Different Anti-Contractile Function and Nitric Oxide Production of Thoracic and Abdominal Perivascular Adipose Tissues

Divergent phenotypes between the perivascular adipose tissue (PVAT) surrounding the abdominal and the thoracic aorta might be implicated in regional aortic differences, such as susceptibility to atherosclerosis. Although PVAT of the thoracic aorta exhibits anti-contractile function, the role of PVAT in the regulation of the vascular tone of the abdominal aorta is not well defined. In the present study, we compared the anti-contractile function, nitric oxide (NO) availability, and reactive oxygen species (ROS) formation in PVAT and vessel walls of abdominal and thoracic aorta. Abdominal and thoracic aortic tissue from male Wistar rats were used to perform functional and molecular experiments. PVAT reduced the contraction evoked by phenylephrine in the absence and presence of endothelium in the thoracic aorta, whereas this anti-contractile effect was not observed in the abdominal aorta. Abdominal PVAT exhibited a reduction in endothelial NO synthase (eNOS) expression compared with thoracic PVAT, without differences in eNOS expression in the vessel walls. In agreement with this result, NO production evaluated in situ using 4,5-diaminofluorescein was less pronounced in abdominal compared with thoracic aortic PVAT, whereas no significant difference was observed for endothelial NO production. Moreover, NOS inhibition with L-NAME enhanced the phenylephrine-induced contraction in endothelial-denuded rings with PVAT from thoracic but not abdominal aorta. ROS formation and lipid peroxidation products evaluated through the quantification of hydroethidine fluorescence and 4-hydroxynonenal adducts, respectively, were similar between PVAT and vessel walls from the abdominal and thoracic aorta. Extracellular superoxide dismutase (SOD) expression was similar between the vessel walls and PVAT of the abdominal and thoracic aorta. However, Mn-SOD levels were reduced, while CuZn-SOD levels were increased in abdominal PVAT compared with thoracic aortic PVAT. In conclusion, our results demonstrate that the anti-contractile function of PVAT is lost in the abdominal portion of the aorta through a reduction in eNOS-derived NO production compared with the thoracic aorta. Although relative SOD isoforms are different along the aorta, ROS formation, and lipid peroxidation seem to be similar. These findings highlight the specific regional roles of PVAT depots in the control of vascular function that can drive differences in susceptibility to vascular injury.

Divergent phenotypes between the perivascular adipose tissue (PVAT) surrounding the abdominal and the thoracic aorta might be implicated in regional aortic differences, such as susceptibility to atherosclerosis. Although PVAT of the thoracic aorta exhibits anti-contractile function, the role of PVAT in the regulation of the vascular tone of the abdominal aorta is not well defined. In the present study, we compared the anti-contractile function, nitric oxide (NO) availability, and reactive oxygen species (ROS) formation in PVAT and vessel walls of abdominal and thoracic aorta. Abdominal and thoracic aortic tissue from male Wistar rats were used to perform functional and molecular experiments. PVAT reduced the contraction evoked by phenylephrine in the absence and presence of endothelium in the thoracic aorta, whereas this anti-contractile effect was not observed in the abdominal aorta. Abdominal PVAT exhibited a reduction in endothelial NO synthase (eNOS) expression compared with thoracic PVAT, without differences in eNOS expression in the vessel walls. In agreement with this result, NO production evaluated in situ using 4,5-diaminofluorescein was less pronounced in abdominal compared with thoracic aortic PVAT, whereas no significant difference was observed for endothelial NO production. Moreover, NOS inhibition with L-NAME enhanced the phenylephrine-induced contraction in endothelial-denuded rings with PVAT from thoracic but not abdominal aorta. ROS formation and lipid peroxidation products evaluated through the quantification of hydroethidine fluorescence and 4-hydroxynonenal adducts, respectively, were similar between PVAT and vessel walls from the abdominal and thoracic aorta. Extracellular superoxide dismutase (SOD) expression was similar between the vessel walls and PVAT of the abdominal and thoracic aorta. However, Mn-SOD levels were reduced, while CuZn-SOD levels were increased in abdominal PVAT compared with thoracic aortic PVAT. In conclusion, our results demonstrate that the anti-contractile function of PVAT is lost in the abdominal portion of the aorta through a reduction in eNOS-derived NO production compared with the thoracic aorta. Although relative SOD isoforms are different along the aorta, ROS formation, and lipid peroxidation seem to be similar. These findings highlight the specific regional roles of PVAT depots in the control of vascular function that can drive differences in susceptibility to vascular injury.

INTRODUCTION
Aortic atherosclerotic lesion and aneurysm are predominant in the abdominal rather than the thoracic portion of the aorta, suggesting inherent properties influencing regional aortic susceptibility to injury; however, the mechanisms involved in this susceptibility are not well understood. Despite being segments of the same artery, elastic features, and collagen contents vary along the aorta, which contribute to the physiological characteristics of pulse wave and blood distribution (Sokolis et al., 2008;Tsamis et al., 2013). Moreover, although the levels of endothelium-dependent relaxation induced by acetylcholine appear to be similar (Oloyo et al., 2012), the mechanisms involved in the alpha-adrenergic-mediated contraction might differ in the thoracic vs. the abdominal aorta (Lamb et al., 1994;Asbún-Bojalil et al., 2002).
Along the aorta, the phenotype of the PVAT is distinguished. Abdominal aorta PVAT resembles the phenotype of white adipose tissue (WAT), while fat surrounding the thoracic aorta shares the characteristics of brown adipose tissue (BAT; Police et al., 2009;Fitzgibbons et al., 2011). In addition, the expression levels of inflammatory genes and markers of immune cell infiltration are greater in abdominal PVAT than in thoracic PVAT, suggesting that the WAT phenotype is more pro-inflammatory and atherogenic than the BAT phenotype in PVAT (Padilla et al., 2013). Accordingly, PVAT from human coronary arteries with the WAT phenotype exhibits a pro-inflammatory profile with reduced adiponectin levels compared to BAT subcutaneous and peri-renal adipocyte depots (Chatterjee et al., 2009).
Although the anti-contractile and anti-inflammatory effects of thoracic aortic PVAT are well-known, the possible role of PVAT regulating the vascular reactivity and redox status of the abdominal aorta is still poorly understood. A previous study demonstrated that the anti-contractile effect of abdominal aortic PVAT is less pronounced when compared to the thoracic portion of the aorta (Watts et al., 2011). However, the mechanism involved in this regional difference remains unclear. This study aimed at investigating the comparative effects of thoracic vs. abdominal aortic PVAT on anti-contractile function, the oxidative profile, and NO synthesis and availability.

Animals
All animal procedures were in accordance with the ethical principles for animal experimentation adopted by the Brazilian Society of Laboratory Animal Science (SBCAL/COBEA) and were approved by the Ethics Committee on Animal Use of the University of Campinas-UNICAMP (protocol number: 3523-1) and of the Institute of Biomedical Science at the University of Sao Paulo (protocol number 53, sheet 19, book 03).
Experiments were conducted in 3-to 4-month-old male Wistar rats (Multidisciplinary Center for Biological Research, UNICAMP) maintained at a constant room temperature (22-24 • C) and light cycle (12:12 h light:dark) with food and water allowed ad libitum to all animals. At the time of the experiments, animals were euthanized under anesthesia (ketamine 80 mg/kg and xylazine 5 mg/kg; i.p.).
After a 30-min equilibration period, all aortic rings were initially exposed twice to 75 mM KCl, the first to check their functional integrity and the second to assess the maximum contractility. Following the wash, vascular reactivity was investigated with cumulative concentration-response curves to acetylcholine (0.1 nM -10 µM) in vessels contracted with phenylephrine (1 µM). Endothelial denudation was confirmed by acetylcholine-induced relaxation <10%. After the samples TABLE 1 | Potency (−LogEC50) and maximal response (Rmax) values to phenylephrine-induced contraction in thoracic and abdominal aortas with (+) or without (−) endothelium (E) and perivascular adipose tissue (PVAT).
At the end of the experiments, the length of each ring was measured. Vasoconstrictor responses to phenylephrine are expressed as mN/mm. Relaxation responses to acetylcholine are expressed as the percentage of relaxation of the contractile response induced by phenylephrine (1 µM).

Nitric Oxide (NO) and Reactive Oxygen Species (ROS) Evaluation In situ
Thoracic and abdominal aortic tissues with their respective coated PVAT were isolated, placed in Petri dishes with cold Krebs-Henseleit solution, cleaned and sectioned into 3-mm rings. Next, in a dark chamber, aortic segments were incubated for 30 min with Krebs-Henseleit solution (pH = 7.4, 37 • C) plus 4,5-diaminofluorescein diacetate (DAF-2, 10 µM) or with dihydroethidium (DHE, 2 µM) for NO and ROS measurement, respectively, as previously described (Gil-Ortega et al., 2014). Some aortic segments were co-incubated with DAF-2 plus  L-NAME (1 mM) to evaluate the specific generation of NO from NO synthase or with DHE plus MnTMPyP (25 µM), a cell-permeant superoxide dismutase (SOD) mimetic, to evaluate superoxide anion formation. Subsequently, the aortic segments with PVAT were fixed in 4% paraformaldehyde for 4 h and then embedded in freezing medium (Tissue-Tek, Sakura Finetek, Torrance, CA). Transverse sections (20 µm thick) of frozen arteries were obtained on a cryostat. Digital images were collected on a microscope (Nikon, Chiyoda-ku, Tokyo, Japan) equipped with epifluorescence and fluorescein/rhodamine filters using a 20X objective. The images were analyzed using ImageJ software (NIH, Bethesda, MD, USA). NO availability was evaluated by DAF-2 mean optical density of the fluorescence in the endothelium and PVAT, and ROS production were analyzed based on the integrated density of the DHE fluorescence normalized by the number of nuclei labeled with ethidium bromide (EB-positive nuclei) in the vascular wall segment of the aorta and its respective PVAT.
After washing, membranes were incubated for 90 min with a peroxidase-conjugated IgG antibody specific for the primary antibody used. Protein expression was detected with Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA), and the membranes were subjected to autoradiography (Amersham Hyperfilm ECL, GE Healthcare). The blots were digitized, and intensity was quantified using ImageJ 1.46p software (National Institutes of Health). Ponceau staining was used to normalize expression of the evaluated proteins in each sample.

Statistical Analysis
Results are expressed as the means ± SEM. Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software Corp., USA). Concentration-response curves were analyzed using two-way ANOVA followed by the Bonferroni's post-test. For comparisons between abdominal and thoracic aortic samples in the same condition, the Student t-test was used. Values of P < 0.05 were considered significantly different.

PVAT Exerts an Anti-Contractile Effect in the Thoracic but Not the Abdominal Aorta
To determine the anti-contractile effects of PVAT in thoracic and abdominal aortic tissues, we performed concentration-response curves to phenylephrine in rings with (open symbols) or without (filled symbols) PVAT in intact (circle) or denuded (triangle) endothelium. Thoracic aortic rings with PVAT and intact endothelium (PVAT+E+) presented a significant reduction in potency and maximal response to phenylephrine when compared to rings without PVAT (PVAT−E+; Figure 1A and Table 1). Although the endothelium damage increased the phenylephrineinduced contraction (compare PVAT−/E+ vs. PVAT−/E−, Figure 1A), the anti-contractile effect of PVAT was still observed in endothelium-denuded rings. Thus, the presence of PVAT (PVAT+E−) in endothelium-denuded rings also reduced both the potency and maximal response to phenylephrine when compared to rings without PVAT and endothelium (PVAT−E−; Figure 1A and Table 1). In contrast, the presence of PVAT did not alter the phenylephrine-induced contraction in either intact or denuded endothelium abdominal aortic rings ( Figure 1B and Table 1).
We also assessed the endothelium-dependent relaxation response to acetylcholine in thoracic and abdominal aorta.  As expected, endothelium damage blocked the vasodilatation induced by acetylcholine in both thoracic and abdominal aorta (Figures 1C,D). However, no effects of PVAT on the acetylcholine-induced relaxation were observed in either thoracic or abdominal aorta (Figures 1C,D).

ROS Production and Lipid Peroxidation Did
Not Vary along the Aorta ROS production was detected based on DHE fluorescence (Figure 3A), and lipid peroxidation was evaluated based on the expression of 4-HNE adducts ( Figure 3B). ROS production was almost fully inhibited by MnTMPyP in abdominal and thoracic aorta, suggesting superoxide as the main ROS evaluated in situ by DHE fluorescence in both the vascular wall and PVAT ( Figure 3A). Both ROS production and lipid peroxidation were similar in abdominal and thoracic aortic tissues and PVAT (Figures 3A,B).

Anti-Oxidative Profiles of Abdominal and Thoracic Aortic Tissue and PVAT
The protein expression levels of SOD isoforms were investigated in abdominal and thoracic tissues. EC-SOD did not differ between abdominal and thoracic aortic tissues and PVAT ( Figure 4A), while reduced Mn-SOD expression was detected in abdominal compared with thoracic PVAT, without changes in the vascular wall ( Figure 4B). In contrast, CuZn-SOD expression was increased in abdominal PVAT compared to thoracic PVAT, with no regional differences in the aortic wall ( Figure 4C).

DISCUSSION
The present results showed that: (1) the anti-contractile effect of PVAT on alpha-adrenergic-induced contraction observed in thoracic aorta is completely lost in the abdominal section, (2) there is a significant reduction in eNOS-derived NO production in PVAT but not in the endothelium of the abdominal vs. the thoracic aorta, and (3) ROS production and lipid peroxidation levels appear to be similar along the aorta, although the relative expression levels of SOD isoforms are different. Therefore, a minor contribution of NO in the abdominal vs. the thoracic adipose depot, rather than changes in redox status, might be involved in the functional regional differences along the aorta.
Anti-contractile effects of murine thoracic periaortic fat have been demonstrated in response to several contractile agonists, including phenylephrine, serotonin and angiotensin II (Löhn et al., 2002;Fésus et al., 2007;Gao et al., 2007;Lee et al., 2011;Sun et al., 2013;Araujo et al., 2015). Importantly, the endothelium contributes to the anti-contractile effect of PVAT in the response to angiotensin II (Gálvez-Prieto et al., 2012), while it only attenuates or does not alter its effect on the alphaadrenergic mediated contraction in the thoracic aorta (Gao et al., 2007;Lee et al., 2009Lee et al., , 2011. In agreement with these data, both endothelium and PVAT, independently of each other, restrain the contractile response to phenylephrine in the thoracic aorta. In contrast, in the abdominal aorta, only the endothelium exerted an anti-contractile effect in the phenylephrine-induced contraction. These data are consistent with previous data demonstrating anticontractile effects of endothelium-derived NO in both thoracic and abdominal aortic tissues (Kleinbongard et al., 2013), while the anti-contractile function of periaortic fat on the angiotensin II-induced contraction is impaired in the abdominal section (Watts et al., 2011).
Although the contractile response to phenylephrine varies along the aorta, endothelium-dependent relaxation levels induced by acetylcholine were similar in abdominal and thoracic aorta. Similar relaxation responses to acetylcholine were also observed in abdominal vs. thoracic sections of aorta in male Sprague-Dawley rats (Oloyo et al., 2012). In addition, PVAT did not play a role in the aortic relaxation response to acetylcholine, as previously demonstrated (Ketonen et al., 2010;Gálvez-Prieto et al., 2012). NO availability using DAF-2 fluorescence was previously demonstrated in PVAT of mouse mesenteric arteries (Gil-Ortega et al., 2010) and thoracic aorta (Xia et al., 2016). Here, for the first time, we demonstrated that NO availability in PVAT was significantly reduced in abdominal vs. thoracic aorta, whereas endothelium-derived NO levels were similar between the two portions of the aorta. The reduction in DAF-2 fluorescence in the abdominal periaortic fat was accompanied by a similar magnitude of reduction in eNOS expression in this tissue, while eNOS expression in the endothelium remained the same along the aorta. Reinforcing these results, it is interesting to observe that in PVAT preserved but endothelium denuded abdominal aortic rings the inhibition of NO synthesis did not change the phenylephrine-induced contraction, while it was increased in thoracic aortic rings. The cell-specific localization of the higher eNOS protein expression in thoracic aortic PVAT remains to be elucidated. Because microvessels are present throughout the thoracic PVAT, we cannot rule out endothelial cells from these vessels as a source of NO in PVAT, albeit the vast majority of eNOS positive cells in PVAT are adipocytes (Xia et al., 2016).
It is known that the abdominal aorta exhibits a different PVAT phenotype compared to the thoracic aorta: while abdominal PVAT exhibits a phenotype of pro-inflammatory fat, thoracic periaortic fat shares characteristics of BAT (Padilla et al., 2013). Interestingly, enlargement of lipid droplet morphology has been observed in thoracic periaortic fat from obese mice (Fitzgibbons et al., 2011). This change in phenotype induced by a high-fat diet is accompanied by a significant reduction in PVAT-derived NO production related to decreased eNOS phosphorylation (Xia et al., 2016). However, aerobic exercise training can overbrowning and enhances the eNOS expression in thoracic PVAT (Araujo et al., 2015). These studies support the hypothesis that physiological white fat depots surrounding the abdominal aorta may not exhibit a significant anti-contractile function due to less pronounced eNOS expression and NO production and/availability.
In the absence of PVAT and endothelium, abdominal and thoracic portions of the aorta exhibit similar raw forces in response to phenylephrine, suggesting both as major mechanisms involved in the regional contractile differences along the aorta. However, we cannot exclude other intrinsic factors. Although contraction in response to phenylephrine of rat abdominal and thoracic aorta is mediated via alpha1D-adrenergic receptor subtype (Asbún-Bojalil et al., 2002), prostanoids derived from smooth muscle cells contribute to contraction only in the abdominal aorta (Lamb et al., 1994). In addition, distal aortic segments are stiffer than proximal ones (Sokolis et al., 2008;Devos et al., 2015), although the mechanisms underlying this regional difference within the aorta is not fully established. Previous study has shown that impairment of eNOS activity is an important mechanism inducing vascular extracellular matrix remodeling in a model of abdominal aortic aneurism (Gao et al., 2012). Therefore, the substantial reduction in NO availability observed in the present study at the abdominal aorta might be a relevant factor implicated in the susceptibility of this part of the aorta to vascular injury.
It is well-known that NO availability is impaired by the spontaneous reaction of NO with superoxide anion causing the generation of peroxynitrite, a potent oxidant with potential cytotoxicity. Gil-Ortega et al. (2014) demonstrated that an enhanced superoxide generation in PVAT surrounding mesenteric arteries of obese mice is associated with a loss of the anti-contractile effect of PVAT on the contractile response of mesenteric arteries to noradrenaline. Therefore, we attempted to investigate whether oxidative differences along the aorta could be involved in the impaired anti-contractile function and NO availability noted in the abdominal aorta. Interestingly, no regional differences were noted in either superoxide generation, based on DHE fluorescence, or lipid peroxidation, assessed based on the presence of 4-HNE-protein adducts between the vascular wall and PVAT of abdominal and thoracic portions of the aorta. Early dysfunction of thoracic aortic PVAT was associated with increased lipid peroxidation, evaluated based on the presence of thiobarbituric acid reactive species (TBARS), in response to high fructose diet (Rebolledo et al., 2010). However, this change reflected those reported in abdominal adipose depots (Alzamendi et al., 2009), suggesting that they are the consequence of general rather than local oxidative stress involved in the PVAT control of vascular function.
The major enzymatic control of superoxide anion levels in the vessel wall is exerted by SOD isoforms (Faraci and Didion, 2004). SOD activity was also detected in PVAT of murine mesenteric arteries and thoracic aorta, playing an important role in the redox status of vascular wall (Rebolledo et al., 2010;Gil-Ortega et al., 2014). SODs convert superoxide anion into hydrogen peroxide, thereby protecting NO availability and signaling. Although the three isoforms of SOD catalyze the same reaction, they differ in both localization and their importance for vascular function.
Here, we noted similar patterns of SOD expression between the abdominal and thoracic aortic walls. However, in comparison with the thoracic PVAT, abdominal PVAT exhibited reduced protein levels of Mn-SOD and enhanced cytosolic CuZn-SOD. Interestingly, CuZn-SOD overexpression protects the aorta from lipid peroxidation and DNA fragmentation, whereas Mn-SOD heterozygous-deficient mice exhibited enhanced aortic lipid peroxidation and apoptosis, mechanisms of importance for the development of atherosclerosis (Guo et al., 2001). However, the functional significance of differences in vascular expression or activity in PVAT is still poorly understood; future studies will be necessary to address this question.
Taken together, the present results demonstrate that, compared with the thoracic aorta, the anti-contractile function of PVAT is impaired in the abdominal portion of the aorta through a reduction in eNOS-derived NO production without changes in ROS generation and lipid peroxidation. Knowing that aortic atherosclerotic lesions and aneurysms are predominant in abdominal rather than thoracic portions of the aorta, these findings highlight a new specific regional role of PVAT depots in the control of vascular function that may drive differences in susceptibility to vascular injury between these two portions of the aorta.