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.
Citation: Azuma J, Wong RJ, Morisawa T, Hsu M, Maegdefessel L, Zhao H, et al. (2016) Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression. PLoS ONE 11(2): e0149288. https://doi.org/10.1371/journal.pone.0149288
Editor: Michael Bader, Max-Delbrück Center for Molecular Medicine (MDC), GERMANY
Received: October 19, 2015; Accepted: January 10, 2016; Published: February 19, 2016
Copyright: © 2016 Azuma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This work was supported in part by research grants from the National Institutes of Health (1R01HL122939 [PST]) and the Department of Veterans Affairs (101BX002641 [PST]).
Competing interests: The authors have declared that no competing interests exist.
Abdominal aortic aneurysm (AAA) disease is a potentially lethal condition that is characterized by the destructive remodeling of the infrarenal (IR) aorta . It is a complex disease process involving the infiltration of inflammatory cells, the production of reactive oxygen species (ROS), upregulation and activation of degradative proteases, inactivation of nascent protease inhibitors, stimulation of apoptosis, degradation of elastin, transmural inflammation, and resistive hemodynamic conditions . Currently, there is no effective medical or pharmaceutical intervention available for small AAAs that might delay or prevent the need for invasive aneurysm repair.
One mechanism of particular interest is the flow-related upregulation of an anti-inflammatory enzyme, heme oxygenase-1 (HO-1). Polymorphisms in the HO-1 promoter region have been linked to AAA disease risk in humans . Three isoforms of HO have been described. Two (HO-2 and HO-3) are constitutively expressed, while HO-1 is inducible by inflammatory stimuli. HO-1 degrades heme and produces equimolar concentrations of the bioactive products: carbon monoxide (CO), which is a vasodilator, ferrous iron, and biliverdin, which is rapidly converted to bilirubin by biliverdin reductase. Biliverdin and bilirubin are potent antioxidants [4,5] and can protect the vascular endothelium and intima-media, and attenuate lesion formation following injury. Other potential protective effects of HO-1 include a reduction of vascular smooth muscle cell (VSMC) proliferation, attenuation of vasoconstriction , scavenging of free radicals, inhibition of platelet aggregation , and up-regulation of transferrin expression with subsequent sequestration of oxidative free iron . Although high levels of HO activity (>15-fold) produce toxic, reactive iron species, moderate increases (<5-fold) appear to be cytoprotective in animal models . It has been shown that heme administration can strongly upregulate HO-1 activity [10,11]. An increase in HO-1 gene expression has been observed under high-flow conditions and associated with experimental AAA attenuation .
3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) inhibitors (or statins) are lipid-lowering agents that are widely used clinically . Reduction of plasma lipids through statins occurs by competitive inhibition of HMG-CoA reductase, an enzyme crucial for cholesterol synthesis. Mechanisms beyond the reduction of plasma cholesterol levels contribute significantly to the anti-atherogenic and tissue protective properties of statins. These pleiotropic, cholesterol-independent benefits include anti-proliferative , immunomodulatory , vascular cytoprotection , angiogenic , plaque stabilizing [17,18], and antioxidative effects [14,19], and anti-inflammatory properties [19,20]. While some trials have seen no improvement in AAA progression in patients undergoing statin therapy , observational studies and a meta-analysis suggest that statin therapy is associated with decreased expansion rates in patients with small AAAs . We previously demonstrated that statins induce HO-1 expression and reduce oxidative stress in vascular cells in vivo [23,24]. We hypothesize that these properties of HO-1 may be one of the therapeutic effects of statins in ameliorating the development of AAA disease.
Materials and Methods
Eight-week-old apolipoprotein E-deficient (ApoE-/-) male mice (n = 18) with a C57BL/6J background were purchased from Jackson Laboratories (Bar Harbor, ME). In our second set of studies, 8-wk-old wild-type (WT, HO-1+/+, n = 14) and HO-1 heterozygous (HO-1 Het, HO-1+/-, n = 13) FVB mice were used and purchased from Jackson Laboratories. For studies investigating the effect of AAA development on HO-1 promoter activity, we used adult HO-1-luc mice (8-wk-old), whose transgene contains the full-length HO-1 promoter fused to the reported gene luciferase. All mice were provided with water and food ad libitum. All studies were approved by the Stanford University Institutional Animal Care and Use Committee.
Angiotensin II-ApoE-/- AAA model
The subcutaneous osmotic angiotensin II (Ang II) infusion model was used to create suprarenal (SR) murine AAAs in the ApoE-/- mice. Under inhaled anesthesia with 2% isoflurane, osmotic mini-pumps (Alzet Model 2004, Durect Corp., Cupertino, CA), prepared in sterile manner with Ang II (Sigma-Aldrich, St. Louis, MO) in saline, were inserted beneath the dorsal skin of each mouse. Pumps were set to deliver Ang II at constant rate of 1000 ng/kg/min [2