論文の公開元へInhibition of microRNA-33b specifically ameliorates abdominal aortic aneurysm formation via suppression of inflammatory pathways
概要
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OPEN
Inhibition of microRNA‑33b
specifically ameliorates abdominal
aortic aneurysm formation
via suppression of inflammatory
pathways
Tomohiro Yamasaki1, Takahiro Horie1*, Satoshi Koyama1, Tetsushi Nakao1, Osamu Baba1,
Masahiro Kimura1, Naoya Sowa2, Kazuhisa Sakamoto3, Kazuhiro Yamazaki3,
Satoshi Obika4,5, Yuuya Kasahara4,5, Jun Kotera6, Kozo Oka6, Ryo Fujita6, Takashi Sasaki6,
Akihiro Takemiya6, Koji Hasegawa2, Kenji Minatoya3, Takeshi Kimura1 & Koh Ono1*
Abdominal aortic aneurysm (AAA) is a lethal disease, but no beneficial therapeutic agents have been
established to date. Previously, we found that AAA formation is suppressed in microRNA (miR)-33deficient mice compared with wild-type mice. Mice have only one miR-33, but humans have two miR33 s, miR-33a and miR-33b. The data so far strongly support that inhibiting miR-33a or miR-33b will be
a new strategy to treat AAA. We produced two specific anti-microRNA oligonucleotides (AMOs) that
may inhibit miR-33a and miR-33b, respectively. In vitro studies showed that the AMO against miR33b was more effective; therefore, we examined the in vivo effects of this AMO in a calcium chloride
(CaCl2)-induced AAA model in humanized miR-33b knock-in mice. In this model, AAA was clearly
improved by application of anti-miR-33b. To further elucidate the mechanism, we evaluated AAA
1 week after CaCl2 administration to examine the effect of anti-miR-33b. Histological examination
revealed that the number of MMP-9-positive macrophages and the level of MCP-1 in the aorta of
mice treated with anti-miR-33b was significantly reduced, and the serum lipid profile was improved
compared with mice treated with control oligonucleotides. These results support that inhibition of
miR-33b is effective in the treatment for AAA.
Abbreviations
AAA Abdominal aortic aneurysm
ABCA1 ATP binding cassette transporter A1
ALT Alanine aminotransferase
AMO Anti-microRNA oligonucleotide
AmNA Amido-bridged nucleic acid
ANOVA Analysis of variance
AST Aspartate transaminase
CaCl2 Calcium chloride
CRE Creatinine
1
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54
Shogoin‑kawahara‑cho, Sakyo‑ku, Kyoto 606‑8507, Japan. 2Division of Translational Research, National
Hospital Organization, Kyoto Medical Center, 1‑1 Fukakusa Mukaihata‑cho, Fushimi‑ku, Kyoto 612‑8555,
Japan. 3Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54
Shogoin‑kawahara‑cho, Sakyo‑ku, Kyoto 606‑8507, Japan. 4Graduate School of Pharmaceutical Sciences, Osaka
University, 1‑6 Yamadaoka, Suita‑shi, Osaka 565‑0871, Japan. 5Center for Drug Design Research, National
Institutes of Biomedical Innovation, Health and Nutrition, 7‑6‑8 Saito‑Asagi, Ibaraki‑shi, Osaka 567‑0085,
Japan. 6Sohyaku. Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Shonan Health Innovation
Park, 2‑26‑1, Muraoka‑Higashi, Fujisawa‑shi, Kanagawa 251‑8555, Japan. *email: thorie@kuhp.kyoto-u.ac.jp;
kohono@kuhp.kyoto-u.ac.jp
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DAPI 4’,6-Diamidino-2-phenylindole
DMEM Dulbecco’s modified Eagle’s medium
EC Endothelial cell
EVG Elastica van Gieson
FBS Fetal bovine serum
HDL-C High-density lipoprotein cholesterol
HE Hematoxylin–eosin
IL Interleukin
JNK C-Jun N-terminal kinase
KI Knock-in
KO Knock-out
KOKI Knock-out, knock-in
LDL-C Low-density lipoprotein-cholesterol
MCP-1 Monocyte chemotactic protein-1
miR MicroRNA
MMP Matrix metalloproteinase
PBS Phosphate-buffered saline
PM Peritoneal macrophage
SEM Standard error of the mean
αSMA α-smooth muscle actin
SREBF Sterol regulatory element-binding transcription factor
T-BIL Total bilirubin
T-CHO Total cholesterol
TG Triglycerides
TNFα Tumor necrosis factor α
VSMC Vascular smooth muscle cell
WT Wild-type
The majority of aortic aneurysms in adults are essentially asymptomatic, but the risk of rupture increases as the
diameter of the aneurysm increases1. Of these, abdominal aortic aneurysms (AAA) have a reported incidence of
1.5 to 2 per 1,000 people per year in the United S tates2. Currently, surgical treatment is considered p
referable3,
4
whereas treatment with various drugs such as beta-blockers , angiotensin-converting enzyme inhibitors5, and
calcium channel b
lockers6 are being investigated. However, large-scale randomized controlled trials using these
drugs have not shown clear effects. Therefore, the development of therapeutic agents that can inhibit the expansion of aortic aneurysms is of great significance.
Using genetically engineered mice, we previously found that the absence of microRNA (miR)-33 suppressed
AAA progression via several anti-inflammatory p
athways7. Therefore, we decided to use these data to develop
a new therapeutic agent for aortic aneurysms. Nucleic acid medicine has been attracting attention as a new
therapeutic agent for hereditary and refractory diseases that have been difficult to treat8,9. In the development
of conventional oligonucleotide therapeutics, there have been problems with stability and efficacy in vivo10,
but advances in modified nucleic acid technology11 and Drug Delivery System technology12 have changed this
situation, and candidates that are highly effective not only after local administration but also after systemic
administration are being d
eveloped13. Oligonucleotide therapeutics are expected to have high specificity and
efficacy similar to antibody drugs and can be produced by chemical synthesis similar to small molecule drugs10.
In fact, oligonucleotides are designed based on the target RNA sequence, and highly effective oligonucleotides
can be obtained in a short time13.
In rodents, there is only one miR-33 (miR-33a) in the intron of sterol regulatory element binding transcription factor 2 (Srebf2), but in humans, in addition to miR-33a, there is another miR-33 (miR-33b) in the intron of
SREBF1. In this study, we developed bridged nucleic acid-modified anti-microRNA oligonucleotides (AMOs)
that individually target miR-33a and miR-33b, which differ by only two bases. As a result, we showed that it is
possible to target aortic aneurysms and large blood vessels and elucidated its mechanism of action in detail using
humanized miR-33b knock-in (KI) mice.
Results
miR‑33b KI mice showed severe calcium chloride‑induced AAA formation. Previously, we cre-
ated a calcium chloride (CaCl2)-induced AAA model in miR-33-deficient (miR-33a−/− miR-33b−/−) and wildtype (WT) mice (miR-33a+/+ miR-33b−/−) and found that AAA formation was suppressed in miR-33-deficient
mice7. This indicated that the amount of miR-33 is related to the worsening of AAA. In the present study, we
investigated the effects of miR-33a and miR-33b on AAA in order to clarify the pathogenesis in humans. We
previously generated miR-33b KI mice (miR-33a+/+ miR-33b+/+), which have human miR-33b in intron 16 of
Srebf114. miR-33b KI mice have both miR-33a and miR-33b as in humans. In these mice, miR-33b is physiologically co-expressed with Srebf1. We next crossed miR-33a knock-out (KO) mice with miR-33b KI mice to generate
mice with only miR-33b (KOKI) (miR-33a−/− miR-33b+/+)15. Then, we compared the severity of CaCl2-induced
AAA formation16 in the WT, KOKI, and KI mouse lines. As shown in Fig. 1a,b, the diameter of the aorta was
significantly increased in KOKI mice and further increased in KI mice compared with WT mice. In addition, the
lesion length of the aorta in KI mice was significantly larger than that in KOKI and WT mice. Next, we measured
the copy numbers of miR-33a and miR-33b in 1 µg of total RNA of the aorta (Fig. 1c). As expected, only miR-33a
was present in WT mice and only miR-33b was present in KOKI mice. miR-33b levels were higher than miR-33a
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Figure 1. Genetic phenotypes of abdominal aortic aneurysm (AAA) formation in wild-type (WT) mice,
miR-33a knock-out and miR-33b knock-in (KOKI) mice, and miR-33b knock-in (KI) mice. (a) Representative
photographs of sham controls and calcium chloride (CaCl2)-induced AAA in WT, KOKI, and KI mice. White
bars indicate 1 mm. (b) Maximum diameter and lesion length of the abdominal aorta between the left renal
artery and the terminal aorta of CaCl2-induced AAA, n = 10 mice in WT, n = 9 mice in KOKI, and n = 7 mice
in KI mice. One-way ANOVA with Holm–Sidak’s multiple comparisons test. *P < 0.05 and ***P < 0.001. (c)
Absolute copy numbers of miR-33a and miR-33b in sham (left), CaCl2-induced AAA (mid), and integrated
graphs, n = 5 mice in each sham, n = 7 mice in WT and KOKI for CaCl2-induced AAA, and n = 7 mice in KI for
CaCl2-induced AAA. One-way ANOVA with Holm–Sidak’s multiple comparisons test (left and mid) and twoway ANOVA with Holm–Sidak’s multiple comparisons test (right). *P < 0.05 and **P < 0.01. All data represent
mean ± standard error of the mean (SEM).
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Figure 2. Selection of effective and specific anti-microRNA oligonucleotide (AMOs) against miR-33a and
miR-33b. (a) Experimental scheme for reporter assay. In the absence of anti-microRNA (miR), intrinsic miR
inhibits luciferase expression (left). Adding anti-miR inhibits intrinsic miR, and the expression of luciferase will
increase. Luciferase intensity is proportional with inhibition efficiency of anti-miRs (right). (b) Reactivities of
miR-33a perfect match (33a PM) and miR-33b perfect match (33b PM) reporter vectors in HepG2 cells (human
hepatocellular carcinoma cell line), n = 3. One-way analysis of variance (ANOVA) with Dunnett’s multiple
comparison test. ***P < 0.001. ...
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