Download S0735109713055897_mmc1

APPENDIX
SUPPLEMENTARY METHODS
Study population. Forty-one unrelated patients diagnosed with primary HCM at the University of Naples
“Federico II”, Naples, Italy, and 41 healthy subjects recruited from the Milan subsidiary of the blood donor
organization Associazione Volontari Italiani Sangue (AVIS) were enrolled in the study. The AVIS subjects were
defined as “healthy” based on the absence of any type of cardiovascular disease or familiarity for it;
biochemical parameters, medical records, and interviews were consensually obtained. All subjects were
investigated using the same study protocol. Control subjects were age- and sex-matched as much as possible
with HCM patients. All probands underwent physical examination, electrocardiogram (ECG), two-dimensional
echocardiography, Doppler studies, and 24-hour ECG Holter monitoring. The diagnosis of HCM was based on
echocardiographic demonstration of a hypertrophic but non-dilated left ventricle (wall thickness >15mm) in the
absence of any other cardiac or systemic disorders producing a comparable grade of LV hypertrophy (1).
Classification parameters defining HCM status were those established by the American Heart Association
guidelines (1). Informed consent was obtained from each patient and the study protocol conformed to the
ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the institution's
human research committee.
Blood collection and RNA isolation. Five ml of peripheral blood was collected in EDTA-containing Vacutainer
tubes. The vials were processed within 2 h of collection by centrifugation at 1100 X g at room temperature in a
bench top centrifuge for 20 minutes to eliminate all blood cells. The plasma-containing supernatant was
extracted without disturbing the interface layer and aliquoted (250 l) into RNase/DNase-free Eppendorf
tubes. All samples were stored immediately at -80 C.
MiRneasy (Qiagen, Hilden, Germany) isolation kit was used among the five kits tested because in our hands
it was the most efficient in terms of RNA recovery and quality. Total RNA from human plasma was then purified
using miRNeasy Mini Kit, following the manufacturer’s instructions. An aliquots of 1.5 l of each sample was
quantified by Nanodrop 1000 spectrophotometer absorbance (Nanodrop, Wilmington, Delaware, USA). In
order to normalize sample-to-sample variation and minimize the spike-in degradation while increasing miRNA
recovery, 6 fmol/l of RNA oligonucleotides synthesized by EXIQON (Exiqon A/S, Vedbaek, Denmark) was
added to each denatured sample; the spike-in probe was selected based on the absence of any sequences
homologous to those of the miRNAs to be measured. In addition, 1l of 1ng/l tRNA was added in the phenol
phase during extraction in order to increase the recovery of the isolated RNA. To avoid the limitation of plasma
RNA quantifications due to the small concentration of miRNAs in plasma, all samples were eluted into 25l of
RNAse-free water; 8 l was then used as a template to generate cDNA.
Assessment of circulating miRNAs. Reverse transcription. Reverse transcription reactions were performed
using the EXIQON miRNA Reverse Transcription Mercury Universal cDNA synthesis kit (Exiqon A/S, Vedbaek,
Denmark) in 20 l as a final volume. PCR System 9700 (Applied Biosystems, Foster City, CA, USA) was used to
1
carry out the RT reactions using the following conditions: 42°C for 60min, 95°C for 5min, hold at 4°C. RT
products were stored undiluted at -20°C prior to running the real-time PCR. The Freedom Evo 150 liquid
handling system (Tecan Group Ltd., Männedorf, Switzerland) was used for reliably aliquoting reaction mixes for
both the generation of reverse transcription and real time PCR detection.
Customized panel for the detection of miRNAs. We selected 21 candidate miRNAs previously associated with
cardiovascular disease. The miRNA-specific primer sequences were obtained from the Exiqon website
(http://www.exiqon.com/microrna-real-time-pcr-primer-sets). In order to perform external normalization, a
specific spike-in miRNA was designed.
microRNA
miR-499-3p
miR-499-5p
miR-199a-3p
miR-199a-5p
miR-1
miR-145
miR-16
miR-133a
miR-143
miR-155
miR-195
miR-126-3p
miR-126-5p
miR-208a
miR-208b
miR-21
miR-26a
miR-29a
miR-27a
miR-30a
miR-214
Primer sequence
5′-AACATCACAGCAAGTCTGTGCT-3′
5′-TTAAGACTTGCAGTGATGTTT-3′
5′-ACAGTAGTCTGCACATTGGTTA-3′
5′-CCCAGTGTTCAGACTACCTGTTC-3′
5′-TGGAATGTAAAGAAGTATGTAT-3′
5′-GTCCAGTTTTCCCAGGAATCCCT-3′
5′-TAGCAGCACGTAAATATTGGCG-3′
5′-TTTGGTCCCCTTCAACCAGCTG-3′
5′-TGAGATGAAGCACTGTAGCTC-3′
5′-TTAATGCTAATCGTGATAGGGGT-3′
5′-TAGCAGCACAGAAATATTGGC-3′
5′-TCGTACCGTGAGTAATAATGCG-3′
5′-CATTATTACTTTTGGTACGCG-3′
5′-ATAAGACGAGCAAAAAGCTTGT-3′
5′-ATAAGACGAACAAAAGGTTTGT -3′
5′-TAGCTTATCAGACTGATGTTGA-3′
5′-TTCAAGTAATCCAGGATAGGCT-3′
5′-TAGCACCATCTGAAATCGGTTA-3′
5′-TTCACAGTGGCTAAGTTCCGC-3′
5′-TGTAAACATCCTCGACTGGAAG-3′
5′-ACAGCAGGCACAGACAGGCAGT-3′
Measurement of miRNA levels. SYBR green qRT-PCR was performed in 96-well plates at a final volume of 20 l.
More precisely, the miRNA level was measured using 1l of forward and reverse miRCURY LNA Universal RT
microRNA PCR primers, 10l of Exiqon Syber Green master mix, and 8 l of diluted cDNA (1:40) per reaction
(Exiqon A/S, Vedbaek, Denmark). Quantitative RT-PCR reactions were performed in triplicate for all samples; a
±0.2 difference between detected threshold cycle values (Ct) was considered acceptable. The Tecan Freedom
Evo 150 system (Tecan Group Ltd. Männedorf, Switzerland) was used for liquid handling. miRNA-208a was also
measured with the TaqMan assay (Applied Biosystems, Foster City, CA, USA), according to manufacturer’s
guidelines. Specifically, the miRNA level was measured using 10l of TaqMan MasterMix, 1l of primers and
1.33l cDNA, and 7.67l DNase/RNase-free water.
qRT-PCR was performed using an ABI Prism 7900 Real-Time PCR instrument with the following
amplification profile: denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 10s, 60°C for 1 min. The
Ct was computed with Sequence Detection System software (Applied Biosystems, Foster City, CA, USA). The
data were analyzed with automatic settings for assigning baseline. The Ct was defined as the fractional cycle
2
number at which the fluorescence exceeded the given thresholds. miRNA expression levels were normalized to
a non-endogenous synthetic spike miRNA (Exiqon ID number: 206999).
Assessment of hypertrophy. Trans-thoracic color Doppler and TDI echocardiography. All echocardiographic
studies were performed in left lateral decubitus, using a commercially available imaging system (IE 33, Philips,
Andover, MA, USA) equipped with a S5 1- to 5-MHz phased array transducer (Philips, Andover, MA, USA). Each
patient underwent standard M-mode, two-dimensional, and Doppler echocardiographic studies. M-mode
images of the left ventricle (LV) were obtained in the parasternal long axis, and LV end-diastolic and endsystolic diameters were measured just below the mitral valve leaflets tips after alignment of the cursor
perpendicular to the LV wall and indexed to the body surface area (LVEDI) according to American Society of
Echocardiography guidelines (2). To estimate the extent and distribution of LV hypertrophy, Wigle’s score was
obtained (3): this method attributes a score from 1 to 4 to the maximal basal septal thickness, and additional
marks if hypertrophy extends to medium and/or apical septum, and/or lateral wall. The extent of LV
hypertrophy (hypertrophy index or Spirito-Maron Index) (3) was calculated from the short-axis view at the level
of both mitral valve and papillary muscle by dividing the left ventricular wall into four segments (anterior
septum, posterior septum, lateral free wall, and posterior free wall), and by adding the maximal wall thickness
measured (at either the mitral valve or papillary muscle level) in each of the four ventricular segments. As an
additional simple estimate of LV hypertrophy, the maximal wall thickness (MWT) measured at any level in the
LV wall was also considered.
Apical-view Doppler echocardiography was used to measured transmitral peak flow velocities: mitral peak
E velocity at rapid ventricular filling and mitral peak A velocity at atrial contraction, their ratio (E/A), and E wave
deceleration time at the mitral tips. At the pulmonary vein level, we measured the ratio between systolic and
diastolic peak flow velocities, and the backward flow velocity (Ar). As an estimate of passive diastolic
dysfunction, the difference in duration between mitral and backward pulmonary flow (A-Ar) at atrial
contraction was calculated. Moreover, pulsed tissue Doppler (TDI) was used to measure the early diastolic
velocity at the lateral corner of the mitral annulus (E’), the ratio of the peak early diastolic filling and E’ (E/E’),
and the peak systolic velocity. Color Doppler flow imaging was used for semiquantitative assessment of mitral
regurgitation, which was graded from mild to severe according to the EAE criteria (4). Left ventricular outflow
tract gradient was calculated with the simplified Bernoulli’s equation (P=4v2, where P represents pressure and v
represents flow velocity). Particular care was taken to avoid contamination of the left ventricular outflow
waveform by the mitral regurgitation jet.
Cardiac magnetic resonance. MR studies were performed using a 1.5 Tesla MRI system (Gyroscan Intera, Philips
Medical System, Best, the Netherlands) equipped with high performance gradients (maximum gradient
amplitude 30 mT/m, maximum slew rate 150 mT/m/ms). Images were acquired with a 5-element cardiac
phased-array coil using a vectorcardiographic method for ECG-gating and respiratory gating. After performing a
survey scan, LV long axis and 4-chamber horizontal long axis images were acquired using a breath-holding 2D
balanced turbo field echo multiphase-multislice sequence (TR/effective TE, 2.8/1.4; matrix, 160X256; slice
thickness, 10-mm; flip angle, 500); subsequently, biventricular short-axis images were obtained using 10 slices
covering the left ventricle from the apex to the base for evaluation of LV mass. Late gadolinium enhancement
images in short-axis orientation were acquired for quantification of myocardial fibrosis 10–15 min after
administration of 0.2 mmol/kg/body weight gadopentetate dimeglumine (Magnevist, Bayer HealthCare
Pharmaceuticals, Berlin, Germany), using a three-dimensional T1-weighted inversion recovery turbo gradient
echo sequence. Echo time was 1.5 ms, repetition time 2.8 ms, flip angle 15°, typical in-plane resolution 1.8 × 2
3
mm, slice thickness 8 mm. Post-processing analysis was performed on a dedicated workstation (Viewforum,
Philips Medical System, Best, the Netherlands). Analysis of LV mass was performed choosing the slice with the
greatest cardiac diameter of the 2D-balanced turbo field echo multiphase-multislice acquisition in the
biventricular short axis; subsequently, the endocardial and the epicardial borders were manually traced,
carefully including the papillary muscles, on each end-diastolic and end-systolic frame for each of 10 slices. To
assess the presence of myocardial fibrosis, a semi-quantitative evaluation was performed by giving a score
from 0 to 4, where 0 = no evidence of fibrosis; 1 = fibrosis between 0–25%; 2 = fibrosis between 25–50%; 3 =
fibrosis between 50–75%; and 4 = transmural fibrosis.
4
Online Table 1 Characteristics of the microRNAs Followed in the Study
microRNA
Cardiomyocyte miRNAs
miR-499-3p
miR-499-5p
miR-1
miR-133a
Putative Role
Dysregulation
Pathology
Regulates myosin isoform
expression in cardiac myocytes
("myo-miRNA")
Upregulated in HCM and HF,
expressed 600-fold below
miR-499-5p
-
Upregulated in HCM and HF
AMI, HF
(10-14)
Downregulated in heart
defects, arrhythmias
Downregulated in HCM,
inversely related to cardiac
fibrosis and cardiomyocyte
hypertrophy
AMI,
hypertrophy
(13,15-17)
Regulates myosin isoform
expression in cardiac myocytes
and skeletal muscle ("myomiRNA")
Involved in cardiac
development and hypertrophy
Involved in cardiac
development and regulation of
myocardial cell size.
Controls myosin heavy chain
miR-208
isoform expression ("myomiRNA") and metabolism
Fibroblast miRNAs
Expressed in human
fibroblasts, control angiotensin
miR-155
II type 1 receptor, activated
during prolonged hypoxia
miR-21
Cardiac fibroblasts survival and
activation
Increases myocardial fibrosis
by de-repressing collagen and
miR-29
elastin translation in cardiac
fibroblasts
Smooth-muscle miRNAs
Modulates vascular smooth
miR-143
muscle cell phenotype
miR-145
Promotes smooth muscle
differentiation
Endothelial miRNAs
Regulates cardiomyocyte cell
size, protects myocytes against
miR-199-3p
ischemia/hypoxia-induced
apoptosis.
Regulates cardiomyocyte cell
miR-199-5p
size, protects myocytes against
Downregulated in cardiac
hypertrophy
Reference
(9-11)
AMI, UAP,
Takotsubo
(13,17-19)
cardiomyopathy
AMI
(11,13,14)
Involved in vascular disease
and inflammation, Friedreich
CAD and T2DM (20-23)
ataxia HCM, and
hypertension
LV fibrosis and a
biomarker of
Upregulated in cardiac
cancers (HCC, (24,25)
fibrosis and HF
glioblastoma,
breast cancer)
Downregulated in cardiac
fibrosis, profibrillatory, atrial T2DM
fibrosis
Downregulated in
atherosclerosis
Downregulated in vascular
injuries and atherosclerosis,
upregulated in PAH
Upregulated in eccentric
HCM, impairs sarcomere
composition and induces
endothelial cell dysfunction
Upregulated in eccentric
HCM, impairs sarcomere
5
(19,26,27)
CAD, ischemic
stroke, PAH
-
T2DM
(28-31)
(20,28-33)
(34-36)
(20,34-36)
miR-195
miR-126-3p
miR-126-5p
miR-16
miR-26
miR-27
ischemia/hypoxia-induced
apoptosis.
Promotes cell division and
apoptosis while inhibiting cell
proliferation.
Controls angiogenesis and
maintains vascular integrity
Controls angiogenesis and
maintains vascular integrity
Regulates cell-autonomous
angiogenic functions in
endothelial cells
Induced under hypoxic
conditions
Promotes pro-angiogenic
growth in endothelial cells
composition and induces
endothelial cell dysfunction
Upregulated in HCM
Downregulated in CAD and
T2DM
Downregulated in CAD and
T2DM
AMI, CAD,
T2DM
AMI, CAD,
T2DM
(37)
(5,26,38)
(5,26,38)
Upregulated in HF, reduces
angiogenesis
-
(39)
Anti-apoptotic effects
-
(40)
Upregulated in angiogenesis,
adipogenesis, inflammation,
Atherosclerosis
lipid metabolism, oxidative
(41-42)
obliterans
stress, insulin resistance and
T2DM
Regulates connective tissue
Downregulated in HCP and
growth factor in
(18)
HF
cardiomyocytes
Protects cardiomyocytes
Upregulated during ischemic
miR-214
against ischemia/reperfusion
(43-44)
injury and HF
injury; inhibits angiogenesis
AMI: acute myocardial infarction; CAD: coronary artery disease HCM: hypertrophic cardiomyopathy; HF: heart
failure; PAH: pulmonary artery hypertension; T2DM: type 2 diabetes mellitus; UAP: unstable angina pectoris.
miR-30
6
Online Figure 1. Relative expression of circulating microRNAs in patients with hypertrophy due to aortic
stenosis. Individual-value plots displaying the variability and differences in the plasma level of each miRNA in
healthy individuals (right side of plots) and patients with aortic stenosis (AS, left side of plots). Asterisks
represent significant p-values (<0.04) obtained with two-sided t-test of miRNA expression in the AS vs. healthy
groups. A crossbar on each plot indicates the mean expression level for each group.
7
Online Figure 2. Scatter plots for the correlation of miR-29a with hypertrophy parameters. The circulating
level of miR-29a (2−ΔΔCt values) in each HCM patient is plotted against maximum wall thickness assessed by
trans-thoracic echography (upper left) or cardiac magnetic resonance (lower left), hypertrophy index (upper
right), and left ventricular mass (lower right). r, Pearson correlation coefficient; p, relative computed p-value.
8
Total fibrosis score
Online Figure 3. Correlation of the circulating miR-29a level with the extent of myocardial fibrosis in HCM
patients. Scatter plot of myocardial fibrosis assessed with gadopentetate–cardiac magnetic resonance. The
total score of 10 sections/heart is given on the ordinate, and plotted against 2−ΔΔCt values for circulating miR29a, on the abscissa. r, Pearson correlation coefficient; p, relative computed p-value.
9
SUPPLEMENTARY REFERENCES
1. Gersh BJ, Maron BJ, Bonow RO, et al. 2011 ACCF/AHA guideline for the diagnosis and treatment of
hypertrophic cardiomyopathy: executive summary: a report of the American College of Cardiology
Foundation/American Heart Association Task Force on Practice Guidelines. Circulation 2011;124:2761-96.
2. Lang RM, Bierig M, Devereux RB, et al. Recommendations for chamber quantification: a report from the
American Society of Echocardiography's Guidelines and Standards Committee and the Chamber
Quantification Writing Group, developed in conjunction with the European Association of
Echocardiography, a branch of the European Society of Cardiology. J Am Soc Echocardiogr 2005;18:144063.
3. Wigle ED, Sasson Z, Henderson MA, et al. Hypertrophic cardiomyopathy. The importance of the site and
the extent of hypertrophy. A review. Prog Cardiovasc Dis 1985;28:1-83.
4. Lancellotti P, Moura L, Pierard LA, et al. European Association of Echocardiography recommendations for
the assessment of valvular regurgitation. Part 2: mitral and tricuspid regurgitation (native valve disease).
Eur J Echocardiogr. 2010;11:307-32
5. Long G, Wang F, Duan Q, et al. Human circulating microRNA-1 and microRNA-126 as potential novel
indicators for acute myocardial infarction. Int J Biol Sci 2012;8:811-8.
6. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the
2(-Delta Delta C(T)) Method. Methods 2001;25:402-8.
7. Westfall P, Young S. Resampling-based Multiple Testing: Examples and methods for p-value adjustment:
John Wiley & Sons, 1993.
8. Šidāk Z. Rectangular confidence region for the means of multivariate normal distributions. Journal of
American Statistical Association 1967;62:626-633.
9. Matkovich SJ, Hu Y, Eschenbacher WH, Dorn LE, Dorn GW, 2nd. Direct and indirect involvement of
microRNA-499 in clinical and experimental cardiomyopathy. Circ Res 2012;111:521-31.
10. Shieh JT, Huang Y, Gilmore J, Srivastava D. Elevated miR-499 levels blunt the cardiac stress response. PLoS
One 2011;6:e19481.
11. van Rooij E, Quiat D, Johnson BA, et al. A family of microRNAs encoded by myosin genes governs myosin
expression and muscle performance. Dev Cell 2009;17:662-73.
12. Olivieri F, Antonicelli R, Lorenzi M, et al. Diagnostic potential of circulating miR-499-5p in elderly patients
with acute non ST-elevation myocardial infarction. Int J Cardiol 2012.
13. Wang GK, Zhu JQ, Zhang JT, et al. Circulating microRNA: a novel potential biomarker for early diagnosis of
acute myocardial infarction in humans. Eur Heart J 2010;31:659-66.
14. Corsten MF, Dennert R, Jochems S, et al. Circulating MicroRNA-208b and MicroRNA-499 reflect myocardial
damage in cardiovascular disease. Circ Cardiovasc Genet 2010;3:499-506.
15. Zhao Y, Ransom JF, Li A, et al. Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice
lacking miRNA-1-2. Cell 2007;129:303-17.
16. Ai J, Zhang R, Li Y, et al. Circulating microRNA-1 as a potential novel biomarker for acute myocardial
infarction. Biochem Biophys Res Commun 2010;391:73-7.
17. Kuwabara Y, Ono K, Horie T, et al. Increased microRNA-1 and microRNA-133a levels in serum of patients
with cardiovascular disease indicate myocardial damage. Circ Cardiovasc Genet 2011;4:446-54.
18. Duisters RF, Tijsen AJ, Schroen B, et al. miR-133 and miR-30 regulate connective tissue growth factor:
implications for a role of microRNAs in myocardial matrix remodeling. Circ Res 2009;104:170-8, 6p
following 178.
19. Castoldi G, Di Gioia CR, Bombardi C, et al. MiR-133a regulates collagen 1A1: potential role of miR-133a in
myocardial fibrosis in angiotensin II-dependent hypertension. J Cell Physiol 2012;227:850-6.
20. Fichtlscherer S, De Rosa S, Fox H, et al. Circulating microRNAs in patients with coronary artery disease. Circ
Res 2010;107:677-84.
10
21. Kelly M, Bagnall RD, Peverill RE, et al. A polymorphic miR-155 binding site in AGTR1 is associated with
cardiac hypertrophy in Friedreich ataxia. J Mol Cell Cardiol 2011;51:848-54.
22. Martin MM, Lee EJ, Buckenberger JA, Schmittgen TD, Elton TS. MicroRNA-155 regulates human angiotensin
II type 1 receptor expression in fibroblasts. J Biol Chem 2006;281:18277-84.
23. Corsten MF, Papageorgiou A, Verhesen W, et al. MicroRNA profiling identifies microRNA-155 as an adverse
mediator of cardiac injury and dysfunction during acute viral myocarditis. Circ Res 2012;111:415-25.
24. Kumarswamy R, Volkmann I, Thum T. Regulation and function of miRNA-21 in health and disease. RNA Biol
2011;8:706-13.
25. Thum T, Gross C, Fiedler J, et al. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase
signalling in fibroblasts. Nature 2008;456:980-4.
26. Zampetaki A, Kiechl S, Drozdov I, et al. Plasma microRNA profiling reveals loss of endothelial miR-126 and
other microRNAs in type 2 diabetes. Circ Res 2010;107:810-7.
27. Kriegel AJ, Liu Y, Fang Y, Ding X, Liang M. The miR-29 family: genomics, cell biology, and relevance to renal
and cardiovascular injury. Physiol Genomics 2012;44:237-44.
28. Cordes KR, Sheehy NT, White MP, et al. miR-145 and miR-143 regulate smooth muscle cell fate and
plasticity. Nature 2009;460:705-10.
29. Elia L, Quintavalle M, Zhang J, et al. The knockout of miR-143 and -145 alters smooth muscle cell
maintenance and vascular homeostasis in mice: correlates with human disease. Cell Death Differ
2009;16:1590-8.
30. Xin M, Small EM, Sutherland LB, et al. MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics
and responsiveness of smooth muscle cells to injury. Genes Dev 2009;23:2166-78.
31. Boettger T, Beetz N, Kostin S, et al. Acquisition of the contractile phenotype by murine arterial smooth
muscle cells depends on the Mir143/145 gene cluster. J Clin Invest 2009;119:2634-47.
32. Gan CS, Wang CW, Tan KS. Circulatory microRNA-145 expression is increased in cerebral ischemia. Genet
Mol Res 2012;11:147-52.
33. Caruso P, Dempsie Y, Stevens HC, et al. A Role for miR-145 in Pulmonary Arterial Hypertension: Evidence
From Mouse Models and Patient Samples. Circ Res 2012;111:290-300.
34. Rane S, He M, Sayed D, et al. Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and
Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circ Res 2009;104:879-86.
35. Song XW, Li Q, Lin L, et al. MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is
essential for the maintenance of cell size in cardiomyocytes. J Cell Physiol 2010;225:437-43.
36. Haghikia A, Missol-Kolka E, Tsikas D, et al. Signal transducer and activator of transcription 3-mediated
regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a key role for
ubiquitin-conjugating enzymes. Eur Heart J 2011;32:1287-97.
37. He JF, Luo YM, Wan XH, Jiang D. Biogenesis of MiRNA-195 and its role in biogenesis, the cell cycle, and
apoptosis. J Biochem Mol Toxicol 2011;25:404-8.
38. Wang S, Aurora AB, Johnson BA, et al. The endothelial-specific microRNA miR-126 governs vascular
integrity and angiogenesis. Dev Cell 2008;15:261-71.
39. Chamorro-Jorganes A, Araldi E, Penalva LO, Sandhu D, Fernandez-Hernando C, Suarez Y. MicroRNA-16 and
microRNA-424 regulate cell-autonomous angiogenic functions in endothelial cells via targeting vascular
endothelial growth factor receptor-2 and fibroblast growth factor receptor-1. Arterioscler Thromb Vasc Biol
2011;31:2595-606.
40. Kulshreshtha R, Ferracin M, Wojcik SE, et al. A microRNA signature of hypoxia. Mol Cell Biol 2007;27:185967.
41. Chen WJ, Yin K, Zhao GJ, Fu YC, Tang CK. The magic and mystery of microRNA-27 in atherosclerosis.
Atherosclerosis 2012;222:314-23.
42 Li T, Cao H, Zhuang J, et al. Identification of miR-130a, miR-27b and miR-210 as serum biomarkers for
atherosclerosis obliterans. Clin Chim Acta 2011;412:66-70.
11
43. Aurora AB, Mahmoud AI, Luo X, et al. MicroRNA-214 protects the mouse heart from ischemic injury by
controlling Ca(2)(+) overload and cell death. J Clin Invest 2012;122:1222-32.
44. van Mil A, Grundmann S, Goumans MJ, et al. MicroRNA-214 inhibits angiogenesis by targeting Quaking and
reducing angiogenic growth factor release. Cardiovasc Res 2012;93:655-65.
12