Abstract
Angiogenesis is critical for tissue repair following myocardial
infarction (MI), which is exacerbated under insulin resistance or
diabetes. MicroRNAs are regulators of angiogenesis. We examined the
metabolic regulation of miR-409-3p in post-infarct angiogenesis.
miR-409-3p was increased in patients with acute coronary syndrome (ACS)
and in a mouse model of acute MI. In endothelial cells (ECs),
miR-409-3p was induced by palmitate, while vascular endothelial growth
factor (VEGF) and fibroblast growth factor (FGF) decreased its
expression. Overexpression of miR-409-3p decreased EC proliferation and
migration in the presence of palmitate, whereas inhibition had the
opposite effects. RNA sequencing (RNA-seq) profiling in ECs identified
DNAJ homolog subfamily B member 9 (DNAJB9) as a target of miR-409-3p.
Overexpression of miR-409-3p decreased DNAJB9 mRNA and protein
expression by 47% and 31% respectively, while enriching DNAJB9 mRNA by
1.9-fold after Argonaute2 microribonucleoprotein immunoprecipitation.
These effects were mediated through p38 mitogen-activated protein
kinase (MAPK). Ischemia-reperfusion (I/R) injury in EC-specific
miR-409-3p knockout (KO) mice (miR-409^ECKO) fed a high-fat,
high-sucrose diet increased isolectin B4 (53.3%), CD31 (56%), and
DNAJB9 (41.5%). The left ventricular ejection fraction (EF) was
improved by 28%, and the infarct area was decreased by 33.8% in
miR-409^ECKO compared with control mice. These findings support an
important role of miR-409-3p in the angiogenic EC response to
myocardial ischemia.
Keywords: MT: Non-coding RNAs, angiogenesis, endothelial cells,
microRNAs, acute myocardial infarction, DNAJB9
Graphical abstract
graphic file with name fx1.jpg
[53]Open in a new tab
__________________________________________________________________
Icli and colleagues examined the metabolic regulation of miR-409-3p in
endothelial cells and how it regulates angiogenesis following acute
myocardial infarction. Endothelial cell-specific genetic deletion of
miR-409-3p in mice improves angiogenesis and heart function in response
to myocardial ischemia and targets the DNAJB9/p38 mitogen-activated
protein kinase (MAPK) signaling pathway.
Introduction
Acute myocardial infarction (MI) is characterized by myocardial
necrosis following coronary artery occlusion, resulting in prolonged
myocardial ischemia.[54]^1^,[55]^2 Over the past several decades,
patient survival rates and recurrent ischemic event rates post MI have
improved because of recent advances in pharmacological management and
interventions.[56]^3 However, long-term morbidity and mortality after
acute MI remains high because of left ventricular (LV) dysfunction and
adverse LV remodeling, consisting of myocardial fibrosis and cardiac
hypertrophy.[57]^2^,[58]^4 Post-MI healing requires angiogenesis early
in the process, beginning in the border zone. Stimulation of this
response to enhance extension of newly formed blood vessels into the
infarcted zone is associated with improved outcomes consisting of less
remodeling, improved heart function, and reduced infarct size in animal
models. These observations indicate that improving the natural
angiogenic response post MI might represent a therapeutic
strategy.[59]^2 However, diabetes impairs the angiogenic response and
post-MI reparative process in human and animal
models.[60]^5^,[61]^6^,[62]^7
Angiogenesis is the formation of new blood vessels from preexisting
vessels. In this highly regulated process, activated endothelial cells
(ECs) proliferate, migrate, and differentiate to form a new vascular
lumen in response to proangiogenic stimuli such as vascular endothelial
growth factor (VEGF), fibroblast growth factor (FGF), or tumor necrosis
factor alpha (TNF-ɑ).[63]^8 After an ischemic event in the heart,
proangiogenic factors are induced to stimulate blood flow to the
infarcted region of the myocardium. Current investigational treatments
focus on use of proangiogenic and anti-apoptotic factors and cell-based
therapies.[64]^9^,[65]^10^,[66]^11^,[67]^12 For example, VEGFA is
induced in infarct regions of the heart and is responsible for
increased microvasculature permeability, granulation tissue thickness,
and vessel formation.[68]^13^,[69]^14 VEGF receptors are expressed in
ECs in the infarct region of mouse models post MI,[70]^2 but the
angiogenic response does not return myocardial health and function back
to pre-MI levels, especially under diabetic conditions. The exact
mechanisms limiting angiogenesis post MI remain poorly understood.
However, impaired endothelial function resulting from diabetes or
obesity could have important implications for this
process.[71]^15^,[72]^16 While current therapies are aimed at limiting
the damage the heart incurs post MI, there is a lack of available
treatments that improve myocardial repair.[73]^1
The above observations suggest that additional factors may limit
angiogenesis post MI and thus contribute to adverse remodeling post
infarct. Many patients with coronary artery disease have concomitant
conditions such as diabetes or obesity, which independently promote
endothelial dysfunction and could therefore adversely affect
angiogenesis.[74]^15^,[75]^16 Thus, understanding the mechanisms
through which these metabolic conditions impair angiogenesis might
identify potential candidate therapeutic targets not only to limit
myocardial damage but to promote cardiac repair after MI.[76]^1
MicroRNAs (miRNAs) are small, non-protein-coding RNAs that are 19–25 nt
in length that are capable of binding the 3′ untranslated region (UTR)
of target mRNA through association with an RNA-induced silencing
complex (RISC).[77]^4 This binding leads to inhibition or degradation
of the target mRNA, making miRNAs powerful regulators of gene
expression with vast potential for therapeutic applications. miRNAs
regulate numerous physiological and pathological cellular functions and
have been implicated in the process of angiogenesis, including in the
myocardium post MI.[78]^15^,[79]^17 We recently identified miR-409-3p
as a regulator of angiogenesis in brown fat and as promoting insulin
resistance with increased expression in the ECs of obese mouse brown
adipose tissue (BAT).[80]^18 As described above, metabolic conditions
promote coronary artery disease and may contribute to insufficient
angiogenesis after MI. Therefore, in the current study, we examined the
regulation of miR-409-3p in the setting of patients with acute MI and
in a mouse experimental MI model. We also investigated the molecular
mechanisms in the EC through which miR-409-3p alters angiogenesis.
DNAJ heat shock protein family member B9 (DNAJB9) is a member of the
molecular chaperone gene family, a group of specialized proteins that
can bind to substrate proteins and assist with folding, unfolding,
degradation, translocation, or disaggregation of protein aggregates.
Although its exact function is not completely understood, DNAJB9 is
known to act as a co-chaperone to major chaperone families, such as
heat shock protein 70s (Hsp70).[81]^19 DNAJB9 is also involved in the
unfolded protein response (UPR), a highly conserved pathway that is
classically activated by endoplasmic reticulum (ER) stress[82]^20 and
is transcriptionally activated when unfolded proteins accumulate in the
ER lumen (ER stress). Other factors are also known to be involved in
activation of the UPR pathway. Plasma levels of palmitate, a saturated
free fatty acid, is increased in obesity or insulin resistance,
adversely affecting many cellular processes and inducing low-level
inflammation. Palmitate can activate the UPR in many cell types,
resulting in associated ER stress.[83]^21
Here, we report that EC-specific deletion of miR-409-3p improves
angiogenesis and heart function in mice post MI and targets the
DNAJB9/p38 mitogen-activated protein kinase (MAPK) signaling pathway.
These findings unveil possible targets for future therapeutic
modulation to improve angiogenesis and remodeling of the heart post MI.
Results
To investigate the regulatory role miR-409-3p may play in acute MI,
miR-409-3p expression was measured in human plasma samples from the
Feiring Heart Biopsy Study 2 in a cohort of 21 adult patients
undergoing coronary bypass surgery (coronary artery bypass grafting
[CABG]) with non-coronary syndrome (NCS) or those with MI or unstable
angina within 30 days prior to admission. As seen in [84]Figure 1A,
circulating levels of miR-409-3p were increased by 83% in acute
coronary syndrome (ACS) patient plasma samples, as measured by
quantitative real-time PCR. Similarly, C57BL6/J wild-type (WT) mice
that underwent left anterior descending (LAD) coronary artery ligation
with 45-min ischemia followed by reperfusion showed increased
circulating miR-409-3p compared with the sham surgery controls
([85]Figure 1B). To elucidate regulation of miR-409-3p expression by
pro-angiogenic factors, human umbilical vein ECs (HUVECs) were treated
with the prototypical pro-angiogenic growth factors VEGF or FGF.
Interestingly, while the pro-angiogenic growth factors VEGF and FGF
significantly reduced miR-409-3p expression in HUVECs over 24 h
([86]Figures 1C and 1D), palmitate significantly increased miR-409-3p
expression over 24 h ([87]Figure 1E). In summary, these data suggest
that miR-409-3p is differentially regulated by pro- and anti-angiogenic
stimuli and that its expression may correlate with acute ischemic
injury.
Figure 1.
[88]Figure 1
[89]Open in a new tab
Expression of miR-409-3p in patients with ACS and in murine myocardial
ischemia
(A and B) Expression of miR-409-3p in plasma of patients with ACS
(A) and plasma of mice that underwent sham surgery or LAD ligation with
45-min ischemia-reperfusion (B). (C–E) Modulation of miR-409-3p
expression in HUVECs treated with 50 ng/mL VEGF (C), 50 ng/mL FGF (D),
or 100
[MATH: μ :MATH]
M palmitate (E). Data are representative of 3–6 replicates per
condition unless indicated otherwise. Statistical significance was
determined by unpaired Student’s t test or two-way ANOVA. ∗p < 0.05,
∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars indicate ± SEM.
Functionally, in the presence of palmitate, overexpression of
miR-409-3p “mimics” (miR-409-3p[m]) in HUVECs inhibited cell growth by
22%, whereas miR-409 inhibitors (miR-409[i], complementary antagonist)
increased EC growth by 22.2% ([90]Figure 2A). miR-409-3p overexpression
in the presence of palmitate decreased wound closure in scratch assays
by 33% compared with the non-specific (NS) control, whereas miR-409-3p
inhibition increased wound closure by 39% compared with the NS control
([91]Figures 2B and [92]S1), suggesting that miR-409-3p retains its
anti-angiogenic function in ECs in the presence of palmitate.
Figure 2.
[93]Figure 2
[94]Open in a new tab
miR-409-3p modulates angiogenesis in ECs in the presence of palmitate
(A and B) Growth (A) and EC migration (B) assays in HUVECs transfected
with NS control (NS[m]) or miR-409-3p mimic (miR-409-3p[m]) or
miR-negative inhibitor control (NS[i]) and miR-409-3p inhibitor
(miR-409-3p[i]) under 16-h 100 μM palmitate treatment. Statistical
significance was determined by unpaired Student’s t test and based on
comparison with the respective control group. ∗p < 0.05, ∗∗p < 0.01,
∗∗∗p < 0.001. ns, not significant. n = 6–8 replicates per condition.
Error bars indicate ±SEM.
To identify a target of miR-409-3p, a systematic approach utilizing RNA
sequencing (RNA-seq) profiling and in silico approaches combining
bioinformatics with prediction algorithms such as miRGator and
TargetScan was endeavored. RNA-seq analysis identified DNAJB9 as one of
the top regulated genes from 32 genes that were repressed by at least a
1.4-fold change (FC) with false discovery rate (FDR)-adjusted p < 0.01
([95]Figure 3A). In accordance with our RNA-seq profiling approach,
multiple independent prediction algorithms also identified DNAJB9 as a
predicted target of miR-409-3p with two potential binding sites in the
3′ UTR ([96]Figures S2A and S2B). Gene Ontology (GO) canonical pathway
analyses, also performed utilizing genes’ FDR-adjusted p < 0.01,
predicted the UPR as the most significantly regulated pathway
([97]Figure 3B). Interestingly, DNAJB9 is known to be a part of the UPR
signaling pathway.[98]^22 Furthermore, GO disease and function analyses
predicted miR-409-3p to be involved in cellular growth and function
([99]Figure 3C), in agreement with our findings in vitro.
Overexpression of miR-409-3p decreased DNAJB9 mRNA expression by 47%
compared with a NS control ([100]Figure 3D) in the presence of
palmitate, while DNAJB9 protein expression was decreased by 31.2%
([101]Figure 3E, top). Conversely, inhibition of miR-409-3p increased
DNAJB9 expression by 24.6% ([102]Figure 3E, bottom). Argonaute2 (AGO2)
microribonucleoprotein (miRNP) immunoprecipitation (IP) studies showed
1.9-fold enrichment of DNAJB9 ([103]Figure 3F), while FEM1C, a gene
that was not predicted by RNA-seq analysis to be a target of
miR-409-3p, was not enriched in the presence of miR-409-3p
([104]Figures S2C and S2D). Furthermore, overexpression of miR-409-3p
inhibited luciferase activity of the reporter construct containing the
3′ UTR sequence of DNAJB9 ([105]Figure 3G). Collectively, these data
indicate that DNAJB9 is a bona fide target of miR-409-3p in ECs.
Figure 3.
[106]Figure 3
[107]Open in a new tab
DNAJB9 is a target of miR-409-3p
(A) RNA-seq analyses of ECs that overexpress miR-409-3p or an NS
control and treated with 100
[MATH: μ :MATH]
M palmitate with FDR-corrected p < 0.01 and fold change (FC) > 1.4. (B
and C) GO analysis of RNA-seq with predicted (B) top 10 ingenuity
canonical pathways and (C) disease/function pathways. (D and E) HUVECs
transfected with NS[m] or miR-409-3p[m] or NS[i] or miR-409-3p[i] and
treated with 100
[MATH: μ :MATH]
M palmitate for 16 h were subjected to (D) quantitative real-time PCR
to measure DNAJB9 mRNA or (E) western blot analysis using antibodies to
DNAJB9 and α-tubulin. (F) AGO2 miRNP IP quantitative real-time PCR
analysis of enrichment of DNAJB9 mRNA in HUVECs transfected with NS[m]
or miR-409-3p[m] and treated with 100
[MATH: μ :MATH]
M palmitate for 16 h. (G) Luciferase activity of the DNAJB9 3′
untranslated region (UTR) normalized to protein concentration was
quantified in HEK293T cells transfected with NS[m] or miR-409-3p[m].
Statistical significance was determined by unpaired Student’s t test or
two-way ANOVA based on comparison with the respective NS control group.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 6 replicates per condition.
Error bars indicate ± SEM.
To identify downstream signaling pathways of miR-409-3p, we performed
GO analyses using the differentially expressed genes (DEGs) with
FC > 1.4 and FDR-adjusted p < 0.01 from the RNA-seq datasets. DEGs were
subjected to GO network analysis, and p38 MAPK was among the
significantly regulated signaling pathways ([108]Figure 4A).
Overexpression of miR-409-3p in ECs significantly reduced the p38
phosphorylation up to 29% in response to 30 and 60 min of palmitate
stimulation ([109]Figure 4B), whereas miR-409-3p inhibition increased
p38 MAPK phosphorylation by up to 47% in response to 30 and 60 min of
palmitate stimulation ([110]Figure 4C). This regulation was specific to
p38 signaling, while other signaling pathways, such as protein kinase B
(AKT) or mammalian target of rapamycin (mTOR) were not found to be
modulated by miR-409-3p ([111]Figures S3A and S3B).
Figure 4.
[112]Figure 4
[113]Open in a new tab
miR-409-3p modulates p38 MAPK signaling
(A) Gene network visualization of the RNA-seq date set from ECs
overexpressing miR-409-3p or the NS control and treated with 100
[MATH: μ :MATH]
M palmitate for 16 h identified P38 MAPK as a potential downstream
signaling pathway. (B and C) Western blot analyses of phosphorylated
p38 (p-p38) and p38 antibodies on HUVECs transfected with NS[m] or
miR-409-3p[m] (B) and NS[i] or miR-409-3p[i] (C) and treated with
100 μM palmitate for the indicated duration. Statistical significance
was determined by unpaired Student’s t test or two-way ANOVA based on a
comparison with the respective NS control group. ∗p < 0.05, ∗∗p < 0.01.
n = 5–6 replicates per condition. Error bars indicate ± SEM.
To determine whether DNAJB9 silencing can functionally recapitulate the
effects of miR-409-3p in EC growth and migration, we employed the small
interfering RNA (siRNA) knockdown strategy to silence DNAJB9 expression
in ECs ([114]Figure S2E). Silencing of DNAJB9 mimicked miR-409-3p′s
effects on EC migration, as measured by wound healing scratch assay
([115]Figure 5A), and inhibited EC growth in the presence of palmitate
([116]Figure 5B), similar to miR-409-3p overexpression. Additionally,
knockdown of DNAJB9 decreased p38 phosphorylation up to 25% after 60
and 90 min of palmitate stimulation ([117]Figure 5C). Collectively,
these data indicate that DNAJB9 knockdown phenocopies the effects of
miR-409-3p in ECs.
Figure 5.
[118]Figure 5
[119]Open in a new tab
Knockdown of DNAJB9 functionally recapitulates miR-409-3p′s effects
in vitro
(A–C) EC migration (A), growth (B), and western blot analysis (C) of
p38 MAPK signaling in HUVECs transfected with negative control siRNA
(Ctrl[siRNA]) or DNAJB9 siRNA (DNAJB9[siRNA]). ECs were treated for
16–18 h with palmitate (A and B) or for the indicated duration (C).
Statistical significance was determined by unpaired Student’s t test or
two-way ANOVA based on comparison with the indicated control group.
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 6–9 replicates per condition.
Error bars indicate ± SEM.
To study the EC-specific effects of miR-409-3p neutralization on MI, an
EC-specific knockout mouse model of miR-409-3p was generated using
homozygous loxP floxed mice (miR409 ^fl/fl) and crossed with mice with
a constitutively active VE-Cadherin promoter to generate EC-specific
miR-409-3p knockout (miR409^ECKO) mice ([120]Figure S4A). Cre-mediated
recombination was confirmed by genomic DNA PCR ([121]Figure S4B).
Compared with the miR409 ^fl/fl control group, miR-409-3p expression in
miR409^ECKO mice was reduced by 80% in ECs isolated from the heart but
not in peripheral blood mononuclear cells (PBMCs) ([122]Figure S4C).
Additionally, miR-409-3p expression was reduced by 88% in ECs compared
with the non-EC fraction of the heart (composed of ∼77% cardiac
myocytes, 21% vascular smooth muscle cells, and 2% fibroblasts) in
miR409^ECKO mice ([123]Figure S4D). To assess whether EC-specific
neutralization of miR-409-3p will induce angiogenesis and ameliorate LV
function decline post acute MI, miR409 ^fl/fl control mice and
miR409^ECKO mice were fed a high-fat and high-sucrose diet for 8 weeks
subjected to 45-min LAD ligation followed by reperfusion
([124]Figure 6A). In response to the high-fat and high-sucrose diet,
plasma free fatty acid (FFA) levels of miR409 ^fl/fl and miR409^ECKO
were increased by 37.9% and 38.3%, respectively, compared with the chow
control group ([125]Figure S4E). Transthoracic echocardiography was
performed on mice prior to ischemia-reperfusion as well as 2 days post
MI to evaluate LV function. Baseline ejection fraction (EF) and
fractional shortening (FS) were normal. Furthermore, there were no
significant differences in EF or FS in baseline echocardiograms of the
MiR409 ^fl/fl or the MiR409^ECKO group ([126]Figure S5B). Infarct area
was 33.8% smaller in the MiR409^ECKO compared to the control group as
measured by triphenyltetrazolium chloride (TTC) staining of the heart
([127]Figures 6B and [128]S5A), while there were no significant changes
observed in the heart weights or total body weights between the groups
([129]Figure S5E). Short-axis M-mode echocardiogram images showed a 28%
improvement in LV EF and a 23% improvement in FS in the miR409^ECKO
group compared with the miR409^fl/f control group 2 days post MI
([130]Figures 6C and [131]S5C). In addition, the miR409^ECKO group also
showed a smaller change in EF and FS 2 days post MI compared with the
baseline measurements ([132]Figures 6C and [133]S5C). In agreement with
these findings, long axis B-mode echocardiogram images showed 32%
improvement of the LV EF in the miR409^ECKO group compared with the
miR409^fl/f control group 2 days post MI ([134]Figure S5D). To evaluate
angiogenesis post MI, heart tissue was harvested 2 days post
ischemia-reperfusion, and the border zone was stained for isolectin B4
and CD31. Interestingly, miR409^ECKO mice showed a 53.3% increase in
isolectin B4 staining ([135]Figure 6D), while CD31 was increased by 56%
([136]Figure 6E), and elimination of the primary antibody did not
result in a significant signal ([137]Figure S5F). Furthermore, in
accordance with our in vitro findings, staining for DNAJB9 exhibited a
41.5% increase in response to EC-specific miR-409-3p neutralization in
miR409^ECKO mice compared with the control group ([138]Figure 6F) as
well as a corresponding 2-fold increase in DNAJB9 expression level in
the ischemic zone of the heart ([139]Figure 6G), while phosphorylation
of p38 MAPK was significantly increased 1.9-fold overall between the
two groups ([140]Figure 6H). In summary, these results identify an
important role of miR-409-3p in improving LV function post MI through
modulation of angiogenesis.
Figure 6.
[141]Figure 6
[142]Open in a new tab
EC-specific knockout of miR-409-3p improves angiogenesis and heart
function in a murine model of acute MI
(A) Ischemia-reperfusion injury was performed on miR409^ECKO and
miR409^fl/fl control mice after 8 weeks of a high-fat, high-sucrose
diet. (B) Infarct size in the left ventricle was calculated with
triphenyltetrazolium chloride (TTC) staining of the heart and
normalized to the AAR. (C) Representative M-mode SAX echocardiograms,
LV ejection fraction (EF), and difference from baseline data 2 days
after ischemia-reperfusion injury. (D and E) Angiogenesis post MI was
quantified on day 2 in heart sections from the border zone stained for
isolectin B4 (D) or CD31 (E) (scale bars, 100 μm; inset, 12.5 μm). (F)
DNAJB9 positivity in the border zone of the heart was measured by
confocal microscopy (scale bars, 100 μm). (G) Relative mRNA expression
levels of DNAJB9 in the ischemic zone. (H) Western blot analysis of the
p38 MAPK signaling pathway from ischemic zone heart samples (n = 4 mice
per group). Statistical significance was determined by unpaired
Student’s t test or two-way ANOVA based on comparison with the
indicated control group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. n = 5–8
mice per condition unless noted otherwise. Error bars indicate ±SEM.
Discussion
In the current study, we investigated the role of miR-409-3p in
angiogenesis after MI. We observed the following: (1) there is
increased circulating miR-409-3p in humans after ACS syndrome or in
mice after ischemia/reperfusion; (2) miR-409-3p opposes endothelial
migration and angiogenesis in vitro; (3) miR-409-3p modulates p38 MAPK
pathway activation through direct targeting of DNAJB9; and (4) genetic
deletion of endothelial miR-409-3p promotes angiogenesis and recovery
of LV function after ischemia/reperfusion in mice. We interpret these
findings to identify miR-409-3p as a novel regulator of post-MI
angiogenesis through the DNAJB9-p38 MAPK signaling axis.
Acute MI remains one of the leading causes of death among ischemic
cardiovascular diseases in the US and around the world. Myocardial
healing after acute ischemia is a complex biological process, and
accumulating studies demonstrate the importance of angiogenesis in
post-MI healing and in improvement of LV
function.[143]^23^,[144]^24^,[145]^25^,[146]^26^,[147]^27 However, this
process is markedly impaired in the context of insulin resistance and
diabetes. In recent years, a number of miRNAs have been reported to
potentially induce therapeutic angiogenesis post MI. For example,
miR-26a has been characterized as an anti-angiogenic miRNA increased in
MI that inhibits EC migration and sprouting angiogenesis through
targeting bone morphogenic protein (BMP)/SMAD1 signaling,[148]^17
whereas miR-210 has been found to promote angiogenesis in the
myocardium post MI, leading to improved cardiac function through
stimulating hepatocyte growth factor.[149]^28 miR-92a is an
anti-angiogenic miRNA that targets mRNAs corresponding to
pro-angiogenic proteins and is inhibited by oxidative stress.[150]^29
However, the exact mechanism by which these miRNAs modulate
angiogenesis and the role of ECs in this process is not completely
understood.[151]^2^,[152]^17^,[153]^30^,[154]^31
Our group recently identified miR-409-3p as an important regulator of
adipose tissue angiogenesis, browning, and metabolism in obese
mice.[155]^18 However, its role in post-MI angiogenesis and heart
function has not been studied before. In this investigation, we show
that neutralization of endothelial miR-409-3p rapidly stimulates
angiogenesis post MI by increasing DNAJB9 expression and p38 MAPK
phosphorylation, building on the growing body of literature showing an
important role of miRNAs in regulating the angiogenic response post MI.
Furthermore, we identified that miR-409-3p expression is increased in
response to acute MI in mice and in human subjects with ACS
([156]Figures 1A and 1B). Overexpression of miR-409-3p impaired EC
angiogenic responses in the presence of palmitate, while its inhibition
had the opposite effects, solidifying its role as a potent
anti-angiogenic miRNA ([157]Figures 2 and [158]S1). EC-specific genetic
deletion of miR-409-3p in mice that consumed a high-fat and
high-sucrose diet for 8 weeks showed robust induction of angiogenesis
within 2 days of MI with improved LV function and reduced infarct size
([159]Figures 6A–6F and [160]S4A). It is important to note that we
observed some variation in infarct size ([161]Figure S5A), which may be
caused by the slight variations of the mouse vascular anatomy as well
as surgical variation during ligation of the LAD. However, the observed
variation in our studies is in accordance with studies utilizing
ischemia-reperfusion[162]^32^,[163]^33 or permanent LAD ligation models
in mice.[164]^34^,[165]^35 Our findings suggest, for the first time,
that deficiency of miR-409-3p improves LV function and angiogenesis in
response to myocardial ischemia.
Vascular endothelium plays an important role in maintaining vascular
homeostasis and regulates vascular tone, permeability, blood fluidity,
inflammation, and angiogenesis.[166]^36^,[167]^37^,[168]^38 Plasma FFA
levels are increased in patients with type 2 diabetes (T2D) and
obesity, resulting in increased prevalence of cardiovascular
events.[169]^39^,[170]^40^,[171]^41^,[172]^42 An increasing number of
studies show a correlation between FFAs and vascular endothelium
dysfunction.[173]^43^,[174]^44 Additionally, miRNAs have been
implicated in improving various aspects of palmitate-induced EC
dysfunction, such as dampening inflammation, apoptosis, and stimulating
EC migration.[175]^45^,[176]^46^,[177]^47^,[178]^48^,[179]^49 Here, we
used palmitate, a 16-carbon saturated fatty acid and a common
circulating saturated FFA,[180]^50 to dissect the role of miR-409-3p
and its targets in EC-driven angiogenic responses. Interestingly, while
palmitate increased miR-409-3p expression in ECs ([181]Figure 1E),
neutralization of miR-409-3p expression in ECs in the presence of
palmitate significantly increased EC growth and migration
([182]Figure 2), overcoming palmitate-induced EC dysfunction
([183]Figure S1). DNAJB9 similarly phenocopied miR-409-3p′s effects on
EC growth and migration in the presence of palmitate through regulation
of p38 MAPK signaling ([184]Figure 5). Mice with genetic EC deletion of
miR-409-3p that were maintained on a high-fat and high-sucrose diet
showed elevated plasma FFA levels comparable with the
literature.[185]^51^,[186]^52 However, LV function and infarct size
were improved in miR409^ECKO mice, suggesting alleviation of EC
dysfunction caused by prolonged exposure to a Western diet. These
findings are in agreement with the literature, where modulation of
other miRNAs has been shown to improve palmitate-induced EC
dysfunction.[187]^53 For example, overexpression of miR-155 has been
shown to inhibit palmitate-induced apoptosis and promote EC
proliferation through regulation of the Wnt signaling pathway,[188]^47
whereas miR-126 modulated EC migration through the ERK (extracellular
signal-regulated kinase)/MAPK signaling axis in the presence of
palmitate[189]^49; however, our study is distinct from others. First,
we identified DNAJB9, a member of the UPR pathway, to be directly
targeted by miR-409-3p to exert its downstream effects on regulation of
EC angiogenesis. Next, we showed that the p38 MAPK signaling pathway
was utilized by the miR-409-3p/DNAJB9 signaling axis in the presence of
palmitate in ECs in vitro and in a genetic deletion of endothelial
miR-409-3p in a mouse model of acute MI in vivo.
Investigative efforts to improve the prognosis of MI patients have
centered around use of angiogenic or anti-apoptotic factors or
cell-based therapies targeting the interface between perfused and
non-perfused areas within the myocardium. Accumulating studies report
the benefits of enhanced angiogenesis by increasing cardiomyocyte
survival within the ischemic zone and subsequent LV
function.[190]^10^,[191]^23^,[192]^24^,[193]^25^,[194]^26 We examined
angiogenesis within the ischemic zone in our genetic deletion of
endothelial miR-409-3p in a mouse model that underwent LAD ligation
with 45-min ischemia-reperfusion and showed that this non-perfused area
is an active site of angiogenesis as early as 2 days after MI.
Consistent with previous reports, confocal immunofluorescence
evaluation of the border zone showed increased angiogenesis, as
measured by CD31 and isolectin B4 staining ([195]Figure 6).
Furthermore, the increase in angiogenesis positively correlated with
improved LV function, as measured by EF and FS ([196]Figures 6 and
[197]S5). One of the mechanisms explaining the increased angiogenesis
observed in miR409^ECKO mice may involve activation of p38 MAPK through
DNAJB9 ([198]Figures 5 and [199]6). Activation of p38 MAPK has been
shown to induce actin organization and endothelial migration.[200]^54
Pharmacological inhibition of p38 MAPK in an experimental prostate
tumor model in vivo was associated with a significant reduction in
tumor growth and vessel density, an effect that was associated with
pathological angiogenesis.[201]^55 Our studies are in agreement with
these findings, where neutralization of miR-409-3p in ECs increased EC
migration in vitro in the presence of palmitate ([202]Figure 2).
Additionally, overexpression of miR-409-3p or siRNA knockdown of DNAJB9
significantly decreased EC migration ([203]Figures 2 and [204]5).
There are a few limitations of this study. First, our in vivo
experiments involved a non-inducible, constitutively active, genetic
deletion of miR-409-3p. We observed that ECs isolated from the heart of
this mouse strain showed an ∼80% decrease in miR-409-3p expression
([205]Figure S4C, left). Although the remaining miR-409-3p expression
could be due to slight contamination of non-ECs, such as fibroblasts,
cardiac myocytes, or other cell types that also express
miR-409-3p,[206]^18 it could also be the result of incomplete
recombination. The UPR pathway has been shown by several
groups[207]^56^,[208]^57 to have cardioprotective effects following
ischemia-reperfusion injury. Therefore, we cannot completely rule out
involvement of non-EC cell types in the phenotype reported in this
study. In total PBMCs, we did not observe a difference in miR-409-3p
expression between Cre+ miR409^ECKO mice and their littermate Cre−
MiR409 ^fl/fl controls, ruling out a possible contribution from immune
cells ([209]Figure S4C, right). We further profiled expression of
miR-409-3p in Cre+ miR409^ECKO mice and showed an ∼89% decrease in
miR-409-3p expression in ECs compared with non-ECs. In an effort to
profile the relative expression of miR-409-3p in non-EC heart extracts,
we measured miR-409-3p expression in the major cardiac cell types, as
documented in the literature.[210]^58^,[211]^59 While ∼77% of the
non-EC cell population obtained was composed of cardiac myocytes, ∼21%
vascular smooth muscle cells, and 2% fibroblasts. Therefore, a
significant majority of miR-409-3p expression in the non-EC heart
extracts was contributed by cardiac myocytes ([212]Figure S4D).
We note that the canonical UPR involves a sophisticated set of
signaling pathways that are activated when altered cellular homeostatic
conditions disrupt protein folding in the ER, causing accumulation of
unfolded/misfolded proteins, which, in turn, leads to activation of the
UPR pathway.[213]^60 Increased ER stress and the UPR are known to have
downstream angiogenic effects. For example, Hsp70 is highly expressed
in ECs and regulates EC proliferation, survival, and migration in tumor
angiogenesis.[214]^61 Interestingly, angiogenic stimuli, such as
hypoxia, ischemia, inflammation, and oxidative stress, can also trigger
the UPR pathway, and this process has been shown to be vital for
endothelial survival and activity. Furthermore, molecular chaperones
that normally facilitate protein folding in the ER can regulate
angiogenic factor production and angiogenic
responses.[215]^61^,[216]^62^,[217]^63^,[218]^64 In this report, we
showed that one of these chaperone molecules, DNAJB9,[219]^65
facilitates EC-mediated angiogenic responses in the heart following MI
through regulation of p38 MAPK signaling. There are several upstream ER
transmembrane protein sensors in the canonical UPR pathway that mediate
UPR signals, such as inositol-requiring 1 (IRE1), PKR-like ER kinase
(PERK), and activating transcription factor 6 (ATF6), which, in turn,
activate transcription factors such as XBP1 (X-box binding protein 1).
Nuclear translocation of XBP1 induces expression of ER chaperones such
as DNAJB9 and calreticulin (CALR)[220]^66 to alleviate ER stress and
prevent chronic UPR activation.[221]^60 In addition, in ECs, UPR can be
activated without accumulation of unfolded proteins in the ER, a
pathway that is associated with VEGF activation of the mTORC1 complex
through the UPR mediators IRE1, PERK, and ATF6.[222]^22 Our studies are
novel and add to these findings by showing that, in the presence of
palmitate, miR-409-3p modulates DNAJB9 expression, which, in turn,
regulates p38 MAPK phosphorylation and angiogenesis. However, whether
such regulation requires ER stress involving the IRE/XBP1 signaling
node or through ER stress-independent activation of UPR signaling by
palmitate remains to be determined.
Despite some of the limitations discussed here, the novel mechanistic
insights from this study have important implications. We demonstrated
here, for the first time, that miR-409-3p is dysregulated after MI and
plays an important role in promoting EC angiogenic processes and the
ischemic angiogenic response to acute MI. Identification of DNAJB9 as a
novel miR-409-3p target provides potential clues regarding the
mechanism by which miR-409-3p modulates EC angiogenic responses.
EC-specific knockout of miR-409-3p in our genetic miR409^ECKO mouse
strain shows that there is a significant increase in phosphorylation of
p38 MAPK signaling, a pathway that modulates EC migration.[223]^54
These findings support the mechanism by which miR-409-3p modulates
angiogenesis through utilization of the UPR in ECs and provide new
opportunities for therapeutic intervention.
Materials and methods
Cell culture and transfection
HUVECs (Lonza, CC-2519) passaged fewer than five times were used for
all experiments and cultured in EGM-2 growth medium (Lonza, cc-3162).
For transfection studies, HUVECs plated at 50,000 cells/mL were
cultured overnight before being transfected with Lipofectamine 2000
transfection reagent (Invitrogen, 52887) in accordance with the
manufacturer’s instructions. For reporter studies, HUVECs were
transfected with 400 ng of the indicated reporter constructs and either
50 nM miR-409-3p mimic (Thermo Scientific, 4464066) or NS control
(Thermo Scientific, 4464058) or 100 nM miR-409-3p inhibitor (Thermo
Scientific, 4464084) or NS control (Thermo Scientific, 4464076). All
knockdown experiments were conducted using the same transfection
protocol with the respective control (Thermo Scientific, 4390843) or
target of interest siRNA at 30 nM, except for the scratch EC migration
assay at 10 nM.
Quantitative real-time PCR
HUVECs were suspended in TRIzol reagent (Invitrogen), and total RNA and
miRNA were isolated according to the manufacturer’s instructions.
Reverse transcriptions were performed using the QIAGEN QuantiTect
Reverse Transcription Kit (205311) or miRCURY LNA RT Kit (339340). The
QuantiTect SYBR Green RT-PCR Kit (204245) or miRCURY LNA SYBR Green PCR
Kit (339345) from QIAGEN, respectively, was used for quantitative
real-time PCR analysis with the CFX Opus 96 real-time PCR system
(Bio-Rad). Gene-specific primers (Invitrogen) were used to detect human
DNAJB9, while specific miRCURY LNA miRNA PCR assays from QIAGEN
(339306) were used to detect miR-409-3p ([224]Table 1). Samples were
normalized to either endogenous human β-actin or 5S rRNA. FCs were
calculated by ΔΔCt method. Significance was determined by Student’s
two-tailed t test or one-way ANOVA; p < 0.05.
Table 1.
Primers for quantitative real-time PCR
Gene Forward sequence (5′-3′) Reverse sequence (5′-3′) Species
β-Actin GGACTTCGAGCAAGAGATGG AGCACTGTGTTGGCGTACAG human
HPRT GCTATAAATTCTTTGCTGACCTGCTG AATTACTTTTATGTCCCCTGTTGACTGG human
DNAJB9 TCTTAGGTGTGCCAAAATCGG TGTCAGGGTGGTACTTCATGG human
DNAJB9 CTCCACAGTCAGTTTTCGTCTT GGCCTTTTTGATTTGTCGCTC mouse
MYC GGCTCCTGGCAAAAGGTA CTGCGTAGTTGTGTGCTGATGT human
β-Actin GAAATCGTGCGTGACATCAAAG TGTAGTTTCATGGATGCCACAG mouse
TNNT2 CAGAGGAGGCCAACGTAGAAG CTCCATCGGGGATCTTGGGT mouse
ACTA2 GTCCCAGACATCAGGGAGTAA TCGGATACTTCAGCGTCAGGA mouse
DDR2 ATCACAGCCTCAAGTCAGTGG TTCAGGTCATCGGGTTGCAC mouse
vWF CTCTTTGGGGACGACTTCATC TCCCGAGAATGGAGAAGGAAC mouse
[225]Open in a new tab
Western blot analysis
HUVECs transfected with miR-409-3p mimic, miR-409-3p inhibitor, NS miR
controls, DNAJB9 siRNA, or control siRNA were cultured for 72 h before
the cells were harvested. Mouse tissue was harvested according to the
experimental protocol, and a mechanical tissue homogenizer was used.
Total cellular protein was isolated by RIPA buffer (50 mM Tris-HCl [pH
7.4], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS;
Boston BioProducts, BP-115) supplemented with 1× Halt protease
inhibitor cocktail (Thermo Scientific, 1861261) and 1× 0.5 M EDTA
(Thermo Scientific, 1861274). Cell or tissue debris was removed by
centrifugation at 15,000 rpm for 15 min. Protein quantification was
performed using the Pierce BCA Protein Assay Kit (Thermo Scientific,
23227). Lysates were separated using 5%–15% gels, transferred to
polyvinylidene fluoride (PVDF) membranes, and subjected to western
blotting using antibodies against DNAJB9 (Abcam, ab118282).
phosphorylated p38 (p-p38; Cell Signaling Technology, 9215), p38 MAPK
(Cell Signaling Technology, 9212), p-AKT (Cell Signaling Technology,
4060), pan-AKT (Cell Signaling Technology, 2920), p-mTOR (Cell
Signaling Technology, 2971), mTOR (Cell Signaling Technology, 2972),
α-tubulin (Cell Signaling Technology, 3873), and β-actin (Cell
Signaling Technology, 4970) were incubated with gentle agitation at 4°C
overnight at a concentration of 1:1,000. Horseradish peroxidase
(HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technology,
7074) at a dilution of 1:2,000, HRP-conjugated horse anti-mouse
antibody (Cell Signaling Technology, 7076) at a 1:5,000 dilution, or
HRP-conjugated mouse anti-goat antibody (Santa Cruz Biotechnology,
sc-2354) at a 1:2,000 dilution were used, depending on the primary
antibody specifications. An enhanced chemiluminescence (ECL) assay was
performed according to the manufacturer’s instructions (Thermo
Scientific, 34095, A38555). Three or more biological replicates were
performed for each experiment. Quantification was conducted using
ImageJ software, and significance was determined by Student’s
two-tailed t test or one-way ANOVA; p < 0.05.
Total RNA extraction and bulk RNA-seq analysis
Total RNA from HUVECs transfected with 30 nM miR-409-3p mimic or NS
control and treated with 100 μM palmitate was purified using the RNeasy
Mini Kit (QIAGEN, 74104). The library preparation was done using the
QIAseq Stranded mRNA Kit (QIAGEN). From 500 ng starting material, the
mRNA was enriched and fragmented using enzymatic fragmentation. After
first and second strand synthesis, the cDNA was end repaired and 3′
adenylated. Sequencing adapters were ligated to the overhangs. Adapted
molecules were enriched using 13 cycles of PCR and purified by
bead-based cleanup. The library size distribution was validated and
quality inspected on a TapeStation (Agilent Technologies). Libraries
are pooled based in equimolar concentrations based on the Bioanalyzer
automated electrophoresis system (Agilent Technologies). The library
pools were quantified using qPCR, and an optimal concentration of the
library pool was used to generate clusters on the surface of a flow
cell before sequencing on a NextSeq500/550 instrument (1× 75 cycles)
according to the manufacturer’s instructions (Illumina). Raw data from
RNA-seq were deposited into GEO: [226]GSE231988.
Pathway enrichment analysis
DEGs were identified using adjusted p < 0.01 (FDR) and FC > 1.3. The
DEGs were visualized using a volcano plot generated with
EnhancedVolcano R package (GitHub). DEGs were also subjected to network
analysis using Ingenuity Pathway Analysis (IPA) software (QIAGEN).
Genes that were identified with p < 0.05 were subjected to gene set
enrichment analyses using IPA software (QIAGEN). The significant values
for the canonical pathways were calculated by Fisher’s exact test, and
the top 10 pathways were visualized.
miRNP IP
miRNP IP was performed as described previously.[227]^67 Briefly,
Myc-tagged Ago-2 was co-transfected with either miR-409-3p or miR
negative control in HUVECs. Cells were washed in ice-cold PBS, released
by scraping, and lysed in buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl,
2 mM EDTA, 0.5% Triton X-100, 100 units/mL of RNase Plus [Promega]
supplemented with 1× protease inhibitor. One-twentieth of the
supernatant volume was collected in TRIzol for use as an extract
control. The remaining portion of the supernatant was pre-cleared with
protein A/G UltraLink Resin (Pierce), to which 2 μg anti c-myc antibody
(Cell Signaling Technology, 2276) was added, and the mixture was
allowed to incubate overnight at 4°C. The following day, protein A/G
UltraLink Resin was added. After 4 h of mechanical rotation at 4°C, the
agarose beads were pelleted and washed four times in wash buffer (50 mM
Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Triton X-100). Finally, 1 mL of
TRIzol was added to the beads, and RNA was isolated as described above.
Total RNA was reverse transcribed into cDNA for quantitative real-time
PCR analysis.
Scratch assay for EC migration
HUVECs plated in 12-well plates at 50,000 cells/well and transfected
with miR-409-3p mimic, miR-409-3p inhibitor, NS miR controls, DNAJB9
siRNA, or control siRNA were cultured for 60 h in 12-well plates. The
cell monolayer was scratched using a 200-μL micropipette tip to form a
750-μm wound, followed by 16–18 h of 100 μM palmitate treatment. Cells
were imaged by a CytoSMART Omni live-cell imaging device (CytoSMART
Technologies) over time to assess wound closure. 3–5 technical
replicates were used per condition.
EC growth assay
HUVECs transfected with miR-409-3p mimic, miR-409-3p inhibitor, NS miR
controls, DNAJB9 siRNA, or control siRNA were cultured for 24 h in
12-well plates. Cells were then plated in 24-well plates at 8,000
cells/well and treated with 100 μM palmitate for 16 h, followed by
tagging with Calcein AM (Thermo Scientific, C3100) at 1 μM for 1 h at
37°C. Imaging was performed using Nikon FL and LI-COR Odyssey M and
analyzed using ImageJ software to assess cell growth. Four technical
replicates and 7 random images were taken blindly per condition.
Histology and immunostaining
Murine heart tissues were embedded in paraffin and sectioned. Sections
were deparaffinized and rehydrated, and antigen retrieval was done by
placing the slides in 10 mM Tris at 85°C for 30 min. Blocking was
performed using 5% normal donkey serum (Jackson ImmunoResearch,
017-000-121) with 0.2% Triton X-100 for 90 min at room temperature.
Streptavidin/biotin blocking (Vector Laboratories, SP-2002) was
performed on sections to be stained for isolectin B4. Sections were
incubated with primary antibodies overnight at 4°C. Primary antibodies
were diluted in 5% normal donkey serum with 0.2% Triton X-100 as
follows: 1:25 goat pAb to DNAJB9 (Abcam, ab118282), 1:25 rat anti-CD31
(Dianova, DIA-310), and 1:100 biotinylated isolectin B4 (Vector
Laboratories, B-1205). Slides were washed with PBS and incubated with
the secondary antibodies DyLight594 horse anti-goat IgG (Vector
Laboratories, DI-3094), Cy3-conjugated AffiniPure donkey anti-rat IgG
(Jackson ImmunoResearch, 712-165-153), or Alexa Fluor 488-conjugated
streptavidin (Jackson ImmunoResearch, 016-540-084), respectively, for
90 min at room temperature. After drying, slides were mounted with
ProLong Gold antifade reagent with DAPI (Invitrogen, [228]P36935).
Quantification was performed using ImageJ software from 6 random areas
obtained by confocal imaging using Olympus FV3000. Hematoxylin and
eosin (H&E)-stained heart sections were imaged using LI-COR Odyssey M,
and total LV area quantification was performed using ImageJ software.
TTC staining and fluorescent microsphere injections
Mice were injected with 75μL of 2-μm FluoSpheres carboxylate-modified
microspheres (F8826, Thermo Scientific) retro-orbitally 48 h post
ischemia-reperfusion. The harvested heart was sliced into 2-mm sections
using heart slicer matrix. Each slice was used to quantify the area at
risk (AAR) and the infarcted zone. The AAR, demarcated by fluorescent
microspheres, was visualized using LI-COR Odyssey M. To outline the
infarcted area, sections were incubated in 1% (w/v) TTC (Sigma) in PBS
(pH 7.4) at 37°C for 20 min. For each section, the AAR and infarct area
were measured using ImageJ.
Luciferase activity assay and cell culture transfection
DNAJB9 3′ UTR (OriGene, SC214927) sequences were PCR amplified with
specific primers and purified, followed by restriction enzyme
digestion. HEK293T cells (a gift from the MCRI Cell Culture Core)
cultured in 12-well plates were transfected in triplicates using
Lipofectamine 2000 transfection reagent with 500 ng of the indicated
reporter construct per well. After 24 h, cells were transfected with
50 nM miR-409-3p mimic and the equivalent NS control. Cells were then
harvested 12 h later in accordance with the Luciferase Reporter Assay
System (Promega, E4530). Each reading of luciferase activity was
normalized to the total protein read for the same lysate as quantified
using the Pierce BCA Protein Assay Kit (Thermo Scientific, 23227).
Experimental ischemia-reperfusion model
MI was performed as described previously.[229]^68 Briefly, 12-week-old
mice were deeply anesthetized and ventilated, and the heart was exposed
through a thoracotomy. A suture was tied around the proximal LAD artery
for 45 min, after which the suture was untied, and the chest was
closed.
Transthoracic echocardiography and analyses
Mice were anesthetized with 2.5% isoflurane, and short-axis (SAX)
M-mode images and long-axis (LAX) B-mode images were acquired through
transthoracic echocardiography before ischemia-reperfusion and 48 h
after ischemia-reperfusion. Analyses were performed using the Vevo LAB
3.1.0 (Fujifilm) cardiac package. EF and FS were measured by averaging
values obtained from 5 cardiac cycles in SAX M-mode and from B-mode LAX
images as described previously.[230]^69^,[231]^70 SAX M-mode images
were obtained at the mid-papillary level, with care taken to measure
only endocardium not overlapping with the papillary muscles.
EC isolation from the heart
Hearts were harvested 48 h post ischemia-reperfusion and digested with
1 mg/mL collagenase and dispase solution in DMEM/F12 at 37°C for
40 min, followed by neutralization with DMEM/F12 with 10% fetal bovine
serum (FBS). The cell suspension was filtrated through a 70-μm strainer
(BD Falcon) and centrifuged at 500 × g for 10 min at 4°C. The resulting
pellet was suspended in RBC lysis solution (eBioscience) for 5 min and
neutralized with DMEM/F12 medium with 10% FBS. The suspension was then
filtered through a 40-μm strainer (BD Falcon), and the pellet was
suspended in PBS with 0.1% BSA and 2 mM EDTA. Later, the suspension was
incubated with sheep anti-rat IgG Dynabeads coated with PECAM-1
antibodies (BD Biosciences, 557,355). The bead-bound cells were
collected using a magnet and washed 3 times. The composition of the
non-EC cell lysates was evaluated using the following primer sets:
TNNT2 (Troponin T2, cardiac type) for cardiac myocytes, DDR2 (discoidin
domain receptor tyrosine kinase 2) for fibroblasts, ACTA2 (actin alpha
2, smooth muscle) for vascular smooth muscle cells, and vWF (von
Willebrand factor) for ECs ([232]Table 1).
PBMC isolation
Murine blood from heart puncture was heparinized and pooled to 2 mice
per sample. Lymphocyte separation media (LSM) 1107 (PromoCell, C-44010)
was used according to the manufacturer’s instructions with 3 mL LSM per
blood sample. PBMCs were washed with PBS three times. The
centrifugation step post first wash was repeated twice to form a
pellet. After three washes, cells were resuspended in 1 mL TRIzol.
FFA analysis
Blood was extracted from mice collected in K2EDTA-coated tubes (BD
Microtainer, 365974) and immediately centrifuged at 2,000 × g for
10 min. The plasma supernatant was collected and stored at −80°C for
later use. Measurement of non-esterified fatty acids (NEFAs) in plasma
by a coupled enzymatic reaction system was conducted using the FFA
Assay Kit (Fluorometric), which contains palmitic acid as a standard
(Cell Biolabs, STA-619). The FFA assay was performed as instructed by
the manufacturer, and 1:5-diluted plasma samples were used. The assay
was performed on black 96-well plates (Brandplates, 781668), and
fluorescence was detected by fluorescence microplate reader (Thermo
Scientific, Varioskan LUX).
Animal studies
C57BL/6 homozygous floxed miR-409 mice (bearing loxP sites flanking the
only exon of the miR-409 gene; [233]Figure S4A) were generated at
Cyagen. Mice bearing the floxed allele were then crossed with
constitutive Cre-expressing mice (kindly provided by Iris Z. Jaffe),
resulting in EC-specific miR-409-3p-deficient mice (miR409^ECKO) and
miR-409-3p^fl/fl Cre^− littermates (miR409 ^fl/fl). The genotypes of
mice were confirmed by PCR genotyping of tail biopsies (Transnetyx)
using the indicated primers ([234]Table 2).
Table 2.
Primers for Cre/lox recombination and PCR genotyping
PCR primer set 1 5′arm forward F1:
5′-CGACCAGCATTTCATCCCGTTTAC-3′ 3′loxP reverse R1:
5′-CTATACGAAGTTATTCTTCCCTGAG-3′
PCR primer set 2 5′loxP forward F2:
5′-ACGTAAACGGCCACAAGTTC-3′ 3′arm reverse R2:
5′-GTTGGAAGAGCATTTTGCTGCTG-3′
[235]Open in a new tab
Male, 4-week-old miR409^ECKO and miR409 ^fl/fl mice were placed on a
high-fat and high-sucrose diet containing 36 kcal % fat and 43.2 kcal %
sucrose (Research Diets, D11092103) and maintained on a 12-h light/dark
cycle in a pathogen-free animal facility for 8 weeks. Food was changed
on a weekly basis in accordance with the manufacturer’s guidelines, and
animals had constant access to food and water. At the endpoint, all
tissues and blood were harvested in accordance with Tufts University
Institutional Animal Care and use (IACUC) standards, and tissues were
either snap-frozen for RNA and protein isolation or fixed in 10%
formalin (Sigma-Aldrich, HT5014) for histology. Blood was collected
into EDTA tubes, and plasma was separated by 15-min 1,500 × g
centrifugation, followed by RNA isolation using the Norgen RNA
Purification Kit (17200) according to the manufacturer’s instructions.
Male mice were age matched in all experiments, and cage-matched
littermates were used for experiments. Analyses of in vivo samples were
performed by blinded observers. Animal protocols were approved by
Laboratory Animal Care at Harvard Medical School and conducted in
accordance with National Institutes of Health guidelines for proper
care and use of laboratory animals.
Circulating miR-409-3p levels in patients with ACS
We examined EDTA plasma from a subgroup of 21 patients from the Feiring
Heart Biopsy Study 2, which included adult patients undergoing CABG in
the Feiring Heart Clinic in Norway between 2011 and 2017. The ACS
diagnosis was defined as ACS in medical records. The study was approved
by the Regional Ethics Committee in Norway. Written informed consent
was obtained from participants or their appropriate surrogates. Plasma
samples were generated from fasting blood collected in EDTA-containing
containers before CABG and stored in small aliquots at −86°C. No
thawing of the specimens was allowed prior to the actual RNA analyses.
The Norgen RNA Purification kit (17200) was used to isolate RNA from
the plasma samples according to the manufacturer’s instructions,
followed by reverse transcription and quantitative real-time PCR as
described under Quantitative real-time PCR).
Statistical analysis
Data are presented as mean ± SEM. All in vitro and in vivo experiments
are representative of 3 independent experiments unless indicated
otherwise. Sample sizes for mouse experiments were chosen based upon
pilot or similar well-characterized studies in the literature. There
were no inclusion or exclusion criteria used. Data were subjected to
unpaired two-sided Student’s t-test, one-way ANOVA with Bonferroni
correction for multiple comparisons, or two-way ANOVA with Šidák’s
multiple-comparisons test. p < 0.05 was considered statistically
significant.
Data availability
The data generated from this study and the associated resources are
available from the corresponding author upon reasonable request.
Acknowledgments