Bmal1 in Perivascular Adipose Tissue Regulates …...10.1161/CIRCULATIONAHA.117.029972 2 Abstract...

10.1161/CIRCULATIONAHA.117.029972

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Bmal1 in Perivascular Adipose Tissue Regulates Resting Phase Blood Pressure

Through Transcriptional Regulation of Angiotensinogen

Running Title: Chang et al.; PVAT Bmal1 Regulates BP During Resting Phase

Lin Chang, MD, PhD1; Wenhao Xiong, MD2; Xiangjie Zhao, MS1; Yanbo Fan, MD, PhD1;

Yanhong Guo, MD, PhD1; Minerva Garcia-Barrio, PhD1; Jifeng Zhang, PhD1;

Zhisheng Jiang, MD, PhD2; Jiandie D Lin, PhD3; Y Eugene Chen, MD, PhD1

1Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan, Ann

Arbor, MI; 2Institute of Cardiovascular Disease, Key Laboratory for Atherosclerology of Hunan

Province, University of South China, Hengyang, Hunan, P.R. China; 3Life Sciences Institute and

the Department of Cell & Developmental Biology, University of Michigan, Ann Arbor, MI

Addresses for Correspondence:Dr. Lin Chang, MD, PhD Dr. Eugene Chen, MD, PhDFrankel Cardiovascular Center Frankel Cardiovascular CenterDepartment of Internal Medicine Department of Internal MedicineUniversity of Michigan University of Michigan2800 Plymouth Road 2800 Plymouth RoadNCRC 026-214N, Ann Arbor, MI 48109 NCRC 026-351S, Ann Arbor, MI 48109Tel: 734-998-7630 Tel: 734-647-5742Fax: 734-763-7097 Fax: 734-763-7097Email: [email protected] Email: [email protected]

1Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan, Ann

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by guest on February 4, 2018http://circ.ahajournals.org/

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Abstract

Background—The perivascular adipose tissue (PVAT), surrounding vessels, constitutes a distinct functional integral layer of the vasculature required to preserve vascular tone under physiological conditions. However, there is little information regarding the relationship between PVAT and blood pressure regulation, including its potential contributions to circadian blood pressure variation. Methods—Using unique brown adipocyte-specific aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and angiotensinogen (Agt) knock out mice we determined the vasoactivity of homogenized PVAT in aortic rings and how brown-adipocyte peripheral expression of Bmal1 and Agt in PVAT regulate the amplitude of diurnal change in blood pressure in mice. Results—We uncovered a peripheral clock in PVAT and demonstrated that loss of Bmal1 in PVAT reduces blood pressure in mice during the resting phase leading to a super-dipper phenotype. PVAT extracts from wild type mice significantly induced contractility of isolated aortic rings in vitro in an endothelium independent manner. This property was impaired in PVAT from brown adipocyte-selective Bmal1 deficient mice (BA-Bmal1-KO). The PVAT contractile properties are mediated by local angiotensin II (Ang II), operating through angiotensin II type 1 receptor-dependent signaling in the isolated vessels and is linked to PVAT circadian regulation of Agt. Indeed, Agt mRNA and Ang II levels in PVAT of BA-Bmal1-KO mice were significantly reduced. Systemic infusion of Ang II, in turn, reduced Bmal1 expression in PVAT while eliminating the hypotensive phenotype during the resting phase in BA-Bmal1-KOmice. Agt, highly expressed in PVAT, shows circadian expression in PVAT and selective deletion of Agt in brown adipocytes recapitulates the phenotype of selective deletion of Bmal1 in brown adipocytes. Furthermore, Agt is a transcriptional target of Bmal1 in PVAT. Conclusions—These data indicate that local Bmal1 in PVAT regulates Agt expression and the ensuing increase in Ang II, which acts on smooth muscle cells (SMCs) in the vessel walls to regulate vasoactivity and blood pressure on a circadian fashion during the resting phase. These findings will contribute to better understand cardiovascular complications of circadian disorders, alterations in the circadian dipping phenotype and the crosstalk between systemic and peripheral regulation of blood pressure.

Key Words: blood pressure; Angiotensin II; circadian; perivascular adipose tissue

aortic rings in vitro in an endothelium independent manner. This property was impairerrr d in PVATttfrom brown adipocyte-selective Bmal1 deficient mice (BA-Bmal1-KO). The PVAAAAAATTTT T T cocococococontntntntntnttrarararararar ctctctctctcttilililililileeeproperties are mediated by local angiotensin II (Ang II), operating through angiotenenenennensisisisisisin IIIIIIIIIIII ttttttypypypypypypeee eee 111111eceptor-dependent signaling in the isolated vessels and is linked to PVAT circadian regulation of

Agt. Indeed, Agt mRNA and Ang II levels in PVAT of BA-Bmal1-KO mice were significantly educececececeed.d.d.d.d.d. SSSSSSysysysysysysystetetetetetemim cc cc c iniiii fusion of Ang II, in turn, reduccucucuced Bmal1 expppprerrr ssioooon nnn in PVAT while

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Clinical Perspective

What is new?

This is the first demonstration of a peripheral clock in PVAT contributing to the

homeostatic regulation of circadian BP variation.

This study uses novel brown adipocyte specific Bmal1- and Agt-KO mouse models to

demonstrate that the PVAT peripheral clock safeguards optimal vascular tone during the

resting phase.

This study uncovered local Agt and ACE circadian expression in PVAT and

Bmal1-dependent transcriptional circadian regulation of Agt which controls local levels of

Ang II during the resting phase.

This study found that deletion of Bmal1 or Agt in PVAT results in a super-dipper

phenotype (exacerbated hypotension) during the resting phase.

What are the clinical implications?

Alterations in the peripheral clock in PVAT regulating BP could contribute to the adverse

clinical cardiovascular outcomes observed in shift workers or upon jet lag.

It is possible that obesity could alter the PVAT peripheral clock to promote abnormal dipper

phenotypes increasing cardiovascular risk.

These findings are likely relevant to the personalized design of clinical studies, including

diagnosis, biopsy collection and chronopharmacology.

The present results can inform the design of novel therapeutic approaches for hypertension

by targeting the PVAT peripheral clock.

Ang II during the resting phase.

This study found that deletion of Bmal1 or Agt in PVAT results in a super-didididididipppppppppppperererererer

phenotype (exacerbated hypotension) during the resting phase.

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Introduction

Hypertension, a disorder of complex pathophysiology, is a major contributor to cardiovascular

and renal diseases. Although the exact intricacies of hypertension etiology still remain unsolved,

it is clear that this is a multi-factorial, complex polygenic disorder with many interacting

mechanisms contributing to its pathophysiology and involving many organ systems, including

the heart, kidney, brain, vessels and the immune system1. Perivascular adipose tissue (PVAT) is

de facto a distinct functional vascular layer and plays an active role in the control of vascular

(dys)function in (patho)physiological conditions such as hypertension2, 3. Current studies show

paradoxical effects of PVAT in the regulation of vascular tone2. On one hand, PVAT exerts

anti-contractile effects in various vascular beds, a phenomenon that seems to be mediated by

some, still elusive, PVAT-derived relaxing factors (PVRF). This anti-contractile effect of PVAT is

impaired in disease conditions such as metabolic syndrome4, 5 and hypertension5-8, further

underscoring the physiological role of PVAT in vascular tone. PVRFs might comprise

adiponectin9, 10, H2S11,NO12, Ang-(1-7)13, palmitic acid methyl ester14 and prostacyclin15. On the

other hand, we previously demonstrated that PVAT dramatically constricted carotid artery rings15

while coronary artery PVAT of obese swine augmented vasoconstriction16. Additionally, PVAT

enhanced the mesenteric artery contractile response to perivascular nerve stimulation through the

production of superoxide mediated by NAD(P)H oxidase17. Chemerin is expressed in PVAT and

induces vasoconstriction18. Thus, apart from the existence of anti-contractile PVRF in PVAT, the

existence of PVAT-derived contractile factors (PVCF) must also be highlighted. A delicate

dys)function in (patho)physiological conditions such as hypertension . Current stududuuu ies show

paradoxical effects of PVAT in the regulation of vascular tone2. On one hand, PVATATATATATAT eeeeeexeeeexeertrtrtrtrtrtsssss s

anti-contractile effects in various vascular beds, a phenomenon that seems to be mediated by

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balance between those two faces of PVAT on the regulation of vascular tone could contribute to

the maintenance of vessel homeostasis and blood pressure (BP) regulation. Currently, direct

evidence derived from animal models to support the notion of an anti-contractile or contractile

role for PVAT on the regulation of BP is largely missing. We reported that a mouse model

lacking PVAT due to peroxisome proliferator-activated receptor gamma (PPARγ) deletion in

vascular smooth muscle cells (VSMCs) presents hypotension in the resting period. However, it is

not clear whether loss of PVAT or PPARγ deletion in VSMCs in these mice causes the

hypotension19.

Circadian variations in BP are among the best recognized circadian rhythms of physiology in

humans20 and rodents21. In humans, the highest BP is observed during the morning hours and the

lowest during the sleep hours. This diurnal-nocturnal cycle of BP in rodents is reversed as the

result of their nocturnal active-phase. It is known that circadian rhythms in BP are controlled by

the "master clock" located in the suprachiasmatic nucleus (SCN)22. The positive (Clock,Npas2,

Bmal1) and negative (Per1, Per2, Per3, Cry1, and Cry2) arms of the clock genes in the SCN

comprise a negative feedback loop, resulting in cyclic activation of the transcriptional machinery

of clock genes in an approximate 24-hour cycle of biological rhythm23. Studies from animals and

humans suggest that Baml1 is associated with hypertension. The Bmal1 gene is located within

hypertension susceptibility loci in rat and maps close to a region genetically divergent between

spontaneously hypertensive rats and their normotensive counterparts24. Genetic association

studies showed that two BMAL1 haplotypes were associated with hypertension24 and that the

rs3816358 SNP in BMAL1 was significantly associated with the non-dipper phenotype in young

Circadian variations in BP are among the best recognized circadian rhythms ofofofofofof pppppphyhyhyhyhyhysisisisisisiiolololololologogogogogogy y y y y y ininiiii

humans20 and rodents21. In humans, the highest BP is observed during the morning hours and the

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hypertensive patients, suggesting a genetic association with diurnal BP changes in essential

hypertension25. Indeed, disruption of master clock genes has been shown to change BP and alter

circadian rhythms of BP. Conventional Cry-knockout mice are hypertensive during the resting

phase and on average normotensive during the active period26. Mice with a mutation of Npas2

are hypotensive27. Clock mutant mice exhibit only a subtle dampening of BP upon light-dark

conditioning27. Notably, mice with global deletion of Bmal1 showed evidence of abolished

circadian rhythms of BP and were hypotensive27. However, the SCN master clock is not solely

responsible for the circadian regulation of BP. Indeed, the components of the circadian clock

genes are found in peripheral tissues, including blood vessels28. Furthermore, the peripheral

circadian clocks can be uncoupled from the central SCN circadian clocks29. A robust diurnal

variation in BP and lower BP during the dark phase was apparent in mice with endothelial

cell-specific deletion of Bmal130. PPARγ deletion in aortas repressed Bmal1 activity and herein

disrupted circadian rhythms of BP31. Also, selective deletion of Bmal1 in VSMCs decreased BP

without affecting SCN-controlled locomotor activity in mice32. Together, these studies suggest

that peripheral circadian clocks contribute to BP circadian rhythmicity.

The components of the renin angiotensin system (RAS), including angiotensinogen (Agt)

-albeit not renin-are present in PVAT33. In fact, pre-treatment of intact vessel rings with the

selective Ang II antagonist, Sari-Ile8-Ang II, significantly attenuated electrical stimulation

elicited contraction in a PVAT-dependent fashion34. Chronic treatment with an angiotensin

converting enzyme (ACE) inhibitor, quinapril, alleviated the potentiation effects of PVAT in

contraction upon electrical stimulation35, further supporting a role for PVAT-derived Ang II in

genes are found in peripheral tissues, including blood vessels28. Furthermore, the ppppppereeeee ipipipipipipheheheheheheh rarararararal l l llll

circadian clocks can be uncoupled from the central SCN circadian clocks29. A robust diurnal

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vasoactivity.

In this study, we report a peripheral circadian clock operating in PVAT-dependent circadian

regulation of BP. Deletion of Bmal1 or Agt in brown adipocytes including PVAT dramatically

reduced BP in the resting phase resulting in a super-dipper phenotype. Furthermore, we found

that Bmal1 positively regulates Agt expression at the transcriptional level in PVAT adipocytes.

Thus, Bmal1, Agt and Ang II in PVAT coordinately contribute to the regulation of homeostatic

circadian rhythmicity of BP during the resting phase.

Methods

Please see detailed experimental methods in online supplemental materials, including the list of

primers in supplemental Table I. Requests to access the data, analytic methods and study

materials for reproducibility purposes should be addressed to the corresponding authors.

Mice

Brown Adipocyte-specific deletion of Bmal1(BA-Bmal1-KO) or Agt (BA-Agt-KO) mice were

generated by crossbreeding Bmal1flox/flox (Jackson Laboratory, stock# 007668)or Agtflox/flox mice

(Jackson Laboratory stock # 018388) with UCP-1 driven Cre mice36, respectively (all in

theC57BL/6 background). Eight-week-old males were used in this study, and age-matched

wild-type littermate mice were used as controls. Mice were housed at the University of Michigan

animal facility ( 22°C and 12/12-hour light/dark cycle) and supplied with rodent diet no. 2918

(18% protein, 6% fat and moderate phytoestrogen; Harlan Laboratories) ad libitum. The Animal

Research Ethics Committee of the University of Michigan approved the study protocol for

Methods

Please see detailed experimental methods in online supplemental materials, including the list of

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animals. Telemetry, Ang II infusion and aortic ring constriction in response to PVAT extracts

were described previously19,37 and are further detailed in the supplemental materials.

Statistical analysis

Mean ± SD. values were determined using GraphPad Prism Software (version 7). Statistical

comparisons between two groups were performed by Student’s t test (Figure 1C, Figure 2C,

Figure 3A, Figure 3D, Figure 4C, Figure 5B Figure 5C, Figure 5D), and more than two groups

were performed by one-way ANOVA (Figure 1E, Figure 2B, Figure 2E, Figure 3F, Figure 4F).

Groups were considered significantly different if p values were <0.05.

Results

Bmal1 deficiency in brown adipocytes reduces BP during the resting phase

Expression of Bmal1 and Clock mRNA in brown PVAT shows circadian rhythmicity (Figure 1A),

higher at 6AM (the onset of resting phase), gradually reduced during the lights-on time (resting

phase) to their lowest level at 6PM, and progressively restored during the lights-off time (active

phase) to their higher level at 6AM. Per1 and Rev-erbα mRNA show the opposite pattern.

Furthermore, at pressor levels (500ng/kg/min) Ang II infusion results in acute and sustained high

levels of systolic BP (SBP), albeit with loss of circadian regulation for the first 3 days followed

by a progressive restoration of the dipping ability during the resting phase by day 4

(Supplemental Figure I A). Remarkably, the acute loss of circadian dipping in SBP is associated

with reduced levels of Bmal1 protein in PVAT when the tissue was collected early in resting

phase (Supplemental Figure IB). Subsequent recovery of circadian SBP variation was associated

Results

Bmalalalalalalal111111 dddddded ficicicicicicienenenenenenencyyyy iiiin brown adipocytes reduces BBBP during the rrrrrestinnnggg g phase

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with a progressive restoration of Bmal1 protein levels in PVAT after day 4 of Ang II infusion.

These data suggest that the peripheral clock in PVAT might contribute to physiological circadian

regulation of BP with Bmal1 expression in PVAT positively associated with the dip in BP during

the resting phase. This led to our hypothesis that Bmal1 and its target genes within PVAT may

communicate timing information within the vessel beds to regulate BP.

Since adipocytes in mouse PVAT are brown adipocytes15, 38, we generated BA-Bmal1-KO

mice to further understand the role of the PVAT peripheral clock on circadian regulation of BP.

Bmal1 protein was detected in all types of adipose tissues and in the whole aorta in the wild-type

mice while Bmal1 was successfully deleted in PVAT and interscapular brown adipose tissue

(BAT) of the BA-Bmal1-KO mice (Figure 1B). During daytime resting phase, Bmal1 mRNA

levels and its target circadian related genes such as Dbp, Per3 and Rev-erbα were reduced, while

Cry1, E4bp4 and Npas2 were increased in PVAT from BA-Bmal1-KO mice (Figure 1C). As

expected, loss of the regulatory circadian feedback loop was observed in the BAT as well, but not

in other organs in BA-Bmal1-KO mice (Supplemental Figure II). To determine the effects of loss

of Bmal1 in brown adipocytes on BP rhythmicity, we used telemetry to measure the BP in mice

kept in a 12/12-hour dark/light cycle. Unlike the global Bmal1 knockout mice in which circadian

rhythms of BP are abolished and the mice are hypotensive27, in a 22oC room temperature

environment, BA-Bmal1-KO mice are only hypotensive during the resting phase (from 6am to

6pm) and on average normotensive during the active period (from 6pm to 6am) (Figure 1D, 1E).

Bmal1-depletion in brown adipocytes results in a ~10% further reduction of BP during the

resting phase (SBP 116.8±7.7 mmHg in wild-type mice, and SBP 104.4±4.5 mmHg in

mice while Bmal1 was successfully deleted in PVAT and interscapular brown adippppipposososooso eeeeee titiiiititissssssssssssueueueueueue

BAT) of the BA-Bmal1-KO mice (Figure 1B). During daytime resting phase, Bmal1 mRNA

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BA-Bmal1-KO mice) with comparable BP during the active phase (SBP 132.6±13.3 mmHg in

wild-type mice, and SBP 131.4±13.5 mmHg in BA-Bmal1-KO mice), thus indicating that the

BA-Bmal1-KO mice present an exacerbated hypotensive (“super-dipper”) phenotype at rest39.

Interestingly, the heart rates of the BA-Bmal1-KO mice were reduced during both the resting and

active phases without affecting the locomotor activity (Figure 1D, 1E). However, the cardiac

function parameters during the resting phase were comparable between wild-type and

BA-Bmal1-KO mice (Supplemental Figure I C).

Since heart rate can be a determinant of diurnal changes in arterial pressure in mice40, we

injected 10mg/kg isoproterenol intraperitoneally to increase the heart rate of BA-Bmal1-KO

mice to match that of the wild-type mice (Supplemental Figure III A). The BP of wild-type and

BA-Bmal1-KO mice was immediately reduced, and restored about 1 hour after injection of

isoproterenol during the resting phase. However, recovery of BP in the BA-Bmal1-KO mice was

significantly lagged. When injection of isoproterenol was performed in the evening (active

phase), recovery of SBP of BA-Bmal1-KO mice was slightly lagged, with comparable DBP

levels between wild-type mice and BA-Bmal1-KO mice despite same levels of heart rates in both

wild-type and BA-Bmal1-KO mice (Supplemental Figure III B).

Additionally, global loss of Bmal1 leads to compensatory thermogenesis in BAT41, which

might be associated with BP and heart rate42. Indeed, selective deletion of Bmal1 in brown

adipocytes significantly increased expression of thermogenic genes such as Ucp1, Cidea and

Elovl3 in PVAT and BAT (Supplemental Figure IV A). However, the whole body metabolic

indexes in BA-Bmal1-KO mice were comparable to those of wild-type when the ambient

njected 10mg/kg isoproterenol intraperitoneally to increase the heart rate of BA-BmBmBmBmBmBmalalalalalal111111--KOKOKOKOKOKOK

mice to match that of the wild-type mice (Supplemental Figure III A). The BP of wild-type and

BA-BmBmBmBmBmBmBmal1-KOKOKOKOKOKOKO mmicicice was immediately reduced, andndnd restored abouuuuttt t 1 hohhoour after injection of

soppppopprrrrorr terenol dududududuurir ngnngng thhhheeeeee resstststsss iininininini g phase. HHHowevveveer, rerererecoveveveveveveveryryryryryryr ofofofofofofof BBBPB innnn theee BABABAAAAA------Bmal1--KOKOKOKOKKK mice wwawww s

iiigngngnifififiiciciicananantltlyy yy lalallllaggggggeeeeed.d.dddd. WhWhWhWWWheenene iinininjejjejejejectctc ioioionn fofofo iiiiiisososoprprprototttotterereee eenenenolollo wawawass s pepepee frfrfrfororormememedddd iinin ttttthehehee eeeeveveveniniiiningngg ((a(a(actcttctiiviviveeee


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temperature was set at 22oC or 4oC (Supplemental Figure IV B) despite lower body temperature

during the resting phase in BA-Bmal1-KO mice at 22oC (35.6±0.4 oC in wild-type vs 35.1±0.3oC

in BA-Bmal1-KO) or 4oC (34.2±0.4 oC in wild-type vs 33.3±0.5oC in BA-Bmal1-KO) (Figure

1D and 1E). This suggests that increasing thermogenesis related genes in PVAT and BAT of

BA-Bmal1-KO mice is likely a compensatory response to impaired thermogenesis in these

tissues. To determine whether compensatory thermogenesis in brown adipocytes affects the

resting phase dipping in BP and bradycardia in BA-Bmal1-KO mice, we further analyzed BP,

heart rate and body temperature of the mice housed either in thermoneutral (30oC) or cold (4oC)

environments. In a thermoneutral environment, the body temperature of wild-type and

BA-Bmal1-KO mice are comparable during resting (35.8±0.2oC vs 35.7±0.3oC) and active

phases (36.5±0.2oC vs 36.6±0.1oC) (Figure 1D and 1E). Interestingly, when the temperature was

switched from 22oC to 30oC, the BP and heart rate of both wild-type and BA-Bmal1-KO mice

significantly dropped. However, compared to wild-type mice, the BP of BA-Bmal1-KO mice

was still lower during the resting phase (SBP:100±4 mmHg in wild-type vs 87±6 mmHg in

BA-Bmal1-KO), while the heart rate of BA-Bmal1-KO mice was higher than that of wild-type

mice (HR:338±32bmp in wild-type vs 365±33bpm in BA-Bmal1-KO). During the active phase,

the BP of wild-type and BA-Bmal1-KO mice were comparable (SBP: 108±7 mmHg in wild-type,

and 106±9 mmHg in BA-Bmal1-KO), while the heart rate of BA-Bmal1-KO mice was still

higher than that of wild-type mice (400±35bpm in wild-type, and 429±38bpm in BA-Bmal1-KO).

Next, we switched the temperature from 30oC to 4oC. Both BP and heart rate were markedly

increased in all mice, accompanied with a decrease in body temperature and locomotor activity

environments. In a thermoneutral environment, the body temperature of wild-typeeeeee anananaana ddd d dd

BA-Bmal1-KO mice are comparable during resting (35.8±0.2oC vs 35.7±0.3oC) and active

phasssssseeeeeesssssss ((((((36.5555555±0±0±0±0±0±0±0.2..... oC CCC vs 36.6±0.1oC) (Figure 1D andndnd 1E). Interestinnnnggglg y, wwwhen the temperature was

wiiiitii ccchccc ed from 222222222ooooCCC toooo 3333330oC,CC,C,C,C, the BP annnddd heart t rrrrateee of bobobobobobobothththththth wwwiwwww llldl -tyyyypppep annddd BABABABABABAB -Bmaalalalalal1111111-KOKOKKO micccce

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(Figure 1D and 1E). However, the heart rate of BA-Bmal1-KO mice was lower than those of

wild-type mice despite comparable BP between the two groups. The steep reduction in body

temperature in the BA-Bmal1-KO mice might be due to loss of compensatory thermogenesis in

brown adipocytes at 4oC. These data are consistent with prior reports that thermogenesis is

associated with the BP and heart rate in mice42. However, the resting phase dipper BP in the

BA-Bmal1-KO mice at 22oC and 30oC is independent of thermogenesis in brown adipocytes.

Brown adipocyte-specific Bmal1 deficiency does not alter the intrinsic constriction ability

of aortic rings.

The constriction levels of aortic rings (PVAT removed) isolated from wild-type and

BA-Bmal1-KO mice during the resting phase were comparable in response to stimulation with

phenylephrine, serotonin or Ang II (Supplemental Figure VA). Additionally,

endothelium-dependent and -independent relaxation was similar in aortic rings of wild-type and

BA-Bmal1-KO mice (Supplemental Figure VB), indicating that the aortic rings of the

BA-Bmal1-KO mice retain an intact ability to contract. Given these findings, it is likely that the

hypotensive phenotype in the BA-Bmal1-KO mice is not caused by changes in the aortic

intrinsic ability to constrict but rather arise from differences in factors secreted by PVAT in vivo.

Bmal1-deficient PVAT is less effective in inducing vasoconstriction

PVAT produces and releases large amounts of adipokines and other undetermined or less

characterized factors that could target endothelial cells (ECs) and VSMCs, and herein contribute

to the maintenance of vessel homeostasis3, 43. We assessed the vasoactivity of thoracic PVAT

extracts from wild-type and BA-Bmal1-KO mice on the wild-type aortic rings in which the

The constriction levels of aortic rings (PVAT removed) isolated from wild-type annnnnnd dd

BA-Bmal1-KO mice during the resting phase were comparable in response to stimulation with

phennnnnnylylylylylyly eeepeee hrininininininne,e,e,e,e,e,e sserrrroooto onin or Ang II (Supplementalll FFFigure VA). Adddddddid tionononnally, yy

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BABABABABA-BBmBmalal111-KOKOKKOKOKOK mimimiceceece (((((SuSuSSSSuppppppleleleememementntntalalall FiFiFigugugureree VBVBVBVBVBVBV ))))),,, iiinindididid cacacatitingngng tttttthahahhhhah ttt ththththhthee e aoaoaortrtttrtiicicc rriinininii gsgsgs ooof f ffff ththththeeee e


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PVAT was removed15. PVAT extracts from 10, 20 and 30mg of adipose tissue from wild-type

mice efficiently constricted recipient thoracic aortic rings harvested from 8-week old C57BL/6J

mice in a dose dependent manner (Figure 2A and 2B). Additionally, we asked whether

PVAT-induced constriction of vessel rings was dependent or independent of the endothelium.

PVAT (10mg) extracts still significantly constricted to the same degree the aortic rings devoid of

endothelium (Figure 2C), indicating that PVAT elicits this response in an

endothelium-independent manner. Consistent with the hypotensive phenotype in BA-Bmal1-KO

mice, PVAT extracts isolated during the resting phase from BA-Bmal1-KO mice show

significantly reduced ability to constrict the wild-type rings (Figure 2D), in the same

experimental conditions. PVAT isolated from the active phase has 29% higher constriction ability

in the wild-type mice (Figure 2E), suggesting an intrinsic circadian association of this PVAT

function with the active phase, likely from overall changes in the balance of PVCF and PVRF.

Furthermore, this circadian difference is preserved in the BA-Bmal1-KO mice although there is a

remarkable overall reduction in the ability of PVAT from BA-Bmal1-KO mice to constrict the

wild-type rings at both time points, indicating that the Bmal1-dependent PVAT contractile

dysfunction is preserved throughout the day, in spite of the BP being normal during the active

phase in the knock out mice. This could be the consequence of systemic factors operating during

the active phase and overriding the loss of constriction ability arising from the tissue-specific

depletion of Bmal144. In that regard, it is noteworthy that sustained systemic infusion of Ang II at

pressor levels abolishes the increased hypotensive phenotype, but not bradycardia during the

resting phase in the BA-Bmal1-KO mice (Supplemental Figure VI A and B). In summary, Bmal1

ignificantly reduced ability to constrict the wild-type rings (Figure 2D), in the samamamamamameeeee e

experimental conditions. PVAT isolated from the active phase has 29% higher constriction ability

n thehehehehehee wiwiwiwiwiwildd-tytytyytytypepepepepepepe mimmmicccec (Figure 2E), suggesting an ininntrinsic circadiaaaannn n assososoociation of this PVAT

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in healthy PVAT contributes to sustain circadian regulation of BP and is required to maintain BP

within physiologic levels during the homeostatic dipper phenotype associated with the resting

phase.

Ang II in PVAT contributes to vasoconstriction

PVAT-derived adipokines such as adiponectin, leptin and chermerin regulate vasoactivity

although our expression data suggests that their contribution may be negligible since they

slightly change but in a direction opposing the observed phenotype (Supplemental Figure VII A).

Additionally, the levels of PVAT norepinephrine (a vasoconstrictor45), and PVAT-derived

prostacyclin (a vasorelaxant15) in BA-Bmal1-KO mice were indistinguishable from those in

wild-type mice (Supplemental Figure VII B and C). These data suggest that these PVAT-derived

factors do not likely contribute to the hypotensive phenotype in BA-Bmal1-KO mice. The local

RAS in adipose tissues at large is one of the critical elements in the development of

hypertension46-49. The majority of the local Agt in the vessel wall is located in PVAT, as opposed

to the arterial muscle layers49. Although renin mRNA was undetectable in mouse PVAT (data not

shown), the Agt mRNA and the Ang II converting enzyme (ACE) mRNA in adipose tissues,

including PVAT, are expressed in a circadian manner (Figure 3A), higher during the resting

phase and lower during the active phase. The Agt circadian variation is lost in PVAT and BAT of

the BA-Bmal1-KO mice showing consistent lower levels (Figure 3A) reflected in the reduced

total Ang II in the Bmal1-deficient PVAT extracts (Figure 3D). Plasma Agt and Ang II levels also

showed a circadian variation (Figure 3B and 3C), although it was not affected in the

BA-Bmal1-KO mice. Subcutaneous WAT is recognized as a beige adipose tissue containing

prostacyclin (a vasorelaxant15) in BA-Bmal1-KO mice were indistinguishable fromomommomom thohohohohoh sesesesesese iiiiiinnnnnn

wild-type mice (Supplemental Figure VII B and C). These data suggest that these PVAT-derived

factororororororrsssss s dddddod nototototototot lililililililikeeelyyly contribute to the hypotensivee ppphenotype in BBBBAAAA-Bmmmal1-KO mice. The local

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some Ucp1-positive adipocytes50 and hence potentially affected by our knockout strategy.

However, in this context, sWAT was more similar to gWAT in that the Agt mRNA circadian

variation is comparable between wild-type and BA-Bmal1-KO mice housed at 22oC (Figure 3A).

Even though the Agt mRNA in BAT of BA-Bmal1-KO mice was reduced, the plasma

concentration of Agt and Ang II were comparable between wild-type and BA-Bmal1-KO mice

(Figure 3B and C), indicating that brown adipocyte-derived Agt has negligible contribution to the

circulating Agt and Ang II pools. Thus, the local RAS-derived components in PVAT might

contribute to circadian rhythmicity of BP in a Bmal1-dependent fashion. We investigated

whether RAS in PVAT is involved in vessel ring constriction induced by PVAT extracts. The

recipient vessel rings that were pretreated in a dose dependent manner with type 1 Ang II

receptor blockers (valsartan or losartan, 10-8M to 10-6M), showed significantly lower response to

PVAT extracts from wild-type mice (Figure 3E and 3F), further supporting that local

PVAT-derived Ang II may be associated with vessel constriction induced by PVAT extracts.

These findings are consistent with the observed hypotensive phenotype during the resting phase

and the reduced vasoconstriction ability of PVAT from theBA-Bmal1-KO mice, suggesting a

direct relationship between Bmal1 and Agt/AngII expression in the peripheral clock in this

tissue.

Deletion of Agt in brown adipocytes leads to hypotension during the resting phase in mice

To further establish that RAS in PVAT contributes to circadian BP regulation, we generated

brown adipocyte-selective Agt knockout mice (BA-Agt-KO). Agt protein was present in all types

of adipose tissues (Figure 4A) from the wild-type mice, but was undetectable in PVAT and BAT

whether RAS in PVAT is involved in vessel ring constriction induced by PVAT exxxxxxtrtrtrtrtrtracacacacacactstststststs...... ThThThThThThT eeeeee

ecipient vessel rings that were pretreated in a dose dependent manner with type 1 Ang II yy

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from BA-Agt-KO mice (Figure 4B). The PVAT Ang II levels in BA-Agt-KO mice were reduced

more than 90% compared to wild-type PVAT (Figure 4C). Consistently, the PVAT extracts from

BA-Agt-KO mice showed a significantly reduced ability to constrict wild-type vessel rings

(Figure 4D), comparable to theBA-Bmal1-KOmice. The daytime resting phase BP at 22oC is

significantly reduced in the BA-Agt-KO mice when compared to the wild-type mice while the

nighttime active phase BP is preserved and comparable to that of the wild-type mice (Figure 4E,

4F), indicating that local deletion of Agt in PVAT recapitulates the BA-Bmal1-KO phenotype of

exacerbated resting phase dipping in BP. Interestingly, and as in the BA-Bmal1-KO mice, the

heart rate in the BA-Agt-KO mice are lower during both daytime and nighttime with preserved

and comparable locomotor activities (Figure 4E, 4F). The response to intraperitoneal injection of

isoproterenol during the resting phase parallels that of the BA-Bmal1-KO mice (Supplemental

Figure VIII). Additionally, the heart rate in the mice was changed by switching the

environmental temperature from 22oC to 30oC or from 30oC to 4oC (Figure 4E and F).At 30oC,

the heart rates of the BA-Agt-KO mice were reduced to the same level as those of wild-type

mice. However, the resting phase BP was still lower in BA-Agt-KO mice than in wild-type mice.

On the other hand, 4oC environmental stimulus significantly elevated both the BP and heart rate

in BA-Agt-KO mice to levels comparable of those of wild-type mice. The body temperature and

locomotor activity are comparable between wild-type and BA-Agt-KO mice in all experimental

conditions (Figure 4E and F). Combined, these results suggest that bradycardia of BA-Agt-KO

mice in the22oC environment is unlikely to drive the super-dipper phenotype in BP. Additionally,

despite the lower heart rate, the cardiac function parameters of BA-Agt-KO mice are similar to

heart rate in the BA-Agt-KO mice are lower during both daytime and nighttime wwwwwwitititititithhhhhh prprprprprpreseseseseseserererererervevevevevevedddddd

and comparable locomotor activities (Figure 4E, 4F). The response to intraperitoneal injection of

sopppppprororororororoteteteteteterenoolllllll dududududududuringngngng the resting phase parallels thaaattt of the BA-Bmmmmaala 1-KOKK mice (Supplemental

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eenene iviviviroroonmnmnmenene tatatatttt lll tetetetempmppeerere atatttatururu eee e ffrfrfromomom 222222222oooCCCCC ttototo 303030ooooooCC CCCCC ororo frfrfromomom 333000ooCCC CCC tttototott 44444ooooooCCCCCCC (F((F(F( iiigigigururureeee 4E4E4E4444 aaandndndddd FFFF)).).).AtAtAAtAt 3030303030ooooCCCC,C,C,


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those of wild-type mice (Supplemental Figure IX A). Furthermore, the aortic rings from

BA-Agt-KO mice isolated during the resting phase (10AM) and devoid from PVAT show

intrinsic constriction properties comparable to those of wild-type mice when vasoactivity is

induced by phenylepherine, serotonin and Ang II and are indistinguishable from wild-type rings

in endothelium-dependent and -independent relaxation assays (Supplemental Figure IX B and C).

This suggests that the hypotensive phenotype during the resting phase in the BA-Agt-KO mice is

caused by the Agt deficiency in PVAT. Additionally, the fact that BA-Agt-KO recapitulates the

BA-Bmal1-KO phenotypes is consistent with a Bmal1-Agt-AngII axis accounting for the

hypotensive phenotype in the resting phase.

Agt is a novel transcriptional target of Bmal1 in PVAT

Bmal1 is expressed in PVAT in a circadian rhythmic manner. Interestingly, Agt expression in

PVAT appears to follow a circadian rhythm as well, although right-shifted when compared to

Bmal1 (Figure 5A), suggesting delayed expression and opening the possibility that Bmal1 may

function as a transcriptional regulator for Agt. We found a putative Bmal1 binding site

(CACATG box) located between -808 to -814 bp upstream of the Agt transcription start site. In a

luciferase reporter assay (Figure 5B), a 1543bp region from the Agt promoter containing that

putative Bmal1 E-box displayed a 4-fold increase in reporter activity relative to the control

vector in response to overexpression of mouse Bmal1 in HEK293 cells while mutation of the

E-box abolished this response. ChIP analysis confirmed Bmal1 protein binding to the Agt

promoter region harboring the proximal CACATG motif located between -808 to -814bp

upstream of the transcription start site (Figure 5C). A negative control, isotypic IgG antibody,

hypotensive phenotype in the resting phase.

Agt is a novel transcriptional target of Bmal1 in PVAT

Bmalalalalalalal111111 iiiisisi expppppprererererereressssssss edeeded in PVAT in a circadian rhythmmmmic manner. Intttterrrer stiniinngly, Agt expression in

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BBmBm lalala 11111 (F(F(F((( igiggurururu eeee 5555AAA)A)),, , sususuggggggeesesestitititingngng dddelelellllayayayedededed exexeexee prprpreeesesessisiis ononon anana ddd dddd opopopenenenee ininiiinii ggg ththththhthee e popopossssssibibibbililililllititiitiii yy y thththhhhatattat BBmBmBm lalal1111 mamamayy y


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does not pull down the promoter, demonstrating that this is a functional Bmal1-binding site in

the Agt promoter. Furthermore, lentivirus-mediated overexpression of Bmal1 significantly

upregulated Agt mRNA (Figure 5D) and protein (Figure 5E) levels in brown adipocytes. These

data indicate that Bmal1 binds to the Agt promoter and functionally upregulates Agt expression

in brown adipocytes and are consistent with the reduced Agt expression we observed in our

BA-Bmal1-KO PVAT mice, thus defining Agt as a novel target of Bmal1 of physiological

relevance for circadian regulation of BP in PVAT.

Discussion

Adipose tissues in the body are essential for physiological homeostasis. Obesity is positively

associated with hypertension and related cardiovascular diseases. Mice lacking all adipose

tissues display hypertension and insulin resistance51. Unlike WATs in visceral and subcutaneous

regions, the PVAT, which surrounds large arteries, like the aorta, is a BAT-like adipose tissue

functionally different from WAT3, 15. However, the nature and extent of functional relationships

between PVAT and the other layers of the blood vessels are still largely unknown. PVAT is

actively involved in regulation of BP by controlled release of contractile and anti-contractile

factors, among a plethora of paracrine mediators ensuring vessel homeostasis3, 52, 53. However,

the specific role of PVAT in regulation of BP is unclear. We showed that selective lack of PVAT

enhanced atherosclerosis15, and those mice were hypotensive during the resting phase19.

The mechanisms for central circadian systems control of BP rhythmicity have been well

reviewed54. However, the peripheral circadian system in blood vessels contributes to BP

Discussion

Adipose tissues in the body are essential for physiological homeostasis. Obesity is positively

assoooooociciciciciciciatattatatatated wwititititititith h hhhhh hyyyypppep rtension and related cardiovaasa cccular diseases.... MMMMice ee lacking all adipose

isssssssueueueueueues display y yy hyhhhhhh peppepertenenenennnsionnnnnnn aaaaaand insulinn resistaaannnnceee551. UUUUUUnlnlnlnlnlnln ikikikikikike ee e e ee WWWAW TsTsTTss in vivviscccccererrrrral and sssububuububu ccuutaneououoouus

eeee igigigiononons,s,s, tthehee PPPPPPVAVAVAVATTT,T,, wwwhhhihichchhch sssurururrororoununu dsdsdsddd lllarara gegege ararartetetettete iiririeeseses,,, lililil kekekeke ththeee aoaoaortrttrta,a,a, iiiiiis ss aa a BABABABABATTTTT-lilililiiikekekkekkk aaadidididididipopoposesese tttiisisissususueeeeee


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circadian rhythmicity as well55. Bmal1 is expressed in both ECs and VSMCs. Bmal1 in the

vasculature is transcriptionally controlled by PPARγ. Knockout of PPARγ in ECs or VSMCs

significantly reduced circadian variation in BP and heart rate31. Bmal1 in VSMCs regulates

Rho-kinase 2 transcription, while deletion of Bmal1 abolished myosin phosphorylation, ROCK2

activation and agonist-induced vasoconstriction. Mice deficient for Bmal1 in ECs or VSMCs had

compromised BP circadian rhythmicity and decreased SBP during the nighttime active phase and

reduced heart rates throughout the day30,32. Ang II regulates circadian clocks in the SCN56 and

VSMCs57. Here we demonstrate that components of the peripheral clock show circadian

regulation in PVAT. Furthermore, the Bmal1 protein in PVAT of mice is acutely repressed by

systemic Ang II infusion. Consistently, the circadian rhythmicity of BP is temporarily abolished

with recovery at day 4 of Ang II infusion, when Bmal1 levels are restored, thus linking Bmal1

expression in PVAT to the circadian regulation of BP.

Selective deletion of Bmal1 in brown adipocytes in mice does not affect the intrinsic ability

of the ECs and VSMCs to respond to contractile stimuli. Unlike Bmal1 deficiency in ECs or

VSMCs30, 32, the BP in BA-Bmal1-KO mice is only reduced during the resting phase and

impaired local RAS, evidenced by strikingly lower levels of Ang II in PVAT can account for that

phenotype in this novel mouse model.

During active phase, when the BP and heart rate are simultaneously increased together with

the SCN-controlled locomotor activity, BP is normal in BA-Bmal1-KO mice. Even though PVAT

extracts collected from BA-Bmal1-KO and wild-type mice during the night active phase

enhanced vasoconstriction differently, it is possible that during the active phase altered local

egulation in PVAT. Furthermore, the Bmal1 protein in PVAT of mice is acutely rrepepepepepeprerererereressssssssssssededededededd bbbbbby y y y yy

ystemic Ang II infusion. Consistently, the circadian rhythmicity of BP is temporarily abolished

withhhhhh rerererererer ccoveryryyyyyy atataaaata ddddaayayay 4 of Ang II infusion, when BmBmBmal1 levels areeee rrrrestoooreerered, thus linking Bmal1

exprprprprprpreeeseee sion in PPPPPPVAVAVAVAVVV TTT toooo tttttthe cccccccirrrrrrcadian regguuulationnn ooof BBBBP.

SeSeSeSelelelelellectctc ivivveeeeee dedededeleleleletttititionono ooofff f BBBBmamamal1l1ll1ll iinn brbrbrbbb owowown n n adadaddipipiiiiipocococytytyteseseees iiinin mimicececee doddodoeeseses nnottoto aaafffffffffeecececttt t thththttt ee e inininiii ttrtrtriinininsisiiisicc c babababililililiiiititityy y


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PVAT-derived vasoconstrictors, such as Ang II, in the BA-Bmal1-KO mice may be overcome by

sympathetic nerve mediated elevation of BP observed in vivo58, thus allowing the BA-Bmal1-KO

(and BA-Agt-KO) mice present normal BP during the active phase.

Systemic deletion of Agt in adipocytes, including white adipocytes, leads to systemic

reduction in Ang II and hypotension59 while our data uncovered that brown adipocyte-selective

Agt deficiency is hypotensive only during the resting phase associated with reduced local Ang II

production, recapitulating the BA-Bmal1-KO phenotype, without affecting systemic levels of

Ang II. Furthermore, we determined that Agt, which depicts circadian expression in PVAT, is a

direct transcriptional target of Bmal1 in PVAT through a functional Bmal1 E-box at -808 to -814

in the cognate Agt promoter. These data are consistent with the loss of PVAT-derived local Ang

II in both BA-Bmal1-KO and BA-Agt-KO mice associated with reduced vascular tone in the

resting phase, suggesting that local Ang II is one of vasoconstrictors contributing to this PVAT

phenotype in a paracrine fashion.

Diurnal variations in BP are essential to cardiovascular homeostasis. Variations in the

physiological drop (“dipping”) in BP during the resting phase are known risk factors for

cardiovascular events60. These phenotypes are referred to as “non-dipper” (failure to

downregulate BP in the resting phase), or “super-dipper” (exacerbated hypotension during the

resting phase) and both are known to be strongly related to individual risk of cardiovascular

morbidity and mortality39. The coordinated effects between the central clock and tissue specific

peripheral clocks may account for these phenotypes, although their crosstalk is not clearly

defined so far. Thus, upregulation of the RAS in rats suffering from severe hypertension results

direct transcriptional target of Bmal1 in PVAT through a functional Bmal1 E-boxx atatatatatat -80808080808088 8 8 8 8 8 totototototo --8181818181814 44444

n the cognate Agt promoter. These data are consistent with the loss of PVAT-derived local Ang

I innnn bobobobobobob thththththth BAAAAAAA--BBBmBBBB alaalal1-KO and BA-Agt-KO mice aasa sociated with rererreeduceceeddd vascular tone in the

esttttsttinnnnnng phase, susususususuugggggeeestttiinnnnngn thhhhhhhatatatatatat local Angg II is ooonnnne ooofff vaaaaasososososososococococococonsnsnsnsnsnsnstttrt icttttoorsr conoonntrrrrribibbbbbuting tototototototo thhihihis PVATATATATTAT

hphphp eenenen ttottototypypypee e inininn aaa papapararaacrcrc iiininineeee ffafafa hhshshshiiioioioi nn...


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in an inverted BP profile61. It was proposed that circulating Ang II mediates this phenomenon62,63.

Central Bmal1 involvement in that model is evidenced by loss of its rhythmic expression64. In

adipocytes of healthy PVAT, during the resting phase, we uncovered a circadian regulated axis

defined by Bmal1 transcriptionally upregulating Agt expression to ensure physiological levels of

Ang II in PVAT to sustain vascular tone during the normal dip in BP associated with the resting

phase. Our brown-adipocyte specific Bmal1 and Agt knockout mice present a dipping profile

consistent with a “super-dipper” phenotype during the resting phase in vivo. Super-dippers are at

risk for silent myocardial ischemia and non-fatal ischemic stroke, while hypertensive patients

with super-dipper BP are likely to have silent cerebral infarct and to be at high-risk for future

stroke65. Whether our novel mouse models reported here will recapitulate such cardiovascular

risks and adverse outcomes remains to be determined. Additionally, it is possible that a phase

shift or change in amplitude in this local PVAT homeostatic control of BP during the resting

phase may be associated with adverse cardiovascular outcomes in shift workers and upon jet

lag66. Furthermore, the PVAT peripheral clock may be altered in the context of obesity, which is

known to cause both PVAT dysfunction67, 68 and overall disruption of systemic circadian cycles69.

Future studies should also address the molecular aspects of cross-communication between

known drivers of systemic oscillation of BP and the synchronization of the local PVAT peripheral

clock. For instance, it is likely that systemic Ang II might be tied in a regulatory loop with PVAT

controlling local Bmal1 expression, phosphorylation, subcellular localization and overall

activation to synchronize the PVAT peripheral clock and prevent ‘super-dipper’ or cause

‘non-dipper’ phenotypes in disease states. Additionally, and from the perspective of experimental

with super-dipper BP are likely to have silent cerebral infarct and to be at high-risksksksksksk fofofofofoforrrr rr fufufufufufuf tttttturururururure e e e eetttttt

troke65. Whether our novel mouse models reported here will recapitulate such cardiovascular

iskssssss anananananana ddddd d addddddveveveveveveersrsrsrsrsrsrse oououououtcomes remains to be determiinnned. Additionalllllyyyy, it iissss possible that a phase

hififfffft tt or changeeeeee iiiiiiin n amamamppplpliiiitii udeeee eee iiiiini this localll PVATTT homomomeooststststststtatatatatatata iciciciciccic cccccccooono trrrrooolo of BBPBPPPP ddduddd ring tttheheheheheheh rrrreeeesting

hphphp asasaseeee mamamayy y bebebebebeb aaassssssococociiaiaatetteteetedd d d iwiwiwithhhththth aaadvdvdvererereee esesese cccararardidid ovovovasasascucuculalallarr ouououtctccomomomeseseeees iiin nn shhhshs iffififtttt wowowo krkrkrkkkkerere ss s anananddd d upupuponono jejejejettt t


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designs to understand the nature of and balance between PVRFs and PVCFs in PVAT, our

findings here underscore the need to factor in the time of day when PVAT samples are collected.

Conclusions

This study uncovered a peripheral circadian clock in PVAT that contributes to safeguard optimal

vascular tone during the resting phase involving an Bmal1-Agt-AngII axis. The results of this

study provide important insights regarding the potential physiological and pathophysiological

roles of PVAT in circadian BP regulation and support that targeting PVAT function may prove

useful in therapeutic or preventive strategies for cardiovascular disease.

Sources of Funding

This work was supported by NIH grants HL122664-01 (to L. Chang), HL088391 (to Y.E. Chen),

HL138094 (to Y. Fan), HL138139 (to J. Zhang), American Heart Association National Scientist

Development grants 15SDG24470155 (to Y. Guo) and the National Natural Science Foundation

of China 81670429 (to Z.S. Jiang).

Disclosures

None

References

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Sources of Funding

Thissssss wwwwwwwooorooo k wawawawaaaas s ss s s susssss pppppppported by NIH grants HL1226646464-01 (to L. Chananaanng), HLHHH 088391 (to Y.E. Chen),

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Berriel Diaz M, Rozman J, Hrabe de Angelis M, Nusing RM, Meyer CW, WWWWWWahahahahahahliliiiii WWWWWWW, Klingenspor M, Herzig S. Cyclooxygenase-2 controls energy homeostasis in mice by denovo recruitment of brown adipocytes. Science. 2010;328:1158-1161.

51. KiKiKiKiKiKim m m m m m m JKJKJKJKJKJK, GaGGaGaGaGavrilova O, Chen Y, Reitman MMMMMML,LLLLL Shulman GI.III. MMecccchahhhh nism of insulin resistannnnnncccccecc iiiiiin nnnn a--zizzzzzz p/p/p/p/p/p/p fffffff-ffff 1 fafafafafafafatlttltltltlesesesesesess s mimimimiceccccc . J BiBBiBiBiolooooo CCChemmmmmm.. 202020202020000000000000;2222757557575:8:8:8:8:: 454545454566666-8460606060606060.

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60. Muller JE. Circadian variation and triggering of acute coronary events. Am Heart J. 1999;137:S1-S8.

61. Lemmer B, Mattes A, Bohm M, Ganten D. Circadian blood pressure variation in transgenic hypertensive rats. Hypertension. 1993;22:97-101.

62. Langheinrich M, Lee MA, Bohm M, Pinto YM, Ganten D, Paul M. The hypertensive ren-2 transgenic rat tgr (mren2)27 in hypertension research. Characteristics and functional aspects. Am J Hypertens. 1996;9:506-512.

63. Mendelsohn FA, Quirion R, Saavedra JM, Aguilera G, Catt KJ. Autoradiographic localization of angiotensin ii receptors in rat brain. Proc Natl Acad Sci U S A. 1984;81:1575-1579.

64. Monosikova J, Herichova I, Mravec B, Kiss A, Zeman M. Effect of upregulated renin-angiotensin system on per2 and bmal1 gene expression in brain structures involved in blood pressure control in tgr(mren-2)27 rats. Brain Res. 2007;1180:29-38.

65. Kario K, Shimada K. Risers and extreme-dippers of nocturnal blood pressure in hypertension: Antihypertensive strategy for nocturnal blood pressure. Clin Exp Hypertens. 2004;26:177-189

66. Ruger M, Scheer FA. Effects of circadian disruption on the cardiometabolic system. Rev Endocr Metab Disord. 2009;10:245-260.

67. Aghamohammadzadeh R, Unwin RD, Greenstein AS, Heagerty AM. Effects of obesity on perivascular adipose tissue vasorelaxant function: Nitric oxide, inflammation and elevated systemic blood pressure. J Vasc Res. 2015;52:299-305.

68. Gomez-Santos C, Gomez-Abellan P, Madrid JA, Hernandez-Morante JJ, Lujan JA, Ordovas JM, Garaulet M. Circadian rhythm of clock genes in human adipose explants. Obesity (Silver Spring). 2009;17:1481-1485.

69. Engin A. Circadian rhythms in diet-induced obesity. Adv Exp Med Biol. 2017;960:19-52.

2004;26:177 18966. Ruger M, Scheer FA. Effects of circadian disruption on the cardiometabollllllicicicicicic ssssssysysysysysy tetetetetetetem.m.m.m.m.m. ReReReReReReR v

Endocr Metab Disord. dd 2009;10:245-260.67. Aghamohammadzadeh R, Unwin RD, Greenstein AS, Heagerty AM. Effects of obesity

on perivascular adipose tissue vasorelaxant function: Nitric oxide, inflammation and elelelelelelevevevevevvvatatatatatata ededeeee sysysysysystemic blood pressure. J Vasc RRRRRReesee . 2015;52:29999-305555.

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Figure Legends

Figure 1. Knockout of Bmal1 in brown adipocytes reduced BP during the light-on resting

phase. A, Realtime PCR showing mRNA levels of circadian genes in wild-type PVAT, relative to

β-actin. Data shown as mean ± SEM, n=5. B, Representative Western blot showing Bmal1 levels

in adipose tissues and aorta in wild-type and BA-Bmal1-KOmice.C, Realtime PCR showing

mRNA levels of circadian genes in wild-type and BA-Bmal1-KO PVAT, relative to β-actin. Data

shown as mean ± SEM, n=5-6, *p<0.05 vs wild-type. D, Systolic (SBP), diastolic BP (DBP),

heart rate (HR), locomotor activity and body temperature of wild-type and BA-Bmal1-KO mice

was measured by radiotelemetry when the temperature of the environmental chamber was

successively set at 22oC, 30oC, or 4oC. Data are shown as mean±SD, n=5/group. The gray

shading represents light-off period from 18:00 to 6:00. E, Average of 2 constitutive circadian

cycles of systolic, diastolic BP, heart rate, locomotor activity and body temperature data during

light-off and light-on periods shown in panel D. Data are shown as mean ± SD, *p<0.05vs

wild-type.

Figure 2. PVAT constricts blood vessels. A, Representative trace showing dose-dependent

aortic ring constriction in response to extracts from 10-30 mg of thoracic PVAT. The thoracic

aortic rings were collected from 8-week old C57BL/6J mice at 10AM. The PVAT adjacent to the

rings was cleaned before they were mounted onto the wire myograph. The donor PVAT extracts

were collected from 8-week old C57BL/6J mice at 10AM. B, Quantitative analysis of

heart rate (HR), locomotor activity and body temperature of wild-type and BA-Bmmmmmmalaaaaa 111111-KOKOKOKOKOKOK mmmmmmicicicicicice e e eee

was measured by radiotelemetry when the temperature of the environmental chamber was

ucccccccesesesesesesssisisisisisivelylyy sssssssetetetetetetet at ttt 2222oC, 30oC, or 4oC. Data are shhhooown as mean±SDSDSD, n=n=n==5/group. The gray

hadadadadadadiiniiii g represenenenenenenntssss lllighthththttt-off ff f ff f ppppepp riod from mm 18:0000 tttto 666:::00.0.... EEEEEEE, , ,,,, AvAvAvAvAvAvveeere agggge of 222 cccononononononstitutivveee eeee ciircrcrccadiannn

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constriction tension of vessel rings in response to PVAT extracts in panel A. The tensions are

shown as absolute tension differential between basal level and plateau level and expressed in

miliNewton (mN). Data are shown as mean ± SD. n=6. *p<0.05 vs. baseline. C, Vasoconstriction

induced by 10mg of C57BL/6J PVAT extracts on vessel rings with the endothelium present (+EC)

or removed (-EC). Data shown as mean ± SD. n=6. D, Vasoconstriction of C57BL/6J rings

induced by increasing amounts of PVAT extracts collected from C57BL/6J or

BA-Bmal1-KOmiceat 10AM (resting phase). Data shown as mean ± SD. n=6, *p<0.05 vs.

wild-type. E, Vasoconstriction induced by 10mg PVAT extracts collected from the indicated

genotype at 10AM (resting phase) and 10PM (active phase) on vessel rings. Data shown as mean

± SD. n=6.

Figure 3. Ang II in PVAT constricts blood vessels. A, Realtime PCR showing the circadian

change in mRNA levels (relative to β-actin) of Agt and ACE in PVAT, BAT, sWAT and gWAT

collected from the indicated genotype mice every 4 hours. Zeitgebertime“0” indicates the

lights-on. Data shown as mean ± SD, n=3. B & C, Plasma of wild-type and BA-Bmal1-KO mice

was collected every 4 hours, and the plasma Agt and Ang II were measured by ELISA. Data

shown as mean ± SD, n=6/group. D, Ang II levels (ELISA) in thoracic PVAT harvested from

wild-type and BA-Bmal1-KO mice at 10AM (resting phase). Data shown as mean ± S.D. n=6. E,

Representative traces showing that blocking Ang II type 1receptors (AT1R) with either valsartan

(Val) or losartan (Los) at the indicated doses for 5min before stimulation with 10mg of wild-type

PVAT extracts blunts the PVAT-induced constriction of wild-type aortic rings. PVAT extracts and

genotype at 10AM (resting phase) and 10PM (active phase) on vessel rings. Dataaaaa sssssshohohohohohownwnwnwnwnwn aaaaaas s s s s s memememememeananananana

± SD. n=6.

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rings were collected at 10AM. F, Quantitative data from E expressed as % of PVAT extracts

alone (control) in the first trace before addition of either inhibitor. Data shown as mean ± SD.

n=6. *p<0.05 vs control.

Figure 4. Agt-deficiency in PVAT impairs PVAT-induced blood vessel contraction. A,

Representative Western blots showing Agt protein levels in adipose tissues from wild-type and

B , BA-Agt-KO mice. C, Ang II levels in PVAT from wild-type and BA-Agt-KO mice at 10AM.

Data shown as mean ± SD. n=6. D, Vasoconstriction of wild-type aortic rings (PVAT removed)

induced by 10 mg PVAT extracts collected at 10AM from wild-type and BA-Agt-KO mice. E,

Systolic, diastolic BP, heart rate, locomotor activity and body temperature of wild-type and

BA-Agt-KO mice as measured by radiotelemetry when the temperature of environmental

chamber was successively switched from 22oC, to 30oC and to 4oC. Data shown as mean ± SD,

n=5 in each group. The gray shading represents the lights-off period from 18:00 to 6:00. F,

Average of the 2 constitutive circadian cycles of systolic, diastolic BP, heart rate, locomotor

activity and body temperature data during the lights-off and lights-on periods shown in panel E.

Data shown as mean ± S.D, *p<0.05 vs wild-type.

Figure 5. Agt is a novel target of Bmal1 in PVAT. A,QRT-PCR showing the relative circadian

variation of Bmal1 and Agt mRNA levels in PVAT from 8-week old male C57BL/6J mice. The

Bmal1 and Agt mRNA levels relative to β-actin at 6AM were set as 1. Gray color background

shows the lights-off phase. Data are shown as mean ± SEM, n=5. B, Luciferase assay using a

nduced by 10 mg PVAT extracts collected at 10AM from wild-type and BA-Agt------KOKOKOKOKOKO mmmmmmicicicicicice.e.e.e.e.e. EEEEEEE,,,,,,

Systolic, diastolic BP, heart rate, locomotor activity and body temperature of wild-type and

BA-AgAgAgAgAgAgAgtttttt-KOKOOOOO mmmmmmmiciiiiii e aaasa measured by radiotelemetry wwwhen the tempeeeerrrar turerere of environmental

chamamamamamamber was susuuuuuccccccccccc eessiiivvveveveely ssssssswwwwwiw tched fromomm 22oC,C,C,, tooo 3330oCCCCCC anananananand d d d d d d totooo 4ooooCCC.C Dataatta shshshshshshown asssssss mmmmmmmeaeeaeaan ± SDSDDDDD,

n=n=5555 iininin eeeeeacacachh grgrgrouououp.p.p. TTThehheheehe gggrararay yy hhhshshshhadadadininingg g erereeprprpreseseseneneeeentttststs ttthehehehehe llliigigighthththhh sss-ofofoffffffff ff f peepepe iiririiriododod ffrfrfromomom 111188:8:8:888 000000 ttttttoo o 6:6:6:6:0000000000... FFFF,,,


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reporter containing the putative Bmal1 CACATG E-box (-808-/-814) or a mutated site

(AGACAT), as indicated in the structural cartoons on the left. pcDNA3.1 expressing mouse

Bmal1 or vector pcDNA 3.1 (control) were co-transfected with the reporters in HEK293 cells.

Luciferase readings were normalized by Renilla. Data are shown as mean ± SD, n=6. **p<0.01

vs. control vector. C, ChIP assay showing enrichment of Bmal1 protein on the mouse Agt

promoter in brown adipocytes transfected with lenti-Bmal1compared to lenti-GFP. Isotypic IgG

was used as negative control. Data are shown as mean ± SD, n=3. D, QRT-PCR showing that

overexpression of Bmal1 increases Agt mRNA in differentiated brown adipocytes. Data are

shown as mean ± SD, n=3. E, Western blot showing increased Agt protein level in differentiated

brown adipocytes with Bmal1 overexpression.

hown as mean ± SD, n=3. E, Western blot showing increased Agt protein level innn nnn diddddd ffffffffffffererererererenenenenenentitititititiiatatatatatatedededededed

brown adipocytes with Bmal1 overexpression.


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Zhang, Zhisheng Jiang, Jiandie D. Lin and Y. Eugene ChenLin Chang, Wenhao Xiong, Xiangjie Zhao, Yanbo Fan, Yanhong Guo, Minerva Garcia-Barrio, Jifeng

Transcriptional Regulation of AngiotensinogenBmal1 in Perivascular Adipose Tissue Regulates Resting Phase Blood Pressure Through

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1

SUPPLEMENTAL MATERIAL

Supplemental Methods

Blood pressure (BP) determination by telemetry

BP was measured by radiotelemetry (HD-X11, Data Sciences International, St. Paul,

Minn.)1. Eight-week-old BA-Bmal1-KO, BA-Agt-KO and wild type mice were

anesthetized with isoflurane. The left common carotid artery was cannulated with the

implant catheter, and the implant was secured in the abdominal cavity. After 1-week

to allow recovery from surgery, systolic, diastolic BP, heart rate, locomotor activity and

body temperature were recorded. Mice were kept on a 12/12-hour light/dark cycle.

When required, Ang II infusion was performed as previously described2.

PVAT extracts

The thoracic PVAT of 8-week-old Bmal1-KO, Agt-KO and wild type control mice were

dissected and washed 3 times in ice-cold phosphate buffered saline (PBS) solution

(10 mM Na2HPO4, 10 mM H2PO4, 0.9 g NaCI/100ml, pH 7.4) to remove blood in the

tissue. Next, the PVAT was homogenized in ice-cold PBS (1 ml/mg of volume/weight

tissue) using a glass Dounce homogenizer for 10 times on ice, and the resulting

extracts were centrifuged at 5,000 rpm at 4oC for 10 min to eliminate lipids and

debris3. The liquid extracts were kept at -80oC until use.

2

Wire myography

The recipient thoracic aortic rings (~3mm in length) were dissected from 8-week old

C57BL/6 wild type, Bmal1-KO or Agt-KO mice, and the attached PVAT was cleaned in

ice-cold PBS solution. To determine endothelium-dependent responses, in addition to

PVAT removal, the endothelium of the aortic rings was completely removed prior to

myography. Then the recipient vessel rings were mounted onto a wire myograph

chamber (DMT 610M, ADInstruments) containing 10 ml physiological solution (118.99

mM NaCl, 25 mM NaHCO3, 4.69 mMKCl, 1.18 mM KH2PO4, 1.17 mM MgSO4, 2.5

mM CaCl2, 0.03 mM EDTA, and 5.5 mM glucose) at 37°C and bubbled with a mixture

of 95% O2 and 5% CO2. After equilibration and normalization procedures as

described in our previous works1, aortic contractility was determined by addition of 60

mM KCI, PVAT extracts, vasoconstrictors phenylephrine, serotonin and Ang II, or the

vasodilators acetylcholine or sodium nitroprusside. For AT1 receptor inhibition,

10-8-10-6 M losartan or valsartan were used4. Constriction is measured as tension and

expressed in miliNewton (mN) relative to the corresponding controls as indicated in

each case.

Ang II content assay

Thoracic PVAT were freshly collected from 8-week-old Bmal1-KO, Agt-KO and wild

type control mice and extracts were prepared as described above. The blood was

collected with EDTA to separate plasma. Ang II levels in PVAT extracts and plasma

were measured by ELISA according to the manufacturer’s instructions (Ang II ELISA

3

kit, Enzo Life Sciences, Cat# ADI-900-204).

Prostacyclin and Noradrenaline content assay

Thoracic PVAT were freshly collected from 8-week-old wild type and BA-Bmal1-KO

mice. PVAT extracts were prepared as described above. The prostacyclin level was

measured according to the instructions (Enzo Life Sciences, Cat# ADI-900-025). The

Noradrenaline level was measured according to the instructions (IB89537,

Immuno-Biological Laboratories).

Echocardiography.

Transthoracic echocardiography was performed in the supine or left lateral position of

mice anesthetized with 3% isoflurane using a Visual Sonics Vevo 770 micro-imaging

system. Two-dimensional, M-mode, Doppler and tissue Doppler echocardiographic

images were recorded. Systolic and diastolic dimensions and wall thickness were

measured in M-mode in the parasternal short axis view at the level of the papillary

muscles. Fractional shortening and ejection fraction were calculated from the

M-mode parasternal short axis view.

Western blot

Western blot analyses were performed as previously described3. Antibodies used in

this study were anti-Bmal1 antibody (1:1000 dilution, Abcam ab93806), rat

monoclonal to mouse anti-Agt antibody (1:1000 dilution, Life Span BioSciences,

LS-C150249).

4

Quantitative Real-time PCR

Total RNA from mice PVAT was extracted with Trizol reagent (Cat# 15596, Life

Technologies, Grand Island, NY), and the first-strand cDNA was synthesized with the

SuperScript® III First-Strand Synthesis System (Cat# 18080, Life Technologies,

Grand Island, NY). Next, levels of multiple mRNAs in PVAT, as indicated in the

corresponding figures, were analyzed by QRT-PCR using the primers listed in the

supplemental table and are expressed relative to -actin.

Plasmids and transient transfection assays

The genomic fragment harboring a putative Bmal1-binding site (-808/-814) in the

mouse Agt promoter was cloned by PCR from the mouse genomic DNA. The

amplified products of 1.5 kbp upstream of the translation start site of mouse Agt gene

were ligated into the pGL4-luciferase reporter vector (Promega) to generate pGL4-luc

plasmids. Promoter activity was further validated by mutation of the putative

Bmal1-binding site on the promoter at –808/–814 by replacing CACATG with

AGACAT using the QuikChange Site-Directed Mutagenesis Kit (Stratagen). All

PCR-generated constructs were verified by DNA sequencing. Luciferase activity was

measured as described before1. In brief, HEK293 cells were transfected with the wild

type or mutant pGL4-luciferase reporter plasmids and pRenilla-null as internal control

(Promega) using Lipofectamine 2000 (Life Technologies) and co-transfected with

either pcDNA3.1 overexpressing Bmal1 or the pcDNA3.1 vector as control. Cells

were cultured for 24 hours after transfection, and cell lysates were measured using

5

the Dual Luciferase Reporter Assay System Kit (Promega).

Chromatin Immunoprecipitation (ChIP) assay

Mouse interscapular brown preadipocytes were isolated and immobilized by Dr.

Jiandie Lin as previously described as described previously5. Preadipocytes were

transfected with lentiviral vectors, lenti-Bmal1 (GFP-tagged, OriGENE Cat #

MR209553L2V) or control lenti-GFP (OriGENECat # PS100071). After grown to

confluence in differentiation medium (high glucose Dulbecco's modified Eagle's

medium containing 10% fetal bovine serum, 20 nM insulin and 1 nM

3,3′,5-triiodo-L-thyronine), preadipocytes were induced by 0.5 mM

isobutylmethylxanthine, 0.5 μM dexamethasone, and 0.125 mM indomethacin for 48

h. Next, the cells were differentiated in differentiation medium for an additional 1 week.

The cells were fixed and ChIP assays were performed using EZ-ChIP assay kit from

Upstate Biotechnology (Lake Placid, NY). The size of the sonicated DNA fragments

subjected to immunoprecipitation was 0.5 to 1 kbp as determined by ethidium

bromide gel electrophoresis. Purified chromatin was immunoprecipitated using

anti-Bmal1 monoclonal antibody. Eluted DNA fragments were purified to serve as

templates for the PCR amplification. The input fraction corresponded to 10% of the

chromatin solution before immunoprecipitation. Primers used to amplify the area

containing this E-Box binding site (-808 to -814 bp upstream of the transcription start

site) in the ChIP assay are: forward primer 5′-AGCCCATCTCAAACACCATCAAG-3′

reverse primer 5′-TGCATCGCATCCACAACTTCAA-3′, resulting in 173 bp fragment.

6

Supplemental Table. Primers used in the study

mRNA Forward primer Reverse primer

Bmal1 AGGCCCACAGTCAGATTGAAA CCAAAGAAGCCAATTCATCAATG

Rev-reb CCCAACGACAACAACCTTTTG CCCTGGCGTAGACCATTCAG

Npas2 GCTGATGTTGGAGGCATTAGATG CATAGATGATGCTGCCGTCTGT

Cry1 TCGCCGGCTCTTCCAA TCAAGACACTGAAGCAAAAATCG

Cry2 CCCACGGCCCATCGT TGCTTCATTCGTTCAATGTTGAG

Per1 TCGAAACCAGGACACCTTCTCT GGGCACCCCGAAACACA

Per2 GCTCGCCATCCACAAGAAG GCGGAATCGAATGGGAGAATA

Per3 GGCTGCTTTGATCCTGAATTCT GAACGCCCTCACGTCTTGAG

Dbp CACCGTGGAGGTGCTAATGA GCTTGACAGGGCGAGATCA

E4bp4 GCTCAAGAGATTCATAGCCACACA GCTCGGTCAGCAGCCATTT

Clock CTGTATGGGGTGACTTGGGGTTG CTTGGGGAATGGTCTTGGTGCTC

Agt GCGGAGGCAAATCTGAACAACAT GAAGGGGCTGCTCAGGGTCACAT

Ace CCACCAGGGCCCACTACACC GACTTCGCCATTCCGCTGATT

Ucp1 AAAAACAGAAGGATTGCCGAAACT TAAGCATTGTAGGTCCCCGTGTAG

Cidea CTGTCGCCAAGGTCGGGTCAAG CGAAAAGGGCGAGCTGGATGTAT

Elovl3 GGGCCTCAAGCAAACCGTGTG GTTTTTCAGCCTTCATAGTGTAGT

Lpn ACATACCGCATTTCAGGGCA CCCAGGTATCCCGTGTCAAC

Adipoq CATTCCGGGACTCTACTACTTCTC GTCCCCATCCCCATACACCT

Rarres2 AGGTGAAGCCATGAAGTGCT AACTGCACAGGTGGGTGTTT

7

Supplemental Figures and Figure Legends

Figure I. Ang II represses Bmal1 expression in PVAT. (A), Systolic blood pressure

of 10-week old C57BL/6J mice was measure by radiotelemetry. The blood pressure

during day (-3) to day 0 shows normal circadian rhythmicity. Continuous infusion of

Ang II by subcutaneous Alzet minipump (day 0 to day 7, red arrow indicates starting

to infusion of Ang II or saline) acutely, albeit transiently, abolishes circadian

rhythmicity of blood pressure with progressive restoration after day 4 (indicated as

blue arrow) when compared to 0.9% saline infusion as control. Gray shade

represents lights-off period from 18:00 to 6:00. Data are shown as mean ± S.D., n=6.

8

(B), Representative Western blot showing Bmal1 levels in PVAT of mice in (A) when

the PVAT was harvested at 10:00AM. (C), Cardiac function in BA-Bmal1-KO mice.

The left ventricular end diastolic and systolic diameters (LVDd and LVDs,

respectively), ejection fraction (EF) and fractional shortening (FS) were measured in

wild type and BA-Bmal1 KO mice during lights-on (resting) phase using

echocardiography as described in the methods section. Data shown as mean ± SD,

n=5/group.

9

Figure II. Specific loss of circadian feedback loops in brown adipose tissues of

BA-Bmal1-KO mice. PVAT, BAT, sWAT, gWAT, liver and brain of wildtype and

BA-Bmal1-KO mice were collected every 4 hours. The mRNA levels of aryl

hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), D-Box binding PAR

BZIP transcription factor (Dbp), Cryptochrome circadian clock 1 (Cry1) and nuclear

receptor subfamily 1 group d member 1 (Rev-erb) were measured by QRT-PCR

relative to -actin. Gray shade represents lights-off period from 18:00 to 6:00. Data

shown as mean ± SD, n=3/group. **p<0.01 vs wild-type.

10

Figure III. Response of BA-Bmal1-KO mice tosystemic isoproterenol injection.

Heart rate (HR), systolic(SBP) and diastolic (DBP) blood pressure of wild type and

BA-Bmal1-KO mice were measured by radiotelemetry when the temperature of the

environmental chamber was set at 22oC. The mice received 10mg/kg isoproterenol

by i.p. injection at ~10AM, resting phase (A) or 10PM, active phase (B). Data are

shown as mean ± SD, n=5/group.

11

Figure IV. Thermogenic genes and metabolism. (A), mRNA levels of

thermogenesis-related genes: uncoupling protein 1 (Ucp-1), cell death-inducing

DFFA-like effector A (Cidea), ELOVL fatty acid elongase 3 (Elovl3) in PVAT and BAT

of wildtype andBA-Bmal1-KO mice were measured by QRT-PCR relative to -actin.

Data shown as mean ± SD, n=3/group. **p<0.01 vs wild-type. (B), Wild-type and

BA-Bmal1-Kcice were single-housed in metabolic cages. The oxygen consumption

(VO2), carbon dioxide (VCO2) production, energy expenditure (EE) and food intake

were recorded when the chamber temperatures were set at 22oC or 4oC. Data shown

as mean ± SD, n=4. Gray shade represents lights-off period from 18:00 to 6:00.

12

Figure V. Vasoactivity of vessel rings of BA-Bmal1-KO mice. (A),

Dose-dependent contraction curves of thoracic aorta rings from wild type and

BA-Bmal1-KO mice in response to phenylephrine (PE), serotonin (5-HT) and

Angiotensin II (Ang II). Results are shown as mean ± SD, n=10 in each group. (B),

Endothelium-dependent relaxation induced by acetylcholine (Ach) in aortic rings with

intact endothelium, and endothelium-independent relaxation induced by sodium

nitroprusside (SNP) in aortic rings with the endothelium removed. Results are shown

as mean ± SD, n=10 in each group. The thoracic aorta rings used for the above

experiments were harvested during the lights-on phase and had the associated PVAT

removed, as indicated in the methods section.

13

Figure VI. Systolic blood pressure and heart rate inBA-Bmal1-KO mice upon

Ang II infusion. SBP (A) and heart rate (B) were measured by radiotelemetry. Day 0

represents the start day of Ang II infusion (500ng/kg/min). Blue dashed lines indicate

the hypotensive phenotype in the resting phase of the BA-Bmal1-KO animals before

Ang II infusion. Data are shown as mean ± SEM, n=6. *p<0.05 vs wild-type. Gray

shade represents lights-off period from 18:00 to 6:00.

14

Figure VII. PVAT-derived factors are unlikely to contribute to the hypotensive

phenotype.(A), mRNA levels of the anti-contractile factor adiponectin (Adipoq),

contractile factors leptin (Lpn) and chemerin (Rarres2) in PVAT and BAT of wild type

and BA-Bmal1-KO mice were measured by QRT-PCR relative to -actin. Data shown

as mean ± SD, n=3/group. *p<0.05 vs wild-type. Gray shade represents lights-off

period from 18:00 to 6:00. (B), adrenalin and (C), prostacyclin levels in PVAT of

wild-type and BA-Bmal1-KO mice. Tissues were harvested at 10AM. Data shown as

mean ± SD, n=6/group.

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15

Figure VIII. Response of BA-Agt-KO mice to systemic isoproterenol injection.

Heart rate (HR), SBP and DBP of wild-type and BA-Agt-KO mice were measured by

radiotelemetry when the temperature of the environmental chamber was set at 22oC.

The mice received 10mg/kg isoproterenol by i.p. injection at ~10AM, resting phase (A)

or 10PM, active phase (B). Data are shown as mean±SD, n=5/group.

16

Figure IX. Cardiac function and vasoactivity of aortic rings of BA-Agt-KO mice.

(A), The left ventricular end diastolic and systolic diameters (LVDd and LVDs,

respectively), ejection fraction (EF) and fractional shortening (FS) were measured in

wild-type and BA-Agt-KO mice during lights-on (resting) phase using

echocardiography as described in the methods section. Data shown as mean±SD,

n=5/group.(B), Dose-dependent contraction curves of thoracic aorta rings from

wild-type and BA-Agt-KO mice induced by phenylephrine (PE), serotonin (5-HT) and

Angiotensin II (Ang II). Results are shown as mean±SD, n=10/group. (C),

Endothelium-dependent relaxation induced by acetylcholine (Ach) in aortic rings with

17

intact endothelium, or endothelium-independent relaxation induced by sodium

nitroprusside (SNP) in aortic rings with the endothelium removed. Results are shown

as mean±SD, n=10/group.

18

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