Lipocalin-2 deficiency prevents endothelial dysfunction associated with dietary obesity: role of cytochrome P450 2C inhibition

Abstract

Background and purpose: Lipocalin-2 is a pro-inflammatory adipokine up-regulated in obese human subjects and animal models. Its circulating levels are positively correlated with the unfavourable lipid profiles, elevated blood pressure and insulin resistance index. Augmented lipocalin-2 has been found in patients with cardiovascular abnormalities.The present study was designed to investigate the role of lipocalin-2 in regulating endothelial function and vascular reactivity.

Experimental approach: Wild-type and lipocalin-2 knockout (Lcn2-KO) mice were fed with either a standard chow or a high-fat diet. Blood pressures and endothelium-dependent relaxations/contractions were monitored at 2 week intervals.

Results: Systolic blood pressure was elevated by high-fat diet in wild-type mice but not in Lcn2-KO mice. Endothelial dysfunction, reflected by the impaired endothelium-dependent relaxations to insulin and augmented endothelium-dependent contractions to ACh, was induced by high-fat diet in wild-type mice. In contrast, Lcn2-KO mice were largely protected from the deterioration of endothelial function caused by dietary challenges. The eNOS dimer/monomer ratio, NO bioavailability, basal and insulin-stimulated PKB/eNOS phosphorylation responses were higher in aortae of Lcn2-KO mice. Administration of lipocalin-2 attenuated endothelium-dependent relaxations to insulin and promoted endothelium-dependent contractions to ACh. It induced eNOS uncoupling and elevated COX expression in the arteries. Treatment with sulphaphenazole, a selective inhibitor of cytochrome P450 2C9, improved endothelial function in wild-type mice and blocked the effects of lipocalin-2 on both endothelium-dependent relaxations to insulin and endothelium-dependent contractions to ACh, as well as eNOS uncoupling.

Conclusions: Lipocalin-2, by modulating cytochrome P450 2C9 activity, is critically involved in diet-induced endothelial dysfunction.

Figure 1

Starting from 1 week after weaning, mice were fed with standard chow (A and C) or high-fat diet (B and D) until 22–23 weeks of age. Systolic (A and B) and diastolic (C and D) blood pressures of mice were measured every 2 weeks. *P < 0.05 versus wild-type mice; n = 8–12.

Figure 2

Cumulative concentration-response curves for endothelium-dependent relaxations to insulin. The measurement was performed using aortae derived from wild-type or Lcn2-KO mice under high-fat diet conditions (A). The ages of mice were indicated within each panel. Area under the curves was calculated and presented for comparing the effects of prolonged high-fat diet and between the two groups of mice (B, upper panel). Endothelium removal or treatment with L-NAME abolished insulin-induced relaxations of aortae collected from mice fed a high-fat diet for 2–3 weeks (B, bottom panel). *P < 0.05 versus wild-type mice; n = 10–15. EC, endothelial cells.

Figure 3

Carotid artery rings were collected from wild-type and Lcn2-KO mice on a high-fat diet and exposed to ACh in the presence of L-NAME (10⁻⁴ M). Cumulative concentration-response curves were collected for mice with different ages (A). The areas under curve of the cumulative concentration-responses were calculated and presented (B, upper panel). Removal of the endothelium (without EC) or treatment with indomethacin, SC560, S18886, but not NS398 abolished ACh-evoked contractions (B, bottom panel). *P < 0.05 versus respective controls; n = 10–15. EC, endothelial cells.

Figure 4

Basal and insulin (10⁻⁷ M, 15 min)-induced PKB and eNOS phosphorylation (A), nitrite and nitrate levels (B) and nitrotyrosine amount (C) were measured in aortic tissues derived from wild-type and Lcn2-KO mice fed a high-fat diet for 2–3 weeks. The protein (D) and mRNA (E) expressions of COX-1 in carotid arteries were compared between wild-type and Lcn2-KO mice. Generation of superoxide anions by NADPH oxidase was determined using the lucigenin-enhanced chemiluminescence assay (F). ACh-induced accumulation of superoxide anions in wild-type mice was abolished by removal of the endothelium but not by indomethacin (10⁻⁵ M). The average readings are expressed as relative luminescence unit (RLU) and normalized against dried tissue masses. *P < 0.05 versus wild-type mice of the same treatment group; #P < 0.05 versus the basal levels in wild-type mice; n = 5. -EC, without endothelial cells.

Figure 5

Lcn2-KO mice (high-fat diet fed for 2–3 weeks) were given recombinant lipocalin-2 (800 µg per mouse, i.p.). The aortae and carotid arteries were harvested at different time points for evaluation of endothelium-dependent relaxations to insulin (A) and endothelium-dependent contractions to ACh (B), respectively. The area under curve was calculated for comparison (bottom panels). Note that the artery responses were constant throughout the experimental period for animals treated with vehicle control (data not shown). *P < 0.05 versus vehicle-treated wild-type mice; #P < 0.05 versus vehicle-treated Lcn2-KO mice (time zero); n = 5.

Figure 6

Lipocalin-2 was administered into Lcn2-KO mice as in Figure 5. Aortae and carotid arteries were collected at different time points for Western blotting and quantitative PCR to analyse the dimers and monomers of eNOS (A), NO production (B) and COX expressions (C), respectively. The % NO of wild-type aortae was presented from the averages of four experiments. *P < 0.05 versus wild-type mice; #P < 0.05 versus Lcn2-KO mice at time zero; n = 5. The quantitative PCR results are expressed as fold changes versus time zero. Carotid artery collected at 6 h after protein injection was subjected to lucigenin-enhanced chemiluminesence assay as in Figure 4. The results are expressed as relative luminescence unit (RLU) normalized against dried tissue masses (D). Note that lipocalin-2 significantly enhanced NADPH oxidase activity stimulated by ACh (10⁻⁶ M). *P < 0.05 ACh + lipocalin-2 versus other groups; n = 3.

Figure 7

Left: SPZ or vehicle was administered to wild-type mice (under high-fat diet) by i.p. injection. At the end of the treatment period (3 weeks), the aortae and carotid arteries were collected for evaluation of endothelium-dependent relaxations to insulin (A) and endothelium-dependent contractions to ACh (B), respectively. The amount of CYP2C9 protein in aortae of wild-type and Lcn2-KO mice was monitored by Western blotting (C). *P < 0.05 versus the other two groups; n = 5. Right: same treatment was performed in Lcn2-KO mice fed a high-fat diet. At the end of the treatment period (3 weeks), mice were injected with lipocalin-2 protein as described in Figure 5. Six hours after injection, the aortae and carotid arteries were collected for evaluation of endothelium-dependent relaxations to insulin (D) and endothelium-dependent contractions to ACh (E), respectively. Data are presented as areas under curve. Western blotting was performed for analysing the dimers and monomers of eNOS in aortae of these mice (F). *P < 0.05 versus vehicle-treated mice; #P < 0.05 versus lipocalin-2-treated mice; n = 5.