Lipoxins Protect Against Inflammation in Diabetes-Associated Atherosclerosis
Article Figures & Tables
Figures
- Figure 1
LXs prevent the development of diabetes-associated atherosclerosis. A: Averaged weekly plasma blood glucose levels in nondiabetic and diabetic ApoE−/− mice administered vehicle (2% ethanol), LXA4, or Benzo-LXA4 from weeks 1 to 16 (n = 8–10, ±SEM). B and C: Sudan IV stain of aortas isolated from 20-week-old diabetic and nondiabetic ApoE−/− mice administered vehicle, LXA4, or Benzo-LXA4, and respective quantification. N = 8–10, ±SEM; *P ≤ 0.05 vs. ApoE−/− + vehicle; φP ≤ 0.05 vs. diabetic ApoE−/− + vehicle. D: Gene expression analysis of markers of vascular inflammation in aortic tissue isolated from 10-week-old diabetic and nondiabetic ApoE−/− mice administered vehicle, LXA4, or Benzo-LXA4. Expression was normalized to 18S for gene expression analysis (n = 8–10, ±SEM; *P ≤ 0.05 vs. ApoE−/− + vehicle; φP ≤ 0.05 vs. diabetic ApoE−/− + vehicle.)
- Figure 2
LXs attenuate established diabetes-associated atherosclerosis. A: Averaged weekly plasma blood glucose levels in nondiabetic and diabetic ApoE−/− mice administered vehicle (2% ethanol), LXA4, or Benzo-LXA4 from weeks 10 to 16 (n = 8–10, ±SEM). B and C: Sudan IV stain of aortas isolated from 16-week-old diabetic and nondiabetic ApoE−/− mice administered vehicle, LXA4, or Benzo-LXA4, and respective quantification (n = 8–10, ±SEM; *P ≤ 0.05 vs. ApoE−/− + vehicle ; φP ≤ 0.05 vs. diabetic ApoE−/− + vehicle). D: Gene expression analysis of markers of vascular inflammation in aortic tissue isolated from 16-week-old diabetic and nondiabetic ApoE−/− mice administered vehicle, LXA4, or Benzo-LXA4 from weeks 10 to 16. Expression was normalized to 18S for gene expression analysis (n = 8–10, ±SEM; *P ≤ 0.05 vs. ApoE−/− + vehicle; φP ≤ 0.05 vs. diabetic ApoE−/− + vehicle.)
- Figure 3
LXA4 suppresses inflammatory cytokine release from human carotid plaques. A–C: ELISA quantification of proinflammatory cytokines released by human carotid plaques in response to LPS priming (1 µg/mL; 24 h) and or LXA4 (1 nmol/L, 100 nmol/L, 1 μmol/L; 30 min) (n = 4; ±SEM; *P ≤ 0.05 vs. LPS; **P < 0.01 vs. LPS; ***P < 0.001 vs. LPS; #P < 0.05 vs. untreated). Data are presented as the percentage of cytokine levels, relative to LPS-treated tissues (100%). D: Cytokine array analysis of secretory protein abundance of human carotid plaques (n = 4) in response to LPS priming (1 µg/mL; 24 h) and or LXA4 (100 nmol/L; 30 min). The heatmap was created by setting the maximal pixel intensity of the reference spots on the array arbitrarily to 100 (red color), to which the abundance of all other analytes is relative. Minimal abundance (0) is encoded by white, maximal abundance (100) by red. E: Quantification of D, indicating fold changes (FC) of cytokines relative to untreated plaque tissue. Cytokines displaying ≥1.5-fold induction in response to LPS priming are shown. ANG, angiotensin; BDNF, brain-derived neurotrophic factor; DPP4, dipeptidyl peptidase 4; FGF, fibroblast growth factor; GRO, growth-regulated oncogene; HGF-SF, hepatocyte growth factor-scatter factor; IFN, interferon; IGF-BP, insulin-like growth factor binding protein; LIF, leukemia inhibitory factor; MIG, monokine induced by interferon-γ; RAGE, receptor for advanced glycation end products; TARC, thymus and activation regulated chemokine; TGF, transforming growth factor; TSP, thrombospondin; uPAR, urokinase plasminogen activator receptor; VDBP, vitamin D binding protein.
- Figure 4
LXA4 regulates vascular smooth muscle activation. A: Gene expression of markers of SMC activation after PDGF treatment (10 ng/mL; 24 h) and/or LXA4 pretreatment (0.1 nmol/L; 30 min). B and C: Representative images and quantification of crystal violet–stained SMC migratory cells after PDGF treatment (10 ng/mL; 6 h) and/or LXA4 pretreatment (0.1 nmol/L; 30 min). D: Cell proliferation as measured by cell number count in SMCs after PDGF treatment (10 ng/mL; 0–72 h) and/or LXA4 pretreatment (0.1 nmol/L; 30 min). E: Expression of VCAM, IL-6, ICAM, and TNFR-A (TNF receptor-α) in SMCs after TNF-Α treatment (1 ng/mL; 24 h) and/or LXA4 pretreatment (0.1 nmol/L; 30 min). F: Luciferase/Renilla ratio results for SMCs transfected with an NF-κB activity reporter plasmid, and subsequently stimulated with TNF-α (1 ng/mL; 24 h) and/or pretreatment with LXA4 (0.1 nmol/L; 30 min) or Benzo-LXA4 (1 nmol/L; 30 min). Experiments were performed three to six times, and are presented as the mean ± SEM. *P ≤ 0.05 vs. vehicle; φP ≤ 0.05 vs. PDGF/TNF-α treatment. PCNA, proliferating cell nuclear antigen; RAGE, receptor for advanced glycation end products.
- Figure 5
LXA4 regulates aortic EC activation. A: Gene expression analysis of markers of vascular inflammation in aortic ECs in response to TNF-α treatment (1 ng/mL; 24 h) and/or LXA4 pretreatment (0.1 nmol/L; 30 min). B and C: Representative images and quantification of labeled THP1 monocytes adhered to ECs stimulated with TNF-α (1 ng/mL; 24 h) and/or LXA4 pretreatment (0.1 nmol/L; 30 min). Expression was normalized to 18S for gene expression analysis (n = 3–4, ±SEM). *P ≤ 0.05 vs. vehicle; φP ≤ 0.05 vs. TNF-α treatment.
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