The Role of miRNA in Stem Cell Pluripotency and Commitment to the Vascular Endothelial Lineage

The Role of miRNA in Stem Cell Pluripotency andCommitment to the Vascular Endothelial Lineage

KATIE WHITE,* NICOLE M. KANE,*,1 GRAEME MILLIGAN,� AND ANDREW H. BAKER*

*BHF Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary, and Life Sciences,

University of Glasgow, Glasgow, UK; �Institute of Molecular, Cellular, and Systems Biology, College of Medical, Veterinary, and Life Sciences,

University of Glasgow, Glasgow, UK

Address for correspondence: Andrew H. Baker, Ph.D., BHF Professor of Translational Cardiovascular Sciences, BHF Glasgow Cardiovascular

Research Centre, Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, 126

University Place, Glasgow G12 8TA, UK. E-mail: [email protected] address: Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK

Received 2 November 2011; accepted 8 January 2012.

ABSTRACT

Vascular endothelial cells derived from human pluripotent stem

cells have substantial potential for the development of novel

vascular therapeutics and cell-based therapies for the repair of

ischemic damage. To gain maximum benefit from this source

of cells, a complete understanding of the changes in gene

expression and how they are regulated is required. miRNAs

have been demonstrated to play a critical role in controlling

stem cell pluripotency and differentiation and are important for

mature endothelial cell function. Specific miRNAs that

determine stem cell fate have been identified for a number of

different cell lineages; however, in the case of differentiation

and specification of vascular endothelial cells, this is yet to be

fully elucidated.

Key words: microRNA, pluripotent stem cells, endothelial cell

differentiation, angiogenesis

Abbreviations used: CTGF, connective tissue growth factor; bFGF,

basic fibroblast growth factor; DGCR8, DiGeorge syndrome critical

region gene 8; eNOS, endothelial nitric oxide synthase; EGF,

epidermal growth factor; ESC, embryonic stem cells; ESCC,

embryonic stem cell cycle promoting microRNAs; FGFR, fibroblast

growth factor receptor; hESC, human embryonic stem cells;

HUVECs, human umbilical vein endothelial cells; iPS, induced

pluripotent stem cells; miR, microRNA; miRNA, microRNA;

mTOR, mammalian target of rapamycin; Nrp1, neurophilin 1;

PDGFR, platelet-derived growth factor receptor; PI3KR2 ⁄ p85, PI3

kinase regulatory subunit 2; SCF, stem cell factor; Sirt1, silent

information regulator 1; SMC, smooth muscle cells; Spred-1,

sprouty-related protein 1; STAT5A, signal transducer and activator

of transcription 5A; TIMP1, tissue inhibitor of metalloproteinase 1;

Tsp, thrombospondin 1; TSR, thrombospondin type 1 repeats;

VCAM1, vascular cell adhesion molecule; VEGF, vascular endothelial

growth factor; VEGFR, vascular endothelial growth factor receptor.

Please cite this paper as: White K, Kane NM, Milligan G, Baker AH. The role of miRNA in stem cell pluripotency and commitment to the vascular endothe-

lial lineage. Microcirculation 19: 196–207, 2012.

INTRODUCTION

The formation of the vascular network is an essential stage

in embryonic development. The process begins with vascu-

logenesis, whereby angioblasts differentiate to endothelial

cells to form the primary vascular network (reviewed in

[8,9,78]). This is followed by angiogenesis, where new

blood vessels sprout from the existing vasculature to form

a complex network of blood vessels. This is mediated by

endothelial cell activation, proliferation, migration, and

maturation. In adults, blood vessels normally exist in a qui-

escent anti-angiogenic state, although angiogenesis can be

activated as part of a wound-healing response or in patho-

logical processes such as tumor growth.

The differentiation of hESC to endothelial cells is a com-

plex process that requires a highly regulated series of

molecular changes that significantly alter the phenotypic

and functional properties of the cell. Although a number of

signaling pathways, transcription factors, epigenetic

changes, and most recently miRNAs have been implicated

in coordinating the necessary changes in gene expression,

we still lack a complete understanding of all the processes

required and the mechanisms by which these regulatory

components are integrated.

The derivation of endothelial cells from ESCs has been

studied most extensively in developmental models based on

zebrafish, chick, and mouse embryos. Recent improvements

in methods to culture, maintain, and differentiate hESCs

DOI:10.1111/j.1549-8719.2012.00161.x

Invited Review

196 ª 2012 John Wiley & Sons Ltd

The Official Journal of the Microcirculatory Society, Inc. and the British Microcirculation Society

have enabled further research into the regulation of human

endothelial cell derivation. Many endothelial cell differenti-

ation protocols have been developed [53,59,99,100]; how-

ever, as yet no standardized protocol has been defined.

Despite recent improvements in these protocols, problems

with purity and efficiency still remain [47,49,57,70,74]. As

primary human endothelial cells are difficult to acquire and

maintain in culture, the ability to efficiently derive large-

scale, pure cultures of clinical grade cells from hESCs may

provide a tool for improving our knowledge of vascular

biology and the testing of vascular therapeutics. Impor-

tantly, this could greatly influence cell therapy for treating

ischemia as hESC-derived endothelial cells have been

shown to integrate into the host circulation [62], and are

capable of inducing re-vascularization in animal models of

ischemia [87], myocardial infarction [61], and cerebral

ischemia [12]. A better understanding of the regulation

and processes involved in deriving endothelial cells from

ESCs and a mechanistic understanding of their effects in

vivo will enable us to develop optimal strategies to progress

from the bench to clinical interventions.

miRNAs are short, single-stranded noncoding RNAs that

control gene expression post-transcriptionally by either

blocking the translation or inducing messenger RNA degra-

dation (reviewed in [44]). Currently, nearly 1500 miRNAs

have been identified in the human genome (http://

www.mirbase.org). As every miRNA can potentially target

hundreds of genes [3] and every gene can potentially be

targeted by multiple miRNAs, they form a complex regula-

tory system that can significantly impact cellular processes.

It has been predicted that 30–60% of mammalian genes are

directly regulated by miRNAs [30]. Due to their important

role in regulating gene expression, it is critical that miRNA

biogenesis is tightly regulated (reviewed in [21,54]).

miRNA AND THE MAINTENANCE OFPLURIPOTENT STEM CELLS

The two key properties of ESCs, pluripotency, and self-

renewal are maintained by stringent regulation of a complex

set of molecular pathways involving chromatin modifiers,

transcription factors, and noncoding RNAs such as miRNAs

(reviewed in [110]). A core group of transcription factors

Oct4, Sox2, and Nanog are essential for maintaining the

stem cell state in combination with other pluripotency fac-

tors such as KLF4, c-Myc, Tcf3, and RNA-binding protein,

Lin28. These core factors form an auto-regulatory loop that

maintains and coordinates their expression (Figure 1). Their

importance in maintaining pluripotency was highlighted by

the demonstration that it is possible to reprogram somatic

cells to iPS cells by overexpressing Oct4 and Sox2 alone,

although the addition of other nonessential factors such as

Klf-4, c-Myc, Lin-28, and Nanog increases the reprogram-

ming efficiency [95]. The core transcription factors have

been shown to be involved in regulating the expression of a

relatively large number of miRNAs. Marson et al. performed

a large-scale study of miRNA promoters and found Oct4,

Sox2, Nanog, and Tcf3 bound to the predicted promoters of

81 miRNAs in mice and a similar number in humans [63].

A significant proportion of these miRNAs are expressed at

more than 100-fold higher levels in murine ESCs compared

with murine embryonic fibroblasts [33,62]. Approximately a

quarter of the miRNAs identified by Marson et al. are not

expressed in ESCs as their promoters were also bound to

Polycomb group proteins, which catalyze histone modifica-

tions that repress transcription [63]. These repressed

miRNAs are expressed in a tissue-specific manner, thought

to be primed for rapid expression, and may be involved in

determining lineage commitment upon differentiation [63].

Figure 1. Key transcription factors and miRNAs involved in maintaining stem cell pluripotency. Positive auto-regulation of the core pluripotency

factors, microRNAs, chromatin modifications, and additional transcription factors (including Tcf3, Smad1, Stat3, Esrrb, Sal4, Tbx3, Zfx, Ronin, Klf2,

Klf4, Klf5, and PRDM14) maintain stem cell proliferation and pluripotency. MicroRNAs that promote differentiation are repressed by binding of the

core transcription factors and Polycomb group chromatin regulators. The core pluripotency factors recruit SetDB1, which catalyzes the repressive

histone modification H3K9me3 to prevent miRNA expression and cellular differentiation.

MicroRNAs and Stem Cells

ª 2012 John Wiley & Sons Ltd 197

Following initial Dicer and DGCR8 knockdown experi-

ments, which showed miRNA expression, is essential for

maintaining pluripotency and differentiation [50,67,103], a

number of specific miRNAs that are important for main-

taining stemness have been identified. These include the

human miR-371 family (miR-371, -372, and -373), which

is homologous to the mouse miR-290 family (miR-290,

-291a, -291b, -292, -293,- 294, and -295) and miR-302

family (miR-302b, -302c, -302a, -302d, and -367). These

miRNAs are highly expressed in ESCs but undetectable in

somatic cells as their expression decreases dramatically dur-

ing differentiation [15]. They have very similar seed

sequences, so they share common targets [31,88], many of

which are involved in cell cycle regulation; hence, they have

been denoted ESCC promoting miRNAs [54,63,102]. Some

of the ESCC miRNAs have also been demonstrated to tar-

get genes involved in regulating chromatin modification

[4,77,83], so may have a role in integrating the different

mechanisms of regulating gene expression.

Unlike the ESCC miRNAs, which are expressed at very

low ⁄ undetectable levels in somatic cells and rapidly

decrease during differentiation [15,63], the miR 17-92 clus-

ter (miR17-5p, -17-3p, -18a, -19a, -19b-1, -20a, and -92a)

is expressed in both ESCs [5] and mature cells [38], and

expression increases during differentiation [29]. Its expres-

sion has been shown to be upregulated by c-myc in hESCs

and several types of tumors. The miRNAs within the miR-

17-92 cluster have a complex role in regulating cell cycle

progression and apoptosis (reviewed in [65]). In stem cells,

this cluster may have an important role in maintaining

self-renewal [85], which may in part be due to miR-92a

regulating G1-S phase transition by repressing the Cdkn1c

checkpoint gene [81].

ESC DIFFERENTIATION

To enable cell differentiation to occur, factors that main-

tain stemness and self-renewal capacity need to be inhib-

ited, and pathways that mediate lineage specification are

required to be activated in a highly regulated series of

events. The importance of miRNAs in initiating differentia-

tion is highlighted by evidence which demonstrated that,

in their absence, pluripotency factors cannot be downregu-

lated, therefore differentiation is inhibited [50,67,103].

Moreover, several studies (reviewed in [45]) have reported

that the regulation of miRNA processing by numerous

stimuli (e.g., differentiation stimuli) may perform as medi-

ators of crosstalk between distinct signaling pathways. A

number of miRNAs with increased expression during dif-

ferentiation are thought to downregulate the core pluripo-

tency factors (Table 1). For example, miR-145 is expressed

at low levels in ESCs and is highly upregulated during dif-

ferentiation to the majority of cell lineages. It has been

shown to directly target and repress Oct4, Sox2, and Klf4

[108]. When overexpressed in hESCs, self-renewal is inhib-

ited and expression of differentiation markers is induced,

whereas antisense miR-145 increases the self-renewal capac-

ity of hESCs [108].

The RNA-binding protein Lin28 and miR-let-7 form a

regulatory loop that has an important role in determining

whether cells remain pluripotent or differentiate. Lin28 is

expressed at a high level in ESCs and downregulated during

differentiation [68], whereas the reverse is true for let-7,

which is highly expressed in the majority of mature cell

types [98], including endothelial cells [73]. Several studies

have demonstrated the importance of let-7 in differentia-

tion. Transfection of let-7 into DGCR8-ablated ESCs

restores their ability to differentiate and knockdown of let-

7 expression in mature fibroblasts was reported to enhance

the reprogramming of somatic cells to generate iPS [64].

Let-7 directly targets several pluripotency factors including

Lin28, c-myc, and Sal4 to promote differentiation

[63,76,80]. Lin28 has been shown to post-transcriptionally

regulate let-7 expression by blocking maturation of the pre-

form and targeting it for degradation [35,39]. Therefore,

the preform of let7 is detectable in ESCs and somatic cells

whereas the mature form is only detectable in differentiated

cells. Inhibition of Lin28 alone does not mediate differenti-

ation, but upon the onset of differentiation levels of Oct4,

Sox2, and Nanog decrease. This in turn elicits a decrease in

Lin28 expression, as Oct4, Sox2, and Nanog bind to the

Lin28 promoter, positively regulating its expression and

consequently increasing pre-miR let-7 processing.

Table 1. miRNAs with a role in downregulating the expression of

the core pluripotency factors

Pluripotency factor miR that mediates downregulation

Oct4 miR-145 [108]

miR-470 [96]

Nanog miR-296 [96]

miR-470 [96]

Sox2 miR-134 [96]

miR-145 [108]

miR-200c [104]

miR-183 [104]

Klf4 miR-145 [108]

miR-200c [104]

miR-183 [104]

Lin28 Let-7 [64]

miR-125 [106].

c-myc Let-7 [6,64]

miR-429 [93]

miR-132[6]

miR-125b-1[6]

miR-154 [6]

K. White et al.


In addition to their role in downregulating pluripotency

factors, a number of miRNAs involved in lineage commit-

ment have been identified for a range of different cell types

(reviewed in [46]). For example, miR-143 and -145 are

important for determining smooth muscle specification

[18], and miR-1 and -133 play a role in cardiac muscle

specification [14,112]. Although many miRNAs have been

shown to have important functions in mature endothelial

cells and are involved in the regulation of angiogenesis, as

yet no miRNAs have been demonstrated to have an essen-

tial role in endothelial cell commitment.

miRNA AND ENDOTHELIAL CELLS

The initial evidence for a role of miRNAs in vascular devel-

opment was provided by the observation that Dicer knock-

out mice have an embryonic lethal phenotype between

E12.5 and E14.5 [109]. Impaired blood vessel formation in

the embryo and yolk -sac was noted in combination with

the altered expression of key angiogenesis-related genes

including VEGF, FLT1, KDR, and TIE1, suggesting that

Dicer is required for the processing of miRNAs that regu-

late embryonic angiogenesis [109]. Conversely, endothelial-

specific inactivation of Dicer was not embryonic lethal;

however, adult mice were reported to have impaired angio-

genesis in response to stimuli such as VEGF [91]. Further

in vitro studies in adult human endothelial cells have

reported that Dicer knockdown impairs endothelial cell

migration, capillary sprouting, and tube formation

[55,82,90,91]. Knockdown of Drosha expression in HU-

VECs was shown to have no effect on migration; however,

the cells demonstrated reduced angiogenic response as

capillary sprouting was attenuated [55], therefore miRNAs

are thought to have an important role in regulating

angiogenesis.

As global disruption of miRNA processing has been

shown to affect vasculogenesis, angiogenesis, and endothe-

lial cell function, further studies have investigated the role

of specific miRNAs involved in these processes. The first

large-scale investigation of miRNA expression in endothe-

lial cells identified 27 miRNAs that are expressed at rela-

tively high levels in HUVECs, of which 15 were predicted

to target the receptors of angiogenic factors [73]. Several

other studies have since identified around 200 miRNAs that

are expressed in endothelial cells; however, of these

only 28 (miR-15b, -16, -20, -21, -231, -23b, -24, -27a, -29a,

-30a, -30c, -31, -100, -103, -106, -125a, -125b, -126, -181a,

-191, -199a, -221, -222, -320, -let7, -let7b, -let7c, and -

let7d) have been consistently detected in more than half of

the studies [40]. Variation in the miRNAs identified by

these studies is likely due to differences in the technologies

used to determine the miRNA profiles, using endothelial

cells isolated from different species and vascular beds and

maintenance in different culture conditions. Table 2 sum-

marizes miRNAs that are associated with either a pro- or

anti-angiogenic role.

miRNAS AND ENDOTHELIAL CELLDIFFERENTIATION

Although many miRNAs that are highly expressed in

mature endothelial cells have been identified and we have

some understanding of their role in endothelial cell biol-

ogy, we still know very little regarding their importance in

deriving endothelial cells from hESCs. Several miRNAs

have been demonstrated to have a role in mediating angio-

genesis-associated endothelial cell activation, proliferation,

and migration but these studies have been performed

in vitro using mature endothelial cells or using pathological

models of angiogenesis. As yet we do not know whether

these findings also reflect the role of miRNAs in the differ-

entiation of pluripotent ESCs to endothelial cells. As sub-

stantial changes in the transcriptome are required to derive

endothelial cells from ESCs, it is likely to involve additional

miRNAs, as well as other noncoding RNA [34].

One recent study has tried to address our lack of under-

standing of this area by investigating changes in miRNA

expression during differentiation of hESC to endothelial

cells. This identified several miRNAs with increased expres-

sion as the differentiation progressed and three of these

(miR-99b, -181a, and -181b) were selected for further

investigation. Results suggest that lentiviral-mediated over-

expression of these miRNAs enhanced the efficiency of the

differentiation protocol, therefore implicating these miR-

NAs as functionally important in endothelial cell lineage

commitment [48]. However, as yet no mechanism for this

action has been demonstrated.

Another study has shown that miR-181b targets Nrp1, a

transmembrane receptor, which is expressed in arterial

endothelium, and plays a role in angiogenesis [89]. Overex-

pression of miR-181b in HUVECs reduced Nrp1 expres-

sion, cell migration, and tube formation, suggesting that it

has anti-angiogenic properties in mature endothelial cells

[19]. Therefore, miRNAs that have a pro-angiogenic role in

ESCs may not have the same function in mediating mature

endothelial cell angiogenesis.

One of the most highly expressed miRNAs in mature

endothelial cells is miR-126. miR-126 is thought to regulate

angiogenesis in response to growth factors such as VEGF

and bFGF by directly targeting Spred-1 and PI3KR2 ⁄ p85

[28,56,101]. In zebrafish embryogenesis, it has been dem-

onstrated that blood flow activates the zinc-finger tran-

scription factor klf2a, which in turn increases miR-126

expression and therefore increases VEGF signaling and

angiogenesis [69]. In the absence of flow, klf2a and miR-

126 levels are low, so Spred-1 inhibits VEGF angiogenesis



Table 2. miRNAs with a (A) pro-angiogenic or (B) anti-angiogenic role in endothelial cells

(A)

miRNA Targets and function

miR-126 Maintains vascular integrity. Inhibition in mice and zebrafish results in leaky vessels and increased vascular hemorrhaging [28,101]

Promotes angiogenesis in response to VEGF and bFGF via direct targeting of PI3KR2 ⁄ p85 to impair the VEGF-activated Akt

pathway and direct repression of Spred-1 to inhibit the Erk pathway [28,56,101]

During zebrafish embryogenesis, its expression is upregulated by blood flow (mediated by the zinc finger-containing transcription

factor klf2a). It was found to regulate both Spred-1 and p21-activated kinase 1, which are known to be required for

maintaining mammalian endothelial cell motility and permeability. However, PI3KR2 was not found to be a target [69,115]

Targets leukocyte adhesion molecule VCAM1, so may have an anti-inflammatory role in reducing leukocyte adhesion [28,36,101]

Expression is upregulated by the endothelial cell-related transcription factors Ets1 and 2 [37,71]

Let-7b Increases endothelial cell proliferation and motility, possibly through directly inhibiting TIMP1 expression [72]

miR-210 Expressed at negligible levels in endothelial cells in normoxic conditions, but is upregulated in response to hypoxia [26,75]

Overexpression in normoxic endothelial cells increases cell migration and capillary-like structure formation in response to VEGF by

directly targeting ephrin-A3. Inhibition of miR-210 was found to block capillary formation in response to hypoxia. In the

developing cardiovascular system, Eph and ephrin molecules control angiogenic remodeling of blood and lymphatic vessels,

therefore miR-210 may have a role in angiogenesis and endothelial cell survival in response to hypoxia [26]

Ischemic preconditioning of mesenchymal stem cells upregulates miR-210 expression. It promotes cell survival by inhibiting

caspase-8-associated protein-2 and therefore reduces apoptosis [105]

miR-130 Expression is upregulated in response to VEGF-A and bFGF [13,40]

Overexpression in HUVECs induces proliferation, migration, and tube formation [13]

Targets two anti-angiogenic homeobox genes, GAX and HOXA5 [13]

miR-296 Expressed in human brain microvascular endothelial cells. Expression increases in response to angiogenic stimuli such as VEGF

and EGF [107]

Inhibition using antagomirs causes a reduction in angiogenesis both in vitro and in vivo [107]

Hepatocyte growth factor-regulated tyrosine kinase substrate (hgs) was identified as a direct target. hgs causes degradation of

growth factor receptors VEGFR2 and PDGFR, therefore miR-296 inhibition of hgs may increase cellular responses to angiogenic

stimuli [107]

miR-23a-27a-

24-2 and

miR-23b-27b-

24-1 clusters

The mature sequences of 23a ⁄ b and 27a ⁄ b differ by 1 nucleotide whereas 24-1 and 24-2 are identical, so the clusters are

predicted to share many of the same targets. Both clusters are expressed at relatively high levels in endothelial cells and are also

found in a range of other cell types where they have been shown to have a role in cell cycle control, proliferation, apoptosis,

and differentiation [17,36,55,73,90,113]

Knockdown of miR-27 and, to a lesser extent, miR-23 using anti-miRs has been shown to reduce the formation of capillary-like

structures from HUVECs and sprouting angiogenesis of aortic rings ex vivo. Overexpression of the miRNAs was found to have

the opposite effect. miRNA knockdown has also been shown to reduce endothelial cell proliferation and migration in response

to VEGF [113]

Anti-angiogenic SPROUTY2 and SEMA6A are targets of both miR-23 and miR-27. They negatively regulate MAPK and VEGFR2

signaling, respectively, in response to VEGF. Overexpression of the two miRNAs is thought to repress these targets and

therefore block their inhibition of VEGF-stimulated angiogenesis [113]

miR-24 expression is upregulated in cardiac endothelial cells by ischemia. It induces endothelial cell apoptosis and inhibits

endothelial cell sprouting by inhibiting the transcription factor GATA2 and p21-activated kinase PAK4. Overexpression in

zebrafish embryos was also found to block angiogenesis [27]

human

miR-424

(murine

homolog

miR-322)

and miR-16

Is increased by hypoxia and directly targets the ubiquitin ligase scaffold protein cullin-2. Degradation of cullin-2 prevents HIF-1adownregulation and therefore enhances pro-angiogenic signaling [32]

Overexpression of miR-424 in HUVECS was demonstrated to cause an increase in migration and proliferation [32]; however, in

another study, overexpression of miR-424 or miR-16 in endothelial cells impaired proliferation, migration, and tube

formation [10]

miR-16 and miR-424 have the same seed sequence so may have a similar function. They are both highly expressed in endothelial

cells [10]. The expression of mature miR-16 and miR-424 is induced by VEGF and bFGF, and they reduce the expression of

VEGF, VEGFR2, and FGFR1, suggesting a regulatory feedback mechanism occurs [10]

miR-132 Activates angiogenesis in quiescent endothelium by downregulating p120RasGAP. p120RasGAP attenuates p21 Ras activity that

leads to an increase in Ras activity, a decrease in the activation of PI3K and Raf-1, which results in increased angiogenesis [2,51]

In response to hypoxia and serum starvation, pericytes express and secrete miR-132 that is taken up by endothelial cells where it

activates angiogenic mechanisms by inhibiting p120RasGAP [51]

Angiogenic factors such as VEGF and bFGF lead to phosphorylation of the transcription factor cAMP-response element-binding

protein (CREB) CREB, which upregulates miR-132 expression [2]

Overexpression increases endothelial cell proliferation, tube formation, and angiogenesis in vivo [2]

K. White et al.


[69]. Although miR-126 has been implicated as having a

role in angiogenesis in mature endothelial cells, and its

expression is upregulated during endothelial lineage com-

mitment, there is no evidence that it plays a role in lineage

commitment of pluripotent stem cells toward endothelial

cells. Moreover, overexpression of miR-126 has been

reported to inhibit differentiation of pluripotent stem cells

toward vascular lineages [28]. Therefore, although evidence

would suggest that miR-126 has an important role in endo-

thelial cell function, and is induced during vascular differ-

entiation, it does not appear to be essential to early lineage

commitment of endothelial cells.

The miR-17-92 cluster is expressed in both stem cells

and mature cells and its expression increases during differ-

entiation [29]. Although it has been associated with angio-

genesis, the cluster is not thought to be essential for

Table 2. Continued.

(A)

miRNA Targets and function

miR-218 Vascular patterning in mouse retinas is modulated by miR-218 [84]

miR-218 is located in an intron of the Slit1 and Slit2 genes and inhibits Roundabout (Robo) 1 and Robo2. Slit ligands bind to

Robo receptors to activate a signaling pathway that directs the migration of endothelial cells during embryogenesis. Knockdown

of miR-218 alters the regulation of the pathway, resulting in abnormal endothelial cell migration and decreased complexity of

the blood vessel network in the mouse retina [84]

(B)

miR Targets and function

Let7f Inhibits endothelial cell sprouting in vitro [55]

miR-221 ⁄ -222

Share the same seed sequence and common putative targets [60,73]

Inhibit endothelial cell migration, proliferation, and angiogenesis in vitro by targeting the SCF receptor c-kit [60]

Treatment of HUVECs with high concentrations of glucose was shown to induce the expression of miR-221 and inhibit

endothelial cell migration in response to SCF. This correlated with a decrease in c-kit expression and could be inhibited by

antisense miR-221 [91]

miR-221 ⁄ -222 are involved in downregulating eNOS expression; however, this is thought to be via an indirect mechanism as no

binding sites for the miRNAs have been identified [91]

miR-222 is involved in inflammation-mediated vascular remodeling by targeting STAT5A [22]

miR-155 miR-155 is expressed in endothelial cells and its expression increases in response to VEGF and hypoxia [16,75,91,114]

It reduces angiotensin II type I receptor and Ets1 expression. Overexpression of miR-155 in angiotensin II-stimulated HUVECs

inhibits migration and is proposed to have an anti-inflammatory role [16,114]

miR-100 Highly expressed in HUVECs and human aortic vascular SMC [33]

Overexpression in HUVECs inhibited cell sprouting and proliferation but had no effect on migration [33]

Directly targets mTOR, which has a role in angiogenesis and cell proliferation in response to hypoxia [33,43]

miR-100 is downregulated in models of hind-limb ischemia [33]

miR-200b Induced by hypoxic conditions [11]

Anti-angiogenic, targets Ets [11]

miR-101 Is downregulated in response to VEGF [86]

It directly targets the histone-methyltransferase EZH2, a member of the Polycomb group. VEGF-mediated downregulation of

miR-101 results in increased expression of EZH2, which increases methylation, alters gene expression, and increases endothelial

cell sprouting and migration. This provides a mechanistic link between miRNAs and chromatin remodeling [86]

miR-217 Expressed in older endothelial cells [66]

Targets Sirt1, which regulates the cell cycle, senescence, and apoptosis [66]

Inhibition of miR-217 increases angiogenesis [66]

Overexpression of miR-217 in young endothelial cells causes premature senescence and a reduction in angiogenesis [66]

miR-34 Impairs angiogenesis in endothelial progenitor cells by inhibiting Sirt1 expression [111]

miR-503 Overexpression inhibits endothelial cell proliferation, migration, and tubule formation [7]

Expressed at a low level in human endothelial cells but was found to be upregulated in an endothelial cell culture model that

mimics diabetes mellitus and in endothelial cells isolated from ischemic limb muscle [7]

Targets cdc25 and cyclin E1 [7]

miR-503 was detected at a higher level in plasma from diabetic patients and in amputated ischemic leg muscle compared with

controls [7]



endothelial differentiation as cluster knockout mice have

no obvious vascular abnormalities, although they suffer

from heart, lung, and immune system defects [97].

Although overexpression of the entire cluster in endothelial

cells was found to reduce cell sprouting and tube forma-

tion, further analysis of the effects of overexpressing the

individual miRNAs suggests that miRs-17-5p, -18a, and -

19a are pro-angiogenic [91]. miR-17-5p is thought to affect

endothelial cell proliferation and motility by directly inhib-

iting anti-angiogenic TIMP1 [72]. miR-18a and -19a target

proteins containing thrombospondin type-1 repeats (TSR),

which are thought to have angiostatic activity [23]. In

tumor cells, increased miRNA expression and angiogenesis

were inversely correlated with expression of the anti-angio-

genic gene Tsp1 and CTGF, which are direct targets of

miR-19 and miR-18, respectively [23]. In contrast to these

studies, individual overexpression of miR-17, -18a, -19a,

and -20a inhibited sprouting of HUVECs and only caused

a small decrease in Tsp1 and CTGF, suggesting that they

were not direct targets [24]. HIF-1a that activates many

angiogenic genes in response to hypoxia may be a target of

the cluster that contributes to its anti-angiogenic properties

[94]. miR-20a seems to have anti-angiogenic activity by

targeting VEGF-A [42]. In endothelial cells, miR-92a has

also been shown to have anti-angiogenic activities as over-

expression impaired cell migration and vascular sprouting.

Overexpression in HUVECs that were subsequently

implanted into a murine hind limb ischemia model was

found to reduce blood vessel formation, whereas the inhi-

bition of miR-92a enhanced blood vessel growth and recov-

ery and improved left ventricular function and reduced

infarct size in a model of myocardial infarction [5]. In ze-

brafish, overexpression of miR-92a also impaired angiogen-

esis. The pro-angiogenic protein integrin a5 has been

identified as a direct target of miR-92a [5]. Investigations

into the role of the individual miRNAs in this cluster have

produced some conflicting results which may be due to the

use of different cell types, species, and experimental proce-

dures, so the role of this cluster in angiogenesis requires

further investigation.

Recent data suggest that signaling pathways may modu-

late miRNA activity by post-transcriptionally regulating

miRNA processing [20]. Dentelli et al. reported that during

neovascualrization of atherosclerotic plaques, interleukin 3

and bFGF downregulated miR-221 and miR-222 expression

in endothelial cells [22]. This resulted in an increase in

STAT5A expression, which was shown to be a result of direct

targeting by miR-222 [22]. Despite an increase in miR-221

expression, it was found not to have an effect on vascular

growth [22]. Interestingly, previous reports have demon-

strated no evidence of miR-222 regulation in response to

different angiogenic or inflammatory stimuli [91,92], indi-

cating the participation of different transduction pathways

on the regulation of miRNA expression. Furthermore,

Chamorro-Jorganes et al. have recently demonstrated a reg-

ulatory feedback miRNA loop that governs the levels of

angiogenic mediators in endothelial cells [10]. Both in vitro

and in vivo, angiogenic growth factors VEGF and bFGF

A

B

C

Figure 2. Summary of miRNAs with a role in endothelial cell

differentiation and angiogenesis. For differentiation to occur, expression

of pluripotency-associated miRNAs are downregulated (A) and miRNAs

that regulate cell lineage specificity are upregulated; however, for

endothelial cells specification, the specific miRNAs are yet to be

identified (B). In mature endothelial cells, a number of pro-angiogenic

miRNAs and their targets have been identified (C).

K. White et al.


stimulated the expression of mature miR-16 and miR-424

in HUVECs, which in turn directly downregulated VEG-

FR2, FGFR1, and VEGF expression [10]. Although growth

factor and cytokine-mediated regulation of miRNA expres-

sion has been demonstrated, it remains unknown whether

this regulation occurs at the transcriptional level or during

the latter steps of miRNA maturation. Owing to the

requirement for a highly orchestrated and complex tran-

scriptional activation of a series of gene networks and sig-

naling pathways to control differentiation to a specified

lineage, it may be necessary to determine miRNA expres-

sion profiles during normal embryogenesis and vascular

development, in order to facilitate correct identification of

miRNAs involved in the fate decision from pluripotency to

endothelial cells.

The specification of endothelial cells to venous, arterial,

or lymphatic cells is an important stage in angiogenesis

and vascular development. Several genes involved in this

process have been identified [1,41,79] but how they are

regulated is less well understood. Recent studies have

shown that miRNAs may play a role in this as Prox1, a

homeobox transcription factor required for lymphatic

endothelial cell specification and maintenance, is a direct

target of miR-181a [52] and miR-31 [58]. Overexpression

of miR-181a reprogrammed lymphatic endothelial cells

toward a blood vascular phenotype in vitro [52]. In vivo

overexpression of miR-31 in Xenopus and zebrafish

embryogenesis impaired development of the lymphatic sys-

tem via direct inhibition of Prox1 [58].

SUMMARY

Although great progress has been made in increasing our

understanding of the importance and function of miRNAs

in both stem cells and endothelial cells, as yet no miRNAs

have been identified as being essential for lineage commit-

ment to endothelial cells (Figure 2). However, as miRNAs

that are required for regulating differentiation toward other

cell lineages have been identified, it can be speculated that

there are miRNAs that will be involved in determining

endothelial cell differentiation. The majority of current

studies have investigated the role of miRNAs in mature

endothelial cell angiogenesis. These have identified many

angiogenesis-associated miRNAs that are thought to regu-

late genes linked to endothelial cell activation, proliferation,

and migration. They may also be involved in endothelial

cell derivation from ESCs but more experimental evidence

is required to demonstrate this.

To successfully identify miRNAs that have an essential

role in endothelial cell lineage commitment, it is important

that an efficient, standardized protocol for deriving endo-

thelial cells from hESCs is developed. The current protocols

produce a mixed population of cells that makes it difficult

to pinpoint the changes in miRNA expression that occur in

the early stages of differentiation. Evseenko et al. have

recently identified a number of molecular changes that

occur during the early stages of commitment of hESCs to

mesodermal lineages and have identified a population of

cells, human mesoderm embryonic precursors that are

capable of producing all mesodermal cell types [25]. An

investigation into changes in miRNAs in these early meso-

dermal committed cells may provide further insight into

the regulation of mesodermal and endothelial lineage com-

mitment. With the development of more efficient protocols

for deriving endothelial cells [49], advances in technology

and bioinformatics to analyze miRNA expression, and

improvements in the tools available to manipulate miRNA

expression, it should become possible to completely define

endothelial lineage commitment miRNAs.

Improvements in our understanding of the regulation

and pathways involved in determining endothelial cell line-

age commitment and differentiation will enable the devel-

opment of efficient protocols for the production of

clinical-grade endothelial cells. Both hESCs and iPS provide

a potential source for the production of endothelial cells,

which may be exploited to produce models of disease to

further enhance our understanding of cardiovascular dis-

ease and enable the development of novel treatments.

PERSPECTIVE

Vascular endothelial cells derived from human pluripotent

stem cells have great potential for the development of novel

therapeutics to repair ischemic damage. miRNAs are

known to play important roles in maintaining stem cell

pluripotency and regulating endothelial cell function. With

further improvement and standardisation of endothelial cell

differentiation protocols, it may be possible to identify spe-

cific miRNAs which promote commitment to endothelial

cell lineages. This will greatly enhance our understanding

of the differentiation process and enable us to maximise

the potential use of this source of cells.

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AUTHOR BIOGRAPHIES

Katie White (B.Sc. 2003, Ph.D. 2007)

is a postdoctoral research associate at

the University of Glasgow, Institute of

Cardiovascular and Medical Sciences.

Her research interests focus on the

development and optimization of

novel gene and cell-based therapies

for cardiovascular disorders and

investigating the role of miRNA in

vascular cells.

Nicole M. Kane obtained her Ph.D.

from the University of Edinburgh

before undertaking a postdoctoral

position at the British Heart Founda-

tion Cardiovascular Research Centre

at the University of Glasgow. Her

research is focused on the genetic

manipulation of mouse and human

embryonic and induced pluripotent

stem cells to further delineate

pluripotency and differentiation

commitments, in particular to a

cardiovascular lineage.

Graeme Milligan is a Professor of

Molecular Pharmacology at University

of Glasgow. His research interests

include the function and regulation of

G-protein-coupled receptors, novel

approaches to small molecule ligand

screening, and gut health and

metabolic disease as well as aspects of

stem cell biology.

Andrew Baker is based at the Uni-

versity of Glasgow, Institute for Car-

diovascular and Medical Sciences, a

translational centre of excellence with

a focus on primary and secondary

prevention of cardiovascular disease.

In 2010, he was awarded a fellowship

from the Royal Society of Edinburgh

and an Outstanding Achievement

Award from the European Society of

Cardiology. In 2011, he received a

Royal Society Wolfson Research Merit

Award and was awarded a British

Heart Foundation Chair of Transla-

tional Cardiovascular Medicine.



The Role of miRNA in Stem Cell Pluripotency and Commitment to the Vascular Endothelial Lineage

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