Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression

Abstract

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

Fig 1. HO activity.

Total HO enzyme activity (pmol CO/h/mg FW) in the (A) livers and (B) aortae of WT (white bars), HO-1 Het (black bars), and heme-treated HO-1 Het (grey bars) mice 28 days after PPE infusion (Post-PPE) and age-matched WT and HO-1 Het controls. *p≤0.006, †p<0.025, **p≤0.05. Number of animals in each group are shown in parentheses.

Fig 2. Profiles of AAA development following PPE infusion.

(A) AAA diameters were measured at baseline and at 3, 7, 14, 21, and 28 days post-PPE infusion by ultrasound in WT (white squares), HO-1 Het (black squares), heme-treated WT (white diamonds), and heme-treated HO-1 Het (black circles) mice. (B) Representative longitudinal ultrasound views of AAAs 28-days post-PPE infusion from HO-1 Het, heme-treated HO-1 Het, WT, and heme-treated WT mice. *p<0.015, †p = 0.0025 compared to WT and heme-treated HO-1 Het mice. n = 7 to 11 for each group.

Fig 3. Immunohistochemical staining.

(A) Immunohistochemical staining for Mac1 in aortae harvested from WT and HO-1 Het mice at 7 and 28 days post-PPE infusion and of heme-treated HO-1 Het mice at 28 days post-PPE infusion. (B) Macrophage infiltration in the aortae of WT (n = 4 and 7) and HO-1 Het (n = 4 and 7) mice at 7 and 28 days post-PPE infusion, respectively, and of heme-treated HO-1 Het mice (n = 5) at 28 days post-PPE infusion (right panel). Data shown as cells per high power field (hpf). *p<0.0001 compared to WT mice.

Fig 4. HO-1 promoter activity in the aorta following PPE infusion in HO-1-luc mice.

Ex vivo images taken 14 days following (A) saline infusion (control) showing weak HO-1 promoter activity (low light intensity), and post-PPE infusion (B) showing high HO-1 promoter activity (strong light intensity) in the aorta.

Fig 5. mRNA levels of the pro-inflammatory cytokines.

Thioglycollate-elicited peritoneal macrophages from HO-1 Het mice exhibited significantly increased mRNA levels of MCP1 (A), TNF-alpha (B), IL-1-beta (C), and IL-6 (D) as measured using RT-PCR. In contrast, mRNA levels of anti-inflammatory cytokines IL-10 (E) and TGF-1 (F) were significantly lower. Moreover, YM-1 (G), one of the M2 macrophage markers, was significantly lower in HO-1 Het mice. As expected, HO-1 (H) levels were lower in HO-1 Het mice. *p<0.05, n = 3 to 5 for each group.

Fig 6. Body weight and lipid profiles.

5 wks after Ang II infusion, body weight (A) and lipid (B) profiles were measured in vehicle- (white squares, n = 10) and rosuvastatin- (black squares, n = 9) treated ApoE^-/- mice. No significant differences were found between the 2 groups.

Fig 7. Effect of rosuvastatin on AAA severity.

(A) Representative ex vivo pictures of aortas harvested from ApoE^-/- mice treated with saline-vehicle (left panel) or rosuvastatin (right panel) following Ang II infusion. (B) Number of pups categorized by AAA severity scores post-Ang II infusion in vehicle- (white squares) or rosuvastatin- (black squares) treated mice. (C) Overall AAA severity in both groups combined. *p = 0.04, n = 7 and 9 for vehicle- and rosuvastatin-treated mice, respectively.

Fig 8. Effect of rosuvastatin on HO activity and AAA severity.

No significant difference in HO activity between aortas from vehicle- (white squares) and rosuvastatin- (black squares) treated ApoE^-/- mice were found 14 days post-Ang II infusion, but when grouped by severity, HO activity in aortas with type-2 expansions was significantly higher in rosuvastatin-treated mice. *p<0.05, n = 3 for each group.