Inhibition of hypoxia inducible factor-1α attenuates abdominal aortic aneurysm progression through the down-regulation of matrix metalloproteinases

Hypoxia inducible factor-1α (HIF-1α) pathway is associated with many vascular diseases, including atherosclerosis, arterial aneurysms, pulmonary hypertension and chronic venous diseases. Significant HIF-1α expression could be found at the rupture edge at human abdominal aortic aneurysm (AAA) tissues. While our initial in vitro experiments had shown that deferoxamine (DFO) could attenuate angiotensin II (AngII) induced endothelial activations; we unexpectedly found that DFO augmented the severity of AngII-induced AAA, at least partly through increased accumulation of HIF-1α. The findings promoted us to test whether aneurysmal prone factors could up-regulate the expression of MMP-2 and MMP-9 through aberrantly increased HIF-1α and promote AAA development. AngII induced AAA in hyperlipidemic mice model was used. DFO, as a prolyl hydroxylase inhibitor, stabilized HIF-1α and augmented MMPs activities. Aneurysmal-prone factors induced HIF-1α can cause overexpression of MMP-2 and MMP-9 and promote aneurysmal progression. Pharmacological HIF-1α inhibitors, digoxin and 2-ME could ameliorate AngII induced AAA in vivo. HIF-1α is pivotal for the development of AAA. Our study provides a rationale for using HIF-1α inhibitors as an adjunctive medical therapy in addition to current cardiovascular risk-reducing regimens.


Preparation of HIF-1α plasmid and transfection of short hairpin RNA. A human HIF-1α open
reading fragment was obtained from the Mammalian Gene Collection and reconstructed into a pOTB7 plasmid vector. The insertion in the new plasmid (pOTB7-HIF-1α ) was confirmed using DNA sequencing. The pOTB7-HIF-1α and empty plasmids were purified using Midi Plasmid Kit PI025 (Geneaid). The purity of the plasmids was verified with the absorbance ratio at 260 and 280 nm and by 1% agarose gel electrophoresis. Short hairpin RNAs (shRNAs) plasmids to knockdown HIF-1α and scrambled control were provided by National RNAi Core Facility of Academia Sinica, Taipei, Taiwan. Transfection was performed using the Lipofectamine ® 3000 (ThermoFisher Scientific), as the recommendation of the manufacturer.
Measurement of ROS production. The homogenates of the cell lysates were stained with 2′ ,7′ -Dichlorofluorescin diacetate (DCFH-DA). DCFH-DA was oxidized by ROS to form the highly fluorescent 2′ ,7′ -dichlorofluorescein. The samples were loaded onto 96-well plates for 30 minutes at 37 °C, and fluorescence intensity was measured with an excitation of 488 nm and an emission of 520 nm.

Measurement of the activities of matrix metalloproteinases.
Gelatin zymography was used to determine the gelatinolytic activities of the MMP-2 and MMP-9 activities of the homogenates of the aorta and conditioned medium as previously described. In brief, equivalent amount of samples were electrophoresed under non-reducing conditions onto 7.5% SDS polyacrylamide gels containing 0.1 mg/ml gelatin as substrate. The gels were washed in a buffer containing 2.5% Triton X-100 for one hour to remove SDS and incubated with a substrate buffer at 37 °C for 18 hours. The MMP activities were then quantified by densitometry scanning.
Chromatin immune-precipitation assay. Chromatin immunoprecipitation (CHIP) assays were performed as previously described 22 . In brief, confluent cells were cross-linked with 4% PFA and then ceased by adding glycerin. Cells were then washed with cold PBS and collected using a FA lysis buffer. After shearing with sonication, the HIF-1α -bound chromatin was immunoprecipitated by rabbit anti-HIF-1α (GeneTex, GTX127309) and mouse IgG (Cell Signaling) linked to protein A/G Dynabeads (Invitrogene). Protein and RNA were then degraded by Proteinase K (100 μ g) and RNase A (1 μ g), respectively. The purified chromatin DNA was subjected to real time-quantitative PCR.
Primer Sequences Used in Chromatin immune-precipitation assay. To predict potential HIF-1 binding sites, hypoxia response element (HRE) on selected human and mouse genes was analyzed using the Scientific RepoRts | 6:28612 | DOI: 10.1038/srep28612 position weight matrix algorithm from TRANSFAC15 to scan the promoter regions of each gene. The promoter region was defined as − 5000 to + 5000 nucleotides from the transcriptional start site. The sequences of the primers used to detect HIF-1α binding to the three HIF-1α binding sites (HRE1, HRE2) in the human MMP-2 and MMP-9 promoter regions are shown in Supplementary Table 4.
Angiotensin II induced abdominal aortic aneurysm model. This is a prospective interventional animal study. Male low-density lipoprotein receptor (LDLR −/− ) mice on a C57BL/6J background were obtained from Jackson Lab. Mice were fed with high fat diet and kept ad libertum. Alzet osmotic minipumps (model 2004; ALZET Scientific Products, Mountain View, California, USA) were implanted into mice at 8-10 weeks of age. Pumps filled with solutions of AngII (Sigma Chemical CO., St. Louis, Missouri, USA) delivered 1000 ng/kg/min of AngII for 28 days as previously described 23 . The pumps were placed into the subcutaneous space of mice through a small incision in the back and were then closed with surgical clips. Avertin (tribromoethanol, 250 mg/kg, intraperitoneally) was used for anaesthesia and mice were considered as adequately anaesthetized when no attempt to withdraw the limb after pressure could be observed. DFO was given 100 mg/kg/day intraperitoneally as previously described in the treatment of atherosclerosis. Digoxin (1.25 mg/kg/day) was given subcutaneously as previously described in inhibiting HIF-1 synthesis and HIF-1α transcriptional activities with the serum concentration of digoxin in at or below the therapeutic range for humans (0.5-2.0 ng/ml) 24-28 . 2-methoxyestradiol (50 mg/kg/day) was given subcutaneously. The animals were monitored during the treatment for their body weight to assess side-effects. At the end of the study, mice were euthanized by exsanguination under anaesthesia. Blood was withdrawn from the right heart ventricle for analysis. All experimental protocols and procedures were approved by the institutional animal care committee of the National Defense Medical Center (Taipei, Taiwan) and complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8 th edition, 2011) 29 .
Determination of blood pressure and serum levels of lipid profiles. Systolic blood pressure and lipid profiles were determined weekly. Systolic blood pressure was measured in conscious mice using tail cuff apparatus (Softron BP-98A tail blood pressure system, Tokyo, Japan). Serum was obtained by centrifugation at 3,000 rpm for 10 minutes at room temperature. Serum total cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, total calcium and creatinine levels were determined enzymatically (Arkray Kyoto, Japan).

Characteristics and quantification of AAA.
After perfusion and fixation with cold 4% paraformaldehyde, the aorta was exposed under a dissecting microscope, and the periadventitial tissue was removed from the aortic wall. The morphology of the aorta was photographed in situ, and the maximal external diameter of the suprarenal aorta was measured using imaging processing software (Image J). A definition of outgrowth of more than 50% indicated the development of aortic aneurysm as previously described. The characteristics of an AAA were scored as following as previously described: Type I represents a small single dilation (1.5-2.0 times of a normal diameter); Type II denotes a large single dilation (> 2 times of a normal diameter); Type III is multiple dilations; and Type IV is an aortic rupture that lead to death due to bleeding into the peritoneal cavity 30 . Histology and Immunohistochemistry. Perfusion-fixed aortas were embedded, cut in cross section (5 μ m) and stained with hematoxylin and eosin. Verhoeff-Van Geisen (VVG) was used for detecting elastin. The severity of elastin degradation was semi-quantified as previously described 31 . Immunohistochemistry staining of HIF-1α was used to reveal the aberrantly increased HIF-α in the aorta.

Statistical analysis.
All experiments were performed at least three times. All continuous variables were presented as the mean ± standard error of the mean (SEM). Prior to statistical analysis data was tested for equality of variance by Bartlett's test. Statistical significance was evaluated using the unpaired Student t-test for comparisons between 2 means. Two-sample nonparametric comparisons were performed using a Chi-square test. Comparisons between multiple groups were analyzed by one-way analysis of variance to assess the significance (ANOVA) and post-hoc analysis was performed using the Bonferroni test. Statistical significance was defined as a p-value of less than 0.05. Analyses were performed using a statistical software package (Prism version 5, GraphPad Software, La Jolla California USA). All images shown are examples of replicates and are representative for the respective groups.

Results
DFO attenuates AngII-induced endothelial cell activation and dysfunction in vitro but does not protect against AngII-induced AAA. The detrimental effects of AngII on accelerated atherosclerosis and aneurysmal formation have been attributed to MAPK JNK and ERK activations 9,11 . We found that DFO attenuated AngII-induced JNK, ERK1/2 and ROS production. In addition, DFO dose-dependently attenuated AngIIinduced down-regulation of the atheroprotective factors eNOS, SIRT1 and KLR4 in vitro (n = 4-5, Supplementary  Figure 1). Despite the promising in vitro effects, DFO unexpectedly had the trend toward increasing the incidence of AngII-induced AAA (0% vs. 75% vs. 85%, p = 0.69) and the external diameter of the aorta (0.67 ± 0.11 vs. 1.62 ± 0.83 vs. 1.98 ± 1.07 mm, p-values: saline vs. AngII, p < 0.05; Saline vs. AngII + DFO, p < 0.05; and AngII vs. AngII + DFO, n.s., Supplementary Figure 2A,B). In addition, we observed a significant increase in the incidence of type III (multiple aneurysms) and type IV (rupture) AAA in DFO treated mice (35% vs. 70% p < 0.05, n = 20 in each group, Supplementary Figure 2C). DFO did not alter systolic blood pressure (SBP), serum cholesterol and creatinine. The dose of DFO used in the present study did not cause significant weight loss (less than 10% of body weight) or changes in BP during the study period. Increased HIF-1α expression was associated with increased MMP-2 and MMP-9 activity in vitro. It is known that DFO could also cause HIF-1α accumulation by inhibiting PHD 32 . The unexpected findings of DFO aggravated the severity of AngII induced AAA, independent of its ability in preventing AngII induced ECs activation had led us to explore the roles of HIF-1α in the pathogenesis of AAA. To further elucidate how DFO treatment could augment AAA severity, we tested HIF-1α expression in the whole aorta lysate and found that compared with the AngII-treated group, DFO treated mice had significantly higher HIF-1α expression in the aorta lysate and significantly increased MMP-2 and MMP-9 activities (n = 5, Supplementary Figure 3).

Discussion
In the present study, we are able to show the pivotal roles of HIF-1α in the pathogenesis of AAA by using the combination of three unspecific clinical-relevant HIF-1α modulators, i.e., DFO, 2-ME and digoxin. Although DFO could attenuate AngII-induced endothelial activation, i.e. the reduction of ROS production as well reduction of MAPK JNK and ERK1/2 activations in vivo and in vitro. DFO, also as a PHD inhibitor, stabilized HIF-1α and augmented MMPs activities. As a result, the promising anti-inflammatory effects could not confer protection against AngII-induced AAA in vivo. The findings promoted us to decipher the roles of aberrantly induced HIF-1α in the progression of AAA. We found that several aneurysmal prone factors can aberrantly induce HIF-1α , leading to the overexpression of MMP-2 and MMP-9, and can promote aneurysmal progression. We further confirmed that two pharmacological HIF-1α inhibitors, digoxin and 2-ME, attenuated AngII-induced AAA, providing a rationale for using HIF-1α inhibitors as an adjunctive medical approach for small AAA.
HIF-1α is the master transcriptional factor for the adaption to hypoxia in vertebrate animals. HIF-1α can upregulate MMP-2 and MMP-9 in many cell types [35][36][37][38] . A positive correlation exists between global trends in systolic blood pressure, cholesterol, smoking prevalences and AAA related mortalities 3 . In the present study, we showed that several representative aneurysmal-prone factors may lead to HIF-1α accumulation. AngII can stimulate HIF-1α accumulation by increasing HIF-1α transcription and by inhibiting PHD activity, which would also lead to HIF-1α accumulation 39 . Chronic infusion of AngII increased HIF-1α levels in the kidney, and hyper-activation of HIF-1α in the kidney plays a crucial role in AngII-mediated chronic kidney injury 40 . In VSMC and macrophages, HIF-1α promotes vascular inflammation and vascular remodeling 41,42 . OxPAPC is reported to induce inflammatory gene expression and stimulate JNK and p38 signaling in a toll-like receptor (TLR) 2-dependent manner 43 . TLR4 activation is necessary for an augmented pulmonary HIF-1α response in hemorrhagic shock-induced lung injury 44 . Nicotine could induce the activation of HIF-1α and VEGF through α 5 and α 7 nicotinic acetylcholine receptors 45,46 . In the current study, we showed that these aneurysmal-prone factors promoted aneurysm progression at least partly through aberrantly increased HIF-1α expression.
Although AAA had previously been described as a form of atherosclerosis, it is now recognized as a distinct process involving all of the layers of the vessel wall. Several cellular types, including VSMCs, macrophages, neutrophils, T cells, mast cells and peri-adventitial fibroblasts have been shown to involve in the pathogenesis of AAA 34,[47][48][49] . However, emerging evidences suggest the pivotal roles of ECs during the progression of AAA by initiating vascular inflammation, expressing adhesion molecules and activating inflammatory cascades, which may further trigger macrophage infiltration and inflammation in the adventitia and media 9,10 . Loss of the endothelium and its replacement by a thick thrombus are also structural features of human AAAs 48,50,51 . Previous studies suggest that AAA could be attenuated by the administration of drugs that reduce endothelial activation and restoring the endothelial lining were efficient therapy against AAA expansion 9,11,50,51 . AngII infusion could induce endothelial miR-712/miR-205 in AAA, both in vitro and in vivo, and the silencing of miR-712/miR-205 by antisense oligonucleotides could decrease inflammation and the activity of endothelail MMPs, thus preventing AAA development in AngII-infused ApoE −/− mice 52 . Exogenous VEGF could further enhace AngII induced AAA in ApoE −/− mice provide some clues for the involvement of ECs in the progression of AAA 53 . Taken together, our current study further revealed that those aneurysmal-prone factors could cause MMP-2 and MMP-9 overexpression through aberrantly increased HIF-1α in the ECs.
Oxidative stress is induced early during the course of AAA formation, and a growing body of evidence suggests that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a source of oxidative stress in the pathogenesis of AAA. AngII can increase ROS production via NADPH and further increase adhesion molecule expression and inflammatory cell recruitment and further activate stress signals 54 . Consistent with our findings in ECs, Kazi., et al. had found that DFO could ameliorate AngII induced JNK activation in THP-1 cells 55 . Although iron restriction diet had been shown to ameliorate AngII-induced AAA in ApoE −/− mice, the in vivo effects of DFO on AAA progression had not been further tested 55 . On contrary to previous studies showing that DFO inhibits inflammation and atherosclerosis in ApoE −/− mice and cholesterol-fed rabbits 19 , the current study demonstrated that DFO potentiated AngII-induced AAA in hyperlipidemic mice through the accumulation of HIF-1α . We highlight that the potential beneficial iron depletion effects could be potentially overwhelmed by the increased HIF-1α accumulation.
Anti-inflammatory therapies using mast cell stabilizers and doxycycline failed to limit the expansion of preexisting AAAs 13,14 . In addition, the enlargement of preexisting AAAs has been observed in patients with heart and abdominal organ transplants treated with immunosuppressant drugs 56,57 . Collectively, although anti-inflammatory therapy has been shown to improve AAA in murine models, an alternative adjunctive therapeutic approach in other pathways is still required. Given the increasing use of HIF-1α inhibitors as an anti-cancer therapy, pharmacological inhibition of aberrantly induced HIF-1α may provide a promising adjunctive medical therapy for patients with small AAA.
Although DFO, digoxin and 2-ME are non-specific HIF-1α modulators, here we have demonstrated the therapeutic potentials of HIF-1a inhibitors as adjunctive medical treatments for AAA. Chemically modified glycosides without cardiac effects have been developed for the anti-cancer therapies 58 . Inhibition of NF-kB/HIF-1 have been shown to decrease IL-17-induced MMP-2 and MMP-9 expression in fibroblasts 59 . Congestive heart failure was independently associated with post-discharge mortality in patients with AAA [60][61][62] . Digitoxin elicits anti-inflammatory and vasoprotective properties by blocking NF-kB and activating PI-3-kinase/Akt signaling as well as Ca(2+ )/Calmodulin-dependent-protein kinase-II in ECs 63 . The dosage used in mice (1-2 mg/kg/day) have not been shown to have adverse effects on the cardiovascular physiology of mice and the serum concentration of digoxin in mice treated at the above-mentioned dose was at or below the therapeutic range for humans (0.5-2.0 ng/ml) 24,26,27 . Thus, we believe that the beneficial effects of the digoxin/digoxin-like compound should be further explored in the management of small AAAs. It is also worth noting that digoxin (40 μ g/day) has been shown to protect against experimental AAA by inhibiting Th17/IL-17A related inflammatory responses 34 . 2-ME is an endogenous natural metabolite of estradiol with HIF-1α inhibition effects 64,65 . 2-ME has been shown to reduce monocyte adhesion to ECs and prevents the development of atherosclerosis in ApoE −/− mice 66,67 . Given that it has been used in several phase II clinical trials for the treatment of prostate cancer, renal cell carcinoma, and metastatic carcinoid tumors; [68][69][70] we believe that the clinical implications could be further expanded in the management of AAA.
Limitations. Although those HIF-1α modulators are not specific; in conjunction with the facts that HIF-1α accumulation in human AAAs in previous studies, we believe that HIF-1α pathway should be further explored as an intervenable target for stabilizing AAA. We aware that the development of AAA are multi-factorial in origin,

Conclusion
HIF-1α is pivotal for the development of AAA. Several aneurysmal prone factors cause up-regulation of MMP-2 and MMP-9 through aberrantly induced HIF-1α . Medications that potentiate HIF-1α should be used with caution in patients with AAA. Pharmacological HIF-1α inhibition could attenuate AngII-induced AAA. Our study provides a rationale for using HIF-1α inhibitors as an adjunctive medical therapy in addition to current cardiovascular risk-reducing regimens.