Modulation of vascular function by perivascular adipose tissue: the role of endothelium and hydrogen peroxide

Abstract

Background and purpose: Perivascular adipose tissue (PVAT) attenuates vascular contraction, but the mechanisms remain largely unknown. The possible involvement of endothelium (E) and hydrogen peroxide (H2O2) was investigated.

Experimental approach: Aortic rings from Wistar rats were prepared with both PVAT and E intact (PVAT+ E+), with either PVAT or E removed (PVAT- E+, or PVAT+ E-), or with both removed (PVAT- E-) for functional studies. Nitric oxide (NO) production was measured.

Key results: Contraction to phenylephrine and 5-HT respectively was highest in PVAT- E-, lowest in PVAT+ E+, and intermediate in PVAT+ E- or PVAT- E+. In bioassay experiments, transferring bathing solution incubated with a PVAT+ ring (donor) to a PVAT- ring (recipient) induced relaxation in the recipient. This relaxation was abolished by E removal, NO synthase inhibition, scavenging of NO, high extracellular K+, or blockade of calcium-dependent K+ channels (K(Ca)). The solution stimulated NO production in isolated endothelial cells and in PVAT- E+ rings. In E- rings, the contraction to phenylephrine of PVAT+ rings but not PVAT- rings was enhanced by catalase or soluble guanylyl cyclase (sGC) inhibitor, but reduced by superoxide dismutase and tiron. In PVAT- E- rings, H2O2 attenuated phenylephrine-induced contraction. This effect was counteracted by sGC inhibition. NO donor and H2O2 exhibited additive inhibition of the contraction to phenylephrine in PVAT- E- rings.

Conclusion: PVAT exerts its anti-contractile effects through two distinct mechanisms: (1) by releasing a transferable relaxing factor which induces endothelium-dependent relaxation through NO release and subsequent K(Ca) channel activation, and (2) by an endothelium-independent mechanism involving H2O2 and subsequent activation of sGC.

Figure 1

Five-micron cross-sections of aorta before (a) and after removal of PVAT (b) sections were stained with Gomori's trichrome. PVAT, perivascular adipose tissue. Magnification bar represents 500 μM.

Figure 2

Concentration–response curves to phenylephrine (a) and to 5-HT (b) in the thoracic aortic rings with (+) or without (−) PVAT or endothelium (E). The presence of PVAT attenuated the contractile response to phenylephrine and to 5-HT in aortic rings with or without E ^**P<0.01, n=5–7.

Figure 3

Typical recording showing that transferring solution incubated with PVAT−intact aortic rings to PVAT-denuded aortic rings induced a relaxation response in the recipient artery with intact endothelium (E+) (a) but not in endothelium-denuded rings (E−) (b). Relaxation induced by this solution transfer was abolished by NO synthase inhibitor (L-NNA, 100 μM) and NO scavenger (carboxy-PTIO, 100 μM) (c). Vessels were precontracted with phenylephrine (0.3 μM). ^**P<0.01 versus before transfer (paired Student t-test, n=5–7).

Figure 4

NO production in isolated aortic endothelial cells induced by solution incubated with PVAT+ aortic rings detected with DAF-2DA (a–d). Images were obtained using fluorescence microscopy before (a), 10 min after exposure to solution incubated with PVAT+ aortic ring (b) and to carbamylcholine (CCh, 1 μM; c) and to the solution in the presence of carboxy-PTIO (100 μM, d). Magnification bar represents 25 μm. CCh and incubation solution from PVAT+ aortic rings induced the production of NO in PVAT− E+ aortic rings (e). ^**P<0.01 versus basal value. (n=4). Solution incubated with PVAT− ring did not change the fluoresence intensity (data not shown).

Figure 5

Relaxation of PVAT-denuded aortic rings induced by transferring solution incubated with PVAT-intact aortic rings was inhibited by K_Ca channel blockers (tetraethylammonium, TEA, 1 mM), and by a combination of charybdotoxin (ChTX, 0.3 μM) and apamin (0.3 μM), but not by 4-aminopyridine (4-AP, 1 mM) or glipizide (10 μM). Vessels were precontracted with phenylephrine (0.3 μM). ^**P<0.01 versus before transfer (paired Student t-test, n=5–7).

Figure 6

(a) Effects of catalase (1000 μ ml⁻¹), superoxide dismutase (SOD, 150 u ml⁻¹), tiron (1 mM), H₂O₂ (30, 100, 300 μM) and ODQ (10 μM) on the contraction of endothelium-denuded aortic rings with/without PVAT (PVAT+ E−, PVAT− E−) to phenylephrine (0.3 μM). Catalase and ODQ enhanced, whereas SOD and tiron reduced the contraction to phenylephrine in PVAT+ E− rings. H₂O₂ had no effect. In PVAT− E− vessels, H₂O₂ concentration-dependently attenuated the contraction to phenylephrine, and this attenuation was blocked by ODQ. (b) Treatment with NO donor (MAHMA NONOate, 100 μM) or H₂O₂ (300 μM), or a combined treatment of these two agents attenuated the contraction to phenylephrine (3 × 10⁻⁷ M) in PVAT− E−rings. ^*P<0.05, ^**P<0.01 versus respective control (paired Student t-test; n=4–6).