Role of Perivascular Adipose Tissue–Derived Methyl Palmitate in Vascular Tone Regulation and Pathogenesis of HypertensionClinical Perspective
Background—Perivascular adipose tissue (PVAT)–derived relaxing factor (PVATRF) significantly regulates vascular tone. Its chemical nature remains unknown. We determined whether palmitic acid methyl ester (PAME) was the PVATRF and whether its release and/or vasorelaxing activity decreased in hypertension.
Methods and Results—Using superfusion bioassay cascade technique, tissue bath myography, and gas chromatography/mass spectrometry, we determined PVATRF and PAME release from aortic PVAT preparations of Wistar Kyoto rats and spontaneously hypertensive rats. The PVAT of Wistar Kyoto rats spontaneously and calcium dependently released PVATRF and PAME. Both induced aortic vasorelaxations, which were inhibited by 4-aminopyridine (2 mmol/L) and tetraethylammonium 5 and 10 mmol/L but were not affected by tetraethylammonium 1 or 3 mmol/L, glibenclamide (3 μmol/L), or iberiotoxin (100 nmol/L). Aortic vasorelaxations induced by PVATRF- and PAME-containing Krebs solutions were not affected after heating at 70°C but were equally attenuated after hexane extractions. Culture mediums of differentiated adipocytes, but not those of fibroblasts, contained significant PAME and caused aortic vasorelaxation. The PVAT of spontaneously hypertensive rats released significantly less PVATRF and PAME with an increased release of angiotensin II. In addition, PAME-induced relaxation of spontaneously hypertensive rats aortic smooth muscle diminished drastically, which was ameliorated significantly by losartan.
Conclusions—We found that PAME is the PVATRF, causing vasorelaxation by opening voltage-dependent K+ channels on smooth muscle cells. Diminished PAME release and its vasorelaxing activity and increased release of angiotensin II in the PVAT suggest a noble role of PVAT in pathogenesis of hypertension. The antihypertensive effect of losartan is attributed partly to its reversing diminished PAME-induced vasorelaxation.
The systemic blood vessels are surrounded by various amounts of perivascular adipose tissue (PVAT). Since the first report by Soltis and Cassis in 1991 that PVAT attenuated contraction of aortic rings to norepinephrine,1 it has been well accepted that the anticontractile effect of PVAT is due to release of relaxing factor(s) from these adipocytes.2 The vasodilation induced by PVAT-derived relaxing factor (PVATRF) is independent of the endothelium, cyclooxygenase, or cytochrome P450 pathway.2,3 The general consensus is that PVATRF-induced vasodilation is due to opening of potassium channels on the smooth muscle cells.2–5 The chemical identity of the PVATRF, however, remains unknown.
Clinical Perspective on p 1171
Our recent studies using superfusion bioassay cascade technique have demonstrated release of an endogenous potent vasodilator, palmitic acid methyl ester (PAME), from the superior cervical ganglion and retina of the rat.6,7 Because PAME is hydrophobic and has relatively small molecular weight, making it easier to diffuse as a vasoactive substance across the vessel wall, it is a likely candidate for the PVATRF.
The cause of hypertension is complicated and, in most cases, remains unclear.8 The PVAT may play significant roles in regulating vascular smooth muscle tone through numerous mechanisms.9 Changes in PVAT mass and function have been suggested to contribute to increased vascular resistance in spontaneously hypertensive rats (SHR) and angiotensin II (AII)–induced hypertension.5,10 Therefore, we examined the possibilities that PAME is the PVATRF and that its release from the PVAT is diminished in established hypertension. The results indicated that PAME and PVATRF exhibited many similar biochemical pharmacological characteristics. Release of both from the PVAT and the vasodilator response to both were diminished in parallel in the SHR.
Full methods and associated references are available in the online-only Data Supplement.
All male Sprague-Dawley rats (SD), Wistar Kyoto rats (WKY), and age-matched spontaneously hypertensive rats (SHR) were anesthetized with pentobarbital (65 mg/kg IP) and exsanguinated. The thoracic aortic ring (3 mm long) and retina7 were dissected and placed in oxygenated Krebs solution. Endothelial cells (ECs) and PVATs of aortic rings were removed mechanically to obtain an exclusive muscle ring preparation. Some PVAT preparations were histologically verified by fixing aorta in 10% formaldehyde and stained with hematoxylin and eosin (Figure 1A; for details, see the online-only Data Supplement).
Superfusion Bioassay Cascade System
Release of PVATRF from isolated aortic PVAT was examined by a superfusion bioassay cascade system.6,7 The PVAT preparation, serving as donor tissue, was superfused with oxygenated Krebs bicarbonate solution at 37°C. The superfusate continued to superfuse an SD aortic muscle ring serving as detector tissue (Figure 1B). Tension changes in the muscle ring were estimated as percent of sodium nitroprusside (0.1 mmol/L)–induced maximum relaxation. Krebs perfusates collected after superfusion of the PVAT and aortic muscle rings were analyzed by gas chromatography/mass spectrometry (see the online-only Data Supplement).
To examine the effects of calcium-free Krebs solution on PVATRF and PAME release, calcium-free Krebs solution after superfusion of the PVAT preparation was mixed with a known concentration of calcium solution to become normal Krebs solution before superfusing the aortic muscle ring (Figure 1C; see also the online Data Supplement).
Effects of Heating on Vasodilatory Activities of Perivascular Adipose Tissue–Derived Relaxing Factor– or Palmitic Acid Methyl Ester–Containing Krebs Solutions and Culture Mediums
Vasodilatory activities of PVATRF- or PAME-containing Krebs solutions after heating (70°C for 10 minutes; details are given in online Data Supplement) were examined by applying heated solution directly onto the aortic muscle rings. Similarly, culture mediums of NIH 3T3 and 3T3 L1 cells at predifferentiation and 4-day or 8-day postdifferentiation stages (see the online-only Data Supplement) were collected, heated at 90°C for 10 minutes, and examined for vasodilatory activities. The PAME concentrations in culture medium and Krebs perfusates collected after superfusion of the PVAT and aortic muscle rings were analyzed by gas chromatography/mass spectrometry (details are given in the online-only Data Supplement).
Tissue Bath Myography
The aortic ring segment (3 mm long) was mounted on a stainless steel rod and a platinum wire in a tissue bath containing 10 mL Krebs solution at 37°C and equilibrated with 95% O2 and 5% CO2. Tension changes in tissue, mechanically stretched to a resting tension of 2 g,6 were measured by an isometric transducer.11 Phenylephrine and KCl in full concentrations were applied by a noncumulative technique to the bath to induce constriction of endothelium-denuded aortic rings with or without PVAT (see the online-only Data Supplement). All chemicals used and statistical analyses are given in the online-only Data Supplement.
Enzyme Immunoassay for Angiotensin II
The PVATs stripped from thoracic aorta were incubated at 37°C in 3 mL Krebs solution for 30 minutes. The supernatants were collected after centrifugation at 3000 rpm for 10 minutes. The AII contents were measured with the AII enzyme immunoassay kit (see the online-only Data Supplement).
Data are expressed as mean±SEM. One-way ANOVA and the paired t test were used to determine the effects of calcium, the calcium ionophore A23187, indomethacin, methyl arachidonyl fluorophosphonate (MAFP), l-NG-nitroarginine, SKF525A, or miconazole on PVATRF release and the effects of the heat- or hexane-treated PVATRF- or PAME-containing Krebs solution on their vasodilatory activities. In these experiments, control and experimental studies were performed in a continuous perfusion manner. The 2-sample t test was used to compare effects of tetraethylammonium, 4-aminopyridine (4-AP), glibenclamide, and iberiotoxin on aortic preparations; the vasodilatory activities and PAME concentrations between medium from the fibroblast stage and adipocyte stage; the vasodilatory activities and PAME concentrations of PVAT between 20-week-old WKY and 20-week-old SHR or between 8-week-old SHR and 20-week-old SHR; and concentrations of AII between 20-week-old WKY and 20-week-old SHR. Repeated measures ANOVA was used to compare the inhibitory effects of 4-AP on vasorelaxation induced by PVATRF, retina-derived relaxation factor, and exogenous PAME and to compare phenylephrine- or KCl-induced constrictions between aortic ring+PVAT-ECs and aortic ring-PVAT-ECs of WKY and SHR. The OriginPro version 7.5 software (OriginLab Corp, Wellesley, MA) was used for statistical analysis. Post hoc analysis was done with SPSS version 13.0 (SPSS Inc, Chicago, IL). The Tukey test was used for multiple-comparison procedures. Values of P<0.05 were considered statistically significant.
Spontaneous Releases of Perivascular Adipose Tissue–Derived Relaxing Factor and Palmitic Acid Methyl Ester From Aortic Perivascular Adipose Tissue of the Wistar Kyoto Rat
In the presence of phenylephrine-precontracted active muscle tone, the SD aortic muscle ring (ie, ring without ECs and PVAT; Figure 1A) serving as detector tissues relaxed on applications of PAME (0.1 μmol/L) directly onto the ring (Figure 2A and 2B). Normal Krebs solution after superfusion of the PVAT preparation of the WKY (8 to 10 weeks old) also caused relaxation of the aortic muscle ring, indicative of PVATRF release (Figure 2A and 2B). Significant concentrations of PAME were found in the perfusates (Figure 2D). Immediately after removal of the PVAT preparation from the perfusation, the aortic muscle tone returned (or contracted) toward the prior level (Figure 2A). Methanol (the vehicle for dissolving PAME) did not cause relaxation of phenylephrine-precontracted aortic muscle rings (Figure 2B). Superfusion with Krebs solution of the WKY aortic vascular smooth muscle preparations as donor tissue did not cause any relaxation of SD aortic muscle rings (Figure 2B).
A strategy was used (Figure 1C) to determine whether Ca2+ is critical for spontaneous release of PVATRF (indicated by aortic relaxation) and PAME from the PVAT of the WKY. The vasodilatory response of detector SD aortic muscle ring induced by calcium-free Krebs superfusates after superfusion of the PVAT decreased drastically (Figure 2C). The relaxation resumed when calcium-free Krebs solution was replaced with normal Krebs solution superfusing the PVAT preparations (data not shown). In parallel, PAME release from the PVAT preparations decreased drastically when Ca2+-free Krebs solution was used to perfuse the PVAT preparations compared with that when normal Krebs solution was used (Figure 2D). In parallel, relaxations of SD aortic muscle ring induced by Krebs superfusates after superfusion the PVAT were enhanced by the calcium ionophore A23187 in a concentration-dependent manner when applied directly onto the PVAT (Figure I in the online-only Data Supplement).
Failure of Indomethacin to Block Perivascular Adipose Tissue–Derived Relaxing Factor– and Palmitic Acid Methyl Ester–Induced Aortic Relaxation
Krebs superfusates after superfusion of the PVAT preparations of 18- to 20-week-old WKY relaxed phenylephrine-precontracted SD aortic muscle ring. This vasorelaxation was not blocked but was significantly enhanced on addition of indomethacin (10 μmol/L) onto the PVAT preparations (Figure 3A). Aortic relaxation induced by PAME (0.1 μmol/L) added directly onto the rings also was enhanced by indomethacin (Figure 3A). Furthermore, relaxations of SD aortic muscle ring induced by Krebs superfusates after superfusion of the PVAT were not affected by MAFP (20 mmol/L; a phospholipase inhibitor) or combined treatment of MAFP (20 mmol/L) and indomethacin (10 mmol/L). The release of PAME was not affected by MAFP either (Figure II in the online Data Supplement).
Failure of l-NG-Nitroarginine, SKF-525A, and Miconazole to Block Perivascular Adipose Tissue–Derived Relaxing Factor–Induced Aortic Relaxation
Relaxation of phenylephrine-precontracted SD aortic muscle rings induced by Krebs superfusates after superfusion of the aortic PVAT preparations of WKY was not affected by l-NG-nitroarginine (100 μmol/L; a NOS inhibitor) or SKF-525A (10 μmol/L) and miconazole (60 μmol/L; inhibitors of epoxyeicosatrienoic acid synthesis) added directly onto either the PVAT preparations or aortic muscle rings (Figure III in the online Data Supplement). These inhibitors applied directly onto the aortic muscle rings did not affect aortic relaxation induced by exogenous PAME (0.1 μmol/L) applied directly onto the rings (data not shown).
Parallel Blockade of Perivascular Adipose Tissue–Derived Relaxing Factor– and Palmitic Acid Methyl Ester–Induced Aortic Relaxations by Inhibitors of Voltage-Dependent K+ Channels
Relaxation of aortic muscle rings induced by Krebs superfusates after superfusion of the WKY aortic PVAT preparations, indicative of PVATRF release, was blocked by tetraethylammonium (5 and 10 mmol/L; Figure 3B) and 4-AP (2 mmol/L; Figure 3C) added directly onto aortic muscle rings. These concentrations of TEA and 4-AP also blocked aortic relaxation induced by exogenous PAME (0.1 μmol/L) applied directly onto the aortic rings (Figure 3B and 3C). Tetraethylammonium 1 or 3 mmol/L (Figure 3B) applied onto the aortic muscle rings did not affect aortic relaxations induced by PVATRF or exogenous PAME (0.1 μmol/L). In the presence of 4-AP, inhibition of concentration-response curves for PVATRF-, exogenous PAME–, and retina-derived PAME7–induced aortic relaxations (Figure 3D) was similar with IC50 values of 0.58±0.24, 0.43±0.24, and 0.29±0.08 mmol/L, respectively. In contrast, glibenclamide (3 μmol/L) and iberiotoxin (100 nmol/L) applied directly onto aortic muscle rings did not affect the PVATRF- or exogenous PAME–induced aortic relaxations (data not shown).
Lack of Effect of Heating on the Vasodilatory Activities of Krebs Solutions Containing Perivascular Adipose Tissue–Derived Relaxing Factor or Exogenous Palmitic Acid Methyl Ester
Krebs solutions containing exogenous PAME (0.1 μmol/L) or PVATRF (by incubating aortic PVAT of 18- to 20-week-old WKY in Krebs solution at 37°C for 1 hour) applied directly onto the phenylephrine-precontracted SD aortic muscle rings caused relaxation of the rings (Figure 4A and 4B). The relaxation (Figure 4A and 4B) and PAME concentrations in the incubated Krebs solutions (Figure 4B) were not affected after these solutions were heated at 70°C for 10 minutes (Figure 4A and 4B).
Parallel Reduction by Hexane Extractions of the Aortic Relaxations Induced by Perivascular Adipose Tissue–Derived Relaxing Factor– and Palmitic Acid Methyl Ester–Containing Krebs Solutions
The Krebs solutions containing PVATRF after incubation of PVAT preparations at 37°C for 30 minutes and those containing exogenous PAME (0.1 μmol/L) were subjected to hexane extractions. After 1:1 hexane extractions (3 mL Krebs solution extracted with 3 mL hexane) 3 times, both PVATRF- and PAME-containing Krebs solution–induced relaxations of phenylephrine-precontracted SD aortic muscle rings were significantly decreased (Figure 4C). The residual relaxations were abolished by 4-AP (2 mmol/L; Figure 4C). After 2:1 hexane extractions (3 mL Krebs solution extracted with 6 mL hexane) 3 times, aortic relaxation induced by both PVATRF- or PAME-containing Krebs solutions was significantly decreased by 87.96±6.5% and 87.14±6.5%, respectively, of their controls (Figure 4D). No PAME was detected by gas chromatography/mass spectrometry in either extracted solutions (data not shown).
Aortic Relaxation Induced By Culture Medium of Differentiated Adipocytes But Not by That of Fibroblasts
The culture mediums of NIH 3T3 cells and 3T3 L1 cells collected before and after adipocyte differentiation were examined for their vasodilatory activities and PAME concentrations. The medium collected at the fibroblast stage of both lineages did not induce appreciable relaxation of phenylephrine-precontracted SD aortic muscle rings or contain PAME (Figure 5A, 5B, and 5D). In contrast, culture medium of differentiated adipocytes of NIH 3T3 (Figure 5A and 5B) contained a significant concentration of PAME and caused aortic relaxation (Figure 5A and 5B), which was inhibited by tetraethylammonium (10 mmol/L; Figure 5C). The culture medium of differentiated adipocytes from 3T3 L1 cells contained less PAME and induced relatively small relaxation of aortic muscle rings (Figure 5D).
Diminished Spontaneous Releases of Perivascular Adipose Tissue–Derived Relaxing Factor and Palmitic Acid Methyl Ester From the Aortic Perivascular Adipose Tissue of the Spontaneously Hypertensive Rat
In contrast to relaxation of phenylephrine-precontracted SD aortic muscle rings after Krebs superfusion of PVAT preparations of normotensive WKY (18 to 20 weeks old) and prehypertensive SHR (8 to 10 weeks old; Figure 6A, 6C and 6D), Krebs superfusation of PVAT preparations of the SHR (18 to 20 weeks old, established hypertension) caused constriction of phenylephrine-precontracted SD aortic muscle rings (Figure 6B through 6D). The spontaneous release of PAME from the PVAT also significantly diminished in hypertensive SHR (Figure 6D, and Figure IV in the online Data Supplement).
Release of Angiotensin II Vasoconstrictor From the Aortic Perivascular Adipose Tissue of the Spontaneously Hypertensive Rat
Similar to that found in phenylephrine-precontracted aortic muscle ring (Figure 6B and 7A), aortic muscle rings in the absence of active muscle tone (Figure 7A) constricted by Krebs perfusates after superfusion of PVAT preparations of the SHR (18 to 20 weeks old). The aortic constriction was reversibly blocked by losartan (1 μmol/L; Figure 7A). In the presence of phenylephrine-precontracted active muscle tone, constriction of aortic muscle rings induced by Krebs perfusates after superfusion of PVAT preparations of the SHR (18 to 20 weeks old), however, was blocked and converted to vasorelaxation by losartan (Figure 7A and 7B). The spontaneous release of AII from PVAT preparations of the SHR significantly increased compared with that from PVAT preparations of the WKY (Figure IV in the online-only Data Supplement).
Diminished Palmitic Acid Methyl Ester–Induced Relaxation of the Aortic Smooth Muscle Ring of the Spontaneously Hypertensive Rat
Aortic muscle rings of normotensive WKY (20 weeks old) and prehypertensive SHR (8 weeks old) relaxed on applications of exogenous PAME in a concentration-dependent manner (Figure 7C) with EC50 values of 29.9±14.2 and 11.8±10.5 pmol/L, respectively. In contrast, aortic muscle rings of the hypertensive SHR (18 to 20 weeks old) almost did not relax on applications of exogenous PAME with concentrations up to 10 nmol/L (Figure 7C). The loss of responsiveness to PAME was ameliorated by losartan (Figure 7C). In addition, the vascular smooth muscle of the SHR (18 to 20 weeks old) as donor tissue caused constriction of SD aortic muscle ring, which was blocked by losartan administered directly onto the aortic ring (Figure 7D).
Greater Influence of Perivascular Adipose Tissue on Phenylephrine-Induced Aortic Constriction in the Wistar Kyoto Than the Spontaneously Hypertensive Rat
Phenylephrine induced concentration-dependent contractions of endothelium-denuded aortic rings of WKY (8 to 10 and 18 to 20 weeks old) and age-matched SHR. The contractions were significantly greater in aortic rings without PVAT than those with PVAT in WKY of both ages (Figure 8A and 8B) and prehypertensive SHR (Figure 8C) with geometric mean EC50 values indicated in Figure 8A through 8C. The contractions, however, were not different between rings with or without PVAT in established hypertensive SHR (18 to 20 weeks old; Figure 8D). KCl-induced aortic contractions were not significantly different between arteries with and those without PVAT in WKY or SHR of all ages examined (Figure 8E through 8H).
Results of the present study provide strong evidence that PAME is a PVATRF. This is based on findings of similar chemical properties and pharmacological actions of PAME and PVATRF. Furthermore, release of PAME and PVATRF (indicated by aortic relaxation) from the PVAT and relaxation of aortic muscle cells induced by PAME decrease drastically in established hypertension.
It is well accepted that the PVAT of several vascular beds releases vasorelaxing factor(s).2–4,12,13 This is supported by results of the present study using a superfusing bioassay cascade method.9,10 The advantage of this method is that released substances from the PVAT, whether hydrophilic or lipophilic, will reach the detector aortic rings. In addition, the use of exclusive smooth muscle rings as detector tissues eliminates confounding effects from the ECs and PVAT,14,15 which are known to release many vasoactive substances.16–18 Therefore, this superfusing bioassay cascade method allows better determination of the direct transmission between the PVAT and the smooth muscle and whether PVATRF acts directly on the smooth muscle to induce vasorelaxation.
It was suggested that PVATRF was neither a prostanoid nor an epoxyeicosatrienoic acid.2,3,12,19 This is supported by present results that the effects of the released PVATRF as indicated by relaxation of the detector aortic muscle rings were not inhibited by blocking synthesis of prostaglandins and epoxyeicosatrienoic acid. The release of PVATRF and PAME from PVAT was not inhibited by MAFP, a PLA2 inhibitor, further supporting that neither PVATRF nor PAME was an arachidonic acid metabolite.
The PVAT, which plays a paracrine role in regulating vascular function in health and disease,5,10,12,20–24 releases several vasoactive molecules, including, among other, leptin, adiponectin, and AII.13 Of these vasoactive substances, leptin, a vasodilator, plays important roles in regulating vascular function.25–29 Leptin-induced vasodilation, however, is endothelium dependent,24 which is different from the endothelium-independent relaxation induced by PVATRF.2,3 These results suggest that leptin is not likely the PVATRF. Furthermore, adiponectin, which relaxes aortic and mesenteric rings by opening voltage-dependent K+ (Kv) channels, was thought to be a potential PVATRF.20 This possibility was ruled out because the anticontractile effect of PVAT still existed in adiponectin-deficient mice.20 The role of peptides as a potential PVATRF as initially proposed by Löhn et al2 becomes tenuous.
We have reported that PAME released in the superior cervical ganglion6 and the retina7 of the rat is a potent vasodilator by opening Kv channels.7 In the present study, the almost identical concentration-response curves, in the presence of 4-AP (2 mmol/L; a specific Kv channel inhibitor),30–33 for vasodilation induced by PVATRF, retina-derived PAME,7 and exogenous PAME prompted us to propose that PAME was likely the PVATRF. This hypothesis is supported by results of the present study.
First, release of PVATRF and PAME decreased drastically when the PVAT preparation was superfused with Ca2+-free Krebs solution. This Ca2+-dependent release is consistent with reports by others.2
Second, the vasorelaxing effect of PVATRF and PAME from the aortic PVAT of the WKY was independent of nitric oxide synthesis or the presence of endothelium. This result is consistent with reports by others2,3,19 in aorta of the SD and in human internal thoracic artery, although this conclusion is not universally supported by others14,15 in the Wistar rat aortic preparations. The exact reason for the different findings is not known. Species variation and/or different PVATRFs may be responsible. Evidence presented in the present study, however, clearly indicates that both PVATRF and PAME acted directly on the smooth muscle to induce vasorelaxation.
Third, our present results indicate that both PVATRF and PAME acted through the Kv channel. This was based on the findings that aortic relaxation induced by PVATRF was inhibited by 4-AP (2 mmol/L; a preferential inhibitor for Kv channels) and tetraethylammonium in concentrations >5 mmol/L, which are known to inhibit Kv channels, in addition to calcium-activated potassium channels and ATP-sensitive K+ channels.17 The PVATRF-induced relaxation was not affected by lower concentrations of tetraethylammonium (1 or 3 mmol/L; a preferential inhibitor for calcium-activated potassium channel30,31,33,34), iberiotoxin (a specific inhibitor for calcium-activated potassium channel30,33), or glibenclamide (a KATP channel inhibitor30,31,33,34). These results suggest that both PVATRF- and PAME-induced aortic relaxations are mediated by opening the Kv channel on the smooth muscle cells of the SD aorta. This result is consistent with reports by many others3,20 in SD aorta and mesenteric arteries. Other types of potassium channels, however, have been proposed to mediate PVATRF-induced vasorelaxation.2,12,14,15 The variations again may be due to different PVATRFs, bioassay methods, vascular beds and/or animal species.
Fourth, the aortic relaxations induced by Krebs solutions containing PAME or PVATRF were decreased to a similar extent after hexane extractions, and the residual relaxations were blocked in parallel by 4-AP. These results suggest that the PVATRF was lipophilic rather than hydrophilic or a peptide. In addition, both the vasorelaxing effect and concentration of PAME in PVAT-incubated Krebs solutions were not affected after the solutions were heated at 70°C for 10 minutes, suggesting that vasorelaxing activities of PVATRF and PAME were heat stable. The lipid nature of PVATRF may partly explain the discrepancy between our results and those of others.2,14,15 For example, the bioassay methods by others2,14,15 included a donor chamber containing PVAT preparations and an acceptor chamber containing vessel rings without PVAT. Their experimental protocols involved transferring aliquots of bath solution from the donor chamber to the acceptor chamber. A hydrophobic substance like PAME, which stays on the surface of Krebs solution, most likely would not be in the transferred aliquots to the acceptor chamber. Even if it was, it most likely did not reach the detector vessel rings in the acceptor chamber because it would quickly come out of the Krebs solution and stay on the surface of the solution. This problem was avoided, as already been stated, by the use of the superfusion bioassay cascade technique.
Fifth, the adipocytes are the cellular origin of PVATRF and PAME. This is consistent with the report by others2 that PVATRF is released from the adipocytes. The present study demonstrated that significant release of PVATRF and PAME was found in the medium of differentiated adipocytes from NIH 3T3 and 3T3 L1, whereas no significant release was found in the mediums of their respective fibroblasts. There was also positive correlation between the degree of relaxation and PAME concentration in the medium (Figure 5B and 5D). As a negative control, aortic smooth muscle of the WKY did not release PVATRF or PAME, further indicating that PVATRF and PAME are released from the PVAT.
Finally, release of PVATRF and PAME was reduced significantly in rats with established hypertension (20-week-old SHR) compared with prehypertensive SHR and normotensive WKY. This is consistent with reports by others that release of PVATRF decreases in different hypertensive animal models.5,10,35 In addition, PAME almost did not induce relaxation of aortic smooth muscle rings of hypertensive SHR, whereas significant vasorelaxation induced by PAME was seen in WKY and prehypertensive SHR. Thus, spontaneous release of PAME in the PVAT and the aortic response to PAME or PVATRF are significantly decreased in established hypertension. This is supported further by the findings that phenylephrine-induced constriction of aortic rings containing PVAT is smaller than that of aortic rings without PVAT in WKY and prehypertensive SHR. This is likely due to negation of phenylephrine-induced constriction by spontaneously released PVATRF/PAME in the PVAT. As expected, the difference in phenylephrine-induced constriction was not detected in hypertensive SHR, probably because of diminished PAME release. KCl-induced constrictions of the aortic ring with or without PVAT, however, were not different in either WKY or SHR. This is consistent with the findings that activities of Kv channels on the smooth muscle cells mediating vasorelaxation are diminished in high potassium concentrations.36
In addition to the loss of PAME release from the PVAT in established hypertension, the aortic PVAT of hypertensive SHR released a substance that constricted rat aortic smooth muscle. Because the vasoconstriction was blocked by losartan (an AII receptor antagonist) and release of AII was significantly increased in PVAT preparations of the SHR compared with that of the WKY, this vasoconstrictor is likely AII or an AII-like vasoconstrictor. This is very likely because release of AII in the PVAT37,38 and the aortic smooth muscle cells39 of hypertensive rats has been reported. Our present results also suggest release of a losartan-sensitive vasoconstrictor from the aortic smooth muscle cells of the SHR (Figure 7D). Angiotensin II is known to inhibit Kv channels.40 Together, these findings may explain the lack of vasodilatory effects of PAME in the aortic muscle ring of the SHR (Figure 7C) and that antihypertensive effect of losartan is due in part to its blocking AII effects and preserving PAME-induced vasorelaxation.
The identical chemical and pharmacological properties between PAME and the PVATRF suggest for the first time that the PVATRF is PAME, which plays an important role in regulating vascular tone by opening the Kv channels on the vascular smooth muscle. Diminished release of PAME, increased release of AII from the PVAT, and decreased relaxation activity of the vascular smooth muscle to PAME may contribute to a new mechanism for increased vascular resistance and hypertension. Because leptin also is reported to cause hypertension via central mechanisms,27–29 this PVAT-initiated pathogenesis of hypertension may be an important target for clinical consideration.
Sources of Funding
This work was supported by National Science Council of Taiwan grants NSC-97-2120-M-259-002, NSC-96-2320-B-320-005-MY3, and NSC-99-2320-B-320-008; Tzu Chi University grants TCMRC-C95005-01, TCMRCC95005-02, TCIRP98005-01, and TCIRP98005-02; Buddhist Tzu Chi General Hospital grants TCRD98-34 and TCRD99-18; and the Tzu Chi Foundation.
We are grateful to Drs A.R. Hu and A.H. Lua for technical advice in using gas chromatography/mass spectrometry and to Dr Tsung-Cheng Hsieh for statistical analysis.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.111.027375/-/DC1.
- Received February 26, 2011.
- Accepted July 6, 2011.
- © 2011 American Heart Association, Inc.
- Löhn M,
- Dubrovska G,
- Lauterbach B,
- Luft FC,
- Gollasch M,
- Sharma AM
- Verlohren S,
- Dubrovska G,
- Tsang SY,
- Essin K,
- Luft FC,
- Huang Y,
- Gollasch M
- Gálvez B,
- de Castro J,
- Herold D,
- Dubrovska G,
- Arribas S,
- González MC,
- Aranguez I,
- Luft FC,
- Ramos MP,
- Gollasch M,
- Fernández Alfonso MS
- Lin HW,
- Liu CZ,
- Cao D,
- Chen PY,
- Chen MF,
- Lin SZ,
- Mozayan M,
- Chen AF,
- Premkumar LS,
- Torry DS,
- Lee TJ
- Lee YC,
- Chang HH,
- Liu CH,
- Chen MF,
- Chen PY,
- Kuo JS,
- Lee TJ
- Si ML,
- Lee TJ
- Furchgott RF,
- Vanhoutte PM
- Malinowski M,
- Deja MA,
- Gołba KS,
- Roleder T,
- Biernat J,
- Woś S
- Fésüs G,
- Dubrovska G,
- Gorzelniak K,
- Kluge R,
- Huang Y,
- Luft FC,
- Gollasch M
- Frühbeck G
- Lembo G,
- Vecchione C,
- Fratta L,
- Marino G,
- Trimarco V,
- d'Amati G,
- Trimarco B
- Shek EW,
- Brands MW,
- Hall JE
- Morgan DA,
- Thedens DR,
- Weiss R,
- Rahmouni K
- Gollasch M,
- Ried C,
- Bychkov R,
- Luft FC,
- Haller H
- Takemori K,
- Gao YJ,
- Ding L,
- Lu C,
- Su LY,
- An WS,
- Vinson C,
- Lee RM
- Rainbow RD,
- Norman RI,
- Everitt DE,
- Brignell JL,
- Davies NW,
- Standen NB
Methyl palmitate or palmitic acid methyl ester is released spontaneously from the perivascular adipose tissue, causing vasorelaxation by opening voltage-dependent K+ channels on vascular smooth muscle cells of normotensive rats. In established hypertensive rats, diminished palmitic acid methyl ester release and its vasorelaxing activity are accompanied by an increased release of angiotensin II from the aortic perivascular adipose tissue. The diminished palmitic acid methyl ester–induced relaxation of aortic arteries from hypertensive rats is significantly reversed by losartan. These findings suggest a noble role of the perivascular adipose tissue in the pathogenesis of hypertension and provide a new mechanism for losartan in that its antihypertensive effect is partly attributed to its reversing the diminished palmitic acid methyl ester–induced vasorelaxation caused by angiotensin II.