Protein analyses were performed by Western blotting, as described (Meijer et al., 2013). Segment of RA from IRS2+/+ and IRS2−/− mice were exposed to solvent or to insulin for 15 min at 37◦C. In order to study the effects of PVAT on the RA, IRS2+/+ and IRS2−/− PVAT were isolated from overnight fasted mice and then incubated in 100 µl MOPS buffer with 1% of bovine serum albumin (BSA). PVAT samples were stimulated with either solvent or 10 nM insulin for 30 min in a 96 wellplate. Thereafter, freshly isolated femoral artery segments from fasted 8 week old Bl6 mice were added to the PVAT wells and incubated at 37◦C for 15 min. Femoral artery segments were used in order to obtain appropriate amounts of protein for Western blotting. The artery segments were snap frozen in liquid nitrogen and savedat −80◦C till further analysis. The protein lysates were stained with a specific primary antibody against Ser 473 phosphorylated Akt (antibodies obtained from Cell Signaling Technology, Boston, MA, USA) and were visualized with a chemiluminescence kit (GE Healthcare, Diegem, Belgium). A specific primary antibody against ERK1/2 (1:1,000; New England Biolabs, Ipswich, USA) was used to examine ERK1/2 activation. Differences in phosphorylated protein of Akt at ser 473 were adjusted for differences in the total Akt protein staining.

#### Adipokine Secretion

PVAT-conditioned media were prepared and the amount of secreted adipokines were quantified using the mouse magneticbead adipokine milliplex multianalyte ELISA kit (Millipore, Amsterdam, the Netherlands) and detected using the Luminex system. Freshly isolated PVAT from fasted IRS2+/+ (n = 7) and IRS2−/− (n = 5) mice in amounts of comparable size were incubated in 100 µl MOPS buffer with 1% of BSA and stimulated with either solvent or 10 nM insulin at 37◦C for 45 min in a 96 well-plate. The conditioned media were snap frozen in liquid nitrogen and saved at −80◦C till further analysis. Adiponectin, IL-6, Leptin, MCP-1, PAI-1 (Total), Resistin, and TNF-α concentrations in the conditioned media were measured in duplicate and averaged. Data are corrected for the PVAT weight that was used in the incubation.

#### Real Time Quantitative Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from PVAT using a miRCURY RNA isolation kit (Exiqon). PVAT RNA was reversely transcribed and amplified using Ovation PicoSL WTA System V2 (Nugen). Quantitative PCR was performed using a commercial SYBR green mastermix (Biorad) and specific primers (available upon request) for pro-inflammatory genes (TNF-α and CCL2) and adipokines (adiponectin, leptin, and endothelin-1). IRS1 expression was evaluated to check for compensation to IRS2 knockout. Data were corrected for the geometric mean of ribosomal protein S15 (Rps15) and expressed relative to the wild type PVAT.

#### Histology

PVAT surrounding the RA was excised together with part of the underlying muscle and stored overnight in buffered formaldehyde (4%) and embedded in paraffin. For histochemical analysis, slices with a thickness of 5µm were dewaxed, rehydrated and stained with hematoxylin and eosin (H&E). The cross-sectional area of adipocytes was analyzed in blinded fashion using Image J software.

### Blood Pressure and Heart Rate Measurements

Blood pressure and heart rate of IRS2+/+ and IRS2−/− were determined under stress-free conditions using radio telemetry as described (Aman et al., 2012).

#### Statistical Analysis

Values are expressed as mean ± SEM. Steady-state responses are reported as mean change from baseline (percentages) ± SEM. Differences in insulin-induced vasoreactivity were performed using two-way ANOVA. Tukey post-hoc test was where appropriate. Differences in IHC analyses and in protein phosphorylation as found by Western blot were determined using unpaired student t-test. Phosphorylation was expressed as the fold increase over the unstimulated controls, assigning a value of 1 to the control. qRT-PCR data was log2 transformed before statistical analysis. Differences with p < 0.05 were considered statistically significant. Analyses were performed with GraphPad Prism 6.0 (GraphPad software, San Diego, CA, USA).

# RESULTS

## General Characteristics of IRS2+/+ and IRS2−/−Mice

IRS2+/+ and IRS2−/−littermates had similar body weights (23 ± 1 and 24 ± 1 g, respectively, **Table 1**). IRS2−/− mice had higher non-fasting levels of plasma insulin (9 ± 1 vs. 20 ± 2 µU/ml, p < 0.01) and exhibited fasting hyperglycemia (5.3 ± 0.57 vs. 12.6 ± 2.7 mMol/l, p < 0.05) than IRS2+/+. IRS2−/− mice showed a small decrease in blood pressure compare to their IRS2+/+ littermates (**Table 1**).

## Insulin-Mediated Vasoreactivity in IRS2+/+ and IRS2−/− Muscle Resistance Arteries

To gain insight into the role of IRS2 in insulin-dependent vasoreactivity in muscle, we first examined the reactivity of IRS2+/+ and IRS2−/− gracilis muscle RA obtained from IRS2+/+ and IRS2−/− mice in response to insulin and other vasoactive molecules.IRS2+/+ and IRS2−/−RA showed normal endothelial and smooth muscle function, as reflected by responses to acetylcholine (**Figure 1A**), sodium nitroprusside (**Figure 1B**), and exogenous ET-1 (**Figure 1C**). The maximal diameter of IRS2−/− RA was reduced compared to IRS2+/+ RA (111 ± 6 vs. 127 ± 5 micron, P < 0.05), and basal vascular tone in the presence of 25 mM potassium was 34 ± 2% in IRS2+/+ and 27 ± 4% in IRS2−/− RA (P = 0.11). Unexpectedly, insulin induced vasoconstriction in isolated IRS2+/+RA, which was inhibited by the non-selective ET-1 receptor antagonist PD142893 (**Figures 2A,B**). This confirms the critical role of endothelin-1 ininsulin's vasoconstrictor effects, and shows a decreased contribution of NO to insulinmediated vasoreactivity in IRS2+/+ RA. In IRS2−/− mice, no vasoconstrictor response was observed during exposure of the isolated arteries to insulin, nor in the presence of the ET-1 receptor antagonist (**Figure 2B**). Mechanistically, the

TABLE 1 | General characteristics of IRS2-deficient and wild-type littermates at 8 weeks of age.

IRS2−/− had similar body weights and higher levels of insulin and glucose than the IRS2+/+ mice. MAP, mean arterial pressure, SBP, systolic blood pressure. Data represent mean ± SEM, IRS2+/+N = 10, IRS2−/−N = 7.

insulin-induced phosphorylation of ERK1/2 was impaired in IRS2−/− RA compared to IRS2+/+ RA (**Figure 2C**), whereas insulin-stimulated activation of Akt was similar (**Figure 2D**). This indicates that the stimulation of ERK1/2-ET-1 activity by insulin requires IRS2.

The observed insulin-induced vasoconstriction of isolated IRS2+/+ RA contrasts with the response of C57Bl/6 mice (Meijer et al., 2013), which vasodilate in response to insulin during ET-1 receptor inhibition and during incubation with PVAT of lean Bl6 mice (data not shown). As the IRS2 mice used in this study are bred on a mixed background of the mouse strains C57Bl/6 and SV129, we tested whether the Sv129 background caused the vasoconstrictor response by studying insulin responses of isolated RA from SV129 mice. Insulin indeed induced vasoconstriction in arteries from SV129 mice, while it had no net effect on the diameter of C57Bl/6 RA (Figure S1A). Endothelial function (response to acetylcholinemediated vasodilation) was not different between the two strains (Figure S1B**)**. Collectively, arteries from different mouse strains can display different responses to insulin. For this reason and to facilitate comparability with other experiments, we proceeded by studying the effect of PVAT obtained from IRS2+/+ and IRS2−/− mice on RA's obtained from C57Bl/6 mice. RA's from C57/Bl6 mice had a maximal diameter of 137 ± 5 microns.

# PVAT From IRS2+/+ but Not IRS2−/−Mice Uncovers Insulin-Induced Vasodilation in Resistance Arteries

To study the interaction between PVAT and insulin-induced vasoreactivity of isolated muscle RA's, PVAT of IRS2+/+ and IRS2−/− littermates was co-incubated with RA's in the pressure myograph for 45 min. Insulin (10 nM) induced vasodilation in the presence of IRS2+/+ PVAT (17.2 ± 4.9%, N = 9; **Figure 3A**). On the other hand, insulin failed to induce diameter change in arteries incubated with IRS2−/− PVAT (−7.3 ± 4.7%, N = 10) or without the presence of PVAT (0.4 ± 4.1%, N = 9). Baseline vessel tone was not different in RA's (n = 28) in the presence of either IRS2+/+ PVAT orIRS2−/− PVAT nor was acetylcholine-induced vasodilation (**Figures 3B**,**C**).

## Adipocyte Size Is Increased in PVAT of IRS2−/− Mice

To quantify the effects of IRS2 deletion on the morphology and inflammatory status of PVAT, adipocyte area and macrophage infiltration were investigated. Adipocyte area was significantly (p = 0.0013) larger in IRS2−/− PVAT (1,314 ± 161 µm2 ) compared to IRS2+/+ (915 ± 63 µm2 ) (**Figure 3D**). Leukocyte infiltration assessed in tissue slices was not quantifiable in PVAT of both genotypes. IRS2 mRNA levels in PVAT were confirmed to be absent in IRS2−/− PVAT (**Figure 4A**). To check for compensation for IRS2 knockout, we measured IRS1 mRNA expression and we found no differences between IRS+/+ and IRS−/− PVAT (**Figure 4B**).

### Adiponectin Expression and Secretion From IRS2−/− PVAT Are Similar to IRS2+/+ PVAT

To elucidate the mechanisms involved in the interaction of intramuscular PVAT with insulin-induced vasoreactivity, we examined the role of several secreted adipokines in this interaction. Adiponectin secretion was similar between IRS2+/+ and IRS2−/− PVAT conditioned media (**Figure 5A**), as was Adiponectin mRNA in PVAT between IRS2+/+ and IRS2−/− mice (**Figure 4C**). Despite the increase in adipocyte size, leptin expression was similar between IRS2+/+ and IRS2−/− PVAT (**Figure 4D**). Other adipokines and inflammatory markers were also studied. Levels of TNF-α and MCP-1 were undetectable in PVAT-conditioned medium (data not shown). Moreover, TNF-α mRNA levels were low in IRS2+/+ and IRS2−/− PVAT (**Figure 4E**) while mRNA levels of CCL2 were similar between IRS2+/+ and IRS2−/− PVAT (**Figure 4F**). There were no differences between IRS2+/+ and IRS2−/− in the amounts of secreted PAI-1, IL-6, Leptin (**Figures 5B,D,E**). Unexpectedly, we found that IRS2 deficiency decreases resistin secretion by PVAT (**Figure 5C**), demonstrating that IRS2 deficiency does alter the secretory function of PVAT.

#### Normal Phosphorylation of Akt in Response to Insulin in Arteries Incubated With IRS2−/− PVAT

To further study the insulin-induced vasodilator pathway, which is mediated through Akt and eNOS phosphorylation, phosphorylation of Akt was studied in the presence of IRS2+/+ and IRS2−/− PVAT. The phosphorylation of Akt in femoral artery segments of C57Bl/6 mice was significantly increased after insulin stimulation in the presence of IRS2+/+ as well as IRS2−/− PVAT (**Figure 6**).

FIGURE 3 | IRS2−/− PVAT does not uncover insulin-mediated vasodilation. (A) Arteries obtained from C57/Bl6 mice were incubated with PVAT from IRS2+/+ and IRS2−/− littermates and stimulated with increasing doses of insulin. Incubation with IRS2+/+ PVAT, allowed the artery to dilate in reaction insulin in dose-dependent manner (17.2 ± 4.9% dilation in response to highest (insulin), p < 0.0001, N = 9,) while arteries that were incubated with IRS2−/− PVAT or were deprived of PVAT did not react to insulin (−7.3 ± 4.7%, N = 10 and 0.4 ± 4.1%, N = 9, respectively; p = 0.17). Data are presented as mean ± SEM, tested with two-way ANOVA with Tukey post-hoc correction. (B) At the end of the experiment, endothelial integrity was examined by stimulating the arteries with acetylcholine. All the arteries that were included in the analyses in (A) had at least 60% dilation in response to acetylcholine—cutoff point to rule out endothelialdamage. (C) PVAT did not affect the percentage of arteriolar preconstriction in the organ bath. (D) Adipocyte size in IRS2+/+ PVAT (412.1 ± 58.7µm) are smaller (p = 0.03) than adipocytes in IRS2−/− PVAT (914.7 ± 63. 2µm). Data are presented as mean ± SEM, tested with unpaired t-test. (D,E): Morphology of IRS2+/+ and IRS2−/− PVAT at the site of the gracilis resistance artery. (E,F) are at the same magnification. M: muscle.

# DISCUSSION

The main findings of this study are fourfold: (1) IRS2 in the vessel wall of muscle RA is more involved in the ERK1/2-Endothelin-1 pathway than in the Akt-NO pathway of insulin signaling, (2) IRS2−/− PVAT loses its ability to induce insulin-mediated vasodilation in skeletal muscle RA, (3) this is accompanied by increased adipocyte area in IRS2−/− PVAT with no signs of increased inflammation, (4) adiponectin secretion is similar between IRS2−/− PVAT and wildtype PVAT. A graphical summary of our findings is depicted in **Figure 7**.

It has been shown that IRS2 is a major IRS isoform expressed in endothelial cells (Kubota et al., 2003). In their important work, Kubota et al. (2003) showed that mice lacking

statistical analysis as described in section Methods. APN, Adiponectin.

IRS2 in endothelial cells had impaired insulin-induced eNOS phosphorylation. This resulted in a reduced insulin-mediated microvascular recruitment and a decrease in muscle glucose uptake. Insulin induces a strong vasoconstriction in the IRS2+/+ arteries which is completely dependent on ET-1 (**Figure 2**). We found out that this is due the difference in mouse strains as the Sv129-strain is less sensitive to insulin-mediated NO production in the vascular wall (Supplemental Figure S1). Importantly, the arteries that displayed vasoconstriction were in an experimental setup that is deprived of IRS2+/+ PVAT. Nevertheless, this insulin-induced vasoconstriction observed in IRS2+/+ RA is different than our earlier observations in human and Bl6 arteries that did not show insulin-induced vasoconstriction. Moreover, these arteries showed insulin-mediated vasodilation after preincubation with PVAT from lean mice. This is why we decided to proceed with our experimental set-up using RA obtained from Bl6 mice and constantly comparing the IRS2−/− PVAT with PVAT obtained from their wildtype littermates. We show in our study that the absence of IRS2 in PVAT is sufficient to abrogate the PVAT-assisted insulin-mediated vasodilation of skeletal muscle RA despite the fact that these arteries were functionally normal (**Figure 3A**). In their seminal work, Abel et al. showed that alterations in adipocyte inherent characteristics resulted in a decreased muscle glucose uptake (Abel et al., 2001). This means that adipose tissue is capable of (in)directly altering the capacity of the body to regulate glucose homeostasis. In their study, however, the authors did not specifically examine the function of PVAT in these mice.

Kubota et al. have reported that the expression of IRS2 in endothelial cells is reduced by high fat diet in mice (Kubota et al., 2003). Conditions of calorie excess lead to a low-grade inflammation and production of pro-inflammatory cytokines (TNF-α and IL-6) from adipose tissue (Yudkin et al., 1999, 2005) which inhibits adiponectin secretion (Tilg and Moschen, 2006). Indeed, PVAT anticontractile properties are lost due to inflammation (Greenstein et al., 2009; Withers et al., 2011). After we observed that IRS2−/− PVAT has lost its vasodilatory capacity, we looked at inflammatory signs in this tissue. Literature has shown that, as fat mass increases in obesity, the size of adipocytes increases. In our study, adipocytes in IRS2−/− PVAT were larger than IRS2+/+ PVAT (**Figure 3D**). PVAT hypertrophy in IRS2−/− mice may be caused by decreased resistin expression, as resistin

stimulates lipolysis (Ort et al., 2005). (Mita et al., 2011) showed that IRS2-deficient macrophages accumulate in the vascular wall; eventual accumulation in PVAT is expected since PVAT is vascularized. In our study, however, we failed to show signs of inflammation in IRS2−/− PVAT. First, macrophage staining in PVAT samples was not quantifiable because of the small size of PVAT samples and low prevalence of leukocytes (data not shown). Second, there were no differences in IL-6 secretion from IRS2−/− and wildtype PVAT and the concentration of TNFα was low (**Figure 5**). Third, we did not find differences in CCL2 and TNF-α mRNA expression levels between IRS2−/− and wild-type PVAT. It would be interesting to quantify markers of inflammation in IRS2−/− PVAT obtained from older mice to study age related changes in PVAT in conditions of IRS2 deficiency. However the functional defects of IRS2-deficient PVAT are unlikely to be caused by exaggerated inflammation.

Adiponectin is an important modulator of many metabolic processes and has been shown to increase insulin sensitivity and improve vascular function (Funahashi et al., 1999; Yokota et al., 2000; Laakso, 2001).The concentration of adiponectin is inversely associated with DM2 (Duncan et al., 2004; Luo et al., 2010). Adiponectin binds its receptor and activates AMPKα1α2 which eventually stimulates Akt phosphorylation and NO production (Eringa et al., 2006; de Boer et al., 2016). In the current study, however, we did not see differences in the secretion of adiponectin (**Figure 5A**) or expression levels thereof in IRS2−/− and IRS2+/+ PVAT (**Figure 4C**). To confirm this, Akt phosphorylation in wild-type arteries incubated with IRS2-deficient PVAT was comparable to incubation with wild-type PVAT after insulin stimulation (**Figure 6**). The failure of arteries to dilate in response to insulin after incubation with IRS2−/− PVAT despite a normal Akt phosphorylation prompted us to measure eNOS phosphorylation. However, we were unsuccessful to this end due to the small amounts of eNOS protein in resistancearteries. Collectively, our results suggest that the failure of the vasodilator capacity in IRS2−/− PVAT stems from changes in molecules other than adiponectin that directly affects eNOS phosphorylation. In previous publications,

we showed that db/db PVAT has lost its vasodilator capacity which was accompanied by a decreased adiponectin secretion; however, adding recombinant adiponectin to db/db PVAT only partially restored insulin-induced vasodilation, suggesting that other adipokines contribute to PVAT vasodilator capacity (Meijer et al., 2013). Previs et al. have shown that IRS2-deficient adipocytes fail to attenuate lipolysis in response to insulin (Previs et al., 2000). Hence, Free Fatty Acids (FFA) are probable contributors to the impaired vasodilatory capacity of PVAT in IRS2-deficient mice. Moreover, we have previously shown (Bakker et al., 2008) that activation of PKCtheta by the FFA palmitic acid reduces insulin-mediated Akt phosphorylation (Ser(473), which is crucial in insulin-mediated activation of eNOS) while increasing ERK1/2 phosphorylation. Based on our data, we propose the following working hypothesis (depicted in **Figure 7**). In the healthy situation, insulin mediates vasodilation by inducing autophosphorylation of its receptor which results in phosphorylation of IRS-1 and -2 and eventually activating Akt and endothelial Nitric Oxide Synthase (eNOS) to produce NO. The PVAT secretes the necessary adipokines (represented by adiponectin) that enhance NO production in endothelial cells.IRS2-deficient PVAT fails to uncover insulin-mediated vasodilation in muscle RA. Probable mechanisms do not involve adiponectin, but rather involve direct mediators that are released from PVAT (such as FFA) that interact with eNOS to reduce NO production.

#### Study Limitations

In parallel with the findings of this study, there are a number of limitations that need to be considered. First, at 8 weeks of age, IRS2−/− mice showed hyperinsulinemia and hyperglycemia (**Table 1**). Hence, it is not possible to delineate whether the dysfunction in IRS2−/− PVAT is due to a direct role of IRS2 absence in PVAT signaling or due to an indirect role of the

FIGURE 7 | Dual role of IRS2 in regulation of insulin-induced vasoreactivity in muscle. (A) In health, insulin induces vasodilation by activating its receptor, resulting in phosphorylation and activation of insulin receptor substrates (IRSs) 1 and 2.both IRS isoforms are involved inAkt and endothelial Nitric Oxide Synthase (eNOS) activation, while IRS2 mediates insulin-induced activation of ERK1/2. Perivascular adipose tissue (PVAT) secretes vasodilator adipokines such adiponectin that reduce endothelin (ET-1) secretion (de Boer et al., 2016) and enhance NO production in endothelial cells through AMPK (Eringa et al., 2010). (B) IRS2-deficient PVAT fails to uncover insulin-mediated vasodilation in muscle arteries. Probable mechanisms aredirect mediators that are released from PVAT that interact with eNOS to reduce NO production, such as free fatty acids (FFA) (Previs et al., 2000).

alteredmetabolic milieu. To account for this, we included young (∼8–10 weeks) mice in our study which have minimal to mild whole-body metabolic dysregulation when compared to older IRS2−/− mice. Previously, we have found that short term exposure of muscle RA to 10 mM of glucose does not alter their functional properties (Eringa et al., 2002). Moreover, we made our observation in an ex vivo set up wherein the PVAT and the arteries were incubated in a physiological buffer before the start of the experimental measurement. Hence, the indirect effects of whole-body metabolic dysregulation on PVAT phenotype in IRS2−/− mice were kept to a minimum. It should be noted that while hyperglycemia-mediated effects of IRS2 deficiency cannot be fully excluded, these effects are relevant to PVAT dysfunction in DM2. Second, the adipokine secretion pattern or IRS2−/− PVAT observed in this study is highlighted by a paradoxical decrease in resistin secretion. We cannot fully explain this finding, but the low inflammation in IRS2−/− PVAT (**Figures 4E,F**) may contribute as macrophages are an important source of resistin (Qatanani et al., 2009). Third, as the PVAT in our study is missing IRS2 in all of its components (adipocytes, endothelial cells, macrophages), further experiments are needed to decipher the role of IRS2 deletion in the endothelium of PVAT. Lastly, it was not possible to measure the amount of Endothelin-1 released from the cannulated vessels in the organ bath; challenges arose in performing the in vitro analyses of this study due to the small amount of PVAT and small-sized arteries obtained from the mice.

In conclusion, we show in this study that in the muscle microcirculation IRS2 directly mediates insulin's vasoconstrictor effects in the vascular wall while indirectly mediating its vasodilator effects by controlling vasodilator actions of PVAT. IRS2 inactivation in PVAT abolishes its vasodilator capacity

#### REFERENCES

independently from adiponectin secretion or inflammation in PVAT. Future research should focus on detailed analysis of the PVAT secretome in IRS2-deficient PVAT to gain further insight into adipose tissue dysfunction in insulin-resistant individuals.

# AUTHOR CONTRIBUTIONS

AT wrote the manuscript and researched and analyzed data. WB gathered part of the data regarding the IRS2 mouse colony. HN, YS, VvH, and ES supervised, contributed to discussions, and edited the manuscript. EE researched data, supervised, contributed to discussions, and edited the manuscript. EE is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

### FUNDING

This work was supported by the Netherlands Organization for Scientific Research (VIDI grant 917.133.72).

#### ACKNOWLEDGMENTS

We acknowledge Zeineb Gam and Elisa Meinster for their dedicated technical assistance in performing the experiments of this study.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2018.00245/full#supplementary-material

identified in a subject with type 2 diabetes impairs metabolic insulin signaling. J. Clin. Endocrinol. Metab. 88, 1468–1475. doi: 10.1210/jc.2002-020933

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Turaihi, Bakker, van Hinsbergh, Serné, Smulders, Niessen and Eringa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Perivascular Adipose Tissue as a Relevant Fat Depot for Cardiovascular Risk in Obesity

Rafael M. Costa1 \*, Karla B. Neves 2 , Rita C. Tostes 1 and Núbia S. Lobato3

1 Department of Pharmacology, Ribeirao Preto Medical School, University of São Paulo, Ribeirao Preto, Brazil, 2 Institute of Cardiovascular and Medical Sciences, British Heart Foundation, Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, United Kingdom, 3 Institute of Health Sciences, Federal University of Goias, Jatai, Brazil

Obesity is associated with increased risk of premature death, morbidity, and mortality from several cardiovascular diseases (CVDs), including stroke, coronary heart disease (CHD), myocardial infarction, and congestive heart failure. However, this is not a straightforward relationship. Although several studies have substantiated that obesity confers an independent and additive risk of all-cause and cardiovascular death, there is significant variability in these associations, with some lean individuals developing diseases and others remaining healthy despite severe obesity, the so-called metabolically healthy obese. Part of this variability has been attributed to the heterogeneity in both the distribution of body fat and the intrinsic properties of adipose tissue depots, including developmental origin, adipogenic and proliferative capacity, glucose and lipid metabolism, hormonal control, thermogenic ability, and vascularization. In obesity, these depot-specific differences translate into specific fat distribution patterns, which are closely associated with differential cardiometabolic risks. The adventitial fat layer, also known as perivascular adipose tissue (PVAT), is of major importance. Similar to the visceral adipose tissue, PVAT has a pathophysiological role in CVDs. PVAT influences vascular homeostasis by releasing numerous vasoactive factors, cytokines, and adipokines, which can readily target the underlying smooth muscle cell layers, regulating the vascular tone, distribution of blood flow, as well as angiogenesis, inflammatory processes, and redox status. In this review, we summarize the current knowledge and discuss the role of PVAT within the scope of adipose tissue as a major contributing factor to obesity-associated cardiovascular risk. Relevant clinical studies documenting the relationship between PVAT dysfunction and CVD with a focus on potential mechanisms by which PVAT contributes to obesity-related CVDs are pointed out.

Keywords: perivascular adipose tissue, obesity, vascular function, cardiovascular risk, adipokine

#### INTRODUCTION

Obesity is a fast-growing problem that is reaching epidemic magnitudes worldwide, affecting both children and adults (Ogden et al., 2016; Lim et al., 2017). This condition is defined as a disproportionate body weight for height with excessive fat accumulation that is frequently accompanied by mild, chronic, systemic inflammation (Gonzalez-Muniesa et al., 2017).

#### Edited by:

Maik Gollasch, Charité Universitätsmedizin Berlin, Germany

#### Reviewed by:

D. Neil Granger, Louisiana State University Health Sciences Center Shreveport, United States Geraldine Clough, University of Southampton, United Kingdom

\*Correspondence: Rafael M. Costa<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Vascular Physiology, a section of the journal Frontiers in Physiology

Received: 09 December 2017 Accepted: 06 March 2018 Published: 21 March 2018

#### Citation:

Costa RM, Neves KB, Tostes RC and Lobato NS (2018) Perivascular Adipose Tissue as a Relevant Fat Depot for Cardiovascular Risk in Obesity. Front. Physiol. 9:253. doi: 10.3389/fphys.2018.00253

**52**

There is emerging body of scientific, medical, and behavioral data showing that central deposition of adipose tissue is associated with elevated risk of morbidity and mortality due to several cardiovascular complications, including stroke, congestive heart failure, myocardial infarction, and cardiovascular death, and this is independent of the association between obesity and the components of the metabolic syndrome and other cardiovascular risk factors (Arnlöv et al., 2010; Williams et al., 2015). Previous support for this understanding was provided by the American Heart Association in 1998, which has reclassified obesity as a major, modifiable risk factor for coronary heart disease (CHD) (Eckel and Krauss, 1998).

Besides being considered the largest energetic reservoir in the body, white adipose tissue (WAT) has been recognized as a remarkably complex endocrine organ that produces and secretes several substances with endocrine, paracrine, and autocrine functions, acting as a major regulator of systemic energy homeostasis (Rosen and Spiegelman, 2014). In obesity, adipose tissue may become dysfunctional and fail to appropriately expand to store the excess energy. This results in ectopic fat deposition in other tissues that regulate metabolic homeostasis (Tchoukalova et al., 2010). WAT expansion has been associated with numerous local consequences, including inflammation (Apovian et al., 2008), fibrosis (Henegar et al., 2008), hypoxia (Jiang et al., 2011), dysregulated adipokine secretion (Jernås et al., 2006; Skurk et al., 2007), and disrupted mitochondrial function (Heinonen et al., 2015). The whole-body consequences of WAT dysfunction include abnormal glucose and lipid metabolism, insulin resistance, increase in blood pressure, coagulation, fibrinolysis, inflammation, and endothelial dysfunction, all of which provide important mechanisms linking obesity to cardiovascular disease (CVD).

In addition to abdominal adiposity, recent evidence indicates that perivascular adipose tissue (PVAT) produces and releases a wide variety of adipokines and other factors that exert a paracrine influence on the vascular function, not only in veins and conductance arteries, but also in vessels of smaller caliber, which are essential in the regulation of blood pressure (Iozzo, 2011; Aghamohammadzadeh et al., 2012; Malinowski et al., 2013; Szasz et al., 2013). Although PVAT has been considered an inherent component that provides structural support to the vessels, now it is clear that this tissue possesses the dynamic capacity to mobilize near vessels with the potential for cellular communication and control of vascular function (Sacks and Fain, 2007; Chatterjee et al., 2009). Local accumulation of perivascular adipocytes has been consistently associated with the development of cardiometabolic complications in obesity (Chang et al., 2013; Lim and Meigs, 2014). In fact, adipose tissue surrounding the heart has been clinically associated with coronary artery disease (Cheng et al., 2008; Clément et al., 2009; Company et al., 2010), which reinforces the evidence that local adipose tissue accumulation can constitute an important regulator of cardiovascular function and a mediator of the development and progression of CVDs. The present review provides a comprehensive overview on the role of PVAT within the scope of adipose tissue as a major contributing factor to obesity-associated cardiovascular risk. We will also highlight the relevant clinical studies documenting the relationship between PVAT dysfunction and CVD and the potential mechanisms by which PVAT contributes to obesity-related cardiovascular complications.

# OBESITY AND CARDIOVASCULAR RISK

Over the past decades, an explosive increase in overweight and obesity prevalence has taken place in most of the high-income countries (Vandevijvere et al., 2015). Several recent reviews have also shown significant increases in the prevalence of these conditions in low- and middle-income countries, affecting both men and women, adults and children (Gupta et al., 2012; Popkin and Slining, 2013; Sayon-Orea et al., 2013; Yatsuya et al., 2014). Although there are great variations in the prevalence and trends of overweight and obesity among different regions, the numbers have been projected to further increase in coming years (Gupta et al., 2012; Popkin and Slining, 2013; Sayon-Orea et al., 2013; Yatsuya et al., 2014; Poobalan and Aucott, 2016). The percentage of adults with a body mass index (BMI) ≥25 kg/m2 between 1980 and 2013 increased in men and women from 28.8% (95% UI: 28.4–29.3) to 36.9% (36.3–37.4), and from 29.8 to 38%, respectively. Among children and adolescents, in 2013, 23.8% (22.9–24.7) of boys and 22.6% (21.7–23.6) of girls were either overweight or obese in developed countries (Ng et al., 2014). The prevalence rate in developing countries elevated from 8.1% (7.7–8.6) to 12.9% (12.3–13.5) for boys and from 8.4% (8.1– 8.8) to 13.4% (13.0–13.9) in girls, in 2013. The World Health Organization (WHO) estimated that more than 2.1 billion adults were overweight or obese globally in 2014. By 2030, estimations predict that 57.8% of the adult population will have a BMI of 25 kg/m2 or higher (Kelly et al., 2008; Finkelstein et al., 2012). As such, the adverse health consequences of obesity, particularly the burden of CVDs are expected to increase in coming years.

In adults, obesity generally presents as an excess of body weight and adipose tissue, which is clinically assessed by the BMI, calculated by the weight in kilograms divided by the height in square meters (Garrow and Webster, 1985). The idea that BMI is associated with higher all-cause morbidity/mortality risk is supported by a wealth of epidemiological and clinical data (Borrell and Samuel, 2014; Yang et al., 2016; Kong et al., 2017). However, a recent comprehensive estimation, resulting from a systematic review on the association of all-cause mortality in adults with BMI categories used in the United States and internationally, demonstrated that while grades 2 and 3 obesity are both associated with significantly higher all-cause mortality, overweight (defined as a BMI of 25 – <30) is associated with significantly lower overall mortality relative to the normal weight category. Furthermore, the authors did not find significant excess mortality associated with grade 1 obesity (BMI of 30 – <35) (Flegal et al., 2013), suggesting that cardiovascular risk and mortality are not simply associated with the amount of adipose tissue.

Adipose tissue is dispersed throughout the body in discrete depots ranging from 5 to 60% of total body weight (Cinti, 2001). More than 80% of the tissue is found subcutaneously, mainly in the abdominal, gluteal and femoral areas. Visceral adipose tissue and smaller depots close to organs represent the remaining 10–20% of total body fat in men and 5–10% in women (Lee et al., 2013). It has become increasingly evident that the regional body fat distribution, the size of each depot, the depot-related differences in adipose tissue function, and the balance between them are important for the individual cardiometabolic risk. For example, a peripheral adiposity in upper and lower extremities is favorable while the truncal adipose tissue deposition, which includes subcutaneous fat in thoracic and abdominal region and the fat in intrathoracic and intraabdominal regions, is detrimental and associated with increased mortality (Garg, 2004; McLaughlin et al., 2011). Moreover, for any given BMIvalue, there is significant variability with some lean individuals developing disease and others exhibiting a better metabolic profile than expected for their adiposity profile (Samocha-Bonet et al., 2012). Lapidus et al. in 1980s, reported a stronger association of waist-to-hip circumference ratio, which reflects abdominal fat, with a 12-year incidence of myocardial infarction, angina pectoris, stroke, and death, when compared to other anthropometric measures (Lapidus et al., 1984). Subsequently, many clinical studies have established the superior capability of waist-to-hip ratio measure to predict CVD risk when compared to BMI.

The large-scale randomized factorial clinical trial study ADVANCE (Action in Diabetes and Vascular disease: Preterax and Diamicron-MR Controlled Evaluation) examined the relative importance of different adiposity markers in predicting CVD risk in a population of individuals with type 2 diabetes (Czernichow et al., 2011). After 9.1 years of follow up, investigators reported that waist-to-hip ratio is the best predictor of cardiovascular events and mortality in patients with type-2-diabetes while the BMI is the worst. The INTERHEART study, a large multiethnic case-control study of acute myocardial infarction, also evidenced a greater contribution for waist-to-hip ratios accounting for most of the risk of myocardial infarction when compared to BMI, in both sexes and at all ages (Yusuf et al., 2004). A meta-analysis using data pooled from 15 prospective studies that included more than 258,000 subjects reported a progressive increase in cardiovascular risk accompanying the increase in waist circumference and waist-to-hip ratios (de Koning et al., 2007). Specifically, every 1 cm of increase in waist circumference was associated with a 2% increased relative risk of cardiovascular event and a 0.01 increase in waist-to-hip ratio was associated with a 5% increase in risk of future CVD for both men and women. The Nurses' Health Study, one of the largest and longest studies to date that measured abdominal obesity, confirmed these findings using the waist circumference measure (Zhang et al., 2008). The investigators reported that during 16 years of follow-up in US women, elevated waist circumference was associated with significantly increased CVD mortality even among normal-weight women.

As obesity is strongly linked to comorbid conditions, including type 2 diabetes, hypertension, hypercholesterolemia, hypertriglyceridemia, and non-alcoholic fatty liver disease, these risk factors have been considered important intermediate steps in the causal relationship between obesity and CVD risk. From this perspective, there has been considerable debate about whether the adjustment for these risk factors in statistical models is necessary for the true absolute risk predicting CVD risk or whether such adjustments, rather than controlling for, may increase the overall risk of bias. In fact, the increased cardiovascular risk attributed to weight gain persists even after adjustment for the frequently observed co-existing risk factors. In 1983, a 26-year follow-up of participants in the Framingham Heart Study reported that this risk was particularly apparent for heart failure, but it was also identified with CHD, stroke, and death from CVD. Kenchaiah and colleagues also provided unequivocal evidence in 2002, using updated Framingham data. According to authors, approximately 11% of heart failure cases among men and 14% among women are attributable to obesity alone, suggesting that efforts to promote optimal body weight may reduce the risk of heart failure (Kenchaiah et al., 2002). A careful review of these studies further supports the idea that composition of adipose tissue is important for cardiovascular risk stratification and characterizes visceral adipose tissue as an incomparable pathogenic depot that confers risk beyond its contribution to overall adiposity. These constraints that govern the outcomes of obesity are described in a recent review by Mahmood et al. (2014) and emphasizes the importance of adipose tissue depots as potential components in assessing and predicting cardiometabolic risk.

## SYSTEMIC ADIPOSE TISSUE DYSFUNCTION AND VASCULAR FUNCTION IN OBESITY

The WAT is no longer considered to be only a passive tissue for the storage of excess energy in the form of fat. There is now compelling evidence that this tissue acts as an endocrine organ that produces and releases biologically active compounds that regulate metabolic and cardiovascular homeostasis and undergoes pathological expansion during obesity (Waki and Tontonoz, 2007; Coelho et al., 2013). In addition to the betterknown WAT, mammals also exhibit brown adipose tissue (BAT). Functional BAT in adult humans is localized to areas close to the clavicular, periaortic, cervical, and suprarenal regions (Cypess et al., 2009). BAT is an energy-expending organ that produces heat, and it is essential for adaptive thermogenesis. Adipocytes from BAT possess large numbers of mitochondria that contain a unique protein called uncoupling protein 1 (UCP1), which is a proton channel within the inner mitochondrial membrane involved in the dissipation of the proton motive force that is normally used to drive the synthesis of cellular ATP. Consequently, the energy in the mitochondrial electrochemical gradient is released in the form of heat (Cannon and Nedergaard, 2004). Indeed, evidence from clinical and experimental studies indicate that BAT activation increases thermogenesis (Lidell and Enerback, 2010). Recent reports have also unequivocally demonstrated that BAT exerts significant impact on whole-body energy homeostasis and that its activity profoundly influences body weight (Fruhbeck et al., 2009). In fact, overweight and obesity have been associated with lower BAT activity (Vijgen et al., 2011), which is increased after weight loss (Vijgen et al., 2012). Together with data obtained from rodent models (Vegiopoulos et al., 2010; Seale et al., 2011; Kiefer et al., 2012), these reports have drawn attention to BAT as an attractive target for the treatment of obesity and associated metabolic diseases. Hence, potent or permanent interventions targeting on induction of brown phenotype in adipose tissue depots will facilitate long term studies to investigate their precise role in driving metabolic diseases and to determine their potential to attenuate cardiovascular risk. However, it is discernible that such a critical approach will require layers of specificity, beyond the targeting of individual components of WAT, considering the possibility that factors involved in browning of specific adipose tissue depots may not share similar regulatory network.

A highly relevant factor for the understanding of the cardiovascular impact of increased adipose tissue mass is the recognition that adipocytes are endocrine and paracrine determinants of vascular function. The great question that arises from these observations is how excessive accumulation of adipose tissue could lead to the development of such dysfunction even at a distance. The answer to this question is provided by the evidence that adipose tissue is involved in the production of various proteins, collectively called adipokines (including chemokines, cytokines, and hormones) (Kershaw and Flier, 2004), which are secreted into the circulatory system and act in several physiological processes, including energy balance, immune responses, blood pressure, vascular homeostasis and angiogenesis, glucose and lipid metabolism, and even insulin sensitivity (Prins, 2002), all of which may contribute to the elevated cardiovascular risk associated with obesity (Weyer et al., 2000; Wang et al., 2005). The positive energy balance induces an expansion and remodeling of the adipose tissue that is initially driven by adipocyte hyperplasia mediated by the recruitment and proliferation of adipogenic progenitors followed by an increase in adipocyte hypertrophy (Hausman et al., 2001; Spalding et al., 2008), which lead to dysregulated secretion of adipokines and increased release of free fatty acids (**Figure 1**).

Although the altered secretory function of adipose tissue is consistent with the idea that chronic obesity-related lowgrade inflammation in adipose tissue is involved in the metabolic complications of obesity, it is important to consider that adipocytes in adipose tissue are not the only culprit. Regardless of the mechanisms of adipose tissue expansion, positive energy balance eventually leads to necrotic and/or apoptotic cell death and this is paralleled by quantitative and qualitative changes in the cellular composition and the phenotype of individual cells within adipose tissue (Weisberg et al., 2003). For example, adipose tissue from obese individuals is infiltrated by numerous activated macrophages, leading to increases in both absolute macrophage number and the relative level of macrophage-to-adipocyte ratio. In addition to this quantitative change, the macrophage phenotype is also altered by the obesity state. Macrophages that accumulate in adipose tissue of obese organisms tend to express genes associated with increased production of pro-inflammatory cytokines, express inducible nitric oxide synthase (iNOS), and produce high levels of reactive oxygen species (ROS), and nitrogen intermediates (Lumeng et al., 2007). Thus, it has become evident that interactions between the different cell types from enlarged and inflamed adipose tissue contribute to its overall impact on obesity-related disorders. In fact, some evidence exists that the release of some adipokines is even higher from the cells of the stroma-vascular fraction than from adipocytes (Chavey et al., 2009; Hamaguchi et al., 2012).

Adipose tissue dysfunction may also promote a state of systemic endothelial cell activation through the endocrine actions of inflammatory adipokines (Skurk et al., 2007; Dulloo et al., 2010). The impact of these adipokines on vascular function is not limited to the momentary regulation of the release of endothelium-derived vasoactive factors or regulation of vascular smooth muscle tone. Some adipokines may also profoundly affect local growth, migration, and inflammatory processes (Miao and Li, 2012). Obviously, these variety of products secreted by the adipose tissue, without even reporting the entirety of released adipokines, have provided promising possibilities for identifying novel biomarkers associated with obesity and the cardiovascular complications associated with this condition (Bagi et al., 2012; Barton et al., 2012). Although these studies set several adipokines into their functional context enhancing our current knowledge of the endocrine function of the adipose tissue, the identified proteins must be further validated regarding their expression, secretion and function. Translation of protein profiling results into clinical use will contribute to validate novel adipokines potentially representing a link between obesity and human disease. In this regard, studies must move from characterizing protein abundances to elaboration of their functional effects within cellular networks. However, since human genetic variability is estimated to contribute variations in the capacity of different adipose tissue depots to store and release fatty acids and to produce adipokines (White and Tchoukalova, 2014), and many of the direct genetic achievements are still being investigated for their causal impact on phenotypic outcomes, it is prudent to interpret both positive and negative results with caution. Integrated approaches including the analysis of multiple scientific fields will certainly enhance the understanding of this new physiological concept of interorgan crosstalk in the context of obesity.

# PERIVASCULAR ADIPOSE TISSUE EFFECTS ON VASCULAR FUNCTION

Besides the endocrine role of adipose tissue, mediated by adipokines, a major emerging concept indicates that ectopic fat depots surrounding the heart and almost all systemic blood vessels, can also directly affect the cardiovascular function (Chang et al., 2013). These include: the PVAT, that is, the fat immediately adjacent to the adventitia of almost all arteries; the epicardial adipose tissue, which refers to the fat depot laying on the surface of the myocardium surrounding the coronary arteries; and the pericardial adipose tissue, the fat depot located between the visceral and parietal pericardium (Iozzo, 2011). Notably, this anatomical proximity of PVAT has an enormous influence on the cardiovascular system and highly vascularized

obesity.

organs (e.g., the kidney). In fact, over the past years, clinical and experimental evidence accumulated recognizing that PVAT not only stores triacylglycerols/triglycerides and free fatty acids, participating in energy metabolism, but also secretes a wide variety of biologically active molecules, including adipokines, such as leptin, adiponectin, chemerin, visfatin, resistin, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-18 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor 1 (PAI-1), which modulate vascular tone (Maenhaut and Van de Voorde, 2011), smooth muscle cells migration and proliferation (Miao and Li, 2012), neointimal hyperplasia and formation (Takaoka et al., 2010; Schroeter et al., 2013), inflammatory responses and oxidative stress (Salgado-Somoza et al., 2010). Most of these properties converge on endocrine-related effects, but also on a direct, paracrine influence in CVD, not only on large arteries and veins, but also in small and resistance vessels, and skeletal muscle microvessels, considered to be of greatest importance in blood pressure regulation, as has been reviewed elsewhere (Szasz et al., 2013; Xia and Li, 2017).

The structural and physiological characteristics of PVAT vary according to its location. In the mesenteric arteries, PVAT resembles WAT, with less differentiated adipocytes, poor vascularization, a specific profile of cytokines production/secretion, and contains infiltrates of macrophages, fibroblasts and cells of the immune system (Guzik et al., 2013). On the other hand, recent studies have demonstrated that PVAT of the thoracic aorta exhibits features that resemble BAT rather than WAT (Fitzgibbons et al., 2011; Chang et al., 2012), including expression of genes highly or solely expressed in the BAT, the presence of multilocular adipocytes and high abundance of mitochondria. Like BAT, PVAT is activated by cold and generates heat. Interestingly, the cold-induced activation of thermogenesis in PVAT is accompanied by attenuation of the atherosclerotic process in apolipoprotein E deficient mice (ApoE−/−), whereas such protection is lost in mice where PVAT is absent (Chang et al., 2012), indicating that the thermogenic properties of PVAT also mediate its vascular protective effects.

Consistent with the findings that thoracic aorta PVAT shares common characteristics with BAT, our group has recently demonstrated a potential role of mitochondria in periaortic adipose tissue as a source of mediators that could be involved in the modulation of vascular contraction along with the PVAT-derived relaxing factors (Costa et al., 2016). These are all exciting developments that have the potential to provide insight into new aspects of the specific nature and function of different adipose tissue depots while also places PVAT as a promising component of investigation regarding brown fat and its potential beneficial effects, including those on the vasculature. Further work is required to resolve interesting questions raised by these findings: are the precursor components of PVAT adipocytes distinct from those of white or BAT? Although the morphology and mRNA/protein profile of PVAT is evocative of classic BAT, is this tissue a true BAT? Recent studies have illustrated a remarkable possibility that the origin of PVAT adipocytes may so far be distinct from either white or brown adipocytes. Using a vascular smooth muscle cell (VSMC) specific nuclear receptor activated by peroxisome proliferator-γ (PPARγ) deletion, Chang group generated mice completely devoid of PVAT in the aortic and mesenteric regions while both interscapular BAT and gonadal/inguinal/subcutaneous WAT remained intact, indicating that BAT, WAT, and PVAT have different origins (Chang et al., 2012). In this regard, the previous observation of Chatterjee's group demonstrated that in vitro differentiated human coronary artery perivascular adipocytes exhibit a distinct state of adipogenic differentiation and adipokine secretion when compared with adipocytes from subcutaneous or visceral adipose depots (Chatterjee et al., 2009), suggesting that PVAT exhibit a unique gene expression profile that underlies its role in vascular function. Although these wellestablished strategies provide extremely promising prospects, an unresolved question is the relative contribution of different brown and brown-like fat depots to metabolic and CVD in humans. Whether PVAT thermogenesis can be targeted for therapeutic purposes needs to be determined. It also seems clear that understanding the differences between PVAT depots, specifically, the functional analysis of bioenergetics in this tissue and its impact on systemic metabolism is a very promising approach in the context of metabolism and the etiology of the spectrum of cardiometabolic disorders.

The concept that PVAT influences vascular function, particularly contractile responses, dates from at least as early as the 1990s, when Soltis and Cassis described that PVAT retained and impaired the diffusion of pharmacological agents, justifying a reduction in the contractile response observed in vessels with this tissue (Soltis and Cassis, 1991). However, subsequent studies using several agonists of low retention in adipocytes also demonstrated such a decrease (Gollasch and Dubrovska, 2004). Löhn and his colleagues found that the vascular responses to angiotensin II (Ang II), serotonin (5-HT), and phenylephrine (PE) are reduced in intact aortas with PVAT, suggesting that periaortic adipose tissue regulates vascular tone by the release of transferable adventitia-derived relaxing factors (ADRF), now defined as PVAT-derived relaxant factors (PVRF) (Lohn et al., 2002). The anti-contractile effect of PVRFs under physiological conditions has been described in several species and is mediated by different mechanisms depending on the vascular bed. Convincing evidence has demonstrated that VSMC potassium (K+) channels play a critical role in mediating the relaxation responses to PVRFs. The study of Löhn showed that the anticontractile effect of periadventitial fat was reduced by inhibition of ATP-dependent K+ channels and by the tyrosine kinase inhibitor genistein (Lohn et al., 2002). Following these findings, subsequent studies demonstrated that PVAT-derived transferable factors produce vasorelaxation by opening of voltage-dependent K + (Kv) channels (Dubrovska et al., 2004; Verlohren et al., 2004; Gao et al., 2005; Fesus et al., 2007). Schleifenbaum's group further explored the involvement of the Kv-subfamily on PVRFs actions. The authors demonstrated that the voltage-activated family of K+ channels KCNQ (Kv7), which are not targeted by endothelium-derived relaxing factors (EDRF), play significant role in the vasorelaxation induced by PVRFs, which is at least in part mediated or modulated by hydrogen sulfide (H2S). Although the KCNQ channel subtype involved in the PVRF effects has not been identified so far and clear evidence for direct activation of KCNQ channels by PVRF is still missing, the opening of these channels by pharmacological agents restores the diminished anti-contractile effects of perivascular fat in spontaneously hypertensive rats, thus suggesting its pivotal role in perivascular regulation of vascular tone (Schleifenbaum et al., 2010).

Although we are unable to cover the topic in detail in this manuscript, it is important to note that many other mechanisms involved in PVRF-induced anti-contractile effects have been described. Lu and collaborators, by using inferior vena cava rings in the absence and presence of PVAT and endothelium, found that endothelium removal abolishes PVAT anti-contractile response. The same group of researchers has suggested that PVAT releases Ang 1–7, which, by acting on receptors in the endothelium, leads to nitric oxide (NO) release and activation of Kv channels with subsequent vascular relaxation (Lu et al., 2011). PVRF may also act through endothelium-independent mechanisms involving hydrogen peroxide (H2O2) production and subsequent activation of guanylyl cyclase (sGC) (Gao et al., 2007).

In addition to the vasodilator effects, there is also considerable evidence of contractile function mediated by PVAT. Soltis and Cassis reported the critical role of adipocyte-derived Ang II in PVAT-mediated potentiation of electrical stimulation-induced contraction in rat mesenteric arteries (Soltis and Cassis, 1991). This effect was subsequently shown in vivo systemically in rat mesenteric PVAT (Lu et al., 2010). Gao et al. also reported that PVAT enhances the contractile response of mesenteric arteries to perivascular nerve stimulation through superoxide anion (O− 2 ) production (Gao et al., 2006). The study of Payne documented that PVAT impairs coronary endothelial function in response to bradykinin both in vitro and in vivo, implicating local adipose tissue in the initiation and pathogenesis of coronary vascular disease (Payne et al., 2008). In particular, the authors documented that adipose tissue-derived factors diminish endothelial NO production through the direct inhibition of the enzyme NOS. Following these findings, the authors demonstrated that periadventitial adipose tissue-derived factors impair coronary endothelial NO production via a PKC-β-dependent, site-specific phosphorylation of endothelial NOS (eNOS) at Thr495 (Payne et al., 2009). Some adipose tissue-derived factors, such as leptin (Knudson et al., 2005), resistin (Dick et al., 2006), and TNF-α (Picchi et al., 2006), secreted under conditions of inflammation, can also attenuate vasodilatation, and these factors are produced by PVAT.

In the last years, several studies identified additional components involved in the contractile effects of PVAT, which have been reported especially in the context of obesity and CVD. One example of this is the study of Meyer's group, which demonstrated that PVAT controls arterial smooth muscle tone by releasing an "adipocyte-derived contracting factor" (ADCF) formed by cyclooxygenase (COX), which mediates the contractile effects of PVAT in obesity (Meyer et al., 2013). Another powerful example is the work of Watts et al. illuminating a potential role for the adipokine chemerin as an endogenous mediator that is responsible for vasoconstriction in obesity (Watts et al., 2013). In line with these observations, our group demonstrated that incubation of isolated vessels with chemerin for 24 h increases arterial sensitivity to endothelin-1 (Lobato et al., 2012). Thus, it is clear that PVAT is a complex, active organ with several functions beyond mechanical protection for the underlying vasculature and the current literature provides substantial evidence that this tissue produces many putative vasoactive factors that may influence vascular function and obesity-related vascular injury. However, most of these characterization studies have been carried out on vessel rings isolated from animal models, in the presence or absence of the PVAT layer. This poses an important and unresolved question regarding how much of these results can be translated in vivo, especially concerning human physiology. A thoughtful consideration of the implications of manipulating PVAT function to treat chronic vascular diseases is therefore warranted.

### PERIVASCULAR ADIPOSE TISSUE DYSFUNCTION AS A MAJOR CONTRIBUTING FACTOR TO OBESITY-ASSOCIATED VASCULAR DYSFUNCTION

As the understanding of the molecular mechanisms that underlie the connections between PVAT and vascular function progress, the ultimate goal is to establish the integrated role of this vascular component for obesity-related vascular dysfunction. Like observed with the total adipose tissue, PVAT mass is increased throughout the vasculature in both animal models and humans with obesity (Greenstein et al., 2009; Marchesi et al., 2009; Ketonen et al., 2010; Lehman et al., 2010). Considering that the anti-contractile influence of PVAT is directly dependent on its volume (Verlohren et al., 2004; Gao et al., 2006), it would be discernible that such an increase would be associated with enhancement of PVAT anti-contractile effects. However, available support for our understanding of the connection between PVAT and vascular dysfunction in obesity comes from the findings that obesity is associated with structural and functional changes in PVAT, leading to an imbalance in favor of vasoconstrictor and pro-inflammatory substances, as well as changes in the signaling pathways in the vessel and that this condition interferes with vascular function (**Figure 2**). Numerous studies have since validated this postulate (reviewed in Xia and Li, 2017). In line with this observation, Gao et al. (2005) demonstrated that increased adiposity causes an alteration in the modulatory function of PVAT on vascular relaxation response. New Zealand Obese (NZO) mice, which present most symptoms of the metabolic syndrome and a greater amount of PVAT, also show a reduction in the anti-contractile effect of PVAT (Fesus et al., 2007). Higher periaortic fat mass in rats treated for 6 months with high fat diet (HFD) lead to reduced endotheliumdependent relaxation due to downregulation of AMP-activated protein kinase (AMPK) and eNOS in the aorta with a concurrent upregulation of the mTOR ripamycin target, which negatively regulates the AMPK-eNOS pathway (Ma et al., 2010). Another important milestone toward our basic understanding of the relationship between PVAT dysfunction and obesity-associated vascular dysfunction was the demonstration of changes in the proteomic profile of 186 proteins in PVAT of coronary arteries, which correlate with increased contractile effect and the activation of calcium (Ca2+)-dependent signaling pathways in VSMCs from Ossabaw pigs (Owen et al., 2013).

These considerations aside, the diverse aspects of PVAT influence on vascular dysfunction in obesity could be also framed by examining its high relationship with oxidative stress. ROS, such as O− 2 and H2O2, play important roles in the modulation of vascular function by the PVAT. H2O2 is a vasoactive substance that induces both contractile and relaxing responses by different mechanisms depending on its concentration, the type and contraction state of the vessel, and the animal species. H2O2-induced relaxation may be endothelium dependent as a result of increased NO release secondary to K+ channel activation. In addition, H2O2 induces endothelium-independent relaxation through the direct opening of K+ channels in VSMC, by the oxidation of its cysteine residues, as well as by the direct activation of the sGC enzyme (Ardanaz and Pagano, 2006). The contractile effect mediated by H2O2 occurs due to the direct activation of the enzyme COX and an increase of intracellular Ca2+. Evidence indicates that ROS accumulation activates contraction pathways related to MAPKs, with increased ERK 1/2 phosphorylation (Peters et al., 2000). In addition, H2O2 activates the Rho kinase pathway, favoring vascular contraction (Ardanaz and Pagano, 2006). In fact, the Rho kinase pathway is important not only in the contraction of VSMC, but also in increased proliferation and cell migration, which has attracted much attention in relation to its role in the pathogenesis of CVDs (Loirand et al., 2006). Considering that O− 2 induces vascular contraction while H2O2 has dual effects, the final result of the

action of these ROS will depend on the relative balance between the production and the release of these factors by the PVAT. Ketonen et al. demonstrated in C57BL/6J mice fed a HFD that the reduction in endothelium-dependent relaxation occurs due to oxidative stress in PVAT, characterized by an increase in O− 2 and H2O2 production (Ketonen et al., 2010).

The NAD(P)H oxidase, which is the major source of O− 2 in the vasculature, is expressed in PVAT of rat mesenteric arteries and contributes to increased contractile response to perivascular nerve stimulation (Gao et al., 2006). Indeed, the expression of p67phox subunit of the NAD(P)H oxidase complex is increased in periaortic adipose tissue from short-term fed HFD obese mice (60% cal from fat) and this is accompanied by enhancement of both O− 2 and H2O2 levels (Ketonen et al., 2010). Increased NAD(P)H oxidase activity and O− 2 production associated with decreased expression of total SOD activity and extracellular superoxide dismutase (ecSOD) were described in the PVAT of these animals. The changes were accompanied by a reduction in eNOS expression and NO production in the PVAT (Gil-Ortega et al., 2014). Similarly, in NZO mice, increased O− 2 formation and reduced SOD expression in PVAT contribute to vascular dysfunction by reducing the anti-contractile effect of this tissue (Marchesi et al., 2009). Gil-Ortega et al. reported that longterm HFD induces substantial reduction in ec-SOD expression and total SOD activity, an increase of NOX activity and O− 2 release from the mesenteric PVAT, suggesting that the imbalance between antioxidant and pro-oxidant mechanisms in PVAT might contribute to vascular oxidative stress, thus aggravating endothelial dysfunction (Gil-Ortega et al., 2014). Marchesi et al. have also shown that the loss of PVAT anti-contractile properties in NZO mice might be associated with increase in both O− 2 and NAD(P)H oxidase activity (Marchesi et al., 2009). Uncoupling of eNOS in PVAT, which contributes not only to increase ROS formation but also to decrease NO bioavailability, was also recently described as a novel mechanism underlying the vascular dysfunction in diet-induced obesity (Xia et al., 2016).

The mitochondrial electron transport chain (mETC), a significant source of ROS, has also been pointed as an integral

Costa et al. PVAT and Cardiovascular Risk in Obesity

component implicated in the physiological regulation of vascular function by PVAT. We recently showed that O− 2 production from the mETC is increased in PVAT during norepinephrine (NE)-induced aortic muscle contraction. O− 2 is subsequently dismutated to H2O2 by manganese SOD (Mn-SOD), which, in turn, modulates VSMC contraction (Costa et al., 2016). Following these findings, we next provided unequivocal evidence linking mitochondria to PVAT-associated oxidative stress and the subsequent loss of the PVAT anti-contractile effects observed in experimental obesity (da Costa et al., 2017). Under these conditions, the increase in PVAT-mediated ROS generation becomes an important sign of increased vascular contraction. As PVAT has a similar phenotype to BAT, including the expression of UCP-1, which is necessary for non-shivering thermogenesis, as discussed above, and considering that local energy metabolism induced by changes in temperature affects vascular function and atherogenesis (Brown et al., 2014), it can be proposed that increased energy production in PVAT under obesity condition also affects vessel biology favoring the development of CVD.

Structural and functional modifications of PVAT in obesity can also induce vascular remodeling. Periaortic adipocytes significantly increase the growth rate of aortic smooth muscle cells from aged and obese Wistar Kyoto rats supplemented with HFD. This effect is abolished in the presence of proteinase K, but is maintained in the presence of filtered proteins with a molecular mass less than 100 kDa, indicating that PVAT releases soluble proteins that stimulate growth of VSMCs, which can further aggravate aging- and obesity-associated vascular diseases (Barandier et al., 2005). Finally, the paracrine actions of factors produced and released by PVAT can also change vascular insulin sensitivity (Meijer et al., 2013). Although the prospective association between PVAT dysfunction and diabetes has not been demonstrated so far, this possibility has been previously postulated by Yudkin et al. and remarkably illustrated by several subsequent studies (Yudkin et al., 2005). Meijer's group showed that local depots of PVAT, which surround resistance arteries in the muscle microcirculation, controls vascular responses to insulin through adiponectin secretion and subsequent activation of AMPK signaling (Meijer et al., 2013). The expression of adiponectin is significantly lower in the epicardial adipose tissue isolated from patients with CAD (Iacobellis et al., 2005). In humans, PVAT accumulation around the brachial artery was negatively correlated with insulin sensitivity and the post-ischaemic increase in blood flow. The association was independent of the presence of other cardiovascular risk factors, including age, sex, liver fat, BMI, and visceral adipose tissue (Rittig et al., 2008). Additionally, PVAT from obese mice inhibits insulin-induced vasodilatation, which can be restored by inhibition of the inflammatory kinase Jun NH (2)-terminal kinase (JNK) (Meijer et al., 2013). JNK may directly induce insulin resistance by a mechanism that involves phosphorylation of the insulin receptor substrate (IRS) 1, blocking the transduction signal produced by the insulin receptor (Sabio et al., 2008). Studies in humans and animal models have demonstrated an important upstream role of this protein in integrating inflammatory and metabolic function that were recently reviewed elsewhere (Hotamisligil, 2017).

These findings, in addition to the observations that local accumulation of adipose tissue is consistently related to decreased flow-mediated vasodilation (Albu et al., 2005), fasting insulin levels and insulin resistance in humans (Rittig et al., 2008), support the involvement PVAT-derived factors as paracrine, rather than endocrine mediators of both microvascular dysfunction and insulin resistance in obesity. There is also tremendous redundancy in these pathways that support the view that the association between PVAT and cardiometabolic risk factors might not simply be the consequence of overall adiposity, but potentially constitutes an additional risk factor. Considering that hyperglycemia and hyperinsulinemia can also directly impair vascular function and consequently glucose disposal, it is also possible to suggest a vicious cycle of PVAT dysfunction that contributes to and is exacerbated by the impairment in insulin homeostasis.

## INFLAMMATION IN PERIVASCULAR ADIPOSE TISSUE: THE LINK BETWEEN OBESITY AND CARDIOVASCULAR DISEASE?

In the last decades obesity has been associated with a moderate degree of inflammation in adipose tissue, a condition that results from chronic activation of the innate immune system. This type of inflammation, called metaflammation or metabolic inflammation is free of pathogens and orchestrated by metabolic cells in response to excess nutrients and energy (Hotamisligil, 2017). Initial support for this understanding came from the following observations: macrophages-derived TNF-α induces insulin resistance in adipocytes (Pekala et al., 1983); adipose tissue from obese individuals displays increased expression of inflammatory mediators (Hotamisligil et al., 1993; Uysal et al., 1997); and inflammation promotes disturbances in glucose metabolism. It is relevant to emphasize here the first demonstrations that macrophage infiltration occurs into the adipose tissue in obesity (Weisberg et al., 2003; Xu et al., 2003). These findings were the basis for elucidating the involvement of adipose tissue macrophages as direct modulators of metabolism, and led to the observations that the increased free fatty acids exposure in obesity promotes the polarization of macrophages resident in the adipose tissue toward a proinflammatory (M1-polarized) phenotype which can activate inflammatory pathways and impair insulin signaling (Hevener et al., 2007; Lumeng et al., 2007; Nguyen et al., 2007). The molecular mechanisms underlying these events include epigenomic alterations that determine macrophage sensitivity to metabolically driven inflammatory (metaflammatory) signals (Fan et al., 2016), and additional macrophage-secreted products, including the potent and pleiotropic immune mediator TNFα, which attenuates insulin signaling in adipocytes (Li et al., 2016). Many other immune cells including dendritic cells, mast cells, eosinophils, and lymphoid cells also contribute to metabolic tissue homeostasis and to the control of glucose metabolism. Regarding the initiation of metaflammation, an emerging concept is that adipocyte hypertrophy causes local hypoxia, which, in turn, causes infiltration of macrophages and other immune cells (CD4+ and CD8+ T cells, natural killer T cells, and mast cells) in visceral adipose tissue (Roemeling-van Rhijn et al., 2013). This increase in cells of the immune system along with the proinflammatory cytokines in adipocytes may negatively regulate PPARγ activity, which is essential for both adipogenesis and maintenance of the tissue gene expression (Guri et al., 2008).

Intermittent inflammatory processes have also been observed in PVAT. These include increased migration of immune cells, altered production of pro- and anti-inflammatory cytokines, adipokines, and lipid mediators, as well as signaling through a plethora of immune receptors and intracellular signaling molecules. Importantly, this entire cascade and mediators have now provided highly promising evidence that PVAT inflammation plays a key role at various stages of CVD. One powerful example of this is the recent work illuminating a role for perivascular inflammation as an alteration that precedes atherosclerotic plaque formation and even the development of endothelial dysfunction and oxidative stress in ApoE−/− mice (Skiba et al., 2017). Specifically, the authors found increased leukocyte infiltration in the perivascular tissue when compared to the vessel wall. Furthermore, it was demonstrated that PVAT or adventitial inflammation and infiltration with macrophages and T cells precede not only significant atherosclerotic plaque development, but also the impairment of endotheliumdependent NO bioavailability. In support of this finding, it was previously shown that major risk factors for atherosclerosis, including hypertension, hyperlipidemia or ty 2 diabetes promote perivascular inflammation before and during development of atherosclerosis (Galkina et al., 2006; Galkina and Ley, 2009; Sagan et al., 2012; Guzik et al., 2013).

Molecular mechanisms linking PVAT inflammation in obesity to CVD indicate several key components. These may include chemotactic migration of PVAT immune cells into adventitia, with release of cytokines, which can alter vascular function (Mikolajczyk et al., 2016). In this regard, it was previously demonstrated that human PVAT exhibits a strong chemotactic activity on monocytes, granulocytes, and T lymphocytes that is mainly mediated by the secretion of IL-8 and MCP-1, which are known proatherogenic chemokines and their production is increased in obesity. These mediators are likely to contribute to the infiltration of leukocytes at the interface between PVAT and adventitia of atherosclerotic aortas (Henrichot et al., 2005). Visceral adipose tissue accumulation also promotes an increase in the secretion of angiopoietin-like protein 2 (Angptl2), a proinflammatory factor derived from adipocytes and considered a key mediator of chronic adipose tissue inflammation and obesity-related systemic insulin resistance (Tabata et al., 2009). In fact, Angptl2 secreted by PVAT accelerates neointimal formation after endovascular injury in mice by increasing inflammationrelated gene expression and by accelerating extracellular matrix degradation (Tian et al., 2013). In line with these experimental findings, the authors provided relevant clinical data that strongly suggest that PVAT-secreted Angptl2 plays a significant role in accelerating vascular inflammation by cooperating with proinflammatory TNF-α or counteracting the anti-inflammatory activity of adiponectin, potentially leading to development of atherosclerosis in humans (Tian et al., 2013).

PVAT inflammation may also be associated with altered release of adipokines and other adipocyte-derived relaxing factors (Antonopoulos et al., 2015; Woodward et al., 2017). Initial support for this understanding came from the findings that perivascular adipocytes from human and mice without atherosclerotic disease exhibit a heightened proinflammatory state and reduced adipocytic differentiation under basal conditions as compared with adipocytes derived from subcutaneous and visceral adipose depots, and that highfat feeding causes further reductions in adipocyte-associated gene expression while upregulates proinflammatory gene expression (Chatterjee et al., 2009). Specifically, secretion of the anti-inflammatory adipokine adiponectin is markedly reduced, whereas that of proinflammatory cytokines IL-6, IL-8, and MCP-1, is markedly increased in perivascular adipocytes. These changes have also direct effects on the PVAT vasoactive properties, as evidenced in animal and human small-artery studies, where hypoxia and inflammation were shown to attenuate the local vasoactive properties of PVAT by oxidative stress (Greenstein et al., 2009).

In addition to the previously-mentioned adipokines, chemerin, a secreted protein originally revealed as a chemoattractant molecule for immature dendritic cells and macrophages (Wittamer et al., 2003) is now considered a novel adipokine that regulates adipogenesis, adipocyte metabolism (Bozaoglu et al., 2007; Goralski et al., 2007) and inflammation (Parolini et al., 2007; Cash et al., 2010). Chemerin acts through CMKLR1 (chemokine-like receptor 1) or ChemR23 (chemerin receptor 23), which is expressed in macrophages, dendritic cells, adipocytes and vascular cells (Zabel et al., 2005, 2006; Parolini et al., 2007). Chemerin is considered a biomarker for adiposity since its plasma levels strongly associate with BMI and is linked to obesity and metabolic syndrome, a cluster of metabolic disorders that increase the risk for diabetes and CVD (Bozaoglu et al., 2009; Li et al., 2014). Furthermore, serum chemerin levels are significantly elevated in morbidly obese patients and reduced with the weight loss after bariatric surgery. The strong decrease of chemerin after surgery was associated with an improvement in insulin sensitivity and blood glucose, which further support the key role of this adipokine in mediating metabolic alterations in obesity (Ress et al., 2010; Sell et al., 2010). Animal studies reported the parallel findings that knockout mice for the primary receptor for chemerin, ChemR23, exhibit reduced food consumption, weight gain, and adiposity (Ernst et al., 2012). Consistent with this phenotype, ChemR23 knockout mice also displayed lower fasting blood glucose and serum insulin levels.

Recent observations have also shown that chemerin is highly expressed in PVAT. Spiroglou et al. first detected chemerin expression in epicardial and periaortic adipose tissue and demonstrated a positive correlation of this adipokine with both aortic and coronary atherosclerosis (Spiroglou et al., 2010). Most recently, Watts and her co-workers reported that chemerin produced by periaortic PVAT stimulates vascular contraction through the receptor typically attributed to function only in immune cells. Moreover, arteries from obese or hypertensive mice or obese humans with dysfunctional endothelium demonstrated amplified contractions in response to chemerin (Watts et al., 2013). Although there is no empirical evidence linking specific chemerin signaling pathways with the PVAT inflammation, a bulk of animal and human studies exploring the immune functions of chemerin make this a very plausible possibility. CMKLR1 is expressed in several immune cell types known to accumulate in obese adipose tissue (Wittamer et al., 2003; Parolini et al., 2007; Cash et al., 2010). Sell and collaborators demonstrated that chemerin activates the NF-κB pathway and impairs glucose uptake in skeletal muscle cells (Sell et al., 2010). In human endothelial cells, it was shown a significant upregulation of chemerin receptor by pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 (Kaur et al., 2010). Accordingly, circulating levels of chemerin are elevated in diseases associated with chronic inflammation, including obesity and the metabolic syndrome (Wittamer et al., 2003; Bozaoglu et al., 2009; Li et al., 2014). Our recent studies also point to a role of chemerin in regulating inflammatory processes. Chemerin was shown to decrease NO-dependent cGMP signaling, thereby reducing vascular relaxation in rat aorta, an effect related to increased generation of O− 2 , an important mediator of inflammation (Neves et al., 2014). In line with these findings, we demonstrated that chemerin, through Nox activation and redox-sensitive MAPKs signaling, exerts proapoptotic, proinflammatory, and proliferative effects in human vascular cells (Neves et al., 2015).

Taken together, these findings not only provide multiple layers of potential mediators for the inflammatory component of PVAT in obesity but also clearly demonstrate that bidirectional interactions between that the systemic metabolic inflammation and local immune components are critical considerations in determining the physiological and pathological vascular outcomes associated with obesity. The proximity of PVAT as a rich source of proinflammatory cytokines and other mediators together with the associated alterations in this tissue support the concept that specific changes of local adipose tissue depots contribute to disease processes in the neighboring vessel wall.

#### CLINICAL MEASURE OF PERIVASCULAR ADIPOSE TISSUE IN OBESITY-ASSOCIATED CARDIOVASCULAR RISK ASSESSMENT: FINDING THE POINT

Some of the initial thoughts and considerations of the reciprocal relationship between PVAT and risk factors for CVD have been provided by clinical findings showing an association between both perivascular and epicardial adipose tissue with the main anthropometric and clinical parameters of the metabolic syndrome (Iacobellis et al., 2003b). In fact, a very good correlation is observed between epicardial adipose tissue, assessed by echocardiography, and waist circumference, diastolic blood pressure, fasting plasma insulin, LDL cholesterol, and plasma adiponectin (Iacobellis et al., 2003b). Importantly, the association of insulin sensitivity and low adiponectin levels with the epicardial fat thickness is independent of BMI, suggesting that PVAT assessment might provide a more sensitive and more specific measure of the true visceral fat content (Iacobellis et al., 2003a). In support of these findings, a recent metaanalysis of published reports concluded that echocardiographic epicardial adipose thickness is significantly higher in patients with metabolic syndrome than in those without it (Pierdomenico et al., 2013).

Echocardiographic evaluation of epicardial adipose tissue thickness is considered a very reliable method to measure visceral adiposity, as first proposed and validated by Iacobellis et al. (2005). It is also relatively cheap and easy to perform as a screening test for assessment of patients suspected to be at risk for cardiovascular or metabolic outcomes. However, the technic provides only measurements of the regional thickness of epicardial adipose tissue and presents the risk to confuse pericardial fluid with adipose tissue since the fluid also shows up as relatively echo-free on an echocardiograph (Singh et al., 2007). The advances in imaging technology have enabled a more direct quantitative assessment of the local fat depots and have also been the basis for investigations into the possibility that PVAT acts as direct modulator of metabolism and cardiovascular function. From this perspective, clinical findings showed that volumetric quantification of epicardial and peri-coronary adipose tissue thickness by computed tomography, which provides a more accurate assessment due to its higher spatial resolution, is also positively related with parameters of obesity, such as BMI, waist circumference and the abdominal visceral adipose tissue mass (Gorter et al., 2008). In line with this, PVAT thickness, measured by computed tomography in the areas of right coronary artery, left anterior descending artery and left circumflex coronary artery is positively associated with waist circumference, waist-tohip ratio, BMI, blood glucose, triglycerides, and systolic blood pressure. Epicardial adipose tissue is also linked to coronary calcification, a known marker of coronary atherosclerosis (de Vos et al., 2008).

In agreement with the above observations, abdominal periaortic adipose tissue and thoracic periaortic adipose tissue volume, measured by computed tomography, are consistently correlated with visceral abdominal fat, subcutaneous abdominal fat, waist circumference, and BMI in a random subset of participants from the Framingham Heart Study (Schlett et al., 2009). Thanassoulis et al. extended these findings by showing that both thoracic and abdominal periaortic adipose tissue volumes, measured using the same approach, are associated with higher aortic dimensions even after adjustment for other vascular and metabolic risk factors including global measures of obesity such as BMI (Thanassoulis et al., 2012). By contrast, the study of Rosito et al. found no significant associations between pericardial fat and CVD risk factors compared with visceral fat when both were considered in the same model. Nevertheless, the authors evidenced strong correlations between pericardial fat volume and metabolic risk factors even after adjusting for many potential confounders, including age, smoking, alcohol use, and physical activity. Furthermore, pericardial fat was found to be associated with coronary artery Ca2+ and abdominal aortic Ca2+ even after metabolic risk factors and visceral adipose tissue were accounted for, which is consistent with the postulation that pericardial fat may in fact exert a harmful perivascular effect on the coronary arteries (Rosito et al., 2008).

#### SUMMARY

There is tremendous wealth in the mechanisms that support the understanding of the relationship between excess adiposity and cardiovascular risk. The strongest evidence comes from the observations of basic science and translational studies regarding important physiological process that occur in adipose tissue and deleterious effects of relevant importance on vascular complications. It is interesting to consider the role that adipokines play among the mechanisms whereby adipocytes influence vascular function. However, there is considerably more work to be performed in both basic science and clinical areas to understand and reduce the enhanced CVD risk that is evident in the obese state. Clearly, these intriguing observations are not sufficient to answer the question whether increased PVAT mass represents a surrogate marker of cardiovascular risk or an independent pathogenic variable. However, outcome studies need to establish whether PVAT depots have prognostic significance and may therefore provide additional evidence for a causal relationship. Although the reduction of whole-body fat would be ideal among individuals who have obesity, a thoughtful consideration of the additional benefits of targeting fat depots at

# REFERENCES

specific locations in individuals at greater cardiometabolic risk represents an alternative approach.

Further dissection of the vascular signaling pathways altered by PVAT-derived factors will likely reveal functional strategies for suppressing the negative effects of abnormal adipose tissue excess on CVD complications without altering the beneficial effects of normal fat depots. The challenges remaining in this field can be identified in two different areas. How does structural and functional changes of PVAT start in obesity and at what point does it become detrimental? What are the appropriate model systems and paths to elucidate the unknown mechanisms to prevent or treat human CVD? Regarding the contribution of new characterized adipokines, there are also several interesting and emerging concepts. Given these challenges, and the need for therapeutic approaches that do not permanently interfere with entire physiological effects of the PVAT, conveying the experimental insights into successful clinical interventions will require collaborative and diverse cross studies, new experimental approaches and integration of genetic variations with other environmental modifiers to establish links with complex cardiovascular phenotypes and facilitate successful translation to human disease.

# AUTHOR CONTRIBUTIONS

RC, KN, RT, and NL equally contributed to the conception of paper, drafting the manuscript, and approved its final version.

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stress induced by obesity and type 2 diabetes. Nat. Med. 22, 780–791. doi: 10.1038/nm.4114

PKC-beta-dependent phosphorylation of nitric oxide synthase. Am. J. Physiol.  Circulation 117, 1658–1667. doi: 10.1161/CIRCULATIONAHA.107.739714

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Costa, Neves, Tostes and Lobato. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Protective Role of Perivascular Adipose Tissue in Endothelial Dysfunction and Insulin-Induced Vasodilatation of Hypercholesterolemic LDL Receptor-Deficient Mice

#### Natali Baltieri, Daniele M. Guizoni, Jamaira A. Victorio and Ana P. Davel\*

*Department of Structural and Functional Biology, Institute of Biology, University of Campinas, Campinas, Brazil*

#### Edited by:

*Stephanie W. Watts, Michigan State University, United States*

#### Reviewed by:

*A. Elizabeth Linder, Universidade Federal de Santa Catarina, Brazil Philip Aaronson, King's College London, United Kingdom*

> \*Correspondence: *Ana P. Davel<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Vascular Physiology, a section of the journal Frontiers in Physiology*

Received: *07 December 2017* Accepted: *01 March 2018* Published: *19 March 2018*

#### Citation:

*Baltieri N, Guizoni DM, Victorio JA and Davel AP (2018) Protective Role of Perivascular Adipose Tissue in Endothelial Dysfunction and Insulin-Induced Vasodilatation of Hypercholesterolemic LDL Receptor-Deficient Mice. Front. Physiol. 9:229. doi: 10.3389/fphys.2018.00229* Background: Endothelial dysfunction plays a pivotal role in the initiation of atherosclerosis. Vascular insulin resistance might contribute to a reduction in endothelial nitric oxide (NO) production, leading to impaired endothelium-dependent relaxation in cardiometabolic diseases. Because perivascular adipose tissue (PVAT) controls endothelial function and NO bioavailability, we hypothesized a role for this fat deposit in the vascular complications associated with the initial stages of atherosclerosis. Therefore, we investigated the potential involvement of PVAT in the early endothelial dysfunction in hypercholesterolemic LDL receptor knockout mice (LDLr-KO).

Methods: Thoracic aortas with and without PVAT were isolated from 4-month-old C57BL/6J (WT) and LDLr-KO mice. The contribution of PVAT to relaxation responses to acetylcholine, insulin, and sodium nitroprusside was investigated. Western blotting was used to examine endothelial NO synthase (eNOS) and adiponectin expression, as well the insulin signaling pathway in aortic PVAT.

Results: PVAT-free aortas of LDLr-KO mice exhibited impaired acetylcholine- and insulin-induced relaxation compared with those of WT mice. Both vasodilatory responses were restored by the presence of PVAT in LDLr-KO mice, associated with enhanced acetylcholine-induced NO levels. PVAT did not change vasodilatory responses to acetylcholine and insulin in WT mice, while vascular relaxation evoked by the NO donor sodium nitroprusside was not modified by either genotype or PVAT. The expression of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), AKT, ERK1/2, phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204), and adiponectin was similar in the PVAT of WT and LDLr-KO mice, suggesting no changes in PVAT insulin signaling. However, eNOS expression was enhanced in the PVAT of LDLr-KO mice, while eNOS expression was less abundant in PVAT-free aortas.

**69**

Conclusion: These results suggest that elevated eNOS-derived NO production in aortic PVAT might be a compensatory mechanism for the endothelial dysfunction and impaired vasodilator action of insulin in hypercholesterolemic LDLr-deficient mice. This protective effect may limit the progression of atherosclerosis in genetic hypercholesterolemia in the absence of an atherogenic diet.

Keywords: perivascular adipose tissue, endothelium, LDL receptor deficiency, hypercholesterolemia, nitric oxide, insulin, adiponectin

#### INTRODUCTION

Hypercholesterolemia is the main risk factor for the development of atherosclerosis by enhancing low-density lipoprotein (LDL) retention within the vessel wall (Stapleton et al., 2010). Atherosclerosis is a progressive disease in which inflammation, fat deposits, and cells and extracellular matrix accumulation in the artery result in the occlusion of the vessel lumen, underlying a major cause of clinical cardiovascular events worldwide (World Health Organization, 2014; Mozaffarian et al., 2016). One of the initial steps of the atherosclerotic process is the development of endothelial dysfunction, which precedes atherosclerosis development in humans (Guzik et al., 2000; Heitzer et al., 2001).

LDL receptor knockout mice (LDLr-KO) is a model of human familial hypercholesterolemia (FH). Although young adult LDLr-KO mice fed with standard low-fat diet only develop small spontaneous atherosclerotic lesions in the aortic root (Dorighello et al., 2016, 2017), these animals present impaired endothelium-dependent relaxation in the aorta (Rabelo et al., 2003; Langbein et al., 2015; Guizoni et al., 2016). This finding is consistent with reduced endothelium-dependent vasodilation in human hypercholesterolemia (Creager et al., 1990, 1992). In this model of genetic hypercholesterolemia, endothelial dysfunction is associated with the reduced gene and protein expression of endothelial nitric oxide synthase (eNOS) (Guizoni et al., 2016; Langbein et al., 2016), and reduced dimerization of this enzyme, resulting in impaired endothelial nitric oxide (NO) release (Guizoni et al., 2016).

Nitric oxide (NO) is an endothelium-derived vasodilator factor with antiatherogenic properties, including the inhibition of platelets, and monocyte adhesion to the endothelium, platelet aggregation, smooth muscle proliferation, and oxidation of LDL (Vanhoutte, 2003). Consistently, the administration of Larginine, as eNOS substrate and NO precursor, improves the endothelial function in hypercholesterolemic patients (Creager et al., 1992; Siasos et al., 2007), whereas the lack of NO accelerates the progression of atherosclerosis (Kauser et al., 2000). Genetic hypercholesterolemia is also associated with the impaired secretion of insulin (Souza et al., 2013), a hormone stimulating NO production (Potenza et al., 2009). In addition to endothelium-derived NO, NO release from perivascular adipose tissue (PVAT) has recently been demonstrated as an important adipocyte-derived relaxation factor (ADRF) in the aorta (Victorio et al., 2016; Xia et al., 2016), but whether or not changes in PVAT eNOS function are involved in hypercholesterolemia-induced vascular dysfunction remains unknown.

Perivascular adipose tissue (PVAT) surrounds most blood vessels and releases numerous factors and adipokines with paracrine effects on both vascular structure and function (Akoumianakis et al., 2017). Higher levels of proinflammatory adipocytokines and lower levels of adiponectin were found within the PVAT after artery injury (Takaoka et al., 2010), and an inflamed PVAT results in neointima formation (Moe et al., 2013). In addition, increased amounts of angiogenic factors release by PVAT may have a pathological relevance for atherosclerosis development (Chang et al., 2013). However, the thermogenic properties of PVAT have been demonstrated as anti-atherogenic (Chang et al., 2012; Brown et al., 2014). Nevertheless, the role of PVAT in the development and progression of this vascular disease is still unclear. Because PVAT regulates endothelial function and NO bioavailability, we hypothesized a role for this fat deposit in the vascular complications associated with the initial stages of atherosclerosis. Therefore, the present study was designed to evaluate the potential influence of PVAT in the early endothelial dysfunction of hypercholesterolemic LDLr-KO mice.

### MATERIALS AND METHODS

#### Animals

LDL receptor knockout (LDLr-KO) mice and respective C57BL6/J wild-type (WT) mice were purchased from the Jackson Laboratory, and the strains are maintained by breeding at the Multidisciplinary Center for Biological Research (CEMIB-UNICAMP, Campinas, SP, Brazil) with genotypic control. The mice were housed at 22 ± 1 ◦C on a 12:12 h light:dark cycle with free access to a standard rodent chow diet (Nuvital CR1, Colombo, Paraná, Brazil) and water. At 4 months old, the mice were weighted and anesthetized with urethane (5 g/kg body weight, i.p.) to collect blood samples and isolate thoracic aorta.

All experimental protocols were approved (protocol # 3639- 1) by the Ethics Committee on Animal Use of the University of Campinas (CEUA-UNICAMP, Campinas-SP, Brazil) and carried out in accordance with the ethical principles for animal experimentation adopted by the Brazilian Society of Laboratory Animal Science (SBCAL/COBEA).

#### Plasma Biochemical Analysis

Blood samples were obtained from the tail vein for blood glucose measurement (Accu-Chek Advantage, Roche Diagnostics, Sao Paulo, Brazil). Subsequently, the mice were anesthetized (5 g/kg of urethane, i.p.), and blood samples were collected by cardiac puncture and centrifuged (8,000 g for 15 min at 4◦C); the serum supernatants were subsequently collected for biochemical analysis. Total cholesterol and triglyceride levels were measured using standard commercial kits (Chod-Pap, Roche Diagnostic GmbH, Mannheim, Germany).

## Vascular Reactivity in the Presence or Absence of PVAT

The thoracic aorta was isolated and cut into cylindrical segments (∼2 mm in length) with or without surrounding PVAT. The segments were mounted in a tissue chamber bath (Panlab Harvard Apparatus, Cornellà-Barcelona, Spain) containing Krebs–Henseleit solution (in mM: 118 NaCl, 4.7 KCl, 25 NaHCO3, 2.5 CaCl2-2H2O, 1.2 KH2PO4, 1.2 MgSO4- 7H2O,11 glucose and 0.01 EDTA; pH = 7.4, 37◦C) with a resting tension of 0.5 g stabilized for 1 h, as previously demonstrated (Davel et al., 2012; Guizoni et al., 2016). Subsequently, aortic rings were exposed to 125 mM of KCl to test vascular integrity and assess maximal contraction, and no differences were found among groups (data not shown). Following washing, the aortic rings were contracted with a submaximal concentration of the thromboxane A2 receptor agonist (U-46619, 70% of maximal contraction to 125 mM of KCl) and relaxation curves to acetylcholine (0.1 nmol/L to 10µmol/L, Sigma-Aldrich, Saint Louis, MO, USA), insulin (0.1 to 10 nmol/L, Humulin R R rDNA origin, Lilly USA, Indianapolis, USA), or the NO-donor sodium nitroprusside (1 pmol/L to 0.1µmol/L Sigma-Aldrich, Saint Louis, MO, USA) were performed.

## PVAT Homogenization and Western Blotting

Thoracic aorta and respective PVAT were separately pulverized in N2 liquid and homogenized in cold RIPA lysis buffer (Merck Millipore, Billerica, MA, USA) containing PMSF (1 mM), Na3VO4 (1 mM) and 2µL/mL of protease inhibitor cocktail (PIC, Sigma-Aldrich) to obtain total protein extract (Victorio et al., 2016). The proteins were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc.) to determine the protein concentration values of the samples (PVAT: WT = 4.49 ± 2.94 and LDLr-KO = 5.74 ± 1.98 mg/ml; aorta: WT = 6.13 ± 3.09 and LDLr-KO = 7.46 ± 1.98 mg/ml). Then, 40 µg of PVAT or aorta extracts and the 1 µL of plasma were electrophoretically separated on 7.5 or 12% acrylamide SDS–PAGE and then transferred to PVDF membranes using a Mini Trans-Blot Cell system. Non-specific binding sites were blocked with 5% non-fat milk solution, and then the membranes were incubated for 12 h at 4◦C with the following primary antibodies: anti-insulin receptor substrate-1 (IRS-1), anti-p85 subunit of phosphatidylinositol 3 kinase (PI3K), anti-Akt1/2/3, anti-phospho (Ser473)-Akt1/2/3 (1:1,000; Santa Cruz Biotechnology), anti-eNOS (1:1,000; BD Transduction, Franklin Lakes, NJ, USA), anti-ERK1/2, anti-phospho (Thr202/Tyr204)- ERK1/2 (1:1,000; Cell Signaling, Danvers, MA, USA), and antiadiponectin (1.5µg/mL, Novus Biologicals, Littleton, CO, USA). Additionally, α-tubulin (1:1,000; Santa Cruz Biotechnology) was used as an internal control protein for PVAT, αactin (1:1,000; Abcam, Cambridge, MA, USA) was used as an internal control protein for aorta, and Ponceau S staining for adiponectin expression in plasma and PVAT. After washing (10 mM of Tris, 100 mM of NaCl and 0.1% Tween-20), the membranes were incubated for 90 min with the specific secondary antibody conjugated to peroxidase. Immunocomplexes were detected using a luminol peroxidase chemiluminescence kit (ECL Plus, Amersham) and visualized using photographic film (Hyperfilm ECL, Amersham) in a dark room. The intensity of the immunoblots was quantified using ImageJ 1.46p software (National Institutes of Health, Bethesda, MD, USA).

# PVAT NO Release

NO in PVAT was detected with 4,5-diaminofluorescein diacetate (DAF-2 DA) (Xia et al., 2016). Aortic PVAT cryostat sections (20µm) were loaded with DAF-2 DA (8µM) in the presence of acetylcholine (10µM) at 37◦C for 30 min. Then, fluorescence imaging was performed with a microscope (Eclipse Ti-S, Nikon, Tokyo, Japan) equipped with a fluorescence filter. DAF-2 DA was excited by an interference filter at 465–495 nm, and fluorescence emitted between 515 and 555 nm was collected (Zhou and He, 2011). The images captured under a 10X objective were analyzed using ImageJ 1.46p software (National Institutes of Health), the mean optical density of the fluorescence was measured, and the results were normalized by the PVAT area (µm2 ). Fluorescence intensity was quantified by the subtraction of acetylcholinetreated samples from basal conditions.

# Statistical Analysis

Data were expressed as the means ± SEM. Two-way ANOVA was used to analyze the vasorelaxation response curves. When ANOVA showed a significant effect, Bonferroni's posthoc test was used to compare individual means. Unpaired Student's t-test was used for two-group comparisons. For each concentration-response curve, Rmax and the negative logarithm of the concentration of the agonist that produced half of Rmax (−LogEC50) were calculated using non-linear regression analysis. GraphPad Prim Software 5.0 (San Diego, CA, EUA) was used for statistical analysis and −LogEC50 and Rmax calculation. P < 0.05 values were considered significant.

TABLE 1 | Body weight and total serum cholesterol, triglycerides, and glycemia in wild-type (WT) and LDLr knockout mice (LDLr-KO).

*Data are expressed as mean* ± *SEM (N* = *10–14). Unpaired Student's t-test:* \**P* < *0.05 vs. WT.*

vs. WT/PVAT–; + vs. LDLr-KO/PVAT+.

### RESULTS

As expected, LDLr-KO mice exhibited dyslipidemia characterized by elevated plasma levels of total cholesterol and triglycerides (Bonfleur et al., 2011), with no changes in blood glucose or body weight (**Table 1**).

The vascular reactivity study revealed an impaired relaxation to acetylcholine and insulin in aorta of LDLr-KO mice in the absence of PVAT (**Figures 1A,B**), with a reduced Rmax to both agonists (**Table 2**). These data suggest an impaired endothelial vasodilatory response and vascular insulin resistance. However, the presence of PVAT prevented the impaired vasodilatory response to both acetylcholine and insulin in LDLr-KO associated with increased Rmax while not affecting vasorelaxation in WT (**Figures 1A,B**; **Table 2**). Changes in acetylcholine- and insulin-induced relaxation were not the result of a smooth muscle defect, as the relaxation response to the NO donor sodium nitroprusside was similar between WT and LDLr-KO mice, with or without PVAT (**Figure 1C**, **Table 2**).

Considering that NO is the main endothelium-derived vasodilator factor mediating acetylcholine- and insulin-induced relaxation in the murine aorta (Wu et al., 1994), we investigated vascular and PVAT eNOS expression. Interestingly, while eNOS expression was reduced in aortic tissue from LDLr-KO mice (**Figure 2A**), aortic PVAT showed more abundant eNOS expression (**Figure 2B**), associated with enhanced PVAT NO levels (**Figure 2C**). These data suggest increased eNOS-derived NO production as a mechanism involved in the protective effect of aortic PVAT in dyslipidemic LDLr-KO mice.

TABLE 2 | Maximal relaxation response (Rmax) and potency for acetylcholine, insulin, and sodium nitroprusside in aortas with (+) or without (–) perivascular adipose tissue (PVAT) from wild-type (WT) and LDLr knockout mice (LDLr-KO).

*Data are expressed as mean* ± *SEM (N* = *4–9). 2-way ANOVA followed by Bonferroni's post-hoc test, P* < *0.05.* \* *vs. WT/PVAT-;* + *vs. LDLr-KO/PVAT*−*.*

To investigate insulin signaling in PVAT, we evaluated the protein expression of IRS-1, PI3K, and total and phosphorylated Akt and ERK. However, as shown in **Figure 3**, no differences between groups were observed.

Finally, because adiponectin is an important adipokine that stimulates NO production and facilitates endotheliumdependent relaxation (Margaritis et al., 2013), we examined PVAT and circulating adiponectin expression (**Figures 4A,B**). No significant changes were found between WT and LDLr-KO mice.

\**P* < 0.05 vs. WT.

# DISCUSSION

The results from the present study showed that in LDLr-KO mice, the presence of PVAT protected against impaired endotheliumdependent relaxation to acetylcholine and insulin, in association with enhanced eNOS protein expression and NO levels in thoracic aortic PVAT. Since this genetic model of FH exhibited only small aortic-root lesions in mice maintained on a standard low-fat diet (Dorighello et al., 2016), these data reveal a protective role for PVAT in an early phase of vascular injury induced by hypercholesterolemia.

We (Guizoni et al., 2016) and others (Rabelo et al., 2003; Langbein et al., 2015) have demonstrated a slight but significant reduction in acetylcholine-induced relaxation in LDLr-KO mice, as evidenced by a reduced maximal response to this agonist (Hofmann et al., 2017). Similarly, we found a reduced maximal relaxation to acetylcholine in aorta without PVAT from LDLr-KO fed a standard diet, indicating endothelial dysfunction in this genetic model of FH. This endothelial dysfunction became more evident in the presence of high fat/high cholesterol diets or the upregulation of lectin-like oxidized LDLr-1 (LOX-1) (Hofmann et al., 2017). However, Western-type diets also induced a range of secondary factors, such as inflammation, insulin resistance, and obesity, which synergistically interact to increase atherosclerosis. A high-fat diet per se upregulates proinflammatory gene expression in PVAT depots (Chatterjee et al., 2009). Inflamed fat surrounding the vessels resulted in enhanced atherosclerotic lesions and exacerbated endothelial dysfunction (Öhman et al., 2011). Therefore, PVAT may have pathological relevance in advanced atherosclerosis, contributing to plaque complications (Chang et al., 2013). In the present study, we investigated the role of PVAT in endothelium-dependent relaxation in the early stages of vascular injury in LDLr-KO mice fed a standard diet. Interestingly, the presence of PVAT improved relaxation responses to acetylcholine and insulin in the aortas of LDLr-KO mice, suggesting a protective role for this tissue in the present genetic model of FH by improving endothelial function. In healthy mouse aorta, the presence of PVAT did not affect the endothelium-dependent relaxation induced by acetylcholine, as previously demonstrated (Ketonen et al., 2010; Li et al., 2011).

Gil-Ortega et al. (2010) showed adaptive NO overproduction in PVAT during the initial steps of high-fat diet-induced obesity in mice. PVAT-derived NO may contribute to the anticontractile effect of PVAT independently of the endothelium (Aghamohammadzadeh et al., 2016). Since eNOS is expressed in aortic endothelium and PVAT (Victorio et al., 2016; Xia et al., 2016), we investigated whether the protective role of PVAT in endothelium-dependent relaxation responses in LDLr-KO mice was associated with changes in eNOS expression. The results demonstrated reduced eNOS expression in the vascular wall of LDLr-KO mice, as previously demonstrated, associated with reduced NO production (Guizoni et al., 2016). Lower eNOS and nNOS activation is also related to

endothelial dysfunction in apolipoprotein E-deficient mice (Capettini et al., 2011). In accordance with these data, lower NO production is observed in endothelial cells exposed to the serum of hypercholesterolemic patients (Feron et al., 1999). However, in contrast to lower endothelial eNOS expression in the vessel wall, increased eNOS expression and NO levels were observed in the aortic PVAT of LDLr-KO mice. Therefore, higher eNOS-derived NO production in surrounding fat may be the mechanism enhancing endothelium-dependent relaxation in the aortas of LDLr-KO mice.

In addition to reduced relaxation to acetylcholine, the aortas from LDLr-KO mice without PVAT exhibited impaired relaxation to insulin. Insulin is an important stimulus in endothelial cells for eNOS-derived NO production (Potenza et al., 2009). Therefore, lower eNOS expression in the vascular wall should be a mechanism involved in the impaired relaxation to this hormone in hypercholesterolemic mice. Since relaxation response to sodium nitroprusside was not different among groups, changes in smooth muscle responsiveness may not be involved in the reduced vasodilatory responses observed in LDLr-KO mice. High plasma levels of cholesterol in LDLr-KO mice impairs beta-cell pancreatic function reducing insulin secretion, even in the absence of metabolic factors induced by Westerntype diets, suggesting that genetic hypercholesterolemia increases the risk of diabetes development (Bonfleur et al., 2011; Souza et al., 2013). This finding is consistent with the slight increase in fasted plasma glucose previously observed in LDLr-KO mice (Bonfleur et al., 2011; Souza et al., 2013), although we did not find significant differences in non-fasted animals. In the present study, the reduced vasodilatory effect of insulin suggests vascular insulin resistance. Insulin enhances the expression and activity of eNOS in endothelial cells by upregulating the PI3K/Akt pathway, which induced endothelium-dependent relaxation (Montagnani et al., 2002; Potenza et al., 2009). However, hyperinsulinemia and insulin resistance favor the insulin-dependent activation of MAP-kinases (MAPK), which is associated with the elevated expression of proinflammatory and atherogenic factors (Eringa et al., 2004; Cersosimo et al., 2012; da Silva Franco et al., 2017). The presence of PVAT prevented the reduced insulininduced relaxation in aorta of LDLr-KO mice. Thus, we hypothesized that an enhanced insulin signaling in PVAT could be a mechanism opposing the vascular insulin resistance in these animals. However, no differences were found for IRS-1, PI3K, and phosphorylated and total Akt and ERK, suggesting that this signaling pathway may not be involved in the protective effect of PVAT. However, we cannot exclude changes in the insulin molecular pathway, other than those evaluated in the present study.

Adiponectin has been described as an ADRF with antiatherogenic properties (Fésüs et al., 2007; Li et al., 2015). The upregulation of adiponectin is observed in PVAT of internal mammary arteries of obese patients with coronary artery disease and might reflect a compensatory mechanism to preserve endothelial function (Cybularz et al., 2017), as previously demonstrated in obese diabetic mice (Liu et al., 2014). In addition, adiponectin improves insulin sensitivity in major insulin target tissues (Ruan and Dong, 2016) and enhances NO production through eNOS activation via PI3K/Akt phosphorylation and eNOS coupling by increasing BH4 bioavailability (Margaritis et al., 2013). Thus, we investigated the expression of adiponectin in PVAT and plasma. However, no differences were observed between WT and LDLr-KO mice, suggesting that the effect of PVAT on improving endothelial function in LDLr-KO mice is not associated with modifications of local or circulating levels of this ADRF.

Thoracic PVAT acts as a buffer against toxic levels of fatty acids in arterial circulation and clears fatty acids via inducing thermogenesis (van Dam et al., 2017). Interestingly, enhanced thermogenic activity of PVAT improves endothelial function by inducing the increased release of prostacyclin, whereas impaired PVAT thermogenesis causes atherosclerosis (Chang et al., 2012). Therefore, PVAT adaptive thermogenesis has a beneficial impact on endothelial function protecting against vascular injury. We cannot exclude that this protection exerted by PVAT on endothelial function could be lost in aging. One limitation of the present study is that we did not evaluate PVAT thermogenesis, which is a potential mechanism of endothelial protection in standard-diet fed LDLr-KO mice, as Western-type diets induced whitening and impaired thermogenesis in PVAT (Chang et al., 2013). Recently, Srikakulapu et al. (2017) demonstrated that harboring of B-1 cells by PVAT provides atheroprotection to the aorta, suggesting an additional mechanism for the beneficial role of PVAT in the vasculature.

# CONCLUSION

Taken together, the results of the present study suggest that aortic PVAT is protective for endothelial dysfunction in LDLr-KO mice, a genetic model of FH. Therefore, this adaptive mechanism in PVAT may protect endothelial function and maintain normal endothelium-dependent relaxation in the early stages of atherosclerotic disease.

# ETHICS STATEMENT

All experimental protocols were approved (protocol # 3639-1) by the Ethics Committee on Animal Use of the University of Campinas (CEUA-UNICAMP, Campinas-SP, Brazil) and carried out in accordance with the ethical principles for animal experimentation adopted by the Brazilian Society of Laboratory Animal Science (SBCAL/COBEA).

# AUTHOR CONTRIBUTIONS

NB designed and executed the experiments, analyzed the data, wrote the manuscript. DG assisted in the experiments, data analysis and interpretation, read and revised the manuscript. JV performed DAF-2 DA fluorescence analysis. AD conceived the study, guided the experimental design, data analysis and interpretation, read and revised the manuscript.

# FUNDING

This work was supported by grant from Sao Paulo Research Foundation - FAPESP (#2014/22506-6 and # 2016/14461-8).

# REFERENCES

thickening via perivascular adipose tissue inflammation. J. Vasc. Res. 50, 228–237. doi: 10.1159/000350542

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Baltieri, Guizoni, Victorio and Davel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Roles of Perivascular Adipose Tissue in the Pathogenesis of Atherosclerosis

Kimie Tanaka1 and Masataka Sata2 \*

*1 Division for Health Service Promotion, The University of Tokyo, Tokyo, Japan, 2 Department of Cardiovascular Medicine, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan*

Traditionally, it is believed that white adipose tissues serve as energy storage, heat insulation, and mechanical cushion, whereas non-shivering thermogenesis occurs in brown adipose tissue. Recent evidence revealed that adipose tissue secretes many types of cytokines, called as adipocytokines, which modulate glucose metabolism, lipid profile, appetite, fibrinolysis, blood pressure, and inflammation. Most of the arteries are surrounded by perivascular adipose tissue (PVAT). PVAT has been thought to be simply a structurally supportive tissue for vasculature. However, recent studies showed that PVAT influences vasodilation and vasocontraction, suggesting that PVAT regulates vascular tone and diameter. Adipocytokines secreted from PVAT appear to have direct access to the adjacent arterial wall by diffusion or via vasa vasorum. In fact, PVAT around atherosclerotic lesions and mechanically-injured arteries displayed inflammatory cytokine profiles, suggesting that PVAT functions to promote vascular lesion formation. Many clinical studies revealed that increased accumulation of epicardial adipose tissue (EAT), which surrounds coronary arteries, is associated with coronary artery disease. In this review article, we will summarize recent findings about potential roles of PVAT in the pathogenesis of atherosclerosis, particularly focusing on a series of basic and clinical studies from our laboratory.

#### Edited by:

*Stephanie W. Watts, Michigan State University, United States*

#### Reviewed by:

*Irena Levitan, University of Illinois at Chicago, United States Klaus Ley, La Jolla Institute for Allergy and Immunology (LJI), United States*

#### \*Correspondence:

*Masataka Sata<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Vascular Physiology, a section of the journal Frontiers in Physiology*

Received: *30 September 2017* Accepted: *03 January 2018* Published: *13 February 2018*

#### Citation:

*Tanaka K and Sata M (2018) Roles of Perivascular Adipose Tissue in the Pathogenesis of Atherosclerosis. Front. Physiol. 9:3. doi: 10.3389/fphys.2018.00003* Keywords: perivascular adipose tissue, atherosclerosis, epicardial adipose tissue, inflammation, adipocytokine

# PERIVASCULAR ADIPOSE TISSUE AND ADIPOCYTOKINES

It has been believed that atherosclerotic process is initiated by endothelial injury, followed by inflammatory cells infiltration into the subendothelial layer. Thus, inflammation spreads from the inside toward the outside of the artery (Ross, 1999). On the other hand, it is assumed that inflammatory process also progresses from the outside toward the inside. Most of the arteries surrounded by perivascular adipose tissue (PVAT). Aorta has abundant PVAT (Szasz et al., 2013), while PVAT is not detected around cerebral arteries and microvessels. There is no distinct borderline between arterial adventitia and PVAT. PVAT has been considered as simply a supporting organ of vasculature.

In 1991, Soltis and Cassis demonstrated that perivascular adipose tissue significantly influences vascular responsiveness to contractile stimuli using rat aortic rings with or without surrounding adipose tissues (Soltis and Cassis, 1991). This study suggested that PVAT potentially serves as a regulator of vascular responsiveness (Soltis and Cassis, 1991). Recently, adipose tissue have received a lot of attention as endocrine organ. It was reported that adipocytes secrete numerous kinds of

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inflammatory and anti-inflammatory cytokines, called as "adipocytokines," such as tumor necrosis factor (TNF)-α and adiponectin (Matsuzawa et al., 2004). Analyses of human samples showed that PVAT around the coronary arteries, like other adipose tissues, expresses inflammatory cytokines, and chemokines (Mazurek et al., 2003; Henrichot et al., 2005). It was also reported that PVAT secretes reactive oxygen spices, nitric oxide, angiotensin II, and free fatty acid (Szasz et al., 2013). Adipocytokines secreted from PVAT appear to have direct access to the adjacent arterial wall via diffusion or vasa vasorum (Sacks and Fain, 2007; Tanaka et al., 2011) (**Figure 1**).

#### INFLUENCE OF PVAT ON ENDOTHELIAL FUNCTION AND VASCULAR LESION FORMATION: LESSONS FROM ANIMAL MODELS

We evaluated whether inflammation in PVAT affects the lesion formation after a mechanical vascular injury in murine femoral artery (Takaoka et al., 2009) (**Figure 2**). Wild-type (C57BL/6) mice received either a standard chow diet or a high-fat highsucrose (HF/HS) diet. The body weight of the wild-type mice increased by 54% with a HF/HS feeding. The number of macrophages accumulated in PVAT increased by HF/HS diet. Expression of adiponectin was down-regulated, while expression of inflammatory cytokines was up-regulated in PVAT of mice fed on HF/HS diet. A wire was inserted into the femoral artery of mice to induce endothelial denudation and over-dilatation of the femoral artery. The changes in cytokine expression in PVAT around the injured artery was associated with exaggerated neointima formation at 4 weeks after the injury, suggesting that PVAT influences pathological vascular remodeling in response to mechanical vascular injury.

We also investigated an atheroprotective role of healthy PVAT by removing PVAT in mice fed on a standard diet. Removal of healthy PVAT markedly enhanced neointima formation, which was attenuated by transplantation of subcutaneous fat tissues from the mice fed on a standard diet. The results suggest an atheroprotective role of healthy PVAT.  To investigate the local effects of PVAT adiponectin on vascular remodeling, recombinant adiponectin was delivered locally to the adventitial space in adiponectin-deficient mice, using gelatin hydrogel. Four weeks after endovascular injury, neointima formation was reduced by perivascular delivery of adiponectin. Taken together, it was suggested that PVAT functions to prevent lesion formation by secreting atheroprotective adipokines, such as adiponectin. However, obesity alters adipocytokine expression profiles of PVAT, resulting in enhanced neointima formation after vascular injury (Takaoka et al., 2009).

We also reported that mechanical endovascular injury alters adipocytokine expression in PVAT (Takaoka et al., 2010). A wire was inserted into the femoral artery of mice to induce endothelial denudation and over-dilatation. We found that this mechanical

HF/HS diet 4 weeks after endovascular injury. Arrows indicate internal elastic lamina. Scale bar: 100µm. Morphometric analysis of injured femoral arteries in lean (*n* = 7) and obese (*n* = 6) mice 4 weeks after wire-induced injury. Results are expressed as means ± SEM. \*\**P* < 0.01. All figures are cited from the reference (Takaoka et al., 2009) with permission.

injury up-regulated inflammatory cytokines and down-regulated adiponectin in PVAT. These changes were attenuated in TNF-α knockout mice, suggesting that TNF-α is important to transmit endovascular injury to adipocytokine changes in PVAT (Takaoka et al., 2010).

Consistent with our studies, others reported that PVAT plays a role in the pathogenesis of vascular lesion formation. Ketonen et al. reported that obesity-induced endothelial dysfunction is caused by increased oxidative stress and enhanced expression of inflammatory cytokine in PVAT (Ketonen et al., 2010). Manka et al. reported that transplantation of PVAT from obese mice to low-density lipoprotein receptor knockout mice enhanced lesion formation with increased inflammatory cell infiltration and pathological angiogenesis in adventitia. Theses pathological effects of PVAT transplantation was attenuated when PVAT from monocyte chemoattractant protein-1 (MCP-1)-deficient mice was transplanted. These results suggest that PVAT promotes vascular lesion formation through MCP-1-dependent mechanisms (Manka et al., 2014). These animal studies indicate that obesity increases expression of inflammatory adipocytokines in PVAT, leading to endothelial dysfunction and enhanced vascular lesion formation.

### POSSIBLE ROLES OF EPICARDIAL ADIPOSE TISSUE IN THE PATHOGENESIS OF HUMAN CORONARY ARTERY DISEASE

Epicardial adipose tissue (EAT) is assumed to secrete abundant cytokines to the adjacent coronary artery (Sacks and Fain, 2007). For example, in the patients undergoing coronary artery bypass graft (CABG) surgery, it was reported that EAT abundantly expressed interleukin (IL)-1β, IL-6, TNF-α, and MCP-1 compared to their subcutaneous adipose tissue (Mazurek et al., 2003). Baker et al. reported that the expression of adiponectin mRNA was significantly lower in EAT than in gluteal and abdominal adipose tissues (Baker et al., 2006).