT were classified into 15 clusters (C0–14) and that C7_(Seurat) mostly consisted of CL316243-treated cells (Fig. 4a, b and Supplementary Fig. 4a). Beige/brown adipocyte-specific genes such as Ucp1, Cidea and Ppargc1a, were predominantly expressed in C7_(Seurat) (Fig. 4c and Supplementary Figs. 4b, 5), indicating that this cluster represents beige adipocytes induced by β3AR activation. The emergence of the beige adipocyte cluster by β3AR activation with CL316243 was also confirmed using another integration method, Harmony (C6_(Harmony), Supplementary Figs. 6a–f and 7). In addition, the cell type deconvolution analysis of the bulk RNA-seq dataset of iWAT (GSE129083)³⁷ showed that the beige adipocyte population appeared in iWAT treated with CL316243 (Supplementary Table 2). Furthermore, the analysis of another dataset (GSE 133486)³⁶ demonstrated that β3AR activation with cold stimuli also induced the emergence of a beige adipocyte population (C9_(cold)), Supplementary Figs. 8a–e and 9). Gene ontology analysis for the significantly regulated genes in C7_(Seurat) identified Nrg4 as a highly expressed secretory factor involved in the molecular function of receptor-binding (Fig. 4c, d). The enrichment of Nrg4 was also detected in C6_(Harmony) and C9_(cold) (Supplementary Figs. 6d, e and 8c, d).

**Fig. 4: Identification of NRG4 as beige PVAT-derived secretory factor that controls macrophage phenotypes and growth.**

Consistent with the findings from the comprehensive data analysis, we observed that Nrg4 was upregulated in PVAT after vascular injury (Fig. 4e) and in cultured PVAT-derived preadipocytes stimulated by beige adipocyte differentiation factors, accompanied by other beige genes in a time-dependent manner (Fig. 4f). The upregulation of Nrg4 in PVAT surrounding injured FAs was also observed 14 days after injury by in situ hybridization analysis (Supplementary Fig. 10a), where Nrg4 was expressed in beige adipocytes of PVAT expressing Ucp1 (Supplementary Fig. 10b). Nrg4 upregulation was significantly attenuated in Adipo^Cre+;Prdm16 mice and PVAT-derived preadipocytes transduced with siRNA against Prdm16 or Nfia (Fig. 4g, h), whereas it occurred independently of UCP1 (Supplementary Fig. 10c). NRG4 has recently emerged as a brown fat-enriched secreted factor that ameliorates diet-induced metabolic disorders, including insulin resistance and hepatic steatosis^38,39,40. We thus examined whether NRG4 was involved in beige adipocytes-induced resolution of macrophage inflammation. Culture media conditioned by PVAT-derived beige adipocytes induced phenotypic changes from classical to alternative activation and downregulation of inflammatory genes in RAW 264.7 macrophages or BMDMs, but these changes were not observed by the culture media conditioned by PVAT-derived beige adipocytes with knockdown of Nrg4 by siRNA (Supplementary Fig. 10d and Fig. 4i–l). Consistently, recombinant murine NRG4 attenuated the Cd86/Mrc1 ratio and inflammatory cytokine levels in macrophages classically activated (Supplementary Fig. 10e, f). The knockdown of Erb-B2 receptor tyrosine kinase 4 (ErbB4), a receptor of NRG4⁴¹, in macrophages abolished the alternative activation and anti-inflammatory effects of culture media conditioned with PVAT-derived beige adipocytes (Supplementary Fig. 10g–i). Furthermore, culture media conditioned by PVAT-derived beige adipocytes with Nrg4 knockdown no longer inhibited the increase in the number of RAW 264.7 cells activated by IFNγ and LPS (Fig. 4m). In mice, Nrg4 siRNA treatment with pluronic gel was found to exacerbate pathological intimal thickening 14 days after vascular injury (Fig. 4n). Taken together, these results suggest that NRG4 secreted from PVAT-derived beige adipocytes inhibits the growth of classically activated macrophages and induces phenotypic shift to an alternatively activated state, leading to accelerated resolution of macrophage inflammation, resulting in attenuation of pathological vascular remodeling after injury.

PVAT beiging is detectable in human AAD

Acute aortic dissection (AAD) is the acute destruction of aortic wall that accompanies severe local inflammation⁴². We examined whether beiging was recognized in PVAT of human aorta with AAD. In addition to the accumulation of macrophages, protein expression levels of markers for beige cells, such as UCP1 and CIDEA, and NRG4 were significantly increased likely as a compensatory response in the PVAT of AAD lesions compared with the PVAT without AAD (Fig. 5a, b and Supplementary Fig. 11a, b). To elucidate the role of PVAT beiging in AAD, we used an AAD murine model with the systemic administration of angiotensin II, β-aminopropionitrile, and N-nitro-L-arginine methyl ester^43,44,45. CL316243 administration significantly reduced death due to dissection or rupture of the aorta (Fig. 5c), concomitant with the shift of macrophage phenotypes in PVAT to the alternatively activated state evaluated by fluorescence-activated cell sorting (FACS) analysis (Fig. 5d, e and Supplementary Fig. 12). These findings suggest that beiging occurs in the PVAT of the human aorta during acute dissection and may regulate the inflammatory response during the development of AAD.

**Fig. 5: PVAT beiging in human and murine aorta with acute dissection.**

Discussion

Vascular damage provokes regional inflammation and prolonged inflammation leads to pathological vascular remodeling^4,5. The balance of pro- and anti-inflammation is critical for successful vascular wound healing and clinical outcomes^46,47. The primary process of inflammation resolution is accomplished by the phenotypic conversion of pro-inflammatory macrophages into anti-inflammatory macrophages⁴⁷. However, the mechanisms involved in this conversion have not yet been fully elucidated. This study revealed that endovascular injury induced ‘beiging’ of regional PVAT, and that inhibition of PVAT beiging exacerbated thickening of the intima after injury accompanied by an increased accumulation of pro-inflammatory macrophages, while activation of beiging conversely attenuated pathological vascular remodeling and accumulation of pro-inflammatory macrophages. These findings suggest that beiging of PVAT plays a pivotal role in changing the initial inflammation phase to its resolution after vascular injury. Various studies have suggested that endothelial dysfunction leads to the recruitment of circulating monocytes to the intima–medial layer^3,6. In our study, the marked accumulation of monocytes was also identified in the outer tissues surrounding the vasculature from the early phase after vascular injury. The infiltrated monocyte-macrophages elicited PVAT beiging, resulting in the suppression of the excessive vascular inflammatory response to the injury. These data indicate a pivotal role of the “outside-in” manner in wound healing after vascular injury.

Local activation of PVAT beiging attenuated pathological vascular remodeling after injury, suggesting that PVAT acts directly on nearby arteries and mediates the pathophysiological response to vascular damage in a paracrine manner. We identified NRG4 as an anti-vascular remodeling factor derived from beige PVAT. NRG4 is a member of the NRG protein family, which acts via ErbB receptor tyrosine kinases. This molecule is highly expressed in the pancreas, skeletal muscles and BAT⁴⁸ and protects against diet-induced insulin resistance and hepatic steatosis through regulating hepatic lipogenic and cytoprotective signaling^38,40. Recent studies have shown the expression of functional ErbB receptors on innate immune cells such as macrophages, dendritic cells and neutrophils⁴⁹, and the NRG4-ErbB4 axis exerts anti-inflammatory effects in macrophages by promoting apoptosis of classically activated macrophages but not alternatively activated macrophages^49,50. Consistent with these findings, medium conditioned by PVAT-derived beige adipocytes significantly inhibited the number of classically activated macrophages, which was abolished by Nrg4 knockdown. Reduced levels of NRG4 have been reported to be associated with increased carotid intimal thickness, increased angiographic severity of coronary artery disease and acute coronary syndrome^51,52,53. In this study, PVAT beiging was detected in human aorta with acute dissection, suggesting that NRG4 secreted from beige PVAT might protect human aorta against AAD. The lineage of the beige cells that emerged in the PVAT was not examined in this study. Reportedly, smooth muscle cell has been considered a possible lineage of origin for PVAT⁵⁴, and Angueira et al. have shown an increase in Myh11⁺ smooth muscle cell-derived adipocytes in thoracic PVAT after treatment with rosiglitazone⁵⁵. Since rosiglitazone reportedly recruits beige cells in fat tissues⁵⁶, the beige cells in PVAT may be originated from smooth muscle cells. Lineage tracing studies using inducible genetic techniques may provide further biological and functional insights into the beiging phenomenon in the PVAT. Our data showed that the beiging after injury was suppressed by the macrophage depletion or the local β3AR inhibition, implying that the PVAT beiging was elicited via β3AR signaling mediated by infiltrated macrophages. Meanwhile, the involvement of other signaling pathways of beiging such as succinate metabolisms and BMP4 in the beiging process in PVAT after injury remains to be elucidated^57,58. In conclusion, the present study revealed that PVAT beiging plays a critical role in the vascular inflammatory response to injury by controlling macrophage inflammation and its resolution and suggests that PVAT beiging and NRG4 may be novel targets of vascular injury including AAD.

Methods

Animal studies

All animal procedures were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly adhered to the guidelines for animal experiments at the University of Tokyo. Male or, unless otherwise specified, ovariectomized female mice were used for the study. All wild type C57BL/6J mice were purchased from CLEA Japan. Adipo^Cre+ mice (B6;FVB-Tg(Adipoq-Cre)1Evdr/J, #10803), Prdm16^flox/flox mice (B6.129-Prdm16^tm1.1Brsp/J, #024992) and Ucp1^−/− mice (B6.129-Ucp1^tm1Kz/J, #003124) were purchased from Jackson Laboratory. Conditional deletion of Prdm16 in adipocytes was achieved by crossing Prdm16^flox/flox homozygous mice with Adipo^Cre+ hemizygous mice. Genotyping PCR was performed according to the protocol from Jackson Laboratory. All mice were maintained in specific pathogen-free conditions in the animal facilities of the University of Tokyo. They were housed in a controlled environment with a 12 h light/12 h dark cycle at a maintained temperature and kept with free access to food and water throughout the whole experiment period. Ambient room temperature was regulated at 73 ± 5 °F and humidity was controlled at 50 ± 10%. Endovascular injury of femoral arteries (FA) with wire insertion was performed at 10–12 weeks of age using a straight spring wire (0.38 mm in diameter, COOK) as previously described⁵⁹. Arteries and outer tissues surrounding the vasculature were collected. Ovariectomies were performed in 8–10-week-old female mice 2 weeks before the endovascular injury, as previously described⁶⁰. To selectively remove the macrophages, mice were given intraperitoneally clodronate liposomes (200 μl/mouse) or control phosphate-buffered saline (PBS) liposomes (Liposoma BV) on the day of the endovascular injury and 7 days after injury⁶¹. AAD was induced in mice by the simultaneous administration of angiotensin II (Ang II), β-aminopropionitrile (BAPN), and N-nitro-l-arginine methyl ester (L-NAME)^43,44,45. In the present study, AngII (1,000 ng/kg/min; Peptide Institute Inc.) and BAPN (150 mg/kg/day; Sigma-Aldrich) were administered simultaneously to 10-week-old male C57BL/6J mice using Alzet osmotic minipumps (Durect Corporation)^43,44,45. L-NAME (100 mg/kg per day; Cayman Chemical Co.) was administered orally with drinking water at the same time as pump implantation until the end of the experiment⁴⁵. For beiging activation studies in AAD murine model, β3AR agonist (CL316243, 1 mg/kg/day; Sigma-Aldrich) or vehicle PBS was administered daily for seven days using the Alzet osmotic minipump⁶².

Local administration of reagents

Pluronic F-127 gel (Sigma-Aldrich) is a thermoreversible gel⁶³, which is liquid at refrigerated temperatures (4–5 °C), but gel upon warming to room temperature⁶⁴. The gelation is reversible upon cooling. For local gene knockdown studies, 0.2 nmol/mouse Ambion in vivo pre-designed siRNA against Prdm16 (s89042), Nrg4 (s206928), or negative control mixed with Invivofectamine 3.0 reagent (Thermo Fisher Scientific) was dissolved in pluronic gel on ice and administered locally in PVAT of the injured artery. For β3AR signal activation or inhibition studies, β3AR agonist (CL316243, 0.01 mg/kg; Sigma-Aldrich), β3AR antagonist (SR59230A, 0.02 mg/kg; Sigma-Aldrich), vehicle PBS, or vehicle dimethyl sulfoxide was dissolved in pluronic gel and administered in the PVAT.

Histology and immunohistochemistry

Arteries including outer tissues fixed in Ufix (Sakura Finetek), were embedded in paraffin and sectioned into 4-μm-thick sections. Specimens were then stained by hematoxylin and eosin (H&E), and Elastica van Gieson (EVG) methods. The lumen, internal elastic lamina (IEL), and external elastic lamina (EEL) were defined, and the intimal (the tissue between lumen and IEL) and medial (tissue between IEL and EEL) areas were recorded using National Institutes of Health (NIH) ImageJ software. To evaluate vessel remodeling, the ratio of intima to media area (intima/media)⁵⁹ and the % intima–medial area = [(EEL area) – (luminal area)/EEL area] ×100 (%) (% area of intima–media)⁶⁵ were calculated as previously described. To evaluate adipocyte area, PVAT samples were stained with H&E and imaged with DP70 (Olympus). Uncompressed tif files were analyzed for adipocyte area by the Adiposoft plugin (v. 1.16) for NIH ImageJ^66,67. Sections were immunohistochemically stained by using primary antibodies against F4/80 (Bio-Rad, MCA497GA, 1:400), UCP1 (Abcam, ab23841, 1:400), iNOS (Abcam, ab15323, 1:200), CD206 (Abcam, ab64693, 1:1600), CIDEA (Abnova, H00001149-M01, 1:50), or NRG4 (Thermo Fisher Scientific, PA5-96184, 1:1000) with Vectastain ABC kit (Vector Laboratories) according to the manufacturers’ instructions. Peroxidase activity was visualized with DAB staining (Vector Laboratories), and sections were counterstained with hematoxylin. Images were acquired using the DP70 (Olympus) or BZ-X810 (KEYENCE) and analyzed using NIH ImageJ.

Single-molecule fluorescence in situ hybridization

In situ hybridization was performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics) according to manufacturers’ instructions. The probes targeting mm-Ucp1 (#455411) or mm-Nrg4 (#493731, Advanced Cell Diagnostics) were hybridized, followed by RNAscope amplification and co-staining with fluorescein-conjugated WGA to detect cell borders. Slides were mounted with ProLong Diamond Antifade Mountant with DAPI (Life Technologies). Fluorescent signals were captured with the ×40 objective lens on a laser scanning confocal microscope (LSM880, Zeiss). The number of positive dots was calculated using NIH ImageJ.

PVAT-derived preadipocyte isolation

Eleven-week-old male wild type mice (for fluorescence-activated cell sorting analysis) or twelve-week-old female wild type mice (for cell culture) were sacrificed, and the PVAT from the thoracic aorta were extracted. PVAT was minced into small pieces using sharp round scissors and digested using gentleMACS (Miltenyi Biotec) and Adipose Tissue Dissociation Kit (Miltenyi Biotec), as previously described⁶⁸. Digestion was performed in a MACSmix Tube Rotator (Miltenyi Biotec) at 37 °C for 40 min.

Immortalization of PVAT-derived preadipocyte

A stromal vascular fraction of PVAT from 12-week-old female wild type mice was isolated and cultured in collagen-coated dishes, as previously described⁶⁸. Preadipocytes isolated from the PVAT stromal vascular fraction were immortalized using the SV40 antigen as described previously¹⁹.

BMDM isolation

Bone marrow cells were flushed from the femurs and tibias of 10-week-old male wild type mice, and cultured at 37 °C in DMEM/F12 containing 1% streptomycin, 1% penicillin, and 10% fetal bovine serum (FBS) (Cosmo Bio) and in 40 ng/mL of recombinant mouse M-CSF (576406; BioLegend) for 7 days, as previously described⁶⁹. The differentiated BMDMs were detached from plates using TrypLE (Thermo Fisher Scientific) and replated into 12-well tissue culture dishes at a density of 5 × 10⁵ cells per well prior to cell stimulation.

Cell culture

RAW 264.7 cells (European Collection of Authenticated Cell Culture), BMDMs, and PVAT-derived preadipocytes were cultured at 37 °C in DMEM/F12 with 1% streptomycin, 1% penicillin, and 10% FBS. For classically or alternatively activation, RAW 264.7 cells or BMDMs were treated with 20 μg/mL IFNγ (Miltenyi Biotec) + 20 μg/mL LPS (Sigma-Aldrich) or 10 μg/mL IL4 (Sigma-Aldrich) for 24 h, respectively, as previously described⁷⁰. Recombinant murine NRG4 (100 ng/ml, Proteintech) or vehicle PBS was added to RAW 264.7 cells after classical activation. The growth of RAW 264.7 cells was measured with the Cell Titer Glo reagent (Promega) according to the manufacturer’s instructions.

Differentiation of PVAT stromal vascular fraction to beige cells

PVAT-derived preadipocytes were differentiated to beige cells according to the previous reports⁶⁸. Briefly, cells that reached confluence were treated for 48 h in medium containing 10% FBS, 850 nM insulin, 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 1 μM dexamethasone, 1 nM 3,3′,5-Triiodo-l-thyronine (T3) and 0.5 μM rosiglitazone. After 48 h treatment, the cell culture media were switched to media containing 10% FBS, 850 nM insulin, 1 nM T3 and 0.5 μM rosiglitazone and incubated for additional 5 days. Six to eight days after adding the induction medium, cells are fully differentiated to mature beige fat cells and are filled with oil droplets. Culture media conditioned by PVAT-derived beige adipocytes were added to RAW 264.7 cells, and mRNA expression of RAW 264.7 cells 24 h after stimulation was examined. For siRNA-mediated gene knockdown experiments in cultured PVAT-derived adipocytes, siRNAs against Prdm16, Nrg4, Erbb4 (s89042, s206928, s201303, respectively, Silencer select pre-designed siRNA, Thermo Fisher Scientific), Nfia (sc-36045, Santa Cruz Biotechnology) and negative control (Thermo Fisher Scientific) were transfected with Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) 2 days before confluence and following every 48 h, according to the manufacturer’s instructions.

Western blotting

Tissues were lysed in RIPA buffer containing 0.1% sodium dodecyl sulfate, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM EDTA supplemented with protease inhibitor (Roche). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membrane, and detected with the antibodies of anti-UCP1 (Abcam, ab23841, 1:1000) and anti-GAPDH (Cell Signaling Technology, #2118, 1:500).

Quantitative reverse transcription-polymerase chain reaction

Total RNA from tissue samples was extracted using the RNeasy Mini Kit (Qiagen). Superscript VILO cDNA synthesis kit (Invitrogen) was used to generate cDNA. qRT-PCR was performed on QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific) using Thunderbird SYBR qPCR Mix (Toyobo). The specific primers are listed in Supplementary Table 1. Relative expression levels of target genes were calculated using the comparative CT method. Each sample was run in duplicate, and the results were systematically normalized using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) for experiments using cultured cells or β-2 microglobulin (B2m) for experiments using mouse tissues.

Bulk RNA-seq data processing

Outer tissues surrounding the FAs 48 h after injury or sham operation (n = 2 for each group) were collected. Total RNA (480 ng) from each sample was used to generate RNA-seq libraries using TruSeq Stranded mRNA Library Prep kit (llumina). 150 bp paired-end sequencing reads were obtained using Illumina HiSeq 2500 platform. The Fastq sequences were initially checked for quality through the FastQC program v0.11.8. Adaptor sequences were removed by Cutadapt v1.18. Sequencing reads from RNA-seq libraries were then trimmed using Trimmomatic v0.39, where reads with alignment quality < Q33 were discarded, and aligned to the mouse reference genome (mm9) using STAR aligner v2.6.0a⁷¹. RSubread-2.0.1-FeatureCounts software for quantification and DESeq2 v.1.28.0 software⁷² for differential analysis were used on R v.3.6.0.

Single-cell RNA-seq data analysis

scRNA-seq data from publicly available datasets of mouse inguinal white adipose tissue (iWAT) nuclei treated with CL316243 (1 mg/kg/day for four days) or cold stimulation (4 °C for four days) (GSE 133486) were used. Among the original database that contains two types of scRNA-seq data (Adipo^Cre-;Il10ra^flox/flox mice and Adipo^Cre+;Il10ra^flox/flox mice)³⁶, the data from Adipo^Cre-;Il10ra^flox/flox mice were used for the present analysis. The standard Seurat (V.4.0.1) for R (V.4.0.2) protocol (“FindIntegrationAnchors” and “IntegrateData”) was used for the integration⁷³. To observe the effects of CL316243 treatment, an alternative integration step was performed with Harmony (V.0.1)⁷⁴. All sequenced cells were projected onto two dimensions using UMAP on R v.4.0.2. The optimal number of principal components used for UMAP dimensionality reduction was determined using the Jackstraw permutation approach and a grid-search of the parameters. Cells were divided into several clusters and color-coded according to cell types. Cells were also color-coded for the treatment group (CL316243 or cold stimulation) (pink) or control (turquoise). Marker genes were searched using the FindMarkers function in Seurat. Gene Ontology analysis was performed using Database for Annotation, Visualization, and Integrated Discovery ver. 6.8 (DAVID 6.8)⁷⁵. Furthermore, the gene signature from the scRNA-seq data was used to deconvolute public bulk RNA-seq data to confirm the validity of the cell type clustering performed in this scRNA-seq re-analysis. We confirmed that the beige adipocytes also exist in iWAT treated with CL316243 in another public bulk RNA-seq study (GSE129083)³⁷ using MuSiC, a method for predicting cell abundance based on single-cell data⁷⁶.

Fluorescence-activated cell sorting analysis

After the isolation of stromal vascular fraction of PVAT, cells were suspended in PBS containing 5% FBS. Zombie NIR Fixable Viability Kit (BioLegend) was used to label dead cells according to the manufacturer’s instructions. Nonspecific binding of the antibodies to Fc receptors was blocked by using Mouse Fc receptor-blocking agent (BioLegend). Cells were incubated at room temperature 30 mins with FITC anti-mouse CD45 Antibody (BioLegend, #103107, 1:50), PE/Cyanine7 anti-mouse F4/80 antibody (BioLegend, #123113, 1:50), PE anti-mouse CD11b antibody (BioLegend, #101207, 1:50), APC anti-mouse CD206 antibody (BioLegend, #141707, 1:50)‚ and Brilliant Violet 421 anti-mouse CD11c antibody (BioLegend, #117329, 1:50), then centrifuged and fixed with PBS containing 2% PFA. Cells were analyzed using a FACSMelody cell sorter (BD Biosciences). For the analysis of flow cytometry data, CD45⁺, CD11b⁺, and F4/80⁺ cells were defined as macrophages, which could be further differentiated into classically (M1; defined as CD11c⁺, CD206⁻) and alternatively activated (M2; defined as CD11c⁻, CD206⁺) macrophages⁷⁷. Data analyses were performed using FlowJo software (Tree Star).

Statistics and reproducibility

Data were presented as means ± standard error of mean (SEM). All statistics are described in figure legends. In general, comparisons between two groups were performed using the two-tailed Student’s t test or Mann–Whitney U-test, and multiple group comparisons were performed by one-way ANOVA followed by Tukey–Kramer post-hoc test. Survival curves were created using the Kaplan–Meier method and compared by a log-rank test. All statistical analyses were performed using the Prism software (GraphPad). P-values of <0.05 were considered statistically significant.

Human studies

Pathological records were reviewed, and formalin-fixed paraffin-embedded descending aorta tissues were obtained from the archives of the Department of Pathology, The University of Tokyo Hospital. The aortic specimens of AAD were obtained by the autopsy of six patients who died of AAD, five males and one female, aged between 45 and 84 years; four of these patients had hypertension, one patient had suffered from a previous stroke, and three had a smoking history. The aortic specimens of controls were autopsy specimens from five patients who died of non-aortic causes, four males and one female, aged between 44 and 78 years; one patient had hypertension, one patient suffered from dyslipidemia, one with chronic kidney disease, one suffered from a previous stroke, and one had a smoking history. Patients with an apparent history of inherited aortic diseases such as Marfan syndrome were not included in the AAD cases and controls. The inclusion and exclusion criteria for patients with AAD were as follows: AAD cases were selected from consecutive pathological autopsies from 2006 to 2017 with a description of aortic dissection as a finding in autopsy report. Controls were selected from consecutive pathological autopsies from 2014 to 2017 with no significant changes in the aortic wall based on the autopsy. The immunostaining using primary antibodies against CD204 (Transgenic, KT022, 1:500) was performed using a Ventana Benchmark automated stainer (Ventana Medical Systems). The study followed the principles outlined in the Declaration of Helsinki. This study was approved by the ethics committee of the University of Tokyo (approval ID-2020019NI), and written informed consent was waived because this is a retrospective study using existing tissue blocks. Instead, we used an opt out approach to provide participants with an informed choice about participation, although no patient in the cohort for screening used the opt out option.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Source data are provided with this paper. The RNA-seq data have been deposited in GSE 206399, GSE133486, and GSE129083. The information of mouse genome (mm9) is available on UCSC Genome Browser (http://genome.ucsc.edu). The authors declare that all data are available in the article file and Supplementary information files, or available from the authors upon reasonable request. Source data are provided with this paper.

References

Ruparelia, N., Chai, J. T., Fisher, E. A. & Choudhury, R. P. Inflammatory processes in cardiovascular disease: a route to targeted therapies. Nat. Rev. Cardiol. 14, 133–144 (2017).
Article CAS PubMed Google Scholar
Libby, P., Ridker, P. M. & Hansson, G. K. Progress and challenges in translating the biology of atherosclerosis. Nature 473, 317–325 (2011).
Article ADS CAS PubMed Google Scholar
Rader, D. J. & Daugherty, A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 451, 904–913 (2008).
Article ADS CAS PubMed Google Scholar
Ross, R. et al. Localization of PDGF-B protein in macrophages in all phases of atherogenesis. Science 248, 1009–1012 (1990).
Article ADS CAS PubMed Google Scholar
Basatemur, G. L., Jørgensen, H. F., Clarke, M. C. H., Bennett, M. R. & Mallat, Z. Vascular smooth muscle cells in atherosclerosis. Nat. Rev. Cardiol. 16, 727–744 (2019).
Article PubMed Google Scholar
Ross, R. & Glomset, J. A. The pathogenesis of atherosclerosis. N. Engl. J. Med. 295, 369–377 (1976).
Article CAS PubMed Google Scholar
Silvestre-Roig, C., Braster, Q., Ortega-Gomez, A. & Soehnlein, O. Neutrophils as regulators of cardiovascular inflammation. Nat. Rev. Cardiol. 17, 327–340 (2020).
Article PubMed Google Scholar
Gimbrone, M. A. Jr. & García-Cardeña, G. Endothelial cell dysfunction and the pathobiology of atherosclerosis. Circ. Res 118, 620–636 (2016).
Article CAS PubMed PubMed Central Google Scholar
Hergenreider, E. et al. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat. Cell Biol. 14, 249–256 (2012).
Article CAS PubMed Google Scholar
Bernelot Moens, S. J. et al. Rapid estrogen receptor signaling is essential for the protective effects of estrogen against vascular injury. Circulation 126, 1993–2004 (2012).
Article CAS PubMed PubMed Central Google Scholar
Libby, P., Ridker, P. M. & Hansson, G. K. Inflammation in atherosclerosis: from pathophysiology to practice. J. Am. Coll. Cardiol. 54, 2129–2138 (2009).
Article CAS PubMed PubMed Central Google Scholar
Aikawa, M. et al. Lipid lowering reduces oxidative stress and endothelial cell activation in rabbit atheroma. Circulation 106, 1390–1396 (2002).
Article CAS PubMed Google Scholar
Kim, H. W., Shi, H., Winkler, M. A., Lee, R. & Weintraub, N. L. Perivascular adipose tissue and vascular perturbation/atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 40, 2569–2576 (2020).
Article CAS PubMed PubMed Central Google Scholar
Sidossis, L. & Kajimura, S. Brown and beige fat in humans: thermogenic adipocytes that control energy and glucose homeostasis. J. Clin. Invest. 125, 478–486 (2015).
Article PubMed PubMed Central Google Scholar
Cypess, A. M. et al. Activation of human brown adipose tissue by a beta3-adrenergic receptor agonist. Cell Metab. 21, 33–38 (2015).
Article CAS PubMed PubMed Central Google Scholar
Seale, P. et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature 454, 961–967 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Ohno, H., Shinoda, K., Spiegelman, B. M. & Kajimura, S. PPARγ agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein. Cell Metab. 15, 395–404 (2012).
Article CAS PubMed PubMed Central Google Scholar
Cohen, P. et al. Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch. Cell 156, 304–316 (2014).
Article CAS PubMed PubMed Central Google Scholar
Hiraike, Y. et al. NFIA co-localizes with PPARγ and transcriptionally controls the brown fat gene program. Nat. Cell Biol. 19, 1081–1092 (2017).
Article CAS PubMed PubMed Central Google Scholar
Rajakumari, S. et al. EBF2 determines and maintains brown adipocyte identity. Cell Metab. 17, 562–574 (2013).
Article CAS PubMed PubMed Central Google Scholar
Angueira, A.R. et al. Defining the lineage of thermogenic perivascular adipose tissue. Nat Metab 3, 469–484 (2021).
Hiraike, Y. et al. NFIA differentially controls adipogenic and myogenic gene program through distinct pathways to ensure brown and beige adipocyte differentiation. PLoS Genet 16, e1009044 (2020).
Article CAS PubMed PubMed Central Google Scholar
Antonopoulos, A. S. et al. Detecting human coronary inflammation by imaging perivascular fat. Sci. Transl. Med. 9, eaal2658 (2017).
Article PubMed CAS Google Scholar
Oikonomou, E. K. et al. Non-invasive detection of coronary inflammation using computed tomography and prediction of residual cardiovascular risk (the CRISP CT study): a post-hoc analysis of prospective outcome data. Lancet 392, 929–939 (2018).
Article PubMed PubMed Central Google Scholar
Iafrati, M. D. et al. Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice. Nat. Med. 3, 545–548 (1997).
Article CAS PubMed Google Scholar
Ueda, K., Lu, Q., Baur, W., Aronovitz, M. J. & Karas, R. H. Rapid estrogen receptor signaling mediates estrogen-induced inhibition of vascular smooth muscle cell proliferation. Arterioscler Thromb. Vasc. Biol. 33, 1837–1843 (2013).
Article CAS PubMed PubMed Central Google Scholar
Simons, M., Edelman, E. R., DeKeyser, J. L., Langer, R. & Rosenberg, R. D. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo. Nature 359, 67–70 (1992).
Article ADS CAS PubMed Google Scholar
Villa, A. E. et al. Effects of antisense c-myb oligonucleotides on vascular smooth muscle cell proliferation and response to vessel wall injury. Circ. Res. 76, 505–513 (1995).
Article CAS PubMed Google Scholar
Liddiard, K. & Taylor, P. R. Understanding local macrophage phenotypes in disease: shape-shifting macrophages. Nat. Med. 21, 119–120 (2015).
Article CAS PubMed Google Scholar
Berbée, J. F. et al. Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development. Nat. Commun. 6, 6356 (2015).
Article ADS PubMed CAS Google Scholar
Sui, W. et al. Bladder drug mirabegron exacerbates atherosclerosis through activation of brown fat-mediated lipolysis. Proc. Natl Acad. Sci. USA 116, 10937–10942 (2019).
Article CAS PubMed PubMed Central Google Scholar
Dong, M. et al. Cold exposure promotes atherosclerotic plaque growth and instability via UCP1-dependent lipolysis. Cell Metab. 18, 118–129 (2013).
Article CAS PubMed PubMed Central Google Scholar
Bernal-Mizrachi, C. et al. Respiratory uncoupling lowers blood pressure through a leptin-dependent mechanism in genetically obese mice. Arterioscler. Thromb. Vasc. Biol. 22, 961–968 (2002).
Article CAS PubMed Google Scholar
Bernal-Mizrachi, C. et al. Vascular respiratory uncoupling increases blood pressure and atherosclerosis. Nature 435, 502–506 (2005).
Article ADS CAS PubMed Google Scholar
Gates, A. C. et al. Respiratory uncoupling in skeletal muscle delays death and diminishes age-related disease. Cell Metab. 6, 497–505 (2007).
Article CAS PubMed Google Scholar
Rajbhandari, P. et al. Single cell analysis reveals immune cell-adipocyte crosstalk regulating the transcription of thermogenic adipocytes. Elife 8, e49501(2019).
Wang, W. et al. A PRDM16-driven metabolic signal from adipocytes regulates precursor cell fate. Cell Metab. 30, 174–189 (2019). e175.
Article CAS PubMed PubMed Central Google Scholar
Wang, G. X. et al. The brown fat-enriched secreted factor Nrg4 preserves metabolic homeostasis through attenuation of hepatic lipogenesis. Nat. Med. 20, 1436–1443 (2014).
Article CAS PubMed PubMed Central Google Scholar
Ma, Y., Gao, M. & Liu, D. Preventing high fat diet-induced obesity and improving insulin sensitivity through neuregulin 4 gene transfer. Sci. Rep. 6, 26242 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Guo, L. et al. Hepatic neuregulin 4 signaling defines an endocrine checkpoint for steatosis-to-NASH progression. J. Clin. Invest. 127, 4449–4461 (2017).
Article PubMed PubMed Central Google Scholar
Harari, D. et al. Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase. Oncogene 18, 2681–2689 (1999).
Article CAS PubMed Google Scholar
Nienaber, C. A. et al. Aortic dissection. Nat. Rev. Dis. Prim. 2, 16053 (2016).
Article PubMed Google Scholar
Hirakata, S. et al. Genetic deletion of Socs3 in smooth muscle cells ameliorates aortic dissection in mice. JACC Basic Transl. Sci. 5, 126–144 (2020).
Article PubMed PubMed Central Google Scholar
Nishida, N. et al. High salt intake worsens aortic dissection in mice: involvement of IL (Interleukin)-17A-dependent ECM (extracellular matrix) metabolism. Arterioscler. Thromb. Vasc. Biol. 40, 189–205 (2020).
Article CAS PubMed Google Scholar
Izawa-Ishizawa, Y. et al. Development of a novel aortic dissection mouse model and evaluation of drug efficacy using in-vivo assays and database analyses. J. Hypertens. 37, 73–83 (2019).
Article CAS PubMed Google Scholar
Bäck, M., Yurdagul, A. Jr, Tabas, I., Öörni, K. & Kovanen, P. T. Inflammation and its resolution in atherosclerosis: mediators and therapeutic opportunities. Nat. Rev. Cardiol. 16, 389–406 (2019).
PubMed PubMed Central Google Scholar
Watanabe, S., Alexander, M., Misharin, A. V. & Budinger, G. R. S. The role of macrophages in the resolution of inflammation. J. Clin. Invest. 129, 2619–2628 (2019).
Article PubMed PubMed Central Google Scholar
Geissler, A., Ryzhov, S. & Sawyer, D. B. Neuregulins: protective and reparative growth factors in multiple forms of cardiovascular disease. Clin. Sci. 134, 2623–2643 (2020).
Article CAS Google Scholar
Schumacher, M. A. et al. NRG4-ErbB4 signaling represses proinflammatory macrophage activity. Am. J. Physiol. Gastrointest. Liver Physiol. 320, G990–G1001 (2021).
Article CAS PubMed PubMed Central Google Scholar
Schumacher, M. A. et al. ErbB4 signaling stimulates pro-inflammatory macrophage apoptosis and limits colonic inflammation. Cell Death Dis. 8, e2622 (2017).
Article CAS PubMed PubMed Central Google Scholar
Rahimzadeh, M., Farshidi, N., Naderi, N., Farshidi, H. & Montazerghaem, H. Clinical significance of serum concentrations of neuregulin-4, in acute coronary syndrome. Sci. Rep. 10, 5797 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Jiang, J. et al. Circulating neuregulin 4 levels are inversely associated with subclinical cardiovascular disease in obese adults. Sci. Rep. 6, 36710 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Tian, Q. P. et al. Association of circulating neuregulin-4 with presence and severity of coronary artery disease. Int. Heart J. 60, 45–49 (2019).
Article CAS PubMed Google Scholar
Chang, L. et al. Loss of perivascular adipose tissue on peroxisome proliferator-activated receptor-gamma deletion in smooth muscle cells impairs intravascular thermoregulation and enhances atherosclerosis. Circulation 126, 1067–1078 (2012).
Article CAS PubMed PubMed Central Google Scholar
Angueira, A. R. et al. Defining the lineage of thermogenic perivascular adipose tissue. Nat. Metab. 3, 469–484 (2021).
Article CAS PubMed PubMed Central Google Scholar
Ohno, H., Shinoda, K., Spiegelman, B. M. & Kajimura, S. PPARgamma agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein. Cell Metab. 15, 395–404 (2012).
Article CAS PubMed PubMed Central Google Scholar
Mills, E. L. et al. Accumulation of succinate controls activation of adipose tissue thermogenesis. Nature 560, 102–106 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Mu, W. et al. BMP4-mediated browning of perivascular adipose tissue governs an anti-inflammatory program and prevents atherosclerosis. Redox Biol. 43, 101979 (2021).
Article CAS PubMed PubMed Central Google Scholar
Sata, M. et al. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J. Mol. Cell Cardiol. 32, 2097–2104 (2000).
Article CAS PubMed Google Scholar
Ueda, K. et al. Membrane-initiated estrogen receptor signaling mediates metabolic homeostasis via central activation of protein phosphatase 2A. Diabetes 67, 1524–1537 (2018).
Article CAS PubMed PubMed Central Google Scholar
Opperman, K. S. et al. Clodronate-liposome mediated macrophage depletion abrogates multiple myeloma tumor establishment in vivo. Neoplasia 21, 777–787 (2019).
Article CAS PubMed PubMed Central Google Scholar
Shao, M. et al. Zfp423 maintains white adipocyte identity through suppression of the beige cell thermogenic gene program. Cell Metab. 23, 1167–1184 (2016).
Article CAS PubMed PubMed Central Google Scholar
Escobar-Chavez, J. J. et al. Applications of thermoreversible pluronic F-127 gels in pharmaceutical formulations. J. Pharm. Pharm. Sci. 9, 339–358 (2006).
CAS PubMed Google Scholar
Jorgensen, E. B., Hvidt, S., Brown, W. & Schillen, K. Effects of salts on the micellization and gelation of a triblock copolymer studied by rheology and light scattering. Macromolecules 30, 2355–2364 (1997).
Article ADS CAS Google Scholar
Korshunov, V. A. & Berk, B. C. Strain-dependent vascular remodeling: the “Glagov phenomenon” is genetically determined. Circulation 110, 220–226 (2004).
Article PubMed Google Scholar
Galarraga, M. et al. Adiposoft: automated software for the analysis of white adipose tissue cellularity in histological sections. J. Lipid Res. 53, 2791–2796 (2012).
Article CAS PubMed PubMed Central Google Scholar
Ferland, D. J., Garver, H., Contreras, G. A., Fink, G. D. & Watts, S. W. Chemerin contributes to in vivo adipogenesis in a location-specific manner. PLoS ONE 15, e0229251 (2020).
Article CAS PubMed PubMed Central Google Scholar
Aune, U. L., Ruiz, L. & Kajimura, S. Isolation and differentiation of stromal vascular cells to beige/brite cells. J. Vis. Exp. 28, 50191 (2013).
Nakayama, Y. et al. A long noncoding RNA regulates inflammation resolution by mouse macrophages through fatty acid oxidation activation. Proc. Natl Acad. Sci. USA 117, 14365–14375 (2020).
Article CAS PubMed PubMed Central Google Scholar
Sumida, T. et al. Complement C1q-induced activation of beta-catenin signalling causes hypertensive arterial remodelling. Nat. Commun. 6, 6241 (2015).
Article ADS CAS PubMed Google Scholar
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Article CAS PubMed Google Scholar
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Article PubMed PubMed Central CAS Google Scholar
Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587.e3529 (2021).
Article CAS PubMed PubMed Central Google Scholar
Korsunsky, I. et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat. Methods 16, 1289–1296 (2019).
Article CAS PubMed PubMed Central Google Scholar
Huang da, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Article PubMed CAS Google Scholar
Wang, X., Park, J., Susztak, K., Zhang, N. R. & Li, M. Bulk tissue cell type deconvolution with multi-subject single-cell expression reference. Nat. Commun. 10, 380 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Braune, J. et al. IL-6 regulates M2 polarization and local proliferation of adipose tissue macrophages in obesity. J. Immunol. 198, 2927–2934 (2017).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

We thank the participants for donating their tissues for this study; R. H. Karas for insightful discussions and suggestions; K. Akiba, Y. Ishiyama, Y. Nakashima, N. Matsuzaki, M. Oguchi, and T. Wada for the technical assistance; T. Kawahara for statistical consultation; and the Center for Disease Biology and Integrative Medicine, University of Tokyo, for mouse breeding and maintenance. This work was supported by the Japan Society for the Promotion of Science KAKENHI (18J13507, 20K22893, and 22K16097 to Y.A., 18K08096 and 21H02908 to K.U.), Japan Heart Foundation Research Grant, Japanese Circulation Society Clinical Research Grant, Japan Foundation for Applied Enzymology, SENSHIN Medical Research Foundation, Japan Arteriosclerosis Prevention Fund (to Y.A.), Japan Cardiovascular Research Foundation, Takeda Science Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Research Grants from Bristol Myers Squibb K.K., Tokyo Biochemical Research Foundation (to K.U.), JST FOREST Program (Grant Number 21466223), UTEC-UTokyo FSI Research Grant Program (to S.N.), and AMED (JP20ek0210152, JP18gm6210010, JP20ek0210141, JP20ek0109440, JP20ek0109487, JP17gm0810013, JP18km0405209, JP19ek0210118, JP19ek0109406, JP21ek0109543, JP21ek0109569, JP21tm0724601, JP22ama121016, JP22ek0210172, JP22ek0210167, JP22bm1123011) (to S.N. and I.K.).

Author information

These authors contributed equally: Yusuke Adachi, Kazutaka Ueda.

Authors and Affiliations

Department of Cardiovascular Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Yusuke Adachi, Kazutaka Ueda, Seitaro Nomura, Manami Katoh, Mikako Katagiri, Shintaro Yamada, Masaki Hashimoto, Bowen Zhai, Genri Numata, Akira Otani, Norifumi Takeda, Hiroyuki Morita, Eiki Takimoto & Issei Komuro
Department of Advanced Clinical Science and Therapeutics, The University of Tokyo, Tokyo, Japan
Yusuke Adachi
Genome Science Division, Research Center for Advance Science and Technology, The University of Tokyo, Tokyo, Japan
Seitaro Nomura, Manami Katoh & Shintaro Yamada
Laboratory for Cardiovascular Genomics and Informatics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan
Kaoru Ito
Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Munetoshi Hinata & Tetsuo Ushiku
Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
Yuta Hiraike, Hironori Waki & Toshimasa Yamauchi

Authors

Yusuke Adachi
View author publications
You can also search for this author in PubMed Google Scholar
Kazutaka Ueda
View author publications
You can also search for this author in PubMed Google Scholar
Seitaro Nomura
View author publications
You can also search for this author in PubMed Google Scholar
Kaoru Ito
View author publications
You can also search for this author in PubMed Google Scholar
Manami Katoh
View author publications
You can also search for this author in PubMed Google Scholar
Mikako Katagiri
View author publications
You can also search for this author in PubMed Google Scholar
Shintaro Yamada
View author publications
You can also search for this author in PubMed Google Scholar
Masaki Hashimoto
View author publications
You can also search for this author in PubMed Google Scholar
Bowen Zhai
View author publications
You can also search for this author in PubMed Google Scholar
Genri Numata
View author publications
You can also search for this author in PubMed Google Scholar
Akira Otani
View author publications
You can also search for this author in PubMed Google Scholar
Munetoshi Hinata
View author publications
You can also search for this author in PubMed Google Scholar
Yuta Hiraike
View author publications
You can also search for this author in PubMed Google Scholar
Hironori Waki
View author publications
You can also search for this author in PubMed Google Scholar
Norifumi Takeda
View author publications
You can also search for this author in PubMed Google Scholar
Hiroyuki Morita
View author publications
You can also search for this author in PubMed Google Scholar
Tetsuo Ushiku
View author publications
You can also search for this author in PubMed Google Scholar
Toshimasa Yamauchi
View author publications
You can also search for this author in PubMed Google Scholar
Eiki Takimoto
View author publications
You can also search for this author in PubMed Google Scholar
Issei Komuro
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Y.A., K.U., and I.K. conceived the project, designed the study, and interpreted the results.; Y.A. and K.U. performed in vivo and in vitro experiments with the help of S.N., M.Katoh, M.Katagiri, M.Hashimoto, B.Z., G.N., A.O., Y.H., H.W., and E.T.; Y.A. and K.U. analyzed RNA-seq data under the supervision of S.N., K.I., and S.Y.; M.Hinata collected the human samples and clinical histories of the patients and helped immunohistochemistry under the supervision of T.U.; Y.A., K.U., and I.K. performed data analysis and wrote the manuscript under the supervision of S.N., K.I., N.T., H.M., T.U., T.Y., and E.T.; All authors contributed to the article and approved the submitted version.

Corresponding authors

Correspondence to Kazutaka Ueda or Issei Komuro.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Wei Kong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Reporting Summary

Source data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Cite this article

Adachi, Y., Ueda, K., Nomura, S. et al. Beiging of perivascular adipose tissue regulates its inflammation and vascular remodeling. Nat Commun 13, 5117 (2022). https://doi.org/10.1038/s41467-022-32658-6

Download citation

Received: 24 May 2021
Accepted: 10 August 2022
Published: 07 September 2022
DOI: https://doi.org/10.1038/s41467-022-32658-6

This article is cited by

Batokine neuregulin 4 promotes atherosclerotic resolution
- Ibrahim AlZaim
- Joanna Kalucka
Nature Metabolism (2022)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Subjects

Abstract

Introduction

Results

Endovascular injury induces PVAT beiging

PVAT beiging alters vascular inflammatory response

NRG4 as an anti-inflammatory factor

PVAT beiging is detectable in human AAD

Discussion

Methods

Animal studies

Local administration of reagents

Histology and immunohistochemistry

Single-molecule fluorescence in situ hybridization

PVAT-derived preadipocyte isolation

Immortalization of PVAT-derived preadipocyte

BMDM isolation

Cell culture

Differentiation of PVAT stromal vascular fraction to beige cells

Western blotting

Quantitative reverse transcription-polymerase chain reaction

Bulk RNA-seq data processing

Single-cell RNA-seq data analysis

Fluorescence-activated cell sorting analysis

Statistics and reproducibility

Human studies

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Supplementary information

Supplementary Information

Peer Review File

Reporting Summary

Source data

Source data

Rights and permissions

About this article

Cite this article

Share this article

This article is cited by

Batokine neuregulin 4 promotes atherosclerotic resolution

Comments

Search

Quick links