Perivascular Adipose Tissue's Impact on Norepinephrine-Induced Contraction of Mesenteric Resistance Arteries

Figures

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  Figure 1. Maoa and Aoc3 are highly expressed in rat mesenteric PVAT. Amine oxidase gene expression of (A) Maoa, (B) Maob, and (C) Aoc3 (gene for SSAO) in mesenteric resistance vessels (MRV) and in mesenteric PVAT (MPVAT) normalized to β-actin as the reference gene. Bars represent means ± SEM. Means were compared with a one-way ANOVA followed by the Holm-Sidak's multiple comparisons test for parametric data sets (Maob) or the Kruskal-Wallis followed by the Dunn's test for multiple comparisons for the non-parametric data sets (Maoa and Aoc3). *p < 0.05, **p < 0.01, ****p < 0.0001. The number above each bar indicates the number of animals used.

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  Figure 2. MAO-A, MAO-B, and SSAO but not COMT are present in rat MRV and MPVAT. (A) Western blot analysis of monoamine metabolism enzymes MAO-A, MAO-B, COMT, and SSAO in mesenteric resistance vessels (MRV) and in mesenteric PVAT (MPVAT) from eight animals. Positive controls were gut mucosa for MAO-A, stomach fundus for MAO-B, Jurkat cells for COMT and aorta for SSAO. Densitometry analysis of Western blot bands for (B) MAO-A, (C) MAO-B, (D) SSAO. Bars represent the means ± SEM. Western blot densitometry was statistically analyzed with a paired Student's t-test. *p < 0.05, **p < 0.01.

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  Figure 3. Tyramine drives amine oxidase activity in the MRV, MPVAT, AF, and SVF. Tyramine, a substrate for amine oxidases, increased the oxidase activity in the mesenteric resistance vessels (MRV), mesenteric PVAT (MPVAT), adipocyte fraction (AF), and the stromal-vascular fraction (SVF). The brain and aorta were also tested as positive controls. Bars represent the means ± SEM for the number of animals indicated above each bar. Means were compared by a Mann-Whitney test. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control of that same sample type.

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  Figure 4. SSAO mediates tyramine-driven amine oxidase activity in the MPVAT and its fractions, the AF and SVF, but not the MRV. Percent inhibition of tyramine driven oxidase activity by pharmacological inhibitors of MAO-A (1 μM clorgyline), MAO-B (1 μM pargyline), both MAO-A and MAO-B (10 μM pargyline) or SSAO (1 mM semicarbazide) in the (A) mesenteric resistance vessels (MRV), (B) mesenteric PVAT (MPVAT), (C) adipocyte fraction (AF), (D) stromal vascular fraction (SVF), and the positive controls (E) brain (positive control for MAO-A/B), and (F) aorta (positive control for SSAO). Bars represent the means ± SEM for the number of animals indicated near each bar. Means were analyzed by a Kruskal-Wallis test followed by the Dunn's test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. vehicle control (no inhibition).

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  Figure 5. Benzylamine drives amine oxidase activity in MRV, MPVAT, AF, and the SVF. MPVAT, mesenteric PVAT; AF, adipocyte fraction; MRV, mesenteric resistance vessels; SVF, stromal vascular fraction. The brain and aorta were also tested as positive controls. Bars represent the means ± SEM for the number of animals indicated above each bar. Means were compared by a two-way ANOVA followed by a Mann-Whitney or a t-test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control of that same sample type.

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  Figure 6. SSAO mediates benzylamine-driven amine oxidase activity in the MRV, MPVAT and its fractions, the AF and SVF. Percent inhibition of benzylamine driven oxidase activity by pharmacological inhibitors of MAO-B (1 μM pargyline), both MAO-A and MAO-B (10 μM pargyline) or SSAO (1 mM semicarbazide) in the (A) mesenteric resistance vessels (MRV), (B) mesenteric PVAT (MPVAT), (C) adipocyte fraction (AF), (D) stromal vascular fraction (SVF), and the positive controls (E) brain (positive control for MAO-A/B), and (F) aorta (positive control for SSAO). Bars represent the means ± SEM for the number of animals indicated above each bar. Means were analyzed by a Kruskal-Wallis test followed by the Dunn's test for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. vehicle control (no inhibition).

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  Figure 7. PVAT exhibits an anti-contractile effect on mesenteric arteries exposed to NE. NE-induced contraction of the rat mesenteric resistance arteries with or without PVAT and with vehicle (no inhibitors present). Bars represent means ± SEM. N = the number of animals used in each group.

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  Figure 8. Metabolism of NE and H2O2 release by PVAT inhibits contraction of rat mesenteric resistance arteries. NE-induced contraction of the rat mesenteric resistance arteries with or without PVAT and with or without (vehicle) the following inhibitors of NE metabolism. (A) pargyline (10 μM; inhibits MAO-A and B), (B) semicarbazide (1 mM; inhibits SSAO), (C) SP = semicarbazide (1 mM) and pargyline (10 μM inhibits both MAO-A/B and SSAO). Arteries +PVAT were incubated with (D) SP and/or catalase (2000 U/ml). Force of contraction was normalized to the percent of the 60 mM KCl contraction. Bars represent means ± SEM. N = the number of animals used in each group.

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  Figure 9. NE-induced contraction of mesenteric resistance arteries with and without PVAT incubated with NE uptake inhibitors. (A) NE-induced contraction of the rat mesenteric resistance arteries + or −PVAT, with corticosterone (100 μM; inhibits OCT3) or vehicle (H2O). (B) NE-induced contraction of the rat mesenteric resistance arteries + or −PVAT, incubated with nisoxetine (1 μM; inhibits NET) or vehicle (H2O). (C) SPC = semicarbazide (1 mM), pargyline (10 μM), and corticosterone (100 μM; used to inhibit SSAO, MAO-A/B and OCT3), and (D) SPN = semicarbazide (1 mM), pargyline (10 μM) and nisoxetine (1 μM; used to inhibit SSAO, MAO-A/B, and NET). The force of contraction was normalized to the percent of the 60 mM KCl contraction. Bars represent means ± SEM. N = the number of animals used in each group.

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  Figure 10. Diagram of NE handling in PVAT. The hypothesized mechanism for handling of NE by MPVAT and the MRV is shown above. PVAT contributes to the uptake and metabolism of NE from the adventitial side of the artery. Without PVAT, more NE reaches the artery to cause contraction.