Perivascular Adipose Tissue–Derived PDGF-DContributes to Aortic Aneurysm Formation During ObesityZe-Bei Zhang, Cheng-Chao Ruan, Jing-Rong Lin, Lian Xu, Xiao-Hui Chen, Ya-Nan Du, Meng-Xia Fu,Ling-Ran Kong, Ding-Liang Zhu, and Ping-Jin Gao

Diabetes 2018;67:1549–1560 | https://doi.org/10.2337/db18-0098

Obesity increases the risk of vascular diseases, includingaortic aneurysm (AA). Perivascular adipose tissue (PVAT)surrounding arteries are altered during obesity. How-ever, the underlying mechanism of adipose tissue, es-pecially PVAT, in the pathogenesis of AA is still unclear.Here we showed that angiotensin II (AngII) infusionincreases the incidence of AA in leptin-deficient obesemice (ob/ob) and high-fat diet–induced obese mice withadventitial inflammation. Furthermore, transcriptomeanalysis revealed that platelet-derived growth factor-D(PDGF-D) was highly expressed in the PVAT of ob/obmice. Therefore, we hypothesized that PDGF-D mediatesadventitial inflammation, which provides a direct linkbetween PVAT dysfunction and AA formation in AngII-infused obese mice. We found that PDGF-D promotesthe proliferation, migration, and inflammatory factors ex-pression in cultured adventitial fibroblasts. In addition,the inhibition of PDGF-D function significantly reducedthe incidence of AA in AngII-infused obese mice. Moreimportantly, adipocyte-specific PDGF-D transgenic miceare more susceptible to AA formation after AngII infusionaccompanied by exaggerated adventitial inflammatoryand fibrotic responses. Collectively, our findings re-veal a notable role of PDGF-D in the AA formationduring obesity, and modulation of this cytokine might bean exploitable treatment strategy for the condition.

A close relationship has been well established betweenobesity and vascular diseases, including hypertension,atherosclerosis, and aortic aneurysm (AA) (1–3). In addi-tion to metabolic effects, adipose tissue–derived adipo-kines perform various important roles in the regulation of

vascular disorders such as neointimal formation, angio-genesis, and vascular remodeling (4–6). Virtually all arter-ies are surrounded by amounts of perivascular adiposetissue (PVAT), which is juxtaposed to the vascular adven-titia. Recently, it has become recognized that PVAT isabundantly expanded with an altered production of adipo-kines and chronic inflammatory response during obe-sity, which have important effects on vascular disease(7–9).

AA, characterized by chronic vascular inflammation anddestructive connective tissue remodeling, tends to expandasymptomatically until a catastrophic event, such as aorticrupture or dissection, occurs (10,11). Multiple risk factorsare associated with the incidence of AA, including advancedage, smoking, hyperlipidemia, and hypertension (12,13).Recently, obesity is reported to increase the risk of ab-dominal AA. A population study of .12,000 men con-firmed that an index of obesity (waist circumference andwaist-to-hip ratio) independently associates with abdom-inal AA formation (14). Another cohort study (15) sug-gested that the risk of abdominal AA increased by 15%per 5-cm increment of waist circumference up to 100 cmfor men and 88 cm for women. A previous study (16) haspointed out increased AA formation in leptin-deficientobese mice (ob/ob) after angiotensin II (AngII) infusion.However, little is known about the detailed mechanismof AA formation during obesity.

Vascular inflammation is a crucial cause of AA, which ishistologically characterized by medial degeneration andvarious degrees of adventitial immune cell recruitment(17). The infiltration of inflammatory cells, includingmacrophages, lymphocytes, and mast cells, is mainly

The State Key Laboratory of Medical Genomics, Shanghai Key Laboratory ofHypertension, Department of Hypertension, Ruijin Hospital and Shanghai Instituteof Hypertension, Shanghai Jiao Tong University School of Medicine, Shanghai,People’s Republic of China

Corresponding author: Ping-Jin Gao, gaopingjin@sibs.ac.cn, and Cheng-ChaoRuan, ccruan@sibs.ac.cn.

Received 22 January 2018 and accepted 11 May 2018.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0098/-/DC1.

Z.-B.Z. and C.-C.R. contributed equally to this work.

© 2018 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and the workis not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 67, August 2018 1549

OBESITY

STUDIES

observed in the tunica adventitia of human aneurismaltissues (18). Recent studies showed that adventitial fibroblastactivation contributes to AA formation via accelerating im-mune cell–mediated vascular inflammation in mice (19,20).It is also well known that chronic low-grade inflammationwithin the adipose tissue is an important causal factor forobesity-related vascular disorders (21–23). Therefore, wehypothesized that PVAT-derived factor mediates adventi-tial inflammation and promotes AA formation in AngII-infused obese mice.

In the current study, by performing cDNA microarrayanalysis, we detected highly expressed platelet-derivedgrowth factor-D (PDGF-D) in the PVAT of AngII-inducedob/ob mice. PDGF-D stimulated adventitial fibroblast mi-gration and proliferation, as well as inflammatory factorexpression. Blockade of PDGF-D function prevented AAformation in obese mice with AngII infusion. More im-portantly, we further demonstrated the pivotal role ofPDGF-D in AA formation by using an adipocyte-specificPDGF-D transgenic mouse model.

RESEARCH DESIGN AND METHODS

Animals and Animal CareMale 8-week-old ob/obmice and wild-type (WT) mice werepurchased from Slac Laboratory Animal Co., LTD. High-fatdiet (HFD)–induced obese mice were fed a diet containing60% kcal from fat for 4 months before and during theinfusion of AngII. Mice in the normal diet group (low-fatdiet) were age matched to the HFD mice to control foreffects of aging. Mice were infused with PBS or 1,000ng/kg/min AngII using ALZET Mini-Pumps for 14 days.For in vivo PDGFR-D inhibition experiment, mice weretreated via oral gavages (CP673451; 40 ng/kg/day) for14 days during AngII infusion. Systolic blood pressurewas measured by the tail-cuff method, and the average ofthree pressure readings was obtained. All animals had freeaccess to water and a standard laboratory diet. All animalprocedures were approved in accordance with the institu-tional guidelines established by the Committee of Ethics onAnimal Experiments at the Chinese Academy of Sciences.

Gene Expression MicroarraysWhole-mouse genome microarray 43 44 K was purchasedfrom Agilent. Data were scanned by an Agilent MicroarrayScanner using the default settings and were analyzed bythe Shanghai Biotechnology Corporation.

Ultrasound ExaminationThe aortic internal diameter of mice was assessed by aVisualSonics Vevo770 Ultrasound biomicroscope (Visual-Sonics Inc., Toronto, ON, Canada) with a 30-MHz lineararray ultrasound transducer, and the inner lumen diameterat the maximal expanded portion of the aorta was quan-tified as the internal maximal diameter.

Generation of Adipocyte-Specific Transgenic MiceA mouse pdgfd open reading frame was inserted intothe PiggyBac transposon gene expression vector after

the adipocyteprotein-2 promoter. Then, transgenic micewere generated by vector microinjection to the zygote.The pups were screened by PCR assay. Of 52 pupsscreened, 13 were identified positive (F0). F1 was gener-ated by each F0 mating with WT mice with the sameC57BL/6 background. F1 was identified by PCR and Westernblot.

Histology and ImmunostainingAortas fixed in formalin and embedded in paraffin weresectioned at 5 mm. Hematoxylin-eosin (H-E) or Masson’strichrome staining was performed using standard proce-dures. Immunohistochemical staining of PDGF-D wasperformed using anti–PDGF-D antibody. Antigen retrievalwas obtained by heating the tissue slides in 0.01 mol/Lcitrate buffer, pH 6.0, at 100°C for 5 min. Immunoflu-orescence staining of Ki67, CD68, a-smooth muscle ac-tin (a-SMA), and fibroblast-specific protein 1 (FSP1)was performed using anti-Ki67 (ab15580; Abcam), anti-CD68 (MCA1957; Bio-Rad), anti–a-SMA (ab21027; Abcam),and anti-FSP1 (ab124805; Abcam) antibodies, and thenfluorescence-conjugated secondary antibodies (Alexa Fluor555, Alexa Fluor 488; Invitrogen). Sections were mountedin Fluorescence Mounting Medium (Dako). Images of H-Eand immunohistochemical staining were obtained byZeiss microscope. Immunofluorescence staining was ex-amined by a laser-scanning confocal microscope (Zeiss).Images were analyzed with ImageJ and Image-Pro Plussoftware.

Cell CultureFor adipocyte culture, stromal vascular fraction cells inthe subcutaneous adipose tissue were separated fromadipocyte-specific PDGF-D transgenic (PA-Tg) mice andWTmice by a collagenase digestion method. Then stromalvascular fraction to adipocyte differentiation assay wasperformed by treatment with DMEM containing 10% FBS,0.5 mmol/L isobutylmethylxanthine, 0.1 mmol/L dexa-methasone, 1 mmol/L rosiglitazone, and 1 mg/mL insulinfor 2 days, followed by treatment with 1 mg/mL insulin for8 days. At day 10, PA-Tg–conditioned medium (PA-Tg-CM)and WT adipocyte–conditioned medium (WT-CM) werecollected to culture adventitial fibroblasts. Rat adventitialfibroblasts were isolated from the aortas of Sprague-Dawley rats (weight 120–150 g) and cultured in condi-tionedmediumat 37°C in a humidified atmosphere containing5% CO2.

Quantitative Real-time PCR and Western Blot AnalysisQuantitative real-time PCR (QPCR) was performed usingthe SYBR Premix Ex Taq kits (TaKaRa) in an ABI PRISM7900HT System (Applied Biosystems). Succinate dehydro-genase complex subunit A was used as a standard refer-ence. Reactions were performed at 95°C for 30 s followedby 40 cycles at 95°C for 5 s, and at 60°C for 30 s. Mousetissue or cell extracts containing equal amounts of totalprotein were resolved by SDS-PAGE followed by immuno-blot with the immunoblotting antibodies (e.g., PDGF-D,

1550 PDGF-D and Aortic Aneurysm in Obesity Diabetes Volume 67, August 2018

transforming growth factor-b [TGF-b], SMAD2/3). Thechemiluminescence was detected using an ECL detectionsystem.

Adventitial Fibroblast Migration and Proliferation AssayMigration assays were performed using 6.5-mm-diameterand 8.0-mm-pore size Transwells (Costar) coated with0.5% gelatin. Adventitial fibroblasts were prepared inserum-free medium, and 4 3 104 cells were added tothe upper chamber in migration buffer (DMEM containing0.1% BSA). After 24 h of AngII or PDGF-D stimulationat 37°C, cells were removed from the upper surface of themembranes with a cotton swab, and cells that migratedto the lower surface were fixed with 4% paraformaldehydefor 30 min and then stained with 0.1% crystal violet(C6158; Sigma-Aldrich) for 10 min. Migrated cells werethen counted under a microscope. Cell proliferation assayswere performed using Cell-Light EdU Apollo594 In VitroImaging Kit (Ribobio Co., LTD.). Images were obtained by alaser-scanning confocal microscope (Zeiss).

ELISAAdventitial fibroblasts were seeded into six-well plates andincubated in serum-free DMEM for 24 h. The cells werestimulated for indicated stimulations. Interleukin-6 (IL-6),MCP-1, and tumor necrosis factor-a in the supernate, andplasma was quantified using sandwich ELISA kits accord-ing to the protocol provided by the manufacturer (Abcam).

StatisticsResults were expressed as the mean 6 SD. Comparisonsof experimental groups were analyzed by Student t test(two groups) or one-way ANOVA followed by the post hocDunnett test for data with more than two groups (Levenetests for equal variance). The Dunnett T3 test was used asa post hoc test comparison for the analysis of unequalvariances (Welch and Brown-Forsythe test). Probabilityvalues,0.05 were considered to be statistically significant.

RESULTS

AngII Induces AA Formation and Adventitia Activationin Obese MiceInitially, we found that ob/obmice manifest a phenotype ofAA when subjected to 2 weeks of infusion of AngII (Fig.1A). The AA incidence in AngII-infused ob/ob mice was65% (13 of 20) compared with 5% (1 of 20) in WT mice.Among the 13 occurrences of AA in ob/ob mice, 7 wereabdominal AA, 5 were thoracoabdominal AA, and 1 wasthoracic AA (Fig. 1B). Approximately 30% (6 of 20) of theob/obmice died because of aortic rupture, whereas none ofthe WT mice died (Fig. 1C). Ultrasound analysis showedaortic expansion in ob/ob mice compared with WT miceafter AngII infusion (Fig. 1D). Furthermore, AngII infusionresulted in a comparative increase of media thickness inWT and ob/ob mice, whereas adventitia in ob/ob mice wasremarkably thickened compared with WT mice after AngIIinfusion (Fig. 1E), which is accompanied with an increasedmacrophage infiltration (Fig. 1F). Next, we also demonstrated

that AngII caused AA formation in HFD-induced obesemice, including abdominal AA, thoracoabdominal AAand thoracic AA (Supplementary Fig. 1A–C). Accord-ingly, AngII-infusion resulted in adventitial thickeningand macrophage infiltration in HFD mice (Supplemen-tary Fig. 1D and E). These indicate that adventitia ac-tivation may be involved in the AA formation duringobesity.

Transcriptome Analysis Reveals That PDGF-D Is HighlyExpressed in the PVAT of Obese MiceTo determine whether obesity-induced PVAT dysfunctionis involved in the regulation of AA formation and associ-ated with adventitial remodeling in obese mice, we per-formed gene expression microarray analysis. As indicatedin the volcano plots and heatmap, the PVAT clustershowed large overlaps between WT mice, with or withoutAngII infusion, and the PVAT of ob/ob mice also shareda similar transcriptional profile, with or without AngIItreatment. In contrast, there was a great difference inPVAT between WT and ob/ob mice regardless of infusionwith saline or AngII (Supplementary Fig. 2A–C). Thesesuggest that obesity, rather than AngII-induced PVATdysfunction contributes to AA formation in our model.Heatmap and Circos plots showed different expressionsin fatty acid metabolism–related, inflammatory response–related, and fibroblast proliferation–related genes (Sup-plementary Fig. 2C and D). Since AngII-infused ob/obmice showed significant adventitial hypertrophy and fibro-blasts were considered as the main component of theadventitia, we analyzed the transcriptional profile inregulating fibroblast proliferation. Intriguingly, gene pdgfd,which encodes PDGF-D and plays an important role inactivating fibroblasts, was highly expressed in the PVAT ofob/obmice. Next, we examined PDGF-D expression in cellsfrom every layer of the vascular wall, including endothelialcells, smooth muscle cells, adventitial fibroblasts, andadipocytes. The result indicates that PDGF-D is mostlyexpressed in the adipocyte (Supplementary Fig. 2E). Fur-ther, QPCR, immunohistochemistry, and Western blotanalysis confirmed the increased expression of PDGF-Din the PVAT of AngII-infused ob/ob mice (Fig. 2A–C) andAngII-infused HFD mice (Fig. 2D–F). According to thephenotype of hypertrophied adventitia in AngII-infusedobese mice, we assumed that PDGF-D may be involved inthe regulation of adventitia activation and therefore par-ticipates in the pathological process of AA formation inobese mice.

Inhibition of PDGF-D Function Reverses AngII-InducedAA in Obese MiceTo verify the effect of PDGF-D on AA formation in obesemice, we treated AngII-infused obese mice with PDGF-Dreceptor antagonist CP673451. As shown, CP673451markedly diminished the incidence of AA and improvedthe survival rate in ob/ob mice after AngII infusion (Fig.3A–C). PDGF-D has been taken as a fibrosis inducer inmany disease models (24,25). In accordance, the thickness

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and fibrosis of adventitia were remarkably reduced withCP673451 treatment in AngII-infused ob/ob mice (Fig.3D and E). Moreover, adventitia proliferation and FSP1-positive fibroblast invasion were also decreased withCP673451 treatment (Fig. 3F and G). CP673451 treatmentalso decreased CD68-positive macrophage infiltrationin the adventitia, as well as plasma IL-6 and MCP-1 con-centration in AngII-infused ob/ob mice (Fig. 3H and I).Consistently, CP673451 treatment also reduced the inci-dence of AA and improved the survival rate in AngII-infusedHFD mice (Supplementary Fig. 3A–C), accompaniedby decreased adventitia fibrosis and inflammation

(Supplementary Fig. 3D–G). Together, these suggest thatthe inhibition of PDGF-D function ameliorates adventitialremodeling–related AA formation in AngII-infused obesemice.

Adipocyte-Specific PDGF-D Transgenic Mice IsSusceptible to AA Formation After AngII InfusionTo establish a direct link between adipocyte-derivedPDGF-D and AA formation, we created PA-Tg mice byusing adipocyteprotein-2 promoter (Supplementary Fig.4A). The mRNA level of pdgfd showed no differences inthe aorta, heart, kidney, liver, and lung in PA-Tg mice

Figure 1—AngII induces AA formation in obesemice.A: Representative aortas ofWT and ob/obmice infusedwith saline or AngII for 2 weeks.Scale bar, 5 mm. B: The incidence of AA in AngII-infused WT mice (n = 20) and ob/ob mice (n = 20). AAA, abdominal AA; TAA, thoracic AA;TAAA, thoracoabdominal AA. C: Survival curve of AngII-infused ob/ob mice (n = 20) and WT mice (n = 20). D: Representative images fromultrasonography (top) and quantification (bottom) of maximal aortic diameter after AngII infusion for 14 days. The arrows indicate maximalaortic diameter. E: Histopathological analysis of representative abdominal aortas by H-E staining (top) and quantification of aortic media andadventitia thickness (bottom). Scale bar, 100 mm (top) and 50 mm (bottom) in magnified photographs. The adventitia area is shown with blackdotted lines. F: Representative immunofluorescent staining (left) and quantitative analysis (right) of CD68-positivemacrophages in abdominalaortas. DAPI indicates the nucleus. Scale bar, 50 mm. N.S. indicates no significant difference. **P , 0.01, ***P , 0.001.

1552 PDGF-D and Aortic Aneurysm in Obesity Diabetes Volume 67, August 2018

compared with WT littermates (Supplementary Fig. 4B). Incontrast, pdgfd mRNA expression increased clearly in theinterscapular brown adipose tissue, subcutaneous whiteadipose tissue, and PVAT of PA-Tg mice (SupplementaryFig. 4C). The increase of PDGF-D protein in PVAT was alsoverified by Western blot (Supplementary Fig. 4D). Inter-estingly, AngII infusion caused a 55% (11 of 20) incidenceof AA in PA-Tg mice compared with zero (0 of 20) in WTmice (Fig. 4A and B). Among the 11 occurrences of AA,6 were abdominal AA and the other 5 were thoracoabdo-minal AA. Fifteen percent (3 of 20) of PA-Tg mice died asa result of aortic rupture after AngII infusion (Fig. 4C).AngII infusion also resulted in a comparative increase ofmedia thickness in WT and PA-Tg mice, whereas thethickness of adventitia showed a greater increase inAngII-infused PA-Tg mice rather than AngII-infused WTmice. In addition, the adventitia of PA-Tg mice was thickerthan WT mice without AngII treatment (Fig. 4D). Theseresults confirm that adipocyte-derived PDGF-D promotesAngII-induced AA formation, including a dramatic adven-titial remodeling.

Adventitial Fibrosis and Inflammation Are Involved inAA Formation in AngII-Infused PA-Tg MiceAccordingly, PA-Tg mice displayed grossly enlarged fibroticadventitial areas, and AngII infusion amplified the adven-titial fibrosis (Fig. 5A). Ki67 staining showed much moreproliferated cells in the adventitia of PA-Tg mice thanin WT mice (Fig. 5B). In the aneurismal section, we also

detected FSP1-positive fibroblast invasion (Fig. 5C). Ofnote, we detected a significant CD68-positive macrophageinfiltration in the adventitia of PA-Tg mice, which wasdramatically aggravated after AngII infusion (Fig. 5D).Consistently, the plasma levels of IL-6 and MCP-1 weresignificantly increased in PA-Tg mice compared withWT mice, and had a much greater increase after AngII infu-sion (Fig. 5E). These suggest that PDGF-D–mediated ad-ventitial fibrosis and inflammation are involved in AAformation in PA-Tg mice after AngII infusion.

PDGF-D Activates Adventitial FibroblastsTo determine the effect of adipocyte-derived PDGF-D onadventitial fibroblasts, we cultured adventitial fibroblastswith WT-CM and PA-Tg-CM. PA-Tg-CM had a threefoldincrease in PDGF-D concentration compared with WT-CM(Fig. 6D). AngII or PA-Tg-CM, respectively, activated ad-ventitial fibroblast proliferation (Fig. 6A), migration (Fig.6B), and collagen I expression (Fig. 6C). The combinedtreatment with PA-Tg-CM and AngII further amplifiedthese effects on adventitial fibroblasts, which indicatedthe synergistic function of adipocyte-derived PDGF-D andAngII. More importantly, CP673451 inhibited PA-Tg-CM–induced adventitial fibroblast activation, whereas thiseffect of CP673451 was blunted in AngII-stimulated ad-ventitial fibroblasts. Consistent with the proinflammatoryeffect in vivo, PA-Tg-CM increased IL-6 and MCP-1 ex-pression in adventitial fibroblasts, and this increase wasabolished after CP673451 pretreatment (Fig. 6E). Next, we

Figure 2—PDGF-D increases in the PVAT of obesemice.A: RelativemRNAexpression of pdgfd in aortas and PVAT of AngII-infusedWTmiceand ob/ob mice. n = 6 each. B: Immunohistochemistry staining of PDGF-D in the abdominal aortas of indicated mice. Scale bar, 50 mm. C:Western blot (top) and quantitative analysis (bottom) of PDGF-D protein expression in PVAT of indicated mice. n = 4 each. D: Relative mRNAexpression of pdgfd in aortas and PVAT of AngII-infused low-fat diet (LFD) and HFD mice. n = 6 each. E: Immunohistochemistry staining ofPDGF-D in the abdominal aortas of indicated mice. Scale bar, 50 mm. F: Western blot (top) and quantitative analysis (bottom) of PDGF-Dprotein expression in PVAT of indicated mice. n = 4 each. N.S. indicates no significant difference. **P , 0.01, ***P , 0.001.

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Figure 3—Inhibition of PDGF-D function ameliorates adventitial remodeling and suppresses AA formation in obese mice. A: Representativeaortas of AngII-infused ob/ob mice with CP673451 (CP) or without CP673451 (Ctrl) treatment. Scale bar, 5 mm. B: The incidence of AA inAngII-infused ob/ob mice with CP673451 (n = 8) or without CP673451 (n = 8) treatment. AAA, abdominal AA; TAA, thoracic AA; TAAA,

1554 PDGF-D and Aortic Aneurysm in Obesity Diabetes Volume 67, August 2018

analyzed the effect of recombinant PDGF-D on culturedadventitial fibroblasts. Likewise, the results confirmed thesynergistic function of AngII and recombinant PDGF-D onthe regulation of adventitial fibroblast proliferation, mi-gration, collagen expression, and IL-6 and MCP-1 expres-sion (Supplementary Fig. 5A–E). These data indicate thatPDGF-D activates adventitial fibroblasts in vitro.

TGF-b Pathway Is Involved in AngII-Induced AAFormation in PA-Tg MiceSince the TGF-b signaling pathway plays a pivotal role inthe regulation of adventitial fibroblast function, as wellas AA formation in vivo (26–28), we detected whetherPDGF-D stimulated the TGF-b pathway in these mice.First, we found increased TGF-b and downstream collagenI expression in the aortas of PA-Tg mice, and AngII in-fusion resulted in a further increase (Fig. 7A and B).Consistently, the downstream Smad2/3 phosphorylationwas increased in PA-Tg mice, which showed a much higher

level after AngII infusion (Fig. 7B). Similarly, recombinantPDGF-D and AngII had a synergistic effect on the TGF-b/Smad signaling pathway in cultured adventitial fibro-blasts (Supplementary Fig. 6A). Accordingly, the inhibitionof PDGF-D function by CP673451 treatment attenuatedthe activation of the TGF-b/Smad signaling pathway inAngII-infused ob/ob mice and PA-Tg mice (SupplementaryFig. 6B and C). These suggest that the TGF-b/Smad path-way activation in adventitial fibroblasts is involved inPDGF-D–mediated AA formation during obesity.

It is well known that blood pressure elevation contrib-utes to AA formation. Herein we showed that bloodpressure had no further elevation in ob/ob mice, HFDmice, and PA-Tg mice after AngII infusion. Meanwhile,CP673451 treatment did not regulate blood pressure inthese mice (Supplementary Fig. 7A–C). Therefore, PDGF-D–mediated AA formation was independent of bloodpressure regulation. We also detected plasma level ofPDGF-D in mice. The results indicated that circulating

Figure 4—AngII induces AA formation in adipocyte-specific PA-Tg. A: Representative aortas of WT and PA-Tg mice infused with saline orAngII for 2 weeks. Scale bar, 5 mm. B: The incidence of AA in AngII-infused WT mice (n = 20) and PA-Tg mice (n = 20). AAA, abdominal AA;TAA, thoracic AA; TAAA, thoracoabdominal AA. C: Survival curve of AngII-infused PA-Tg mice (n = 20) and WT mice (n = 20). D:Histopathological analysis of representative abdominal aortas by H-E staining (left) and quantification of aortic media and adventitiathickness (right). Scale bars, 100 mm (top) and 50 mm (bottom) in magnified photographs. Adventitia area is shown with black dotted lines.N.S. indicates no significant difference. ***P , 0.001.

thoracoabdominal AA. C: Survival curve of indicated mice. n = 14 each. D: Histopathological analysis of representative abdominal aortas byH-E staining (top) and quantification of aortic media and adventitia thickness (bottom). Scale bars, 100 mm (top) and 50 mm (bottom) inmagnified photographs. Adventitia area is shownwith black dotted lines. E: Representative images (top) and quantitative analysis (bottom) ofMasson’s trichrome staining of abdominal aortas. Scale bar, 100 mm (top) and 50 mm (bottom) in magnified photographs. F: Representativeimmunofluorescent staining (top) and quantitative (bottom) analysis of Ki67-positive proliferated cells in abdominal aortas. Scale bar, 50 mm.G: Representative immunofluorescent staining of a-SMA and FSP1 in abdominal aortas (left), and quantitative analysis of FSP1-positive cellinvasion into the media of vessel wall (right). The arrows indicate FSP1-positive cell invasion. Scale bar, 50 mm. H: Representative immu-nofluorescent staining (left) and quantitative analysis (right) of CD68-positive macrophages in abdominal aortas. Scale bar, 50 mm. I: Plasmaconcentration of IL-6, MCP-1, and tumor necrosis factor-a (TNF-a). n = 8 each. N.S. indicates no significant difference. **P, 0.01, ***P, 0.001.

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Figure 5—PDGF-D promotes adventitial fibrosis and inflammation. A: Representative images (left) and quantitative analysis (right) ofMasson’s trichrome staining in abdominal aortas of indicated mice. Scale bars, 100 mm (top) and 50 mm (bottom) in magnified photographs.B: Representative immunofluorescent staining (left) and quantitative analysis (right) of Ki67-positive proliferated cells in abdominal aortas.Scale bar, 50 mm. C: Representative immunofluorescent staining of a-SMA and FSP1 in abdominal aortas (left), and quantitative analysis ofFSP1-positive cells invasion into the media of vessel wall (right). The arrows indicate FSP1-positive cell invasion. Scale bar, 50 mm. D:Representative immunofluorescent staining (left) and quantitative analysis (right) of CD68-positive macrophages in abdominal aortas. Scalebar, 50 mm. E: Plasma concentration of IL-6 and MCP-1. n = 6 each. *P , 0.05, **P , 0.01, ***P , 0.001.

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levels of PDGF-D increased in ob/ob, HFD, and PA-Tg mice,but it is not influenced by AngII or CP673451 treatment(Supplementary Fig. 7D–F). Together, our data illustratethat PVAT-derived PDGF-D contributes to AA formationvia adventitial remodeling during obesity (SupplementaryFig. 8).

DISCUSSION

This study mainly reveals that obesity-related PVAT dys-function contributes to AngII-induced AA formation bysecreting PDGF-D, which is identified by transcriptomeanalysis of PVAT in ob/ob mice. Pharmacological blockade

of PDGF-D function successfully reduced AngII-inducedAA in obese mice. More importantly, we used PA-Tg miceto confirm the direct role of PDGF-D in AA formation viamediating adventitial fibrosis and inflammation.

Obesity is one of the major causes of morbidity andmortality worldwide, and it is a significant public healthburden affecting human beings (1,29,30). The effect ofobesity on cardiovascular disease mostly depends on thedysfunction of adipose tissue, especially PVAT (31,32).Herein, we showed that the dysfunction of PVAT contrib-utes to AngII-induced AA in obese mice, which is mediatedby PDGF-D–induced adventitial fibrosis and inflammation.

Figure 6—PDGF-D activates adventitial fibroblasts in vitro. Primary adventitial fibroblasts were cultured with WT-CM or PA-Tg-CM, thenstimulated with AngII (1027 mol/L), treated with CP673451 (300 mmol/L) (CP), or not stimulated or treated (Ctrl). A: Representative images(top) and quantitative analysis (bottom) of EdU proliferation staining of adventitial fibroblasts. Scale bar, 50 mm. n = 6 each.B: Representativeimages (top) and quantitative analysis (bottom) of transwell migration assay of adventitial fibroblasts. Scale bar, 50mm. n = 6 each.C: Westernblot (top) and quantitative analysis (bottom) of Col1a1 expression in adventitial fibroblasts. n = 4 each. D: ELISA analysis of PDGF-Dconcentration inWT-CM and PA-Tg-CM. E: QPCR analysis of IL-6/MCP-1mRNA levels in adventitial fibroblasts. n = 6 each. *P, 0.05, **P,0.01, ***P , 0.001.

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PDGF-D is a recently identified member of the PDGFfamily and participates in the regulation of cardiovasculardiseases (33). The PDGF protein family is a potent stim-ulator for cell proliferation and chemotaxis and is docu-mented to play a major role in cell-cell communication fornormal development and pathogenesis (34). Among theseproteins, PDGF-D has been well documented to regulateorgan fibrosis in different animal models (35). In thecardiovascular system, heart-specific overexpression ofPDGF-D induces vascular remodeling, resulting in cardiacfibrosis, dilated cardiomyopathy, and cardiac failure (24).Herein, in addition to promoting adventitial fibrosis, weprovided definitive evidence that PDGF-D accelerates ad-ventitial inflammation in obese mice and contributes to AAformation after AngII infusion. Although the mechanismof the differential expression of PDGF-D in lean controland obese mice is unclear, we assumed that the differentialexpression is a result of the different types of adipocytes(36,37). Chronic inflammation is one of the prime reasonsfor obesity-related adipose tissue dysfunction, where mac-rophage activation–mediated inflammatory factor releasehas been attributed to a central function for fat celldisorders (21,38). Thus, the inhibition of PDGF-D functionmight be a possible intervention to prevent obesity-relatedmetabolic syndrome.

It is increasingly being accepted that adventitial in-flammation plays a key role in the pathogenesis of AA,including infiltration of inflammatory cells in the adven-titia and activation of adventitial fibroblasts (39,40). It hasbeen reported that leukocyte-fibroblast interactions in theadventitia potentiate local monocyte recruitment andactivation, resulting in AA and aortic dissection (19).Even more, local neutrophil recruitment and activation inadventitia facilitate aortic expansion and lead to aorticrupture eventually (18). Herein we are the first to showa direct link between perivascular adipocytes and adven-titial fibroblasts in AA formation. In general, PVAT plays

a proinflammatory role in AA development. PVAT-derivedproinflammatory factors accelerate the recruitment ofmacrophages, lymphocytes, and mast cells in the vascularwall (41,42). Although another study (43) revealed thathigher thoracic and abdominal aortic dimensions are as-sociated with PVAT volume in a cohort study of 3,001individuals, supporting the notion that PVAT volume maycontribute to aortic remodeling. In our study, a PDGF-Dsupplement activates primary cultured adventitial fibro-blasts and increases the production of IL-6 and MCP-1in vitro. Accordantly, adipocyte-specific overexpression ofPDGF-D in vivo aggravates adventitial fibrosis and in-flammation. These indicate a crucial molecular mechanismfor adipocyte-derived PDGF-D in adventitial inflammationand AA formation.

Obesity could potentially be an independent risk factorfor AA, and meanwhile obesity also has been implicatedin the pathogenesis of diabetes. Nevertheless, data fromlarge-scale screenings have shown a paradoxically lowerprevalence of AA in patients with diabetes, even those whohave been reported as having a negative risk factor forhuman abdominal AA formation (44). Recently, a report(45) showed that vascular cell division autoantigen1 (CDA1) plays a role in diabetes to reduce susceptibilityto aneurysm. It appears that CDA1 reduces aneurysmseverity, but not the onset of aneurysm in this model.A CDA1-mediated TGF-b/Smad pathway may limit thegrowth of the aneurysm via increasing accumulation ofextracellular matrix. On the other hand, our study providesan explanation of how the PDGF-D–induced TGF-b/Smadpathway contributes to the onset of aneurysms via pro-moting vascular fibrotic response and inflammation duringobesity. This explanation is in accordance with the resultsof a previous study (16) showing that AngII inducesabdominal AA in diet-induced obese mice as well as geneticobese mice. It has shown that body weight, but not insulinsensitivity, was a significant predictor of abdominal AA

Figure 7—TGF-b and SMAD2/3 pathway is involved in aortas of AngII-infused PA-Tg mice. A: Relative TGF-b and COL1a1 mRNAexpression in aortas from saline or AngII-infused WT and PA-Tg mice. n = 6 each. B: Western blot (left) and quantitative analysis (right)of TGF-b, Col1a1, total-SMAD2/3 (T-SMAD2/3), and phosphorylated-SMAD2/3 (P-SMAD2/3) protein expressions in aortas. n = 6 each.*P , 0.05, **P , 0.01, ***P , 0.001.

1558 PDGF-D and Aortic Aneurysm in Obesity Diabetes Volume 67, August 2018

formation in the mouse model (16). Consistently, ourresults provided experimental evidence for obesity-relatedAA formation, and a molecular mechanism study proposedthat obese fat-derived PDGF-D plays a pivotal role in AAformation. Although epidemic study and basic researchhave claimed the correlation between obesity and AA, theparadox of obesity and diabetes in AA formation still needsfurther investigation.

In conclusion, this study shows that obesity-inducedPDGF-D in PVAT contributes to AA formation. We illus-trate the molecular mechanism that adipocyte-derivedPDGF-D promotes adventitial fibrosis and inflammation,which contribute to AA formation during obesity.

Acknowledgments. The authors thank Professor Jiqiu Wang (Departmentof Endocrinology and Metabolism, Ruijin Hospital, Shanghai, People’s Republic ofChina) for providing HFD mice.Funding. This work was supported by the National Natural Science Foundationof China (grants 91539202, 81570221, 81770495, and 91739303), the ShanghaiMunicipal Commission of Health and Family Planning (grants 2017YQ076 and201540222), the Shanghai Sailing Program (grant 17YF1415900), and the ChinaPostdoctoral Science Foundation (grant 2017M621504).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. Z.-B.Z. and C.-C.R. performed the AngII infusionanimal study and wrote the manuscript. J.-R.L. and L.X. performed animaltissue collection and analysis. X.-H.C. carried out ultrasound imaging. Y.-N.D.and M.-X.F. analyzed survival curve data. L.-R.K. and D.-L.Z. performed theprimary cell culture. P.-J.G. designed and initiated the experiments. P.-J.G. isthe guarantor of this work and, as such, had full access to all the data in thestudy and takes responsibility for the integrity of the data and the accuracy ofthe data analysis.

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