Yu-Jing Gao

Endothelium-dependent relaxation factor released by perivascular adipose tissue.

Objective Recent studies have demonstrated that perivascular adipose tissue (PVAT) releases vascular relaxation factor(s), but the identity of this relaxation factor remains unknown. Here, we examined if angiotensin 1-7 [Ang-(1-7)] is one of the relaxation factors released by PVAT. Method Morphological and functional methods were used to study aorta from adult Wistar rats. Results Immunohistochemical staining showed abundant presence of Ang-(1-7) in aortic PVAT. In vessels with PVAT removed but intact endothelium (PVAT − E+), contraction induced by phenylephrine was attenuated by preincubation with Ang-(1-7). PVAT − E+ vessels precontracted with phenylephrine showed a concentration-dependent relaxation response to Ang-(1-7), and this response was abolished by the removal of endothelium. Relaxation response induced by Ang-(1-7) was also prevented by Ang-(1-7) receptor (Mas) antagonist (A779), nitric oxide synthase inhibitor, and nitric oxide scavenger. Ang-(1-7) did not cause a relaxation response in aorta precontracted with KCl, and the relaxation response to Ang-(1-7) was also blocked by calcium-dependent potassium (KCa) channel blockers. Incubation of PVAT + E+ vessels with A779 or angiotensin-converting enzyme 2 inhibitor DX600 or angiotensin-converting enzyme inhibitor enalaprilat increased the contraction induced by phenylephrine. Transfer of donor solution incubated with PVAT + E+ vessel to recipient PVAT − E+ vessel caused a relaxation response. This relaxation response was abolished when donor vessels were incubated with DX600 or enalaprilat or when recipient vessels were incubated with A779. Conclusion Ang-(1-7) released by PVAT acts on the endothelium to cause the release of nitric oxide, and nitric oxide acts as a hyperpolarizing factor through KCa channels to cause relaxation of the blood vessel.

Mechanisms of hydrogen-peroxide-induced biphasic response in rat mesenteric artery

In phenylephrine (PHE) (1 μM)‐precontracted superior mesenteric arteries from adult rats, low concentration of hydrogen peroxide (H2O2, 10–100 μM) caused only contraction, while high concentration of H2O2 (0.3–1 mM) caused a biphasic response: a transient contraction followed by a relaxation response. Endothelium removal did not affect the biphasic response. 7,7‐Dimethyl‐(5Z,8Z)‐eicosadienoic acid, diclofenac, furegrelate, or SQ 29548 greatly inhibited the contraction but did not affect the relaxation. 17‐Octadecynoic acid, eicosatriynoic acid, ICI 198615, SQ 22536, or ODQ did not inhibit the biphasic response. KCl at 40 mM inhibited the relaxation response to H2O2 by 98±24%. 4‐Aminopyridine (4‐AP) inhibited while tetraethylammonium chloride (TEA), charybdotoxin, or glibenclamide attenuated the relaxation response. A combination of 4‐AP, TEA and glibenclamide mimicked the effects of 40 mM KCl. Iberiotoxin, apamin, or barium chloride did not inhibit the relaxation response. H2O2 at 1 mM hyperpolarized membrane potential and reversibly augmented K+ current in smooth muscle cells of mesenteric artery. These effects of H2O2 were attenuated significantly by 4‐AP. In summary, in PHE‐precontracted rat mesenteric artery: (1) the response to H2O2 shifted qualitatively from contraction to a biphasic response as H2O2 increased to 0.3 mM or higher; (2) the relaxation response is caused by the activation of K+ channels, with voltage‐dependent K+ channels playing a primary role; and the contraction is likely to be mediated by thromboxane A2; (3) the K+ channel activation by H2O2 is independent of phospholipase A2, cyclooxygenase, lipoxygenase, cytochrome P450 monooxygenase, adenylate or guanylate cyclase.

Hydrogen peroxide induces a greater contraction in mesenteric arteries of spontaneously hypertensive rats through thromboxane A2 production

Hydrogen peroxide (H2O2) caused a transient contraction in endothelium‐intact (E+) and ‐denuded (E−) mesenteric arteries (MA) from 8 – 10‐month‐old spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto rats (WKY) in a concentration‐dependent manner (10−5 M to 10−3 M). The contraction to H2O2 in MA (E+ or E−) was greater in SHR than in WKY. Removal of endothelium potentiated the contraction to H2O2 in WKY but not in SHR. Tachyphylaxis to H2O2 was less prominent in SHR than in WKY. The contraction of aorta to H2O2 (5×10−4 M), expressed as a percentage of 80 mM KCl‐induced contraction, was approximately half of that found in the MA. A greater contraction was found in E+ but not E− SHR aortic rings. The contraction of MA to H2O2 (5×10−4 M) was greatly inhibited by SQ 29548 and ICI 192605 (thromboxane A2 (TXA2)/prostaglandin H2 receptor antagonists), quinacrine (a phospholipase A2 (PLA2) inhibitor), indomethacin and diclofenac (cyclooxygenase (COX) inhibitors), and furegrelate (a TXA2 synthase inhibitor). Production of thromboxane B2 induced by H2O2 (5×10−4 M) was greater in SHR MA than in WKY, and was inhibited by quinacrine, indomethacin and diclofenac, and furegrelate, but not by SQ 29584 and ICI 192605. These results suggested (1) that SHR MA exhibits a higher contraction involving an increased smooth muscle reactivity and less tachyphylaxis to H2O2 than WKY; (2) that a greater production of TXA2 through activation of PLA2‐COX‐TXA2 synthase pathway appeared to be responsible for the enhanced contraction in SHR MA. The enhanced vascular response to H2O2 may be related to hypertension in SHR.

Hydrogen peroxide is an endothelium‐dependent contracting factor in rat renal artery

In addition to endothelium‐derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium‐derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H2O2) in endothelium‐dependent contraction (EDC) of rat renal artery to acetylcholine (ACh). ACh (10−6, 10−5, and 10−4 M) induced a transient contraction of rat renal artery with intact endothelium in a concentration‐related manner, but not in the artery with endothelium removed. In phenylephrine‐precontracted renal arteries, ACh induced an endothelium‐dependent relaxation response at lower concentrations (10−8–10−6 M), and a relaxation followed by a contraction at higher concentrations (10−5 M). Inhibition of nitric oxide synthase by Nω‐nitro‐L‐arginine (10−4 M) enhanced the EDC to ACh. Catalase (1000 U ml−1) reduced the EDC to ACh. H2O2 (10−6, 10−5, and 10−4 M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium‐independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh‐induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17‐octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H2 receptors (SQ 29548) and thromboxane A2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H2O2. In isolated endothelial cells, ACh (10−5 M) induced a transient H2O2 production detected with a fluorescence dye sensitive to H2O2 (2′,7′‐dichlorofluorescein diacetate). The peak concentration of H2O2 was 5.1 × 10−4 M at 3 min and was prevented by catalase. Taken together, these results show that ACh triggers H2O2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H2O2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H2O2 is most likely the EDCF in rat renal artery in response to ACh stimulation.

Mechanisms for perivascular adipose tissue-mediated potentiation of vascular contraction to perivascular neuronal stimulation: The role of adipocyte-derived angiotensin II

In rat mesenteric arteries we have recently found that perivascular adipose tissue (PVAT) promoted vasoconstriction to perivascular neuronal activation (by electrical field stimulation, EFS) through generation of superoxide. In this study, we examined the role of adipocyte-generated angiotensin II in PVAT-mediated potentiation of contraction to nerve stimulation. In rat mesenteric PVAT, the presence of angiotesinogen and angiotensin I-converting enzyme (ACE) mRNA was confirmed by RT-PCR. Immunohistochemical staining showed the presence of angiotensin II in mesenteric PVAT. In rat mesenteric arteries, treatment of the vessels with an ACE inhibitor (enalaprilat) or angiotensin II type 1 receptor antagonist (candesartan) reduced PVAT-mediated potentiation of EFS-induced contraction. Exogenously applied angiotensin II enhanced EFS-induced contraction in arteries without PVAT, but not in the arteries with intact PVAT. Chronic treatment with an ACE inhibitor quinapril (14 days) lowered blood pressure and alleviated the potentiation effects of PVAT in EFS-induced contraction. Mesenteric arteries from quinapril-treated group now exhibited the potentiation response to exogenously applied angiotensin II in arteries with intact PVAT to a comparable level as in arteries with PVAT removed. Treatment with hydralazine reduced blood pressure to the same level as quinapril treatment, but did not affect PVAT-associated potentiation of vasoconstriction to EFS and the response to exogenously applied angiotensin II in PVAT-intact arteries. These results showed that adipocyte-derived angiotensin II is critically involved in PVAT-mediated potentiation of EFS-evoked contraction in rat mesenteric arteries.

Alterations in perivascular adipose tissue structure and function in hypertension.

We studied the structural and the functional alterations of perivascular adipose tissue (PVAT) in hypertension with spontaneously hypertensive rats (SHR). Measured with dual energy X-ray absorptiometry, a smaller body fat mass and a greater lean mass were found in SHR than in Wistar-Kyoto (WKY) rats, while body weight was comparable between them. In the thoracic PVAT, the density and the total number of brown adipocytes were greater in SHR than in WKY rats, while the cross section area of PVAT was similar between them. In functional assessment, four types of vessel preparations (with either intact PVAT or intact endothelium, or with both, or without both) were employed. Vessels with intact PVAT from SHR contracted more to phenylephrine than that from WKY rats, while vessels without PVAT exhibited comparable contractile response to phenylephrine between SHR and WKY rats. Both endothelium-dependent and -independent components of PVAT-associated attenuation of phenylephrine-induced contraction were reduced in SHR as compared with that of WKY rats. Bioassay experiments were carried out to assess the transferable relaxation factor from the PVAT. Transfer of bathing solution incubated with PVAT-intact vessel caused less relaxation in SHR recipients than in WKY rats, and the relaxation response was abolished by D-Ala(7)-angiotensin-(1-7), an angiotensin-(1-7) receptor antagonist. In summary, PVAT-associated inhibition of vessel contractile response to agonist was impaired in SHR, and the impairment involved both endothelium-dependent and -independent mechanisms. The functional impairment observed in SHR PVAT may be related to changes in adipocyte composition but not to reduced PVAT mass in SHR.

Elevated Blood Pressure in Transgenic Lipoatrophic Mice and Altered Vascular Function

The role of perivascular fat in the control of vascular function was studied using lipoatrophic A-ZIP/F1 transgenic mice. Only a small amount of brown fat was found around the aorta but not around mesenteric arteries. Blood pressure of A-ZIP/F1 mice became higher than wild-type (WT) mice from 10 weeks of age. The presence of perivascular fat reduced the contraction of WT aorta to phenylephrine and serotonin, whereas this effect was either absent or less prominent in A-ZIP/F1 aorta. In WT mice, transfer of solution incubated with aorta with fat to aorta with fat removed caused a relaxation response, but not in A-ZIP/F1 mice, indicating the release of a relaxation factor from perivascular fat in WT aorta. This factor was acting through the activation of calcium-dependent potassium channels. Perfusion of phenylephrine to the isolated mesenteric bed caused a higher increase in perfusion pressure in A-ZIP/F1 than in WT mice. Contractile response of aorta to angiotensin II (Ang II) was mediated by Ang II type 1 receptors and was higher in A-ZIP/F1 than in WT mice. Expression of Ang II type 1 receptors but not Ang II type 2 receptors was higher in aorta of A-ZIP/F1 than WT mice. Treatment with an Ang II type 1 receptor antagonist (TCV 116, 10 mg/kg per day) for 2 weeks normalized the blood pressure of A-ZIP/F1 mice. These results suggest that the absence of perivascular fat tissue, which enhances the contractile response of the blood vessels to agonists, and an upregulation of vascular Ang II type 1 receptors in A-ZIP/F1 mice, are some of the mechanisms underlying the blood pressure elevation in these lipoatrophic mice.

Modulation of vein function by perivascular adipose tissue

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-Pro(7)-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 (K(v)) 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 K(v) channels.

Effects of hyperglycemia on the modulation of vascular function by perivascular adipose tissue.

Objective To study the acute and chronic effect of hyperglycemia on perivascular adipose tissue (PVAT) function in rat aorta. Method Alterations in PVAT function in rat aorta incubated with 22 mmol/l D-glucose for 30 min and in aorta from streptozotocin (STZ)-induced diabetic rats were studied. Results Incubation with D-glucose caused an attenuation of contraction in response to phenylephrine, both in the presence and absence of endothelium, whereas removal of PVAT eliminated this attenuation effect. The presence of PVAT did not affect concentration-related relaxation response of the aorta to carbamylcholine in STZ rats. There was also no difference in the relaxation response of the aorta to carbamylcholine between STZ and control rats. The presence of PVAT, however, caused a higher attenuation of the concentration-dependent contraction to phenylephrine in aorta from STZ rats with intact endothelium as compared with that from control rats. Incubation of the aorta from control rats with Nω-nitro-L-arginine or carboxy-2-phenyl-4,4,5,5-tetra-methyl-imidazoline-1-oxyl-3-oxide potentiated the contraction of the vessels to phenylephrine, and this potentiation effect was higher in the vessels from STZ rats than control rats when Nω-nitro-L-arginine was used. Removal of PVAT reduced this potentiation effect and eliminated the difference between the vessels from control and STZ rats. Conclusion Under both acute and chronic conditions, hyperglycemia enhanced the relaxation response of the vessels mediated by PVAT. These new findings provide important information on the mechanism underlying the postprandial effect of hyperglycemia on blood pressure control and the presence of hypotension under chronic hyperglycemia in a type-1 model of diabetes.

Mas receptors in modulating relaxation induced by perivascular adipose tissue.

AIMS Perivascular adipose tissue (PVAT) is known to secrete vascular relaxation factors, and angiotensin 1-7 [Ang-(1-7)] acting on the endothelium is one of the endothelium-dependent relaxation factors. Mas protein is the receptor for Ang-(1-7). Using aorta from Mas-knockout (K/O) and wild type (FVB) mice, we wished to establish the essential role of Mas receptors in mediating the endothelium-dependent relaxation response induced by relaxation factors from PVAT. MAIN METHODS Thoracic aortic rings from K/O and FVB mice were prepared with or without PVAT (PVAT+ and PVAT-) and/or intact endothelium (E+) or with the endothelium removed (E-) for functional studies. The contraction and relaxation responses of these vessels to agonist in the presence of different receptor antagonists were studied. KEY FINDINGS PVAT attenuated the contraction induced by phenylephrine (PHE) in the presence of endothelium only in vessels from FVB mice. Mas receptor antagonists D-Ala-Ang-(1-7) (A779) or D-Pro(7)-Ang-(1-7) enhanced the contraction induced by PHE only in vessels from FVB mice. Ang-(1-7) caused a relaxation response only in E+vessels from FVB mice. Transfer of donor solution from PVAT+ vessels to PVAT- recipient vessels caused a relaxation response among FVB vessels and not among vessels from K/O mice. SIGNIFICANCE Mas receptors are essential in mediating the endothelium-dependent relaxation response induced by PVAT, therefore highlighting the important role of Ang-(1-7) in the control of vascular functions through PVAT.