RESEARCH ARTICLE

FGF2 inhibits endothelial–mesenchymal transition throughmicroRNA-20a-mediated repression of canonical TGF-β signalingAna C. P. Correia*, Jan-Renier A. J. Moonen, Marja G. L. Brinker and Guido Krenning‡

ABSTRACTEndothelial-to-mesenchymal transition (EndMT) is characterizedby the loss of endothelial cell markers and functions, and coincideswith de novo expression of mesenchymal markers. EndMT isinduced by TGFβ1 and changes endothelial microRNA expression.We found that miR-20a is decreased during EndMT, and thatectopic expression of miR-20a inhibits EndMT induction. TGFβ1induces cellular hypertrophy in human umbilical vein endothelialcells and abrogates VE-cadherin expression, reduces endothelialsprouting capacity and induces the expression of the mesenchymalmarker SM22α (also known as TAGLN). We identified ALK5 (alsoknown as TGFBR1), TGFBR2 and SARA (also known as ZFYVE9)as direct miR-20a targets. Expression of miR-20a mimics abrogatethe endothelial responsiveness to TGFβ1, by decreasing ALK5,TGFBR2 and SARA, and inhibit EndMT, as indicated by themaintenance of VE-cadherin expression, the ability of the cells tosprout and the absence of SM22α expression. FGF2 increasesmiR-20a expression and inhibits EndMT in TGFβ1-stimulatedendothelial cells. In summary, FGF2 controls endothelial TGFβ1signaling by regulating ALK5, TGFBR2 and SARA expressionthrough miR-20a. Loss of FGF2 signaling combined with a TGFβ1challenge reduces miR-20a levels and increases endothelialresponsiveness to TGFβ1 through elevated receptor complexlevels and activation of Smad2 and Smad3, which culminates inEndMT.

KEYWORDS: Endothelial cell, Endothelial dysfunction, Endothelial–mesenchymal transition, EndMT, Fibroblast growth factor, FGF,MicroRNA, miRNA, Transforming growth factor beta, TGFβ

INTRODUCTIONEndothelial–mesenchymal transition (EndMT) is a process whereendothelial cells lose their endothelial characteristics and start todifferentiate into mesenchymal cells (Krenning et al., 2008, 2010;Moonen et al., 2010; Maleszewska et al., 2013). EndMT isevidenced by the phenotypic and functional alteration of endothelialcells, which results in the repression of endothelial cell markers [e.g.VE-cadherin and CD31 (also known as PECAM1)] and functions(e.g. sprouting ability and thrombolytic properties) and the de novoexpression mesenchymal proteins [e.g. SM22α (also known asTAGLN) and αSMA (also known as ACTA2)] and acquirement ofmesenchymal cell functions (i.e. contractile behavior and collagen

production) (Krenning et al., 2008; Moonen et al., 2010;Maleszewska et al., 2013).

Phenotypic transitions are pivotal to embryonic processes suchas tissue differentiation and morphogenesis, and, of these, EndMTis essential in the formation of the cardiac valves (Krenning et al.,2010). However, postnatally, phenotypic transitions are linked topathological processes, such as tumor metastasis (Potenta et al.,2008) and the occurrence of fibroproliferative diseases. EndMThas been shown to contribute to the formation of neointimal lesions(Cooley et al., 2014; Moonen et al., 2015), vascular calcifications(Yao et al., 2013), and fibrosis of the lungs (Hashimoto et al.,2010), heart (Zeisberg et al., 2007) and kidneys (Zeisberg et al.,2008), as well as heterotypic ossifications (Medici andOlsen, 2012).

In recent years, it has become clear that signaling through thetransforming growth factor-β (TGFβ) superfamily is key in EndMT(Goumans et al., 2008). TGFβ binds to the heteromeric receptorcomplex formed by the activin-like kinase 5 (ALK5, also known asTGFβ type I receptor, TGFBR1) and the TGFβ type II receptor(TGFBR2). Phosphorylation of ALK5 by the kinase TGFBR2,activates its catalytic kinase domain, allowing the activation of thereceptor-regulated Smads (i.e. Smad2 and Smad3) (Goumans et al.,2008; Yoshimatsu and Watabe, 2011).

Smad anchor for receptor activation (SARA; also known asZFYVE9), functions to recruit Smad2 and Smad3 to the TGFβreceptor complex, by controlling their subcellular localization andinteracting with TGFBR1 (Tsukazaki et al., 1998). Activation ofSmad2 and Smad3 causes their dissociation from SARA, aconcomitant complexation with Smad4 and finally nucleartranslocation and transcriptional regulation of target genes such asthat encoding SM22α (Qiu et al., 2003). Although a clear role forTGFβ has been established in the induction of EndMT, less isknown about the signaling mechanisms that modulate or inhibit theinduction of EndMT.

Non-coding microRNAs (miRs) are post-transcriptionalrepressors of gene function and are often dysregulated inpathological processes (reviewed in Quiat and Olson, 2013).MiRs have diverse functions, including the regulation of cellulardifferentiation, proliferation and apoptosis. We have previouslyperformed microRNA expression analysis by microarray to identifymiRs that are deregulated during EndMT, and found that miR-20aexpression is abolished during EndMT (our unpublished data). Wealso previously found that exogenous expression of miR-20a inendothelial cells potently inhibited EndMT induction.

MicroRNA-20a is encoded on chromosome 13 and part of themiR-17-92 cluster. Here, we show that fibroblast growth factor-2(FGF2) induces the endothelial expression of miR-20a, whichtargets ALK5, TGFBR2 and SARA to inhibit canonical TGFβsignaling. Ectopic expression of miR-20a by endothelial cellsblocks Smad2 and Smad3 activation and protects the endotheliumfrom EndMT.Received 24 June 2015; Accepted 22 December 2015

From the Cardiovascular Regenerative Medicine Research Group, Dept. Pathologyand Medical Biology, University of Groningen, University Medical CenterGroningen, Hanzeplein 1 (EA11), Groningen 9713 GZ, The Netherlands.*Present address: Center for Experimental and Molecular Medicine, Dept.Gastroenterology and Hepatology, Academic Medical Center, University ofAmsterdam, Amsterdam, The Netherlands.

‡Author for correspondence (g.krenning@umcg.nl)

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RESULTSTGFβ1 induces endothelial–mesenchymal transition in anALK5-dependent mannerEndothelial cells stimulated with TGFβ1 drastically changedtheir morphology and showed signs of cellular hypertrophy andreduced cell numbers (Fig. 1A,B). These morphological changeswere accompanied by phenotypic alterations. TGFβ1-stimulatedendothelial cells had a pronounced decrease in VE-cadherinexpression (6.8-fold decrease, P<0.001, Fig. 1D–G) and increasedtheir expression of the mesenchymal marker SM22α (7.9-fold increase, P<0.001, Fig. 1H–K), suggestive of EndMT.Endothelial cells that underwent EndMT lost the endothelialsprouting behavior (>60-fold reduction, P<0.001, Fig. 1L–O),corroborating functional alterations previously described duringEndMT (Krenning et al., 2008; Moonen et al., 2010). Blockade ofcanonical TGFβ signaling with the small-molecule ALK5 inhibitorSB431542 inhibited these morphological, phenotypical andfunctional alterations (Fig. 1).

We analyzed the expression of proteins involved in canonical TGFβsignaling (Fig. 1P,Q). Stimulation of endothelial cells with TGFβ1increased the expression of proteins in the TGFβ receptor complex, thatis, ALK5 (2.6-fold, P<0.01), TGFBR2 (2.7-fold, P<0.01) and SARA(2.1-fold, P<0.001) and increased activation (i.e. phosphorylation) ofthe Smad2 and Smad3 transcription factors [phosphorylated (p)Smad2: 2.9-fold, P<0.01; pSmad3: 1.8-fold, P<0.01]. Activation ofTGFβ signaling was reduced to baseline levels by the addition of 5 µMof the small-molecule ALK5 inhibitor SB431542 (Fig. 1P,Q).

These data indicate that active endothelial TGFβ signalingthrough ALK5 and the Smad2 and Smad3 transcription factors isassociated with EndMT and confirms previous data reported by usand others (Kokudo et al., 2008; Krenning et al., 2008; Moonenet al., 2010; Medici et al., 2010, 2011; Yoshida et al., 2012).

miR-20a targets TGFβ signaling at the receptor levelWe have previously observed that miR-20a is a potent inhibitor ofEndMT and questioned its mechanism of inhibition. Online

Fig. 1. TGFβ1 induces EndMT in amanner that is dependent onALK5, Smad2 and Smad3. (A–C) Light microscopy images of endothelial cells. (A) Untreatedcells. Treatment of endothelial cells with TGFβ1 (B) induces hypertrophy, which is counteracted by treatment with the ALK5 inhibitor SB431542 (5 µM, C).Immunofluorescence analysis of VE-cadherin (D–F) and SM22α (H–J). (D,H) Untreated cells. TGFβ1 treatment of endothelial cells decreases VE-cadherinexpression (E) and induces SM22α expression (I). The addition of SB431542 to the TGFβ1-treated cells maintains VE-cadherin expression (F) and inhibitsSM22α expression (J). (G,K) Quantification of immunofluorescence analysis (n=5 per group). Matrigel sprouting of endothelial cells (L–N) is inhibited by TGFβ1treatment (M) and restored by the addition of SB431542 (N). (L) Untreated cells. (O) Quantification of Matrigel sprouting ability of endothelial cells (n=5 per group).(P,Q) Representative immunoblots of components of endothelial TGFβ signaling (P) and their quantification (n=5 per group, Q). Graphical results are mean±s.e.m.*P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA followed by Bonferroni post-tests).

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bioinformatics tools (i.e. the miRanda algorithm; Grimson et al.,2007; Betel et al., 2008) indicate that all components of thecanonical TGFβ pathway are putative targets of miR-20a (Fig. 2A)because they contain one or more conserved binding sites formiR-20a. However, putative miR-20a targets in the TGFβ pathwayvary highly in their miRSVR score, which indicates the likelihoodof actual downregulation (Fig. 2A) (Betel et al., 2008, 2010).Reporter assays, wherein the 3′UTR regions of miR-20a gene

targets are coupled to a luciferase-encoding gene, revealed that theTGFβ signaling members ALK5, TGFBR2 and SARA are genuinemiR-20a target genes (Fig. 2B) as co-transfection of these reporterplasmidswithmiR-20amimics reduced luciferase activity by 1.4-fold(P<0.05), 1.7-fold (P<0.01) and 1.6-fold (P<0.001), respectively. Incontrast, co-transfection of reporter plasmids with a scrambledmiR-20a sequence did not alter luciferase activity (Fig. 2B).Smad3 and Smad4 are putative gene targets of miR-20a (Fig. 2A);

however, co-transfection of their respective reporter constructs withmiR-20a mimics in COS7 cells, did not affect luciferase activity(Fig. 2B) suggesting that Smad3 and Smad4 are not genuine targetsofmiR-20a. Smad2 reporters had reduced luciferase activity upon co-transfection of the 3′UTR reporter constructwith themiR-20amimic,however only to a minimal extend (1.1-fold; P<0.05).Combined, these data indicate that miR-20a affects canonical

TGFβ signaling in endothelial cells at the level of the receptors,which might reduce downstream signaling.

miR-20a gain-of-function inhibits canonical TGFβ signalingTo validate that miR-20a affects endogenous TGFβ signaling, andthus EndMT, in endothelial cells, we transfected endothelial cellswith miR-20a mimics (∼4-fold increase, P<0.001) or expressedscrambled control sequences (Fig. 3A). Endothelial cells treatedwith scrambled sequences and TGFβ1 underwent EndMT, asindicated by changes in cell morphology (Fig. 3C), waning ofVE-cadherin expression (4.9-fold decrease, P<0.001; Fig. 3G,J)and the induction of SM22α (6.8-fold increase, P<0.001;Fig. 3L,O). Gain-of-function of miR-20a in endothelial cells didnot affect the endothelial morphology or phenotype (Fig. 3D,H,M).Notably, miR-20a mimics did affect the endothelial responsivenessto the TGFβ challenge. Cells with ectopic expression of miR-20a

were irresponsive to TGFβ1 and did not undergo EndMT, asindicated by the absence of hypertrophy (Fig. 3E), maintenance ofVE-cadherin expression (Fig. 3I) and absence of SM22α expression(Fig. 3N). Moreover, miR-20a gain-of-function in TGFβ1-treatedendothelial cells partially rescued the endothelial sprouting ability(2.4-fold increase, P<0.001) compared to scrambled controls(Fig. 3Q,S,T). TGFβ-induced EndMT was associated withincreased mRNA expression of the mesenchymal transcriptionfactors Snai1 (2.9-fold increase, P<0.001), Snai2 (4.5-fold increase,P<0.001) and Twist1 (4.4-fold increase, P<0.001), which wereabsent in the miR-20a-treated cells (Fig. 3U).

Gain-of-function of miR-20a in endothelial cells inhibited theTGFβ1-induced increase in protein expression of ALK5 (2.3-foldreduction, P<0.01), TGFBR2 (1.6-fold reduction, P<0.01) andSARA (2.8-fold reduction, P<0.01), which remained equal to theexpression levels in untreated endothelial cells. As a consequence ofdecreased receptor availability in TGFβ1-treated endothelial cells,TGFβ1 was no longer able to activate Smad2 (2.4-fold reduction,P<0.05) and Smad3 (2.8-fold reduction, P<0.001) in miR-20aefficient cells (Fig. 3V,W). Interestingly, expression levels ofALK5, TGFBR2 and SARA showed a tendency to decrease(P<0.10) in endothelial cells treated with miR-20a mimics, but didnot alter the levels of Smad2 and Smad3 activation (Fig. 3V,W).

Combined, these data show that miR-20a targets TGFβ signalingat the receptor level and the decreased availability of the TGFβreceptor complex results in the absence of Smad2 and Smad3activation, thereby limiting EndMT induction.

FGF2 induces miR-20a and inhibits EndMTEndothelial mitogens inhibit EndMT (Medici and Kalluri, 2012;Okayama et al., 2012; Ichise et al., 2014; Zhang et al., 2015). Wequestioned whether this inhibitory effect might be (in part) due tothe induction of miR-20a. Endothelial cells treated with TGFβ1decreased the expression of miR-20a (2.6-fold, P<0.01, Fig. 4A).All endothelial mitogens tended (P<0.1) to increase the expressionof miR-20a in TGFβ1-treated endothelial cells; however, FGF2increased miR-20a expression to a level above that of non-treatedendothelial cells (2.5-fold versus control, 6.5-fold versus TGFβ1treatment, P<0.01; Fig. 4A).

Fig. 2. miR-20a targets TGFβ signaling at the receptor level. (A) In silico analysis (microrna.org) of the TGFβ receptors and downstream mediators identifiesmultiple putative miR-20a-binding sites in the 3′UTR of these genes (red). Putative targeting efficiency is summarized in the miRSVR Score as previouslydescribed (Betel et al., 2008, 2010). (B) Luciferase reporter assays (n=6 per group) for putative miR-20a target genes identifies ALK5, TGFBR2 and SARA asgenuinemiR-20a targets, as co-transfection of these reporter constructs withmiR-20amimics in COS-7 cells reduces luciferase activity. Results aremean±s.e.m.*P<0.05; **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA followed by Bonferroni post-tests).

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We investigated through whichmolecular pathway FGF2 inducedthe expression of miR-20a using small-molecule inhibitors to thedownstream mediators of FGF2 signaling (Fig. 4A). Inhibition ofRas (4.3-fold decrease, P<0.001), phosphoinositide 3-kinase(PI3K) (3.4-fold decrease, P<0.01), Erk1 and Erk2 (Erk1/2, 7.4-

fold decrease, P<0.001) and JNK1–JNK3 (7.1-fold decrease) allabrogated the FGF2-induced expression of miR-20a, whereasinhibition of the p38 MAPK family and phospholipase C (PLC)had no effect of miR-20a expression in FGF2- and TGFβ1-treatedendothelial cells. As Erk1/2 and JNK are both downstream

Fig. 3. See next page for legend.

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mediators of Ras signaling in endothelial cells, these data indicatethat FGF2 induces the expression ofmiR-20a through Ras signaling.Combined treatment of endothelial cells with TGFβ1 and FGF2

inhibited EndMT induction by TGFβ1, as indicated by the absenceof cellular hypertrophy (Fig. 4B–E) and maintenance of VE-cadherin expression (Fig. 4F–I), although VE-cadherin expressionwas reduced compared to FGF2-treated control cells (P<0.01,Fig. 4J). Notably, FGF2 treatment abolished the ability of TGFβ1 toinduce the expression of SM22α in endothelial cells (Fig. 4K–O).TGFβ-induced EndMT, which is associated with increased geneexpression of the mesenchymal transcription factor factors Snai1,Snai2 and Twist1, was abrogated by the addition of FGF2 (allP<0.001; Fig. 4P).We analyzed the expression levels of proteins involved in

canonical TGFβ signaling in TGFβ1 and FGF2-treated endothelialcells. FGF2 signaling inhibited the TGFβ1-induced increase inprotein expression of ALK5 (3.7-fold reduction, P<0.001),TGFBR2 (3.3-fold reduction, P<0.001) and SARA (2.3-foldreduction, P<0.05), which were expressed at levels equal to thosein untreated endothelial cells (Fig. 4Q,R). Endogenous levels ofALK5 decreased (2-fold, P<0.05) in endothelial cells treated withFGF2, whereas expression levels of TGFBR2 and SARA were notaffected. Decreased receptor availability in TGFβ1- and FGF2-treated endothelial cells limited the endothelial ability to activateSmad2 (2.2-fold reduction, P<0.001) and Smad3 (2.4-foldreduction, P<0.01; Fig. 4Q,R).These data suggest that FGF2 inhibits TGFβ1-induced EndMT

in part by the Ras-dependent expression of miR-20a and thesubsequent reduction in TGFβ receptor complex formation.

microRNA-20a loss-of-function inhibits the FGF2-mediatedprotection from EndMTTo address whether the induction of miR-20a expression is requiredfor FGF2-mediated protection from EndMT, we inhibited itsexpression using anti-miRs (Stenvang et al., 2012). Anti-miR-20adecreased miR-20a levels 2.5-fold (P<0.05) compared to untreatedendothelial cells and 8.8-fold (P<0.001) compared to endothelial

cells treated with both TGFβ and FGF2, reducing miR-20aexpression to the level of TGFβ-treated cells (Fig. 5A).

The loss of miR-20a was associated with morphological changes(Fig. 5C,E) loss of VE-cadherin expression (2.8-fold decrease,P<0.001; Fig. 5G,I) and induction of SM22α expression (7.0-foldincrease, P<0.001; Fig. 5L,N) compared to endothelial cells treatedwith both TGFβ and FGF2s (Fig. 5D,H,M). Moreover, anti-miR-20a-expressing cells had similar levels of Snai1, Snai2 and Twist1mRNAs to TGFβ-treated cells, despite the presence of FGF2(Fig. 5P).

We analyzed the expression levels of proteins involved incanonical TGFβ signaling in TGFβ1- and FGF2-treated endothelialcells, wherein FGF2 signaling inhibits the TGFβ1-induced increasein expression of ALK5, TGFBR2 and SARA (Fig. 5Q,R). The lossof miR-20a expression in cells treated with both TGFβ and FGF2increased the expression of ALK5 (3.5-fold, P<0.01), TGFBR2(1.9-fold, P<0.05) and SARA (3.0-fold, P<0.001) to the level ofTGFβ-treated cells (Fig. 5Q,R).

Thus, FGF2 inhibits TGFβ-induced EndMT through theinduction of miR-20a expression. miR-20a limits the expressionof the TGFβ receptor complex (i.e. ALK5, TGFBR2 and SARA).The loss of miR-20a expression is associated with increased Smad2and Smad3 activity, and thus the loss of the FGF2-mediatedprotection from EndMT.

MicroRNA-20a inhibits the induction, but not the progressionof TGFβ-mediated EndMTWe questioned whether miR-20a was able to reverse TGFβ-inducedEndMT by transfecting endothelial cells with miR-20a mimics72 h after TGFβ treatment (Fig. 6A). Interestingly, miR-20a gain-of-function limited the number of endothelial cells undergoingEndMT, as indicated by the increase in cells that expressed VE-cadherin (Fig. 6E–H) and decrease in the number of cells thatexpress SM22α (Fig. 6J–M) compared to TGFβ-treated cells (allP<0.001). However, in cells that had dim expression of VE-cadherin, miR-20a gain-of-function did not increase VE-cadherinexpression to above that of TGFβ-stimulated cells (EndMT cells)and the expression of VE-cadherin in the highly expressingpopulation was not different from untreated endothelial cells(Fig. 6I). Similarly, SM22α expression in the dim population wassimilar to untreated, whereas SM22α expression in the highlyexpressing population was indistinguishable from TGFβ-treatedcells (Fig. 6N). These data imply that miR-20a reduces the numberof cells entering EndMT, but does not affect TGFβ signaling in cellsthat have already entered the EndMT program.

DISCUSSIONHere, we show that FGF2-induced activation of Ras signalinginduces the expression of miR-20a. MiR-20a targets multipleproteins in the TGFβ receptor complex, namely ALK5 (TGFBR1),TGFBR2 and SARA, thereby reducing their expression levels andinhibiting TGFβ1-induced activation of Smad2 and Smad3 and theresulting expression of mesenchymal genes. Corroboratively,endothelial cells show reduced susceptibility to TGFβ1 andmaintain expression of endothelial cell markers and functions.Remarkably, when administered 3 days post TGFβ treatment, miR-20a limited the number of cells that entered EndMT, but did notaffect cells that had already entered the EndMT program.Importantly, our data implies that miR-20a is a new mediator ofendothelial TGFβ1 responsiveness and EndMT.

Postnatal EndMT is associated with fibroproliferative diseases,such as cancer progression and metastasis (Potenta et al., 2008),

Fig. 3. miR-20a gain-of-function inhibits TGFβ1, ALK5 and Smad2 andSmad3 signaling. (A) HUVECs were transfected with miR-20a mimics orscrambled (Scr) control sequences and challengedwith TGFβ1. TGFβ1 readilydecreased miR-20a levels in control cells, but not in cells transfected with miR-20a mimics. (B–E) Light microscopy images of endothelial cells. (B) Untreatedcells. Treatment of endothelial cells with TGFβ1 (C) induces hypertrophy,which is counteracted by miR-20a mimics (E). Treatment of endothelial cellswith miR-20a mimics did not alter cellular morphology (D).Immunofluorescence analysis of VE-cadherin (F–I) and SM22α (K–N).(F,K) Untreated cells. TGFβ1 treatment of endothelial cells decreases VE-cadherin expression (G) and induces SM22α expression (L). Transfection ofHUVECs with miR-20a mimics maintains VE-cadherin expression (I) andinhibits SM22α expression (N) in TGFβ1-treated endothelial cells. Treatment ofendothelial cells with miR-20a mimics alone did not alter VE-cadherin(H) expression nor SM22α expression (M). (J,O) Quantification ofimmunofluorescence analysis (n=5 per group). (P–T) Matrigel sprouting ofendothelial cells (P–S) is inhibited by TGFβ1 treatment (Q) and restored by theaddition of miR-20a mimics in endothelial cells (S). (P) Untreated cells.(T) Quantification of Matrigel sprouting ability of endothelial cells (n=5 pergroup). (U) Gene expression data on mesenchymal transcription factors,Snai1, Snai2 and Twist1. Expression is induced by TGFβ and inhibited by theaddition of miR-20a. (V,W) Representative immunoblots of components ofendothelial TGFβ signaling (V) and their quantification (n=5 per group, W).TGFβ1 increases expression of the TGFβ receptor complex and activatesSmad2 and Smad3. These expression and activity changes are inhibited bythe addition of miR-20a mimics to endothelial cells. Graphical results aremean±s.e.m. *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA followed byBonferroni post-tests).

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cardiac (Zeisberg et al., 2007) and kidney fibrosis (Zeisberg et al.,2008), and has received a large research interest over the past decade.TGFβ signaling is recognized as the key driving force of EndMT(Derynck and Akhurst, 2007; Pannu et al., 2007; van Meeteren and

ten Dijke, 2012) and inhibitors of TGFβ signaling, such as BMP7(Zeisberg et al., 2007), receive increasing therapeutic interest as anti-fibrotic compounds. However, little is known about endogenousinhibitors of TGFβ signaling in endothelial cells during EndMT.

Fig. 4. Endothelial FGF2 signaling through RAS induces microRNA-20a expression and inhibits EndMT. (A) Endothelial cells were stimulated with TGFβ1and endothelial mitogens and assayed for miR-20a expression (n=5 per group). The addition of FGF2 increases miR-20a expression to above the level ofcontrols. miR-20a expression is dependent on Ras and PI3K signaling (n=5 per group), as the addition of inhibitor to Ras (FTS, 5 µM) or PI3K (LY294002, 10 µM)blocks the FGF2-induced expression of miR-20a. Blockage of downstream mediators of Ras and PI3K, JNK (SP600125, 1 µM) and Erk1/2 (U0126, 5 µM)also blocks the FGF2-induced expression of miR-20a. (B–E) Light microscopy images of endothelial cells. (B) Untreated cells. Treatment of endothelial cellswith TGFβ1 (C) induces hypertrophy, which is counteracted by FGF2 (E). Treatment of endothelial cells with FGF2 did not alter cellular morphology (D).Immunofluorescence analysis of VE-cadherin (n=5 per group, F–I) and SM22α (n=5 per group, K–N). (F,K) Untreated cells. TGFβ1 treatment of endothelial cellsdecreases VE-cadherin expression (G) and induces SM22α expression (L). Treatment of HUVECs with FGF2 maintains VE-cadherin expression (I) and inhibitsSM22α expression (N) in TGFβ1-treated endothelial cells. Treatment of endothelial cells with FGF2 alone does not alter VE-cadherin (H) or SM22αexpression (M). (J,O) Quantification of immunofluorescence analysis (n=5 per group). (P) Gene expression data on the mesenchymal transcription factors Snai1,Snai2 and Twist1. Expression is induced by TGFβ and inhibited by the addition of FGF2 (n=5 per group). (Q,R) Representative immunoblots of components ofendothelial TGFβ signaling (Q) and their quantification (n=5 per group, R). TGFβ1 increases expression of the TGFβ receptor complex and activates Smad2 andSmad3. These expression and activity changes are inhibited by the supplementation of FGF2 to endothelial cells. Graphical results are mean±s.e.m. *P<0.05;**P<0.01; ***P<0.001 (one-way ANOVA followed by Bonferroni post-tests).

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Fig. 5. MicroRNA-20a loss-of-function inhibits FGF2-mediated protection from EndMT. (A) Endothelial cells were transfected with anti-miR-20aoligonucleotides or scrambled control (indicated by – in the anti-miR-20a labels) sequences and challenged or not with TGFβ1 and FGF2 (n=5 per group). Theaddition of FGF2 increasesmiR-20a expression to above the level of controls. The addition of anti-miR-20a oligonucleotides blocks the FGF2-induced expressionof miR-20a. (B–E) Light microscopy images of endothelial cells. (B) Untreated cells. Treatment of endothelial cells with TGFβ1 (C) induces hypertrophy, whichis counteracted by FGF2 (D). Treatment of endothelial cells with anti-miR-20a in the presence of TGFβ and FGF2 alters cellular morphology resulting inhypertrophy (E). Immunofluorescence analysis of VE-cadherin (n=5 per group, F–I) and SM22α (n=5 per group, K–N) expression. (F,K) Untreated cells. TGFβ1treatment of endothelial cells decreases VE-cadherin expression (G) and induces SM22α expression (L). Treatment of HUVECwith FGF2maintains VE-cadherinexpression (H) and inhibits SM22α expression (M) in TGFβ1-treated endothelial cells. Treatment of endothelial cells with anti-miR-20a in combination with TGFβand FGF2 decreases VE-cadherin (I) and induces SM22α expression (N). (J,O) Quantification of immunofluorescence analysis (n=5 per group). (P) Geneexpression data on the mesenchymal transcription factors Snai1, Snai2 and Twist1 (n=5 per group). Expression of Snai1, Snai2 and Twist1 is induced by TGFβand inhibited by the addition of FGF2. miR-20a loss-of-function restores the expression of these proteins back to the level of that after TGFβ treatment. (Q,R)Representative immunoblots of components of endothelial TGFβ signaling (Q) and their quantification (n=5 per group, R). TGFβ1 increases expression of theTGFβ receptor complex and activates Smad2 and Smad3. These expression and activity changes are inhibited by the supplementation of FGF2 to endothelialcells. The loss-of-function of miR-20a abolishes the protective effects of FGF2. Graphical results are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001 (one-wayANOVA followed by Bonferroni post-tests).

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MicroRNAs are involved in EndMT (Bijkerk et al., 2012; Fangand Davies, 2012; Ghosh et al., 2012; Kumarswamy et al., 2012;Zhang et al., 2013) and fibroproliferative diseases (Thum et al.,2008; van Rooij et al., 2008; Kato et al., 2009; Chung et al., 2010)by targeting genes that are crucial for endothelial homeostasis oractivating fibroblasts. However, the role of microRNAs that are lostduring EndMT and fibroproliferative diseases remains elusive.Results from the present study indicate that miR-20a plays animportant role in the endothelial susceptibility to TGFβ by activelysuppressing expression of components of the TGFβ receptorcomplex in TGFβ-stimulated cells. Hence, in order to react to andpropagate TGFβ signals in endothelial cell, miR-20a needs todecrease. Indeed, TGFβ1-stimulated endothelial cells had a reducedexpression of miR-20a. Notably, endothelial cells transfected withmiR-20a mimics had a reduced sensitivity to TGFβ1 and wereresistant to EndMT. Interestingly, miR-20a mimics efficientlyinhibited ALK5, TGFBR2 and SARA expression in TGFβ-treatedcells, but did not affect the expression of these proteins in nativeendothelial cells. As the efficacy of a microRNA in decreasing theexpression its target genes relies on the expression level not only ofthe target gene under investigation, but also on the presence of othermRNAs that are targeted by the same microRNA [known ascompeting endogenous RNAs; ceRNAs (Salmena et al., 2011)], ourdata might indicate the presence of ceRNAs in unstimulatedendothelial cells, which inhibits the decrease in ALK5, TGFBR2and SARA.In fibroproliferative diseases, the endothelium shows resistance

to a number of mitogens, such as FGF2, vascular endothelial growthfactor A (VEGFa) and insulin-like growth factor-1 (IGF1) (Baeldeet al., 2007; Cheng et al., 2014; Stiedl et al., 2015). We thereforewondered whether these mitogens could relay some protectiveeffects at non-affected sited versus affected sites where the responseto these mitogens is lost. Hence, we assessed whether thesemitogens might induce the expression of miR-20a and counter

TGFβ-induced EndMT. Indeed, in healthy endothelial cells, FGF2strongly induces the expression of miR-20a in a Ras-dependentmanner, reducing the endothelial expression of the TGFβ receptorcomplex, and prevents EndMT induction by TGFβ1. This impliesthat therapeutic induction of miR-20a expression or the targeteddelivery of miR-20a mimics might pose a novel therapy to treatEndMT in fibroproliferative diseases. Thus, miR-20a links twocrucial growth factor signaling pathways in endothelial homeostasisand dysfunction (Fig. 7). Recent investigations using endothelial-specific deletion of FGF receptor 1 (FGFR1) (Cheng et al., 2014) orFGF receptor substrate 2α (Frs2α) (Chen et al., 2015), corroboratethe importance of FGF2 signaling in the inhibition of EndMT. Theloss of FGFR1 activity aggravates atherosclerosis development andEndMT progression, in part by increased Smad2 activity (Chen etal., 2014; Chen et al., 2015). Moreover, FGFR1 expression is highlyreduced during human atherosclerosis development, a processcharacterized by EndMT (Chen et al., 2015). Interestingly, theexpression of another microRNA that inhibits ALK5 expression,let-7b, is also highly dependent on FGF2 signaling, as the loss ofFGFR1 results in the ablation of let-7b levels and an increase TGFβactivity and EndMT (Chen et al., 2012). These data corroborate andextend earlier reports where FGF2 was reported to reduceendothelial sensitivity to TGFβ (Kawai-Kowase et al., 2004;Ichise et al., 2014).

Interestingly, although we and others describe a protective effectof FGF2 against EndMT induction in mature and progenitorendothelial cells (Moonen et al., 2010; Ichise et al., 2014), othershave linked FGF2 signaling to EndMT induction (Ghosh et al.,2010; Lee et al., 2012). Indeed, FGF2 is known to inhibit EndMT inmacrovascular endothelial cells, whereas it induces EndMT is somemicrovascular endothelial cell types (Lee et al., 2004; Lee and Kay,2006). Whether this difference in EndMT induction is dependent onthe organ-of-origin, as not all microvascular endothelial cellsundergo EndMT following FGF2 stimulation (Ichise et al., 2014),

Fig. 6. MicroRNA-20a inhibits the induction but not the progression of TGFβ-mediated EndMT. (A) Schematic of the experimental procedure. Endothelialcells were treated with TGFβ for 3 days and subsequently transfected with miR-20a mimics or scrambled control sequences (n=4 per group). (B–D) Cellsuninterruptedly treated with TGFβ showed signs of hypertrophy (C), whereas cells with gain-of-function for miR-20a had a diverse phenotype, i.e. some cells werehypertrophic and others were not (D). Untreated cells (B). (E–H) TGFβ treatment decreased VE-cadherin expression (F), whereas miR-20a inhibited thisdecrease in ∼50% of the cells (G). A quantification is shown in H (n=4 per group). (E) Untreated cells. (J–M) SM22α expression was induced by TGFβ treatment(K) and reduced by the addition of miR-20a (L). Untreated cells (J). A quantification is shown in M (n=4 per group). (I,N) Cells that expressed VE-cadherin aftertreatment with both TGFβ and miR-20a mimics (high), expressed VE-cadherin at levels similar to native endothelial cells, whereas dim cells expressed VE-cadherin at similar levels to TGFβ-treated cells (n=4 per group, I). Cells that expressed SM22α after treatment with TGFβ and miR-20a mimics (high), expressedSM22α at levels similar to cells that had undergone EndMT, whereas dim cells expressed SM22α at similar levels to native endothelial cells (n=4 per group, N).Graphical results are mean±s.e.m. ***P<0.001; ns, not significant (one-way ANOVA followed by Bonferroni post-tests).

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or is derived from some microenvironmental cue is currentlyunknown and warrants more investigation.Moreover, although multiple receptor tyrosine kinases (RTKs)

are implemented in the protection against EndMT (Medici et al.,2010; Okayama et al., 2012), only FGF2 signaling induced miR-20a. RTKs depend on scaffold molecules that organize theirdownstream effects. Therefore, it is appealing to hypothesize thatFGF2 utilizes a unique scaffold protein for its signaling, distinctfrom other RTKs. Whether FGF2 functions through a uniquescaffold protein is currently unknown and warrants furtherinvestigation.In summary, FGF2 regulates endothelial TGFβ1 signaling by

controlling ALK5, TGFBR2 and SARA expression, through controlof miR-20a levels. Loss of FGF2 signaling combined with a TGFβ1challenge decreases miR-20a levels and increases endothelialresponsiveness to TGFβ1 through elevated receptor complex levelsand activation of Smad2 and Smad3. TGFβ1 treatment culminates inEndMT, which is abrogated by either FGF2 or exogenous miR-20a.These data suggest that miR-20a is a new regulator of endothelialTGFβ signaling and might be a new target for therapeutic silencingof TGFβ activity in fibroproliferative diseases.

MATERIALS AND METHODSEndothelial cell culture, transfection and stimulationsHuman umbilical vein endothelial cells (HUVECs; Lonza, Verviers,Belgium) were cultured in gelatin-coated culture flasks in endothelial cellmedium containing RPMI 1640 (Lonza, Verviers, Belgium) supplementedwith 20% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA),50 µg/ml bovine pituitary extract (Lonza, Verviers, Belgium), 2 mML-glutamine, 1% penicillin-streptomycin (both Sigma-Aldrich, St Louis,MO) and 5 U/ml heparin (Leo Pharma, Amsterdam, The Netherlands).HUVECs were used between passages 3 and 6.

HUVECs were stimulated with TGFβ1, FGF2, hepatocyte growth factor(HGF), IGF1 or VEGFa (all 10 ng/ml, all Peprotech, Rocky Hill, NJ) for72 h. Small-molecule inhibitors to ALK5 (SB431542, 5 µM), p38 MAPK(SB203580, 1 µM, both Sigma-Aldrich), Ras [farnesyl thiosalicylic acid(FTS); 5 µM, Cayman Chemical, Ann Arbor, MI], PI3K (LY294002,10 µM, SelleckChem, Munich, Germany), Erk1/2 (U0126, 5 µM, Promega,Madison, WI) or JNK (SP600125, 1 µM, EMD Millipore, Darmstadt,Germany) were used where indicated for 72 h.

HUVECs were transfected with microRNA-20a (PM10057), anti-miR-20a (AM10057) or a scrambled control sequence (AM17110, both 100 nM,all AMBION/Life Technologies, Carlsbad, CA), using the siRNA ReagentSystem (Santa Cruz Biotechnology, Heidelberg, Germany) according tomanufacturer’s instructions. After overnight incubation, HUVECs werechallenged with the appropriate stimuli.

3′UTR reporter assaysIsolation of 3′UTR fragments was performed from a cDNA pool derivedfrom various human tissues using specific primers for the ALK5 3′UTR(sense 5′-TTCTACAGCTTTGCCTGAAC-3′, antisense 5′-GTCTGGGAA-TGTCTTTAATT-3′), the TGFBR2 3′UTR (sense 5′-CTCTTCTGGGGCA-GGCTGGG-3′, antisense 5′-AGCTACTAGGAATGGGAACAG-3′), theSARA 3′UTR (sense 5′-ACAGAGAAGACTTCATTTTT-3′, antisense 5′-CAGTGTGGAATTATCCTTTT-3′), the SMAD2 3′UTR (sense 5′-AGCT-TCACCAATCAAGTCCC-3′, antisense 5′-AACATGGTAAACAACTCA-AA-3′), the SMAD3 3′UTR (sense 5′-AGACATCAAGTATGGTAGGG-3′,antisense 5′-CAGACTGAGCTCCTGGCACA-3′) or the SMAD4 3′UTR(sense 5′-GGTCTTTTACCGTTGGGGCC-3′, antisense 5′-AGTTGGCTT-TCTCTTTTAAT-3′) (all 0.6 µM, Biologio, Leiden, The Netherlands). Senseand antisense primers were extended with SgfI (GCGATCGC) and NotI(GCGGCCGC) restriction sequences, respectively. Amplification wasperformed using the DyNAzyme EXT PCR kit (Finnzymes, Vantaa,Finland) according to the manufacturer’s instructions. Amplicon size wasvalidated by gel electrophoresis on 1% agarose gels.

Ampliconswere purified using the QIAquick PCRPurification kit (Qiagen,Venlo, The Netherlands) and cloned into the dual luciferase reporter vectorpsiCHECK-2 (Promega, Madison, WI) using T4 DNA Ligase (Fermentas/Thermo Fisher Sci., Waltham, MA) according to standard protocols.

HEK293 cells (Sigma-Aldrich, St. Louis,MO) were maintained in DMEM(Lonza, Verviers, Belgium) containing 10%FBS, 2 mML-glutamine and 1%Penicillin/Streptomycin. HEK293 cells were transfected with 100 ng/ml UTRreporter plasmid and 50 nM miR-20a mimic or scrambled control (Ambion/Life Technologies, Carlsbad, CA) using Endofectin (GeneCopoeia,Rockville, MD). 48 h post-transfection, luciferase activity was assayedusing the DualGlo Luciferase assay system (Promega, Madison, WI) andrecorded for 1 s on a Luminoskan ASCENT (Thermo Scientific, Waltham,MA) according to manufacturer’s instructions.

MicroRNA and mRNA expression analysisTotal RNA was isolated using TRIzol Reagent (Invitrogen, Carlsbad, CA)according to the manufacturer’s instructions. RNA quantity and purity were

Fig. 7. Schematic representation of the FGF2-mediated regulation of the endothelial TGFβ1 susceptibility by controlling miR-20a levels. (A) In theabsence of FGF2 signaling, TGFβ1 binds to its heteromeric receptor formed by ALK5 and TGFBR2. SARA recruits the R-Smads to the ALK5 kinase. Activation ofSmad2 and Smad3, and concomitant complexation with Smad4, results in nuclear translocation and transcription of mesenchymal genes and repression ofendothelial genes. (B) FGF2 signaling activates its downstream mediators Ras and PI3K, resulting in the expression of miR-20a. miR-20a represses theexpression of the TGFβ receptor complex (ALK5 and TGFBR2) and SARA, thus inhibiting Smad2 and Smad3 activation and the culminating induction of EndMT.

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assessed by spectrophotometric analysis (Nanodrop, Wilmington, DE)wherein both the ratios of absorbance at 260 nm to that at 230 nm (A260/230)and absorbance at 260 nm to that at 280 nm (A260/280) were >1.8. RNAintegrity was assessed by gel electrophoresis on a 2% agarose gel. FormicroRNA expression analysis, 20 ng total RNA was reversely transcribedusing the Taqman MicroRNA Reverse Transcription kit (AppliedBiosystems, Carlsbad, CA) using specific miR-20a (5′-GTCGTATCCAG-TGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCTGC-3′)or RNU6B (U6; 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGC-ACTGGATACGACAA AAATATGG-3′) stem loop primers. QuantitativePCR expression analysis was performed on a reaction mixture containing10 ng cDNA equivalent, 0.5 µM miR primers (miR-20a; 5′-TGCGGTTA-AAGTGCTTATAGT-3′, RNU6B; 5′-TGCGGCTGCGCAAGGATGA-3′,antisense 5′-GTGCAGGGTCCGAGGT-3′, all Biolegio, Leiden, TheNetherlands) and FastStart SYBR Green (Roche, Almere, TheNetherlands). For mRNA expression analysis, 500 ng total RNA wasreversely transcribed using the RevertAid First Strand cDNA SynthesisKit (Applied Biosystems, Carlsbad, CA) using specific Snai1 (sense 5′-GCTGCAGGACTCTAATCCAGA-3′; antisense 5′-ATCTCCGGAGGT-GGGATG-3′), Snai2 (sense 5′-TGGTTGCTTCAAGGACACAT-3′; anti-sense 5′-GTTGCAGT-GAGGGCAAGAA-3′), Twist1 (sense 5′-AAGGCATCACT-ATGGACTTTCTCT-3′; antisense 5′-GCCAGTTTG-ATCCCAGTATTTT-3′) and GAPDH (sense 5′-AGCCACATCGCTCAG-ACAC-3′; antisense 5′-GCCCAATACGACCAAATCC-3′) primers.Quantitative PCR expression analysis was performed on a reactionmixture containing 10 ng cDNA equivalent, 0.5 µM sense primers and0.5 µM antisense primers (all Biolegio, Leiden, The Netherlands) andFastStart SYBR Green (Roche, Almere, The Netherlands). Analyses wererun on a Viia7 real-time PCR system (Applied Biosystems, Carlsbad, CA).

ImmunofluorescenceCells were fixed using 4% paraformaldehyde in PBS at room temperature for15 min. For intracellular staining, fixed cells were permeabilized using 0.5%Triton X-100 in PBS (Sigma-Aldrich) at room temperature for 10 min.Blocking of specific antibody activity was performed using 2% bovine serumalbumin (BSA) in PBS for 10 min. Samples were incubatedwith antibodies toVE-cadherin (1:200, Cell Signaling #2500, Danvers, MA) or SM22α (1:250,Abcam #14106, Cambridge, UK) in PBS containing 2% BSA at 4°Covernight. Samples were washed extensively with 0.05% Tween-20 in PBSand incubatedwithAlexa-Fluor-594-conjugated antibodies to rabbit IgG (LifeTechnologies, Carlsbad, CA, #A21207) in DAPI with PBS with 2% BSA atroom temperature for 1 h. Image analysis was performed on TissueFAXS(TissueGnostics, Vienna, Austria) in fluorescencemode, in combinationZeissAxioObserver Z1 microscope. Data analysis was performed usingTissueQuest fluorescence (TissueGnostics, Vienna, Austria) software. Forall immunofluorescence analyses, 1000–2000 individual cells were analyzed.

ImmunoblottingCells were lysed in RIPA buffer (Thermo Scientific, Waltham, MA)supplemented with 0.1% proteinase inhibitor cocktail (Sigma-Aldrich).Samples (30 µg/lane) were loaded on 10% SDS-PAGE gels and blotted ontonitrocellulose membranes. Membranes were incubated with antibodies toALK5 (1:500, #31013), TGFBR2 (1:500, #61213), SARA (1:1000,#124875, all Abcam, Cambridge, UK), pSmad2 (Ser465 and Ser467,1:500, #3108), pSmad3 (Ser423 and Ser 425, 1:500, #9520), Smad2 andSmad3 (1:500, #3102, all Cell Signaling, Danvers, MA) and GAPDH(1:2000, #9485, Abcam, Cambridge, UK) in Odyssey Blocking Bufferovernight at 4°C. Next, membranes were incubated with IRDye680-conjugated antibodies to rabbit IgG (LI-COR Biosciences, Bad Homburg,Germany) diluted 1:10,000 in Odyssey Blocking Buffer at roomtemperature for 1 h. Proteins were visualized using the Odyssey® InfraredImaging System (LI-COR Bioscience). Densitometric analysis wasperformed using TotalLab TL120 1D (Nonlinear Dynamics, Durham, NC).

Matrigel sprouting10 µl of MatriGel (BD Biosciences, San Jose, CA) was solidified in µ-SlideAngiogenesis (Ibidi, Martinsried, Germany). 15,000 cells per well werecultured on the solidified MatriGel in endothelial growth medium,

overnight. Formation of sprouts was analyzed by conventional lightmicroscopic analysis.

Statistical analysisAll experimental data were obtained from at least five independentexperiments. Data is expressed as mean±s.e.m. Data were analyzed usingone-way ANOVA followed by Bonferroni post-tests. P<0.05 wasconsidered to be statistically significant.

AcknowledgementsWe acknowledge Mr. K. Sjollema (UMCG, UMIC) for expert technical assistancewith fluorescence imaging. Imaging was performed at the UMCG Imaging Center(UMIC), supported by the Netherlands Organization for Health Research andDevelopment (ZonMW grant 40-00506-98-9021).

Competing interestsThe authors declare no competing or financial interests.

Author contributionsG.K. and J.-R.A.J.M. conceived and coordinated the study. G.K. and A.C.P.C. wrotethe paper. G.K., A.C.P.C. and M.G.L.B. designed, performed and analyzed theexperiments shown in Figs 1–6. G.K. and A.C.P.C. prepared the figures. All authorsreviewed the results and approved the final version of the manuscript.

FundingThis work was supported by the Groningen University Institute for Drug Exploration(GUIDE) (to G.K.); and a ZonMW and NWO Innovational Research Incentive grant[grant number 916.11.022 to G.K].

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