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Modulation of vein function by perivascular adipose tissue
European Journal of Pharmacology 657 (2011) 111–116
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Modulation of vein function by perivascular adipose tissue Chao Lu, Ashley X. Zhao, Yu-Jing Gao, Robert M.K.W. Lee ⁎ Smooth Muscle Research Program and Department of Anesthesia, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
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Article history: Received 19 August 2010 Received in revised form 25 November 2010 Accepted 19 December 2010 Available online 13 January 2011 Keywords: Vein Perivascular adipose tissue Endothelium Relaxation factor Angiotensin Potassium channel
a b s t r a c t Although a number of studies have shown that perivascular adipose tissue (PVAT) attenuates arterial contraction through the release of perivascular-derived relaxation factors (PVRF), the role of PVAT in modulating venous function and its mechanism(s) remained unknown. Here we examined the role of PVAT in the modulation of vascular function in the inferior vena cava. Venous rings from male Wistar rats were prepared with both endothelium and PVAT intact, with either PVAT or endothelium removed, or with both endothelium and PVAT removed for functional studies. Contractile response to phenylephrine, U 46619, or 5-hydroxytryptamine was significantly attenuated in PVAT+ as compared with PVAT− veins. PVAT− vessels with intact endothelium (E+) pre-contracted with phenylephrine showed a concentration-dependent relaxation response to angiotensin 1–7 [Ang-(1–7)], and this response was abolished by the removal of endothelium, and by Ang-(1–7) (Mas) receptor antagonists D-Ala-Ang-(1–7) (A779) or D-Pro7-Ang-(1–7). Donor solution incubated with a PVAT+ ring induced a relaxation response in the E+ recipient vessel but not in E− recipient vessel. The use of specific channel blockers and enzyme inhibitors showed that Ang-(1–7) is a transferable PVRF that induces endothelium-dependent relaxation through NO release and activation of voltage-dependent potassium (K+) channels (Kv) channels. We conclude that venous PVAT attenuates agonist-induced contraction by releasing Ang-(1–7), which causes relaxation of smooth muscle through endothelial NO release and activation of Kv channels. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Adipose tissue was traditionally thought to function only as an energy storage site. Recent studies have shown that adipose tissue secretes a significant number of biologically active substances, including leptin, adiponectin, angiotensinogen, resistin, and steroid hormones (Engeli et al., 2003; Guerre-Millo, 2004). In particular, adipose tissue surrounding most of the systemic arteries, known as perivascular adipose tissue (PVAT), has been shown to attenuate vessel contraction to various agonists including phenylephrine, thromboxane A2 mimetic U 46619, 5-hydroxytryptamine (5-HT), and angiotensin II through the release of perivascular-derived relaxation factors (PVRF)(Gao et al., 2005b, 2007; Lohn et al., 2002). The presence of PVRF is demonstrated in the aorta and mesenteric arteries of rats (Dubrovska et al., 2004; Gao et al., 2005a; Lohn et al., 2002), and in the internal thoracic arteries of human (Gao et al., 2005b). We have recently established that angiotensin 1–7 [Ang-(1–7)] is one of the endothelium-dependent PVRF in rat aorta (Lee et al., 2009b). The mechanism of this PVRF was found to act through activation of potassium (K+) channels which hyperpolarizes smooth muscle cells. This is shown by observing hyperpolarization in rat mesenteric arterial ⁎ Corresponding author. Department of Anesthesia, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Tel.: + 1 905 521 2100x75177; fax: + 1 905 523 1224. E-mail address: [email protected] (R.M.K.W. Lee). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.12.028
smooth muscle when PVAT was preserved; abolishment of PVRFinduced relaxation by applying high K+, which reduces K+ gradient across the cell membrane; and blockage of K+ channels which abolished the relaxation effect of PVAT (Dubrovska et al., 2004; Galvez et al., 2006; Gao et al., 2005b; Lohn et al., 2002). It also appeared that PVRF act through different K+ channels depending on the type of artery and type of animal species, including ATPdependent K+ (KATP) channels in rat aorta (Lohn et al., 2002), calcium-dependent K+ (KCa) channels in human internal thoracic artery (Gao et al., 2005b) and rat aorta (Gao et al., 2007), and voltage-dependent K+ (Kv) channels in rat mesenteric arteries (Galvez et al., 2006). Strikingly, all of these studies regarding PVAT function were carried out using arteries from various species. Indeed, comparing with the intensity of studies on arteries, the regulation of vein function has been less focused in general. While there is no doubt about the pivotal role that arteries play in the regulation of blood pressure and flow, the essential role of the venous vessels in the circulatory system should not be neglected because approximately 70% of the circulated blood is inside the veins. Therefore any significant changes in venous caliber may greatly influence cardiac function and tissue blood distribution by altering circulating blood volume. Chronic changes in venous tension may also result in structural alterations such as adaptive remodeling from the corresponding arterial side that can drive a sustained increase in arterial blood pressure (Szasz et al., 2007). PVAT of saphenous veins
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from humans contains nitric oxide (NO) synthase, suggesting that PVAT may play a role in the regulation of venous function (Dashwood et al., 2007). In this study, we examined the role of PVAT in modulating contraction of rat inferior vena cava. 2. Materials and methods
Recipient vessels were incubated with Ang-(1–7) (Mas) receptor antagonists D-Ala-Ang-(1–7) (A779) or D-Pro7-Ang-(1–7) for 25– 30 min before transfer of solution was carried out to study the involvement of Ang-(1–7). Similarly, an equal amount of the Mas receptor antagonists was added to the donor solution as well to avoid any dilution in recipient chamber when donor solution was introduced.
2.1. Animals 2.4. Chemicals Male Wistar rats (300–350 g, Harlan, Indianapolis, IN, USA) were used for this study. The care and the use of these animals were in accordance with the guidelines of the Canadian Council on Animal Care, and approved by the Animal Research Ethics Board of McMaster University. The procedure for the preparation of blood vessels has been described in previous studies (Gao et al., 2005a; Gao and Lee, 2001) and the same procedures were used in the preparation of vena cava. Briefly, the rat was anaesthetized by an overdose of sodium pentobarbital (60 mg/kg, i.p.), and abdominal segment of the inferior vena cava was collected in oxygenated physiological salt solution (PSS) with the following composition (in mM): NaCl, 119; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; CaCl2, 1.6; and glucose, 11. 2.2. Vessel preparation and contractility study Four types of venous rings were prepared: vessels with both PVAT and endothelium intact (PVAT+E+), with either PVAT or endothelium removed (PVAT-E+, or PVAT+E−), or with both PVAT and endothelium removed (PVAT-E−). Care was taken to maintain the consistency in the length of the vein segments in all the experiments. PVAT was removed by dissection under a microscope. Endothelium was removed by gently rolling the vein over the wire in the organ bath before threading of the second wire and the application of tension. Successful removal of endothelium was confirmed by the absence of relaxation response to carbachol (10 μM) in rings precontracted with phenylephrine (10 μM). A computerized myograph system was used to record the isometric tension of the rings. After equilibration for at least 90 min at 0.5 g (4.9 mN) of preload, which is the optimal preload defined in our preliminary experiment, the vena cava rings were challenged with 60 mM KCl twice at an interval of 30 min to establish a baseline contractile response. Contractile response to agonists was expressed as a percentage of KCl-induced contraction. Cumulative concentration-dependent response curves for phenylephrine, U 46619 and 5-HT were constructed. A final contractile response to 60 mM KCl was performed at the end to ensure that contractile response had not deteriorated during the time course of the experiment.
The following chemicals were used: 4-aminopyridine (4-AP), carbachol, tetraethylammonium (TEA), glybenclamide, N(omega)nitro-L-arginine (L-NNA), phenylephrine, U 46619, 5-HT, A779 and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxyPTIO) (Sigma USA); Ang-(1–7), D-Pro7-Ang-(1–7) (Phoenix Pharmaceutical CA USA). Glybenclamide was dissolved in DMSO, U 46619 was dissolved in ethanol; and all other agents were dissolved in deionized water and prepared fresh daily. 2.5. Statistics Results are expressed as mean ± S.E.M where n represents the number of rats. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by post hoc t test for concentration-dependent effects, or by Student's unpaired or paired t-test for comparison between control and treated vessels using the SigmaStat software (SPSS, Inc., Chicago, USA). The differences were considered significant when P ≤ 0.05. 3. Results 3.1. PVAT surrounding inferior vena cava As in the case of most veins, rat inferior vena cava consisted of a thin layer of medial smooth muscle cells, a thick layer of adventitia composed mostly of collagen fibres, and PVAT containing both brown and white adipocytes (Fig. 1). 3.2. Effects of PVAT on contractile responses to agonists Maximal tension induced by 60 mM KCl was similar among the four types of vessel preparations (Fig. 2A), showing that neither the presence of PVAT, nor the procedure to remove PVAT and/or endothelium affected the contractile property of the vein. Phenylephrine induced a concentration-dependent contractile response in
2.3. Bioassay experiments To examine the effects of transferable PVRF, bioassay experiments were carried out using PVAT+ vena cava rings as donors and PVAT− rings as recipients. The donor and recipient vessels were incubated in organ baths for 30 min and the vessels were pre-contracted with phenylephrine (10 μM). Three ml of the solution from donor chamber was transferred to the recipient chamber when the pre-contraction had reached its plateau (usually within 3–5 min), as described in previous studies (Gao et al., 2005a,b), to examine the relaxation effects of a transferable PVRF. Half of the amount of donor solution (1.5 ml) was also transferred into the recipient chamber to study the concentration-response relation of the transferable PVRF. To test the role of NO and K+ channels in PVRF-induced relaxations, recipient vessels were incubated with respective enzyme inhibitors or K+ channel blockers for 25–30 min before transfer of donor solution was carried out. An equal amount of inhibitors or blockers was added to the donor chamber before the transfer to avoid any dilution of blocking agents in the recipient chamber during the transfer.
Fig. 1. Cross-section of rat inferior vena cava with surrounding PVAT. Hematoxylin/ eosin stain. SMC = smooth muscle cells, Adv = adventitia, B = brown adipocytes, W = white adipocytes. Bar: 100 μm.
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Fig. 3. Concentration-dependent response to U 46619 or 5-hydroxytryptamine (5-HT). In vessels with intact endothelium, contractile response to U 46619 (A) or 5-HT (B) was lower in vena cava rings with intact (PVAT+) as compared with vessels with PVAT removed (PVAT−) (n = 9). *P b 0.05, **P b 0.01 (ANOVA).
3.4. Involvement of endothelium and NO
Fig. 2. KCl-induced contraction and concentration-dependent response to phenylephrine (PHE). KCl-induced contraction was similar among the four types of vessel preparations (A). In the presence of endothelium (E+), the presence of PVAT (PVAT+) attenuated the contractile response to phenylephrine (B), whereas this attenuation effect was not seen in veins with endothelium removed (E−) (C). n = 9, *P b 0.05 (ANOVA).
Removal of endothelium in the recipient vessel abolished the relaxation response induced by donor solution from PVAT+ vena cava (Fig. 4A). Incubation of recipient vessels with, L-NNA (100 μM, a NO synthase inhibitor), and carboxy-PTIO (1 mM, a NO scavenger) abolished the relaxation induced by the donor solution from PVAT+ vena cava (Fig. 4B).
vena cava rings with or without PVAT or endothelium (Fig. 2B and C). Contraction was markedly attenuated in veins with intact PVAT and endothelium (PVAT+E+) as compared with those with PVAT removed (PVAT-E+) (Fig. 2B). This attenuation of the contraction effect in PVAT intact vessels was not seen in vessels with endothelium removed (Fig. 2C). Contractile response to U 46619 (Fig. 3A) or 5-HT (Fig. 3B) showed a similar attenuation of contraction effect in the presence of PVAT and endothelium. Removal of endothelium abolished the attenuation effect of PVAT on agonist-induced contraction (data not shown).
3.5. Relaxation responses to Ang-(1–7)
3.3. Relaxation induced by PVAT-generated transferable relaxation factor(s) and the involvement of K+ channels
In vessels with intact PVAT, incubation with A779 significantly increased the contraction induced by phenylephrine in a concentration-dependent manner (Fig. 6A). A779 (1 μM) did not cause any increase in the contraction to phenylephrine in PVAT removed vessels (Fig. 6B). D-Pro7-Ang-(1–7) also increased the contraction induced by phenylephrine in a concentration-dependent manner (Fig. 7A). A combination of A779 (1 μM) and D-Pro7-Ang-(1–7) (0.1 μM) did not cause further increase of phenylephrine-induced contraction than by either A779 or D-Pro7-Ang-(1–7) alone (Fig. 7A). A779 and D-Pro7-Ang-(1–7) (either alone or in combination) did not cause any increase in the contraction to phenylephrine in PVAT removed vessels (Fig. 7B).
Transfer of donor solution incubated with PVAT+ vena cava rings induced a relaxation response in the recipient ring with PVAT removed (Fig. 4A). Transferring half of the amount of this donor solution showed a smaller but significant relaxation response in the recipient ring with PVAT removed. Incubation with Kv channel blocker 4-AP (1 mM) abolished the relaxation effect in the recipient vessels induced by donor solution from PVAT+ vena cava (Fig. 4B). In contrast, KCa channel blocker TEA (1 mM) or KATP channel blocker glybenclamide (10 μM) did not abolish the relaxation effect (Fig. 4B).
Ang-(1–7) induced a concentration-dependent relaxation response in PVAT-E+ venous rings pre-contracted with phenylephrine (10 μM) but not in the preparations with endothelium removed (PVAT-E−, Fig. 5A). Incubation of recipient vessels (PVAT-E+) with Ang-(1–7) receptor (Mas) antagonists (A779, 1 μM) reduced the relaxation response induced by Ang-(1–7) (Fig. 5B). 3.6. Effects of Ang-(1–7) receptor antagonists on phenylephrine-induced contraction
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Contraction (% of KCI)
A 150
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120 90 60 30 0 100% MEDIA E+
50% MEDIA E+
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Contraction (% of KCl)
B 200
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Fig. 4. Relaxation of PVAT-denuded vena cava rings induced by transferring solution incubated with PVAT+ rings (donor). A comparison of percent contraction of the vein before and after the transfer of donor solution showed that the donor solution caused a relaxation response in PVAT− recipient vessel in the presence of endothelium (E+) (A, 100% Media E+). Diluting the donor solution to 50% still caused a relaxation response in the recipient vessel, and this relaxation response was abolished after the removal of endothelium (A). The use of various blockers (B) showed that relaxation induced by the donor solution was inhibited by voltage-dependent K+ channel blocker 4-aminopyridine (4-AP), NO synthase inhibitor (L-NNA), NO scavenger (carboxy-PTIO) and Ang-(1–7) receptor (Mas) antagonist (A779), but not by calciumdependent K+ channel blocker tetraethylammonium (TEA) or ATP-dependent K+ channel blocker glybenclamide. n = 6, *P b 0.05, **P b 0.01 (paired t-test).
Fig. 5. Relaxation response induced by Ang-(1–7). In veins with PVAT removed (PVAT−) and precontracted with phenylephrine (PHE), Ang-(1–7) induced a concentrationdependent relaxation response only in vessels with intact endothelium (E+) (A). This relaxation response in PVAT-E+ rings was reduced by incubation with Ang-(1–7) (Mas) receptor antagonist A779 (1 μM)(B). n = 8, **P b 0.01 (paired t-test).
4. Discussion To our knowledge, this is the first report to show the mechanism involved in the modulation of vein contraction by PVAT, which extends our understanding about PVAT-related modulation to another type of vessel, the vein. The mechanisms of the modulation involve the release of a transferable relaxation factor, which is most likely Ang-(1–7), and subsequent NO release from the endothelium and activation of K+ channels, as in the case of rat aorta. Despite these similarities, there are distinct differences between veins and arteries. First, the subtype of K+ channels involved is Kv in veins in contrast to KCa or KATP subtypes in rat aorta and human internal thoracic artery. Second, the endothelium-independent mechanisms in PVATmediated inhibition of contraction, which are clearly shown in arteries, were not found in veins. These findings clearly demonstrate that PVAT may also be a modulator of venous function with distinct mechanisms involved. Retention of perivascular tissue on saphenous vein prepared for coronary artery bypass surgery by the “no touch” technique protects against distension-induced damage, preserves vessel morphology, and maintains endothelial NO synthase/NO synthase activity (Dashwood et al., 2009). The maintenance of the graft's endogenous eNOS activity and NO production may be highly relevant for the prevention of vein graft occlusion (Dashwood et al., 2009). The potential important role of PVAT to maintain vessel patency was also found in human internal thoracic artery, where PVAT was found to release a transferable relaxation factor that acts through the activation of KCa channels (Gao et al., 2005b). In human saphenous vein, the presence of PVAT increased the sensitivity of the vein to 5-HT which could be
Fig. 6. Incubation with Mas receptor blocker A779 increased the contraction induced by phenylephrine (PHE) in a concentration-dependent manner only in veins with intact PVAT (PVAT+) (A) and not in the veins with PVAT removed (PVAT−) (B)(n = 6). *P b 0.05 (ANOVA).
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A Contraction (% of KCl)
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150
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120
7
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90
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Contraction (% of KCl)
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D-Pro7-Ang-(1-7)
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PHE -Log [M] Fig. 7. Incubation with different concentrations of Mas-receptor antagonist D-Pro7-Ang-(1–7) individually or combined with another antagonist A779 increased the contraction induced by phenylephrine (PHE) in PVAT intact rings (PVAT+) (A) but not in the veins with PVAT removed (B) (n = 7). *P b 0.05 (unpaired t-test).
related to the release of relaxation factor from PVAT (Ford et al., 2006). Our observation that PVRF is produced by PVAT in vena cava is based on our results showing that contractile response to agonists such as phenylephrine, U 46619, and 5-HT was attenuated in the presence of PVAT. We have used different agonists that operated through different mechanisms to cause contraction to ensure that the release of PVRF was not related to the use of a specific agonist. We also carried out bioassay experiments to confirm that a relaxation factor was indeed released by PVAT. We found that the transfer of bathing solution from vena cava with intact PVAT to vena cava with PVAT removed indeed caused a relaxation response. These results clearly demonstrate that PVAT releases a transferable relaxation factor that is able to attenuate vena cava contraction in response to various agonists. In rat aorta, we have previously reported that PVAT caused relaxation of the vessel through two mechanisms: an endotheliumdependent mechanism due to a transferable relaxation factor that causes NO release and subsequent KCa channel activation, and by an endothelium-independent mechanism involving H2O2 and subsequent activation of soluble guanylyl cyclase (Gao et al., 2007). In vena cava, we also found that this transferable relaxation factor acted through the endothelium because removal of endothelium abolished this relaxation effect of the donor solution, and NO synthase inhibitor L-NNA and NO scavenger carboxy-PTIO were able to abolish the relaxation effect induced by donor solution from PVAT+ vessel, which is similar to the findings in rat aorta (Gao et al., 2007). One major difference between artery and vein is that endothelium-independent relaxation response induced by PVAT is absent in the vein, because the attenuation of contraction to various agonists due to the presence of PVAT was not observed after removal of the endothelium. Numerous studies with a variety of vascular tissues have pointed out the key role of K+ channel activation in NO-induced relaxation, which includes both KCa and Kv (Bychkov et al., 1998; Irvine et al., 2003; Tanaka
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et al., 2006; Zhao et al., 1997). In rat aorta, we have shown that the subtype of K+ channels involved in the endothelium-dependent relaxation response to a transferable PVRF was KCa (Gao et al., 2007). However, in rat vena cava, this transferable relaxation factor is acting through Kv channels to cause endothelium-dependent relaxation of the smooth muscle. This is evidenced by our finding that Kv channel blocker (4-AP) was able to abolish the relaxation effect induced by PVRF. In the studies on PVAT associated with arteries, activation of different subtypes of K+ channels has been reported, which includes KATP in rat aorta (Lohn et al., 2002) and Kv in rat mesenteric arteries (Galvez et al., 2006). The differences in the involvement of the subtypes of K+ channels involved in PVAT-associated vascular effects may reflect tissue-specific variation in K+ channel distribution of different subtypes or other mechanisms that warrant further investigation. We have recently reported that in rat aorta, one of the transferable endothelium-dependent relaxation factors released by PVAT is Ang-(1–7) that caused the release of NO to induce vascular relaxation. We therefore used a similar approach to determine if Ang-(1–7) is the transferable PVRF in the vena cava. Our results showed that Ang-(1–7) was indeed involved in rat inferior vena cava, because the relaxation response caused by donor solution was abolished when the recipient vessels were treated with Ang-(1–7) (Mas) receptor antagonists A779 or D-Pro7-Ang-(1–7), and that Ang-(1–7) caused a concentrationdependent relaxation response that was dependent on endothelium, which mimics the response to transfer of donor solution. In the aorta of Sprague–Dawley rats, vasodilator effect of Ang-(1–7) which is dependent on endothelium-derived NO, was found to be mediated by Ang-(1–7) receptors sensitive to D-Pro7-Ang-(1–7) but not to A779, suggesting the presence of a different Ang-(1–7) receptor subtype (Silva et al., 2007). Our results with inferior vena cava showed that both D-Pro7-Ang-(1–7) and A779 were equally effective in blocking the effects of the transferable PVRF and there was no additive inhibitory effect of these two antagonists. It therefore appears that Ang-(1–7) receptors in the vein consisted of one type of receptor sensitive to these two antagonists. In arteries, PVRF not only attenuates contractile responses to various agonists, but may also be involved in reducing basal arterial tone, because in perfused rat mesenteric arteries, lumen diameter was larger in vessels with intact PVAT as compared with those with PVAT removed (Lee et al., 2009a). This suggests that there is a basal release of PVRF under resting conditions in the arteries. Confirmation under in vivo condition is needed to show that PVAT is involved in the maintenance of basal vascular tone. Our study on vein shows that PVAT similarly reduces venous contractile responses to various agonists as in arteries, but whether venous basal tone is regulated by PVAT remains to be determined. Since venous tone is a key regulator of circulating filling pressure, PVAT-mediated regulation of venous contraction may play a role in maintaining hemodynamic homeostasis. In summary, PVAT surrounding the vena cava attenuates agonistinduced contraction by releasing Ang-(1–7) as one of the PVRF. Ang-(1–7) acting through Mas receptors in endothelium causes the release of NO leading to the relaxation of the smooth muscle cells via the activation of Kv channels. In contrast with arteries where PVRF also acts through an endothelium-independent mechanism to attenuate contraction in response to agonists, this mechanism is absent in inferior vena cava. We therefore conclude that PVAT is also a modulator of venous function.
Acknowledgements This study was supported by a grant from the Heart and Stroke Foundation of Ontario, Canada. We thank Dr. Li-Ying Su and Mr. Howard Tse for their technical assistance.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Cardiovascular Pharmacology
Modulation of vein function by perivascular adipose tissue Chao Lu, Ashley X. Zhao, Yu-Jing Gao, Robert M.K.W. Lee ⁎ Smooth Muscle Research Program and Department of Anesthesia, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
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Article history: Received 19 August 2010 Received in revised form 25 November 2010 Accepted 19 December 2010 Available online 13 January 2011 Keywords: Vein Perivascular adipose tissue Endothelium Relaxation factor Angiotensin Potassium channel
a b s t r a c t Although a number of studies have shown that perivascular adipose tissue (PVAT) attenuates arterial contraction through the release of perivascular-derived relaxation factors (PVRF), the role of PVAT in modulating venous function and its mechanism(s) remained unknown. Here we examined the role of PVAT in the modulation of vascular function in the inferior vena cava. Venous rings from male Wistar rats were prepared with both endothelium and PVAT intact, with either PVAT or endothelium removed, or with both endothelium and PVAT removed for functional studies. Contractile response to phenylephrine, U 46619, or 5-hydroxytryptamine was significantly attenuated in PVAT+ as compared with PVAT− veins. PVAT− vessels with intact endothelium (E+) pre-contracted with phenylephrine showed a concentration-dependent relaxation response to angiotensin 1–7 [Ang-(1–7)], and this response was abolished by the removal of endothelium, and by Ang-(1–7) (Mas) receptor antagonists D-Ala-Ang-(1–7) (A779) or D-Pro7-Ang-(1–7). Donor solution incubated with a PVAT+ ring induced a relaxation response in the E+ recipient vessel but not in E− recipient vessel. The use of specific channel blockers and enzyme inhibitors showed that Ang-(1–7) is a transferable PVRF that induces endothelium-dependent relaxation through NO release and activation of voltage-dependent potassium (K+) channels (Kv) channels. We conclude that venous PVAT attenuates agonist-induced contraction by releasing Ang-(1–7), which causes relaxation of smooth muscle through endothelial NO release and activation of Kv channels. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Adipose tissue was traditionally thought to function only as an energy storage site. Recent studies have shown that adipose tissue secretes a significant number of biologically active substances, including leptin, adiponectin, angiotensinogen, resistin, and steroid hormones (Engeli et al., 2003; Guerre-Millo, 2004). In particular, adipose tissue surrounding most of the systemic arteries, known as perivascular adipose tissue (PVAT), has been shown to attenuate vessel contraction to various agonists including phenylephrine, thromboxane A2 mimetic U 46619, 5-hydroxytryptamine (5-HT), and angiotensin II through the release of perivascular-derived relaxation factors (PVRF)(Gao et al., 2005b, 2007; Lohn et al., 2002). The presence of PVRF is demonstrated in the aorta and mesenteric arteries of rats (Dubrovska et al., 2004; Gao et al., 2005a; Lohn et al., 2002), and in the internal thoracic arteries of human (Gao et al., 2005b). We have recently established that angiotensin 1–7 [Ang-(1–7)] is one of the endothelium-dependent PVRF in rat aorta (Lee et al., 2009b). The mechanism of this PVRF was found to act through activation of potassium (K+) channels which hyperpolarizes smooth muscle cells. This is shown by observing hyperpolarization in rat mesenteric arterial ⁎ Corresponding author. Department of Anesthesia, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Tel.: + 1 905 521 2100x75177; fax: + 1 905 523 1224. E-mail address: [email protected] (R.M.K.W. Lee). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.12.028
smooth muscle when PVAT was preserved; abolishment of PVRFinduced relaxation by applying high K+, which reduces K+ gradient across the cell membrane; and blockage of K+ channels which abolished the relaxation effect of PVAT (Dubrovska et al., 2004; Galvez et al., 2006; Gao et al., 2005b; Lohn et al., 2002). It also appeared that PVRF act through different K+ channels depending on the type of artery and type of animal species, including ATPdependent K+ (KATP) channels in rat aorta (Lohn et al., 2002), calcium-dependent K+ (KCa) channels in human internal thoracic artery (Gao et al., 2005b) and rat aorta (Gao et al., 2007), and voltage-dependent K+ (Kv) channels in rat mesenteric arteries (Galvez et al., 2006). Strikingly, all of these studies regarding PVAT function were carried out using arteries from various species. Indeed, comparing with the intensity of studies on arteries, the regulation of vein function has been less focused in general. While there is no doubt about the pivotal role that arteries play in the regulation of blood pressure and flow, the essential role of the venous vessels in the circulatory system should not be neglected because approximately 70% of the circulated blood is inside the veins. Therefore any significant changes in venous caliber may greatly influence cardiac function and tissue blood distribution by altering circulating blood volume. Chronic changes in venous tension may also result in structural alterations such as adaptive remodeling from the corresponding arterial side that can drive a sustained increase in arterial blood pressure (Szasz et al., 2007). PVAT of saphenous veins
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from humans contains nitric oxide (NO) synthase, suggesting that PVAT may play a role in the regulation of venous function (Dashwood et al., 2007). In this study, we examined the role of PVAT in modulating contraction of rat inferior vena cava. 2. Materials and methods
Recipient vessels were incubated with Ang-(1–7) (Mas) receptor antagonists D-Ala-Ang-(1–7) (A779) or D-Pro7-Ang-(1–7) for 25– 30 min before transfer of solution was carried out to study the involvement of Ang-(1–7). Similarly, an equal amount of the Mas receptor antagonists was added to the donor solution as well to avoid any dilution in recipient chamber when donor solution was introduced.
2.1. Animals 2.4. Chemicals Male Wistar rats (300–350 g, Harlan, Indianapolis, IN, USA) were used for this study. The care and the use of these animals were in accordance with the guidelines of the Canadian Council on Animal Care, and approved by the Animal Research Ethics Board of McMaster University. The procedure for the preparation of blood vessels has been described in previous studies (Gao et al., 2005a; Gao and Lee, 2001) and the same procedures were used in the preparation of vena cava. Briefly, the rat was anaesthetized by an overdose of sodium pentobarbital (60 mg/kg, i.p.), and abdominal segment of the inferior vena cava was collected in oxygenated physiological salt solution (PSS) with the following composition (in mM): NaCl, 119; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; CaCl2, 1.6; and glucose, 11. 2.2. Vessel preparation and contractility study Four types of venous rings were prepared: vessels with both PVAT and endothelium intact (PVAT+E+), with either PVAT or endothelium removed (PVAT-E+, or PVAT+E−), or with both PVAT and endothelium removed (PVAT-E−). Care was taken to maintain the consistency in the length of the vein segments in all the experiments. PVAT was removed by dissection under a microscope. Endothelium was removed by gently rolling the vein over the wire in the organ bath before threading of the second wire and the application of tension. Successful removal of endothelium was confirmed by the absence of relaxation response to carbachol (10 μM) in rings precontracted with phenylephrine (10 μM). A computerized myograph system was used to record the isometric tension of the rings. After equilibration for at least 90 min at 0.5 g (4.9 mN) of preload, which is the optimal preload defined in our preliminary experiment, the vena cava rings were challenged with 60 mM KCl twice at an interval of 30 min to establish a baseline contractile response. Contractile response to agonists was expressed as a percentage of KCl-induced contraction. Cumulative concentration-dependent response curves for phenylephrine, U 46619 and 5-HT were constructed. A final contractile response to 60 mM KCl was performed at the end to ensure that contractile response had not deteriorated during the time course of the experiment.
The following chemicals were used: 4-aminopyridine (4-AP), carbachol, tetraethylammonium (TEA), glybenclamide, N(omega)nitro-L-arginine (L-NNA), phenylephrine, U 46619, 5-HT, A779 and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxyPTIO) (Sigma USA); Ang-(1–7), D-Pro7-Ang-(1–7) (Phoenix Pharmaceutical CA USA). Glybenclamide was dissolved in DMSO, U 46619 was dissolved in ethanol; and all other agents were dissolved in deionized water and prepared fresh daily. 2.5. Statistics Results are expressed as mean ± S.E.M where n represents the number of rats. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by post hoc t test for concentration-dependent effects, or by Student's unpaired or paired t-test for comparison between control and treated vessels using the SigmaStat software (SPSS, Inc., Chicago, USA). The differences were considered significant when P ≤ 0.05. 3. Results 3.1. PVAT surrounding inferior vena cava As in the case of most veins, rat inferior vena cava consisted of a thin layer of medial smooth muscle cells, a thick layer of adventitia composed mostly of collagen fibres, and PVAT containing both brown and white adipocytes (Fig. 1). 3.2. Effects of PVAT on contractile responses to agonists Maximal tension induced by 60 mM KCl was similar among the four types of vessel preparations (Fig. 2A), showing that neither the presence of PVAT, nor the procedure to remove PVAT and/or endothelium affected the contractile property of the vein. Phenylephrine induced a concentration-dependent contractile response in
2.3. Bioassay experiments To examine the effects of transferable PVRF, bioassay experiments were carried out using PVAT+ vena cava rings as donors and PVAT− rings as recipients. The donor and recipient vessels were incubated in organ baths for 30 min and the vessels were pre-contracted with phenylephrine (10 μM). Three ml of the solution from donor chamber was transferred to the recipient chamber when the pre-contraction had reached its plateau (usually within 3–5 min), as described in previous studies (Gao et al., 2005a,b), to examine the relaxation effects of a transferable PVRF. Half of the amount of donor solution (1.5 ml) was also transferred into the recipient chamber to study the concentration-response relation of the transferable PVRF. To test the role of NO and K+ channels in PVRF-induced relaxations, recipient vessels were incubated with respective enzyme inhibitors or K+ channel blockers for 25–30 min before transfer of donor solution was carried out. An equal amount of inhibitors or blockers was added to the donor chamber before the transfer to avoid any dilution of blocking agents in the recipient chamber during the transfer.
Fig. 1. Cross-section of rat inferior vena cava with surrounding PVAT. Hematoxylin/ eosin stain. SMC = smooth muscle cells, Adv = adventitia, B = brown adipocytes, W = white adipocytes. Bar: 100 μm.
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Fig. 3. Concentration-dependent response to U 46619 or 5-hydroxytryptamine (5-HT). In vessels with intact endothelium, contractile response to U 46619 (A) or 5-HT (B) was lower in vena cava rings with intact (PVAT+) as compared with vessels with PVAT removed (PVAT−) (n = 9). *P b 0.05, **P b 0.01 (ANOVA).
3.4. Involvement of endothelium and NO
Fig. 2. KCl-induced contraction and concentration-dependent response to phenylephrine (PHE). KCl-induced contraction was similar among the four types of vessel preparations (A). In the presence of endothelium (E+), the presence of PVAT (PVAT+) attenuated the contractile response to phenylephrine (B), whereas this attenuation effect was not seen in veins with endothelium removed (E−) (C). n = 9, *P b 0.05 (ANOVA).
Removal of endothelium in the recipient vessel abolished the relaxation response induced by donor solution from PVAT+ vena cava (Fig. 4A). Incubation of recipient vessels with, L-NNA (100 μM, a NO synthase inhibitor), and carboxy-PTIO (1 mM, a NO scavenger) abolished the relaxation induced by the donor solution from PVAT+ vena cava (Fig. 4B).
vena cava rings with or without PVAT or endothelium (Fig. 2B and C). Contraction was markedly attenuated in veins with intact PVAT and endothelium (PVAT+E+) as compared with those with PVAT removed (PVAT-E+) (Fig. 2B). This attenuation of the contraction effect in PVAT intact vessels was not seen in vessels with endothelium removed (Fig. 2C). Contractile response to U 46619 (Fig. 3A) or 5-HT (Fig. 3B) showed a similar attenuation of contraction effect in the presence of PVAT and endothelium. Removal of endothelium abolished the attenuation effect of PVAT on agonist-induced contraction (data not shown).
3.5. Relaxation responses to Ang-(1–7)
3.3. Relaxation induced by PVAT-generated transferable relaxation factor(s) and the involvement of K+ channels
In vessels with intact PVAT, incubation with A779 significantly increased the contraction induced by phenylephrine in a concentration-dependent manner (Fig. 6A). A779 (1 μM) did not cause any increase in the contraction to phenylephrine in PVAT removed vessels (Fig. 6B). D-Pro7-Ang-(1–7) also increased the contraction induced by phenylephrine in a concentration-dependent manner (Fig. 7A). A combination of A779 (1 μM) and D-Pro7-Ang-(1–7) (0.1 μM) did not cause further increase of phenylephrine-induced contraction than by either A779 or D-Pro7-Ang-(1–7) alone (Fig. 7A). A779 and D-Pro7-Ang-(1–7) (either alone or in combination) did not cause any increase in the contraction to phenylephrine in PVAT removed vessels (Fig. 7B).
Transfer of donor solution incubated with PVAT+ vena cava rings induced a relaxation response in the recipient ring with PVAT removed (Fig. 4A). Transferring half of the amount of this donor solution showed a smaller but significant relaxation response in the recipient ring with PVAT removed. Incubation with Kv channel blocker 4-AP (1 mM) abolished the relaxation effect in the recipient vessels induced by donor solution from PVAT+ vena cava (Fig. 4B). In contrast, KCa channel blocker TEA (1 mM) or KATP channel blocker glybenclamide (10 μM) did not abolish the relaxation effect (Fig. 4B).
Ang-(1–7) induced a concentration-dependent relaxation response in PVAT-E+ venous rings pre-contracted with phenylephrine (10 μM) but not in the preparations with endothelium removed (PVAT-E−, Fig. 5A). Incubation of recipient vessels (PVAT-E+) with Ang-(1–7) receptor (Mas) antagonists (A779, 1 μM) reduced the relaxation response induced by Ang-(1–7) (Fig. 5B). 3.6. Effects of Ang-(1–7) receptor antagonists on phenylephrine-induced contraction
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Contraction (% of KCI)
A 150
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120 90 60 30 0 100% MEDIA E+
50% MEDIA E+
100% MEDIA E-
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Fig. 4. Relaxation of PVAT-denuded vena cava rings induced by transferring solution incubated with PVAT+ rings (donor). A comparison of percent contraction of the vein before and after the transfer of donor solution showed that the donor solution caused a relaxation response in PVAT− recipient vessel in the presence of endothelium (E+) (A, 100% Media E+). Diluting the donor solution to 50% still caused a relaxation response in the recipient vessel, and this relaxation response was abolished after the removal of endothelium (A). The use of various blockers (B) showed that relaxation induced by the donor solution was inhibited by voltage-dependent K+ channel blocker 4-aminopyridine (4-AP), NO synthase inhibitor (L-NNA), NO scavenger (carboxy-PTIO) and Ang-(1–7) receptor (Mas) antagonist (A779), but not by calciumdependent K+ channel blocker tetraethylammonium (TEA) or ATP-dependent K+ channel blocker glybenclamide. n = 6, *P b 0.05, **P b 0.01 (paired t-test).
Fig. 5. Relaxation response induced by Ang-(1–7). In veins with PVAT removed (PVAT−) and precontracted with phenylephrine (PHE), Ang-(1–7) induced a concentrationdependent relaxation response only in vessels with intact endothelium (E+) (A). This relaxation response in PVAT-E+ rings was reduced by incubation with Ang-(1–7) (Mas) receptor antagonist A779 (1 μM)(B). n = 8, **P b 0.01 (paired t-test).
4. Discussion To our knowledge, this is the first report to show the mechanism involved in the modulation of vein contraction by PVAT, which extends our understanding about PVAT-related modulation to another type of vessel, the vein. The mechanisms of the modulation involve the release of a transferable relaxation factor, which is most likely Ang-(1–7), and subsequent NO release from the endothelium and activation of K+ channels, as in the case of rat aorta. Despite these similarities, there are distinct differences between veins and arteries. First, the subtype of K+ channels involved is Kv in veins in contrast to KCa or KATP subtypes in rat aorta and human internal thoracic artery. Second, the endothelium-independent mechanisms in PVATmediated inhibition of contraction, which are clearly shown in arteries, were not found in veins. These findings clearly demonstrate that PVAT may also be a modulator of venous function with distinct mechanisms involved. Retention of perivascular tissue on saphenous vein prepared for coronary artery bypass surgery by the “no touch” technique protects against distension-induced damage, preserves vessel morphology, and maintains endothelial NO synthase/NO synthase activity (Dashwood et al., 2009). The maintenance of the graft's endogenous eNOS activity and NO production may be highly relevant for the prevention of vein graft occlusion (Dashwood et al., 2009). The potential important role of PVAT to maintain vessel patency was also found in human internal thoracic artery, where PVAT was found to release a transferable relaxation factor that acts through the activation of KCa channels (Gao et al., 2005b). In human saphenous vein, the presence of PVAT increased the sensitivity of the vein to 5-HT which could be
Fig. 6. Incubation with Mas receptor blocker A779 increased the contraction induced by phenylephrine (PHE) in a concentration-dependent manner only in veins with intact PVAT (PVAT+) (A) and not in the veins with PVAT removed (PVAT−) (B)(n = 6). *P b 0.05 (ANOVA).
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A Contraction (% of KCl)
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D-Pro7-Ang-(1-7)
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PHE -Log [M] Fig. 7. Incubation with different concentrations of Mas-receptor antagonist D-Pro7-Ang-(1–7) individually or combined with another antagonist A779 increased the contraction induced by phenylephrine (PHE) in PVAT intact rings (PVAT+) (A) but not in the veins with PVAT removed (B) (n = 7). *P b 0.05 (unpaired t-test).
related to the release of relaxation factor from PVAT (Ford et al., 2006). Our observation that PVRF is produced by PVAT in vena cava is based on our results showing that contractile response to agonists such as phenylephrine, U 46619, and 5-HT was attenuated in the presence of PVAT. We have used different agonists that operated through different mechanisms to cause contraction to ensure that the release of PVRF was not related to the use of a specific agonist. We also carried out bioassay experiments to confirm that a relaxation factor was indeed released by PVAT. We found that the transfer of bathing solution from vena cava with intact PVAT to vena cava with PVAT removed indeed caused a relaxation response. These results clearly demonstrate that PVAT releases a transferable relaxation factor that is able to attenuate vena cava contraction in response to various agonists. In rat aorta, we have previously reported that PVAT caused relaxation of the vessel through two mechanisms: an endotheliumdependent mechanism due to a transferable relaxation factor that causes NO release and subsequent KCa channel activation, and by an endothelium-independent mechanism involving H2O2 and subsequent activation of soluble guanylyl cyclase (Gao et al., 2007). In vena cava, we also found that this transferable relaxation factor acted through the endothelium because removal of endothelium abolished this relaxation effect of the donor solution, and NO synthase inhibitor L-NNA and NO scavenger carboxy-PTIO were able to abolish the relaxation effect induced by donor solution from PVAT+ vessel, which is similar to the findings in rat aorta (Gao et al., 2007). One major difference between artery and vein is that endothelium-independent relaxation response induced by PVAT is absent in the vein, because the attenuation of contraction to various agonists due to the presence of PVAT was not observed after removal of the endothelium. Numerous studies with a variety of vascular tissues have pointed out the key role of K+ channel activation in NO-induced relaxation, which includes both KCa and Kv (Bychkov et al., 1998; Irvine et al., 2003; Tanaka
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et al., 2006; Zhao et al., 1997). In rat aorta, we have shown that the subtype of K+ channels involved in the endothelium-dependent relaxation response to a transferable PVRF was KCa (Gao et al., 2007). However, in rat vena cava, this transferable relaxation factor is acting through Kv channels to cause endothelium-dependent relaxation of the smooth muscle. This is evidenced by our finding that Kv channel blocker (4-AP) was able to abolish the relaxation effect induced by PVRF. In the studies on PVAT associated with arteries, activation of different subtypes of K+ channels has been reported, which includes KATP in rat aorta (Lohn et al., 2002) and Kv in rat mesenteric arteries (Galvez et al., 2006). The differences in the involvement of the subtypes of K+ channels involved in PVAT-associated vascular effects may reflect tissue-specific variation in K+ channel distribution of different subtypes or other mechanisms that warrant further investigation. We have recently reported that in rat aorta, one of the transferable endothelium-dependent relaxation factors released by PVAT is Ang-(1–7) that caused the release of NO to induce vascular relaxation. We therefore used a similar approach to determine if Ang-(1–7) is the transferable PVRF in the vena cava. Our results showed that Ang-(1–7) was indeed involved in rat inferior vena cava, because the relaxation response caused by donor solution was abolished when the recipient vessels were treated with Ang-(1–7) (Mas) receptor antagonists A779 or D-Pro7-Ang-(1–7), and that Ang-(1–7) caused a concentrationdependent relaxation response that was dependent on endothelium, which mimics the response to transfer of donor solution. In the aorta of Sprague–Dawley rats, vasodilator effect of Ang-(1–7) which is dependent on endothelium-derived NO, was found to be mediated by Ang-(1–7) receptors sensitive to D-Pro7-Ang-(1–7) but not to A779, suggesting the presence of a different Ang-(1–7) receptor subtype (Silva et al., 2007). Our results with inferior vena cava showed that both D-Pro7-Ang-(1–7) and A779 were equally effective in blocking the effects of the transferable PVRF and there was no additive inhibitory effect of these two antagonists. It therefore appears that Ang-(1–7) receptors in the vein consisted of one type of receptor sensitive to these two antagonists. In arteries, PVRF not only attenuates contractile responses to various agonists, but may also be involved in reducing basal arterial tone, because in perfused rat mesenteric arteries, lumen diameter was larger in vessels with intact PVAT as compared with those with PVAT removed (Lee et al., 2009a). This suggests that there is a basal release of PVRF under resting conditions in the arteries. Confirmation under in vivo condition is needed to show that PVAT is involved in the maintenance of basal vascular tone. Our study on vein shows that PVAT similarly reduces venous contractile responses to various agonists as in arteries, but whether venous basal tone is regulated by PVAT remains to be determined. Since venous tone is a key regulator of circulating filling pressure, PVAT-mediated regulation of venous contraction may play a role in maintaining hemodynamic homeostasis. In summary, PVAT surrounding the vena cava attenuates agonistinduced contraction by releasing Ang-(1–7) as one of the PVRF. Ang-(1–7) acting through Mas receptors in endothelium causes the release of NO leading to the relaxation of the smooth muscle cells via the activation of Kv channels. In contrast with arteries where PVRF also acts through an endothelium-independent mechanism to attenuate contraction in response to agonists, this mechanism is absent in inferior vena cava. We therefore conclude that PVAT is also a modulator of venous function.
Acknowledgements This study was supported by a grant from the Heart and Stroke Foundation of Ontario, Canada. We thank Dr. Li-Ying Su and Mr. Howard Tse for their technical assistance.
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