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Perivascular mesenchymal cells control adipose-tissue macrophage accrual in obesity

Abstract

Chronic low-grade white adipose tissue (WAT) inflammation is a hallmark of metabolic syndrome in obesity. Here, we demonstrate that a subpopulation of mouse WAT perivascular (PDGFRβ+) cells, termed fibro-inflammatory progenitors (FIPs), activate proinflammatory signalling cascades shortly after the onset of high-fat diet feeding and regulate proinflammatory macrophage accumulation in WAT in a TLR4-dependent manner. FIPs activation in obesity is mediated by the downregulation of zinc-finger protein 423 (ZFP423), identified here as a transcriptional corepressor of NF-κB. ZFP423 suppresses the DNA-binding capacity of the p65 subunit of NF-κB by inducing a p300-to-NuRD coregulator switch. Doxycycline-inducible expression of Zfp423 in PDGFRβ+ cells suppresses inflammatory signalling in FIPs and attenuates metabolic inflammation of visceral WAT in obesity. Inducible inactivation of Zfp423 in PDGFRβ+ cells increases FIP activity, exacerbates adipose macrophage accrual and promotes WAT dysfunction. These studies implicate perivascular mesenchymal cells as important regulators of chronic adipose-tissue inflammation in obesity and identify ZFP423 as a transcriptional break on NF-κB signalling.

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Fig. 1: FIPs are activated in association with adipose-tissue inflammation.
Fig. 2: FIPs are more responsive than APCs to the activation of NF-κB signalling.
Fig. 3: Loss of Tlr4 in PDGFRβ+ cells attenuates WAT macrophage accumulation in obesity.
Fig. 4: Zfp423 suppresses the proinflammatory phenotype of FIPs.
Fig. 5: Zfp423 overexpression in PDGFRβ+ cells limits LPS-induced WAT inflammation.
Fig. 6: Zfp423 overexpression in PDGFRβ+ cells attenuates WAT inflammation in obesity.
Fig. 7: ZFP423 suppresses the DNA-binding capacity and activity of NF-κB in FIPs.
Fig. 8: ZFP423 induces a NF-κB p65 coregulator switch.

Data availability

All animal models, reagents and datasets are freely available to investigators upon reasonable request. ChIP–seq data have been deposited to Gene Expression Omnibus (GEO accession GSE134868). RNA-seq data have been deposited to Gene Expression Omnibus (GEO accession GSE151092). Mass spectrometry data has been deposited to MAssIVE (http://massive.ucsd.edu/ProteoSAFe/status.jsp?task=af5cbb331b004854ac6c182936148168). Source data are provided with this paper.

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Acknowledgements

The authors are grateful to P. Scherer and C. Kusminski for critical reading of the manuscript and members of the UTSW Touchstone Diabetes Center for useful discussions. The authors thank C. Lee, the UTSW Animal Resource Center, Metabolic Phenotyping Core, Pathology Core, Live Cell Imaging Core, Flow Cytometry Core, McDermott Sequencing Center and Proteomics Core, for excellent guidance and assistance with experiments performed here. This study and/or personnel were supported in part by the NIH NIDDK F31DK113696 to C.H., NIDDK R01 DK104789, R56 DK119163 and R01 DK119163 to R.K.G., the American Heart Association postdoctoral fellowship 16POST26420136 to M.S, NIDDK R01 DK115477 to D.W.S, NIDDK R01 DK108773 and AHA 14SDG19880020 to D.O., American Diabetes Association 1-18-PMF-030 to V.A.P., NIH R03 DK101865 to O.T.G, the Cancer Prevention and Research Institute of Texas (RP150596) to S.D.B and V.S.M, NIH NIAAA K01AA024809 to L.J. and NIDDK PO1 DK088761to J.K.E.

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Contributions

B.S., M.S. and R.K.G. conceived the study and wrote the manuscript. B.S., M.S., D.O. and R.K.G. designed experiments. B.S., M.S., Q.Z., C.H., L.V., V.A.P. and Y.A.A, performed experiments and analysed the data. S.D.B., M.S. and V.S.M. designed and analysed ChIP–seq experiments. D.W.S., O.T.G, J.K.E. and L.J. provided key reagents.

Corresponding author

Correspondence to Rana K. Gupta.

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Extended data

Extended Data Fig. 1 Thermoneutral housing amplifies the pro-inflammatory phenotype of FIPs.

a, Experimental design: 6 weeks-old male C57BL/6 mice were transferred to thermoneutral housing conditions (TN, 30 °C) or maintained at room temperature (RT, 22 °C) for two months. Then animals were fed HFD for 3 days prior to gene expression analysis of FIPs and APCs. b, mRNA levels of indicated genes in freshly isolated FIPs and APCs obtained following 3 days of HFD. For each experiment, n = 8 for FIPs and APCs at 22 °C. n = 4 for FIPs and APCs at 30 °C. Data presented were reproduced in two independent experiments. Bars represent mean ± s.e.m., * denotes p< 0.05, ** denotes p<0.01, and *** denotes p<0.001 by two-way ANOVA. c, mRNA levels of TNF-signaling signature genes (see Supplementary Table 1) in freshly isolated FIPs (n=8) and APCs (n=8) obtained following 1 month of HFD at 30 °C. Bars represent mean ± s.e.m., * denotes p< 0.05, ** denotes p<0.01, and *** denotes p<0.001 by unpaired two-tailed Student’s t-test. Exact p values and numbers of repetitions can be found in Source Data Extended Data Figure 1.

Source data

Extended Data Fig. 2 Derivation of Mural-Tlr4KO mice and systemic phenotype following high fat diet feeding.

a, Mural-Tlr4KO (PdgfrbrtTA; TRE-Cre; Tlr4loxP/loxP) mice were generated by breeding the PdgfrbrtTA transgenic mice to animals expressing Cre recombinase under the control of the tetracycline-response element (TRE-Cre) and carrying floxed Tlr4 alleles (Tlr4loxP/loxP). Littermates carrying only PdgfrbrtTA and Tlr4loxP/loxP alleles (that is Cre-) were used as the control animals (Control). The addition of doxycycline (Dox) leads to inactivation of Tlr4 in Pdgfrb-expressing cells. b, Intraperitoneal glucose tolerance tests (GTT) of RT-housed Control (n=9) and Mural-Tlr4KO (n=6) mice after HFD feeding. * denotes p< 0.05 by unpaired two-tailed Student’s t-test. c, Intraperitoneal insulin tolerance tests (ITT) of RT-housed Control (n=9) and Mural-Tlr4KO (n=6) mice after HFD feeding. * denotes p< 0.05 by unpaired two-tailed Student’s t-test. df, Levels of total monocytes and pro-inflammatory monocytes (LY6C+) in blood (d), bone marrow (e), and spleen (f) of Control (n=8) and Mural-Tlr4KO (n=8) mice after 5 months of HFD feeding. Bars represent mean ± s.e.m. Exact p values can be found in Source Data Extended Data Figure 2. Data were reproduced two times in independent experiments.

Source data

Extended Data Fig. 3 Inactivation of Zfp423 exacerbates FIPs inflammatory responses.

a, Mural-Zfp423KO (PdgfrbrtTA; TRE-Cre; Zfp423loxP/loxP) mice were generated by breeding the PdgfrbrtTA transgenic mice to animals expressing Cre recombinase under the control of the tetracycline-response element (TRE-Cre) and carrying floxed Zfp423 alleles (Zfp423loxP/loxP). Littermates carrying only PdgfrbrtTA and Zfp423loxP/loxP alleles (that is Cre-) were used as the control animals (Control). The addition of doxycycline (Dox) leads to inactivation of Zfp423 in Pdgfrb-expressing cells. b, mRNA levels of Zfp423 in FIPs of Control (n=3) and Mural-Zfp423KO (n=3) mice fed Dox-containing chow diet for 10 days. c, mRNA levels of indicated pro-inflammatory genes in indicated FIPs treated with vehicle (veh.) (n=4) or 100 ng/ml LPS (n=8) for 2 hours. d, mRNA levels of genes associated with macrophage activation in cultured BMDMs following exposure to indicated FIPs conditioned media (CM) for 1.5 hours. n=4 (for groups with vehicle treatment) or n=5 (for groups with LPS treatment) independent wells of macrophages examined per experiment. e, Macrophage migration following exposure to FIPs conditioned media (CM): cell counts of migrated macrophages following exposure to indicated CM for 3 hours. n=4 (for groups with vehicle treatment) or n=9 (for groups with LPS treatment) independent wells of macrophages examined per experiment. For panels d, e FIPs (isolated from pooled depots of 6-8 mice per genotype) were treated with vehicle or LPS (100 ng/ml) for 2 hours and then incubated in serum-free medium for an additional 24 hours to produce conditioned media. Experiments in this figure were independently repeated three times. Data in this figure are shown as the mean ± s.e.m., *p< 0.05, **p<0.01 or ***p<0.001 by two-tailed unpaired Student’s t-test (b) or two-way ANOVA (ce). Exact p values can be found in Source Data Extended Data Figure 3.

Source data

Extended Data Fig. 4 Adipogenesis from PDGFRβ+ progenitors is not impacted in Mural-Zfp423TG mice.

a, Representative 20x magnification images of APCs from gonadal WAT of Control (pooled from 4 mice) and Mural-Zfp423TG (pooled from 4 mice) animals maintained for 8 days in growth media. No overt difference in the degree of spontaneous adipogenesis is observed. b, mRNA levels of key regulators of adipogenesis (Cebpa and Pparg2) in freshly isolated FIPs and APCs from gonadal WAT of Control (n=8) and Mural-Zfp423TG (n=12) mice after 1-month HFD feeding. c, Average adipocyte size within gonadal WAT sections of control (n=8) and Mural-Zfp423TG (n=8) mice after 5-month Dox-HFD feeding. d, mRNA levels of adipocyte-selective genes and fibrogenic genes in whole gonadal WAT of control (n=8) and Mural-Zfp423TG (n=11) mice after 5-month Dox-HFD feeding. In bd, bars represent mean ± s.e.m. ** denotes p <0.01 by one-way ANOVA (b). e, MuralChaser-Zfp423TG (PdgfrbrtTA; TRE-Cre; TRE-Zfp423; Rosa26RmT/mG) mice were generated by reconstituting the Rosa26RmT/mG allele into the Mural-Zfp423TG background. The addition of doxycycline results in overexpression of Zfp423 and indelible labeling of Pdgfrb-expressing cells with mGFP expression. f, Representative 63x magnification confocal immunofluorescence images of gonadal WAT sections from MuralChaser (PdgfrbrtTA; TRE-Cre; Rosa26RmT/mG) and MuralChaser-Zfp423TG mice after HFD feeding. 8 weeks-old animals were maintained on doxycycline-containing HFD feed for 8 weeks. Sections were stained with anti-PERILIPIN (PERILIPIN; red) and anti-GFP (green) antibodies and counterstained with DAPI (blue; nuclei). Composite images were generated by digital overlay. g, Quantification of de novo adipogenesis: the frequency of GFP+ PERILIPIN+ cells observed by immunostaining was quantified by assaying ~ 3000-4000 adipocytes total from 5 mice per genotype. Bars represent mean ± s.e.m. Exact p values can be found in Source Data Extended Data Figure 4. Data were reproduced two times in independent experiments.

Source data

Extended Data Fig. 5 Zfp423 overexpression in PDGFRβ+ cells confers metabolic benefits in obesity.

a, Western blot of phosphorylated AKT (pAKT), total AKT, and β-Actin protein expression in tissue extracts of gonadal WAT from Control (n=3) and Mural-Zfp423TG (n=3) mice after 5-month HFD feeding. Tissues were collected before (- insulin) and after insulin stimulation (+ insulin). b, Intraperitoneal glucose tolerance tests (GTT) of thermoneutral-housed Control (n=9) and Mural-Zfp423TG (n=11) mice after HFD feeding. * denotes p< 0.05 by unpaired two-tailed Student’s t-test. c, Intraperitoneal insulin tolerance tests (ITT) of thermoneutral-housed Control (n=9) and Mural-Zfp423TG (n=11) mice after HFD feeding. * denotes p< 0.05 by unpaired two-tailed Student’s t-test. d, Levels of serum ADIPONECTIN and serum INSULIN (six-hour fasted) in Control and Mural-Zfp423TG mice maintained on doxycycline-containing chow diet or doxycycline-containing HFD for 1 month or 5 months. For control mice, n=3 of chow, n=9 of 1-month HFD, n=9 of 5-month HFD; for Mural-Zfp423TG mice, n=3 of chow, n=10 of 1-month HFD, n=7 of 5-month HFD. eanalysed using flow cytometry Serum levels of indicated pro-inflammatory cytokines (CCL2, IL-6, TNFα) in Control (n=8) and Mural-Zfp423TG (n=8) mice maintained on doxycycline-containing HFD for 5 months. f, mRNA levels of pro-inflammatory genes in skeletal muscle of Control (n=8) and Mural-Zfp423TG (n=8) mice after 5-months HFD feeding. g, mRNA levels of pro-inflammatory genes in livers of Control (n=8) and Mural-Zfp423TG (n=8) mice after 5-months HFD feeding. hj, Levels of total monocytes and pro-inflammatory monocytes (LY6C+) in blood (h), bone marrow (i), and spleen (j) of Control (n=6) and Mural-Zfp423TG (n=7) mice after 5 months of HFD feeding. For panels dj, bars represent mean ± s.e.m., and * denotes p< 0.05 by unpaired two-tailed Student’s t-test. Exact p values, numbers of repetitions, and uncropped western blots can be found in Source Data Extended Data Figure 5.

Source data

Extended Data Fig. 6 Zfp423 inactivation in PDGFRβ+ cells exacerbates chronic WAT inflammation.

a, Approach: 6 weeks-old mice were preconditioned at thermoneutrality for two weeks then switched to a doxycycline-containing HFD for indicated period. b, Control (n=8; black) and Mural-Zfp423KO (n=9; red) body weights following the onset of HFD feeding. c, WAT mass mice after 1 month (Control, n=10; Mural-Zfp423KO, n=8) and 5 months (Control, n=8; Mural-Zfp423KO, n=8) of HFD feeding. d, Frequency of total PDGFRβ+ cells, FIPs, and APCs within gonadal WAT (gWAT) of Control (n=10) and Mural-Zfp423KO (n=9) mice after 1 month of HFD feeding. e, Zfp423 mRNA levels within freshly isolated FIPs and APCs from gWAT of Control (n=10) and Mural-Zfp423KO (n=9) mice after 1 month of HFD feeding. f, Pro-inflammatory gene expression within freshly isolated FIPs and APCs from gWAT of Control (n=10) and Mural-Zfp423KO (n=9) mice after 1 month of HFD feeding. g, h, 4x magnification images of H&E stained gWAT sections from Control mice maintained on HFD for 5 months. i, j, 10x magnification images of PERILIPIN (red) and MAC-2 (green) expression within gWAT sections from Control mice maintained on HFD for 5 months. k, l, 4x magnification images of H&E stained gWAT sections from from Mural-Zfp423KO mice maintained on HFD for 5 months. m, n, 10x magnification images of PERILIPIN (red) and MAC-2 (green) expression within gWAT sections from Mural-Zfp423KO mice maintained on HFD for 5 months. o, Pro-inflammatory- and macrophage-selective gene expression in gWAT of Control (n=9) and Mural-Zfp423KO (n=8) mice after 5-months HFD feeding. p, Protein levels of indicated cytokines in gWAT od Control (n=8) and Mural-Zfp423KO (n=8) mice after 5-months HFD feeding. q, Frequency of total adipose tissue macrophages (ATMs) within gWAT of Control (n=8) and Mural-Zfp423KO (n=8) mice after 5 months of HFD-feeding. r, Frequency of pro-inflammatory ATMs within gWAT of Control (n=8) and Mural-Zfp423KO (n=8) mice after 5 months of HFD-feeding. s, Frequency of tdTomato+ macrophages within gWAT SVF of obese mice following injection of tdTomato+ monocytes. n=8 for Control; n=8 for Mural-Zfp423TG. Bars represent mean ± s.e.m., *p< 0.05, **p<0.01 or ***p<0.001 by unpaired two-tailed Student’s t-test (os) or one-way ANOVA (e, f). Exact p values and numbers of repetitions can be found in Source Extended Data Figure 6.

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Extended Data Fig. 7 Vascular integrity and adipocyte differentiation from PDGFRβ+ perivascular progenitors are not impacted in Mural-Zfp423KO mice.

a, Representative images of gonadal and inguinal WAT depots of Control and Mural-Zfp423KO mice after Evans blue injection. Fixed tissues were imaged for Evans Blue auto fluorescence at 700 nm using a LI-COR Odyssey infrared imaging system. b, Representative 10x magnification images of APCs from gonadal WAT of Control (pooled from 4 mice) and Mural-Zfp423KO (pooled from 4 mice) animals maintained for 8 days in growth media. No overt difference in the degree of spontaneous adipogenesis is observed. c, mRNA levels of key regulators of adipogenesis (Cebpa and Pparg2) in freshly isolated FIPs and APCs from gonadal WAT of control (n=10) and Mural-Zfp423KO (n=9) mice after 1-month HFD feeding. Bars represent mean ± s.e.m., * denotes p<0.05, *** denotes p<0.001 by one-way ANOVA. d, Average adipocyte size within gonadal WAT sections of control (n=11) and Mural-Zfp423KO (n=8) mice after 5-month Dox-HFD feeding. e, mRNA levels of adipocyte-selective genes and fibrogenic genes in whole gonadal WAT of control (n=9) and Mural-Zfp423KO (n=8) mice after 5-month Dox-HFD feeding. f, MuralChaser-Zfp423KO (PdgfrbrtTA; TRE-Cre; Zfp423loxP/loxP; Rosa26RmT/mG) mice were generated by reconstituting the Rosa26RmT/mG allele into the Mural-Zfp423KO background. The addition of doxycycline results in inactivation of Zfp423 and indelible labeling of Pdgfrb-expressing cells with mGFP expression. g, Representative 63x magnification confocal immunofluorescence images of gonadal WAT sections from MuralChaser (PdgfrbrtTA; TRE-Cre; Rosa26RmT/mG) and MuralChaser-Zfp423KO mice after HFD feeding. 8 weeks-old animals were administered doxycycline-containing chow diet for 7 days and then switched to HFD feed (without doxycycline) for another 8 weeks. Sections were stained with anti-PERILIPIN (PERILIPIN; red) and anti-GFP (green) antibodies and counterstained with DAPI (blue; nuclei). Composite images were generated by digital overlay. h, Quantification of de novo adipogenesis: the frequency of GFP+ PERILIPIN+ cells observed by immunostaining was quantified by assaying ~3000-4000 adipocytes total from 5 mice per genotype. Bars represent mean ± s.e.m. Exact p values and numbers of repetitions can be found in Source Data Extended Data Figure 7.

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Extended Data Fig. 8 Systemic consequences of inactivating mural cell Zfp423 in obese mice housed at thermoneutrality.

a, Western blot of phosphorylated AKT (pAKT), total AKT, and β-actin protein expression in tissue extracts of gonadal WAT from Control (n=3) and Mural-Zfp423KO (n=3) mice after 5-month HFD feeding. Tissues were collected before (- insulin) and after insulin stimulation (+ insulin) of anesthetized animals. b, Intraperitoneal glucose tolerance tests (GTT) of thermoneutral-housed Control (n=12) and Mural-Zfp423KO (n=16) mice after HFD feeding. * denotes p< 0.05, ** denotes p< 0.01 by unpaired two-tailed Student’s t-test. c, Intraperitoneal insulin tolerance tests (ITT) of thermoneutral-housed Control (n=8) and Mural-Zfp423KO (n=14) mice after HFD feeding. * denotes p< 0.05 by unpaired two-tailed Student’s t-test. d, Serum levels of ADIPONECTIN (left) and INSULIN (six-hour fasting) (right) in Control and Mural-Zfp423KO mice maintained on doxycycline-containing chow diet or doxycycline-containing HFD for 1 month or 5 months. For control, n=5 of chow, n=8 of 1-month HFD, n=8 of 5-month HFD; for Mural-Zfp423KO, n=6 of chow, n=11 of 1-month HFD, n=9 of 5-month HFD. e, Serum levels of indicated pro-inflammatory cytokines in Control (n=8) and Mural-Zfp423KO (n=8) mice maintained on doxycycline-containing HFD for 5 months. f, mRNA levels of pro-inflammatory genes in skeletal muscle of Control (n=8) and Mural-Zfp423KO (n=6) mice after 5-months HFD feeding. g, mRNA levels of pro-inflammatory genes in livers of Control (n=8) and Mural-Zfp423KO (n=9) mice after 5-months HFD feeding. hj, Levels of total monocytes and pro-inflammatory monocytes (LY6C+) in blood (h), bone marrow (i), and spleen (j) of Control (n=6) and Mural-Zfp423KO (n=7) mice after 5 months of HFD feeding. Bars represent mean ± s.e.m. * denotes p< 0.05 by unpaired two-tailed Student’s t-test (d, e, g). Exact p values, numbers of repetitions, and uncropped western blots can be found in Source Data Extended Data Figure 8.

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Extended Data Fig. 9 Modulating Zfp423 expression impacts the activation of NFκB signaling in FIPs.

a, Mural-Ikk2CA/Zfp423TG (PdgfrbrtTA; TRE-Cre; TRE-Zfp423; Rosa26RIkk2CA) mice were generated by breeding the Rosa26RIkk2CA allele into the Mural-Zfp423TG background. The addition of doxycycline (Dox) leads to overexpression of Zfp423 and simultaneous activation of constitutively active (CA) IKK2 in Pdgfrb-expressing cells. b, mRNA levels of Zfp423 in cultured FIPs from Control (n=4), Mural-Ikk2CA (n=4), and Mural-Ikk2CA/Zfp423TG (n=4) mice following exposure to doxycycline (1 μg/ml) in vitro for 1 day. c, mRNA levels of indicated pro-inflammatory genes in cultured FIPs from Control (n=4), Mural-Ikk2CA (n=4), and Mural-Ikk2CA/Zfp423TG (n=4) mice following exposure to doxycycline (1 μg/ml) in vitro for 1 day. d, Mural-Ikk2CA/Zfp423KO (PdgfrbrtTA; TRE-Cre; Zfp423loxP/loxP; Rosa26RIkk2CA) mice were generated by breeding the Rosa26RIkk2CA allele into the Mural-Zfp423KO background. The addition of doxycycline (Dox) leads to inactivation of Zfp423 and simultaneous activation of constitutively-active (CA) IKK2 in Pdgfrb-expressing cells. e, mRNA levels of Zfp423 in cultured FIPs from Control (n=4), Mural-Ikk2CA (n=4), and Mural-Ikk2CA/Zfp423KO (n=4) mice following exposure to doxycycline (1 μg/ml) in vitro for 1 day. f, mRNA levels of indicated pro-inflammatory genes in cultured FIPs from Control (n=4), Mural-Ikk2CA (n=4), and Mural-Ikk2CA/Zfp423KO (n=4) mice following exposure to doxycycline (1 μg/ml) in vitro for 1 day. Experiments in this figure were independently repeated three times. Data in this figure are shown as the mean ± s.e.m., *p< 0.05 or ***p<0.001 by one-way ANOVA. Exact p values can be found in Source Data Extended Data Figure 9.

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Extended Data Fig. 10 Proposed Model: PDGFRβ+ perivascular cells regulate WAT macrophage accumulation in obesity.

FIPs are a subpopulation of PDGFRβ+ perivascular cells that are capable of exerting a pro-inflammatory phenotype. In lean (chow-fed) mice, the expression of ZFP423 in FIPs serves to moderate the activation of NFκB signaling and adipose tissue inflammation, through a molecular mechanism depicted in Fig. 8g. In response to high fat diet feeding, Zfp423 expression is reduced, facilitating the activation of NFκB signaling in FIPs and the production of pro-inflammatory cytokines/chemokines required to drive the chronic macrophage accumulation and activation that occurs during prolonged high fat diet feeding.

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Shan, B., Shao, M., Zhang, Q. et al. Perivascular mesenchymal cells control adipose-tissue macrophage accrual in obesity. Nat Metab 2, 1332–1349 (2020). https://doi.org/10.1038/s42255-020-00301-7

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