Endothelial loss of Fzd5 stimulates PKC/Ets1-mediated transcription of Angpt2 and Flt1

Endothelial loss of Fzd5 stimulates PKC/Ets1-mediated transcription of Angpt2 and Flt1 Aims Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. Methods and results Using short interference RNA-mediated loss-of-function assays, the function and mechanism of sign- aling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neoves - sel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G /G cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in 0 1 enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of 2+ canonical Wnt signaling, non-canonical Wnt/Ca -mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. Conclusions The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2. Keywords Endothelial cells · Angiogenesis · Fzd5 · Wnt signaling Introduction New formation of blood vessels from pre-existing vessels, a process called angiogenesis, is a critical step in embryogen- esis and continues to play important roles throughout life in Electronic supplementary material The online version of this both health and disease [1]. It is a dynamic process that is article (https ://doi.org/10.1007/s1045 6-018-9625-6) contains tightly regulated by a diverse range of signal transduction supplementary material, which is available to authorized users. cascades, and imbalances in these pathways can be a causa- * Caroline Cheng tive or a progressive factor in many diseases [2]. K.L.Cheng-2@umcutrecht.nl Multiple studies suggest an important role for endothelial signal transduction via Frizzled (Fzd) receptors in angio- Experimental Cardiology, Department of Cardiology, genesis [3–5]. The Fzd receptors belong to a family of 10 Thoraxcenter, Erasmus University Medical Center, Rotterdam, The Netherlands transmembrane receptors (Fzd1–10), which can initiate Fzd/ Wnt canonical and non-canonical signaling upon binding Department of Nephrology and Hypertension, Division of Internal Medicine and Dermatology, University Medical with one of the 19 soluble Wnt ligands. Canonical Wnt sign- Center Utrecht, Utrecht, The Netherlands aling depends on Fzd receptor and LRP 5/6 co-activation, Department of Pediatric Surgery of the Erasmus initiating Disheveled (Dvl) to stabilize β-catenin, followed Medical Center, Sophia Children’s Hospital, Rotterdam, by β-catenin-mediated transcriptional regulation [6–8]. In The Netherlands Vol.:(0123456789) 1 3 Angiogenesis contrast, non-canonical Wnt signaling also involves Dvl, (supplemented with 100  U/ml penicillin/streptomycin; 2+ but proceeds via Wnt/Ca -mediated activation of nuclear Lonza, and 10% FCS; Lonza), respectively, in 5% C O factor of activated T-cells (NFAT) or Wnt/planar cell polar- at 37  °C. The experiments were performed with cells ity (PCP)-mediated activation of c-JUN N-terminal Kinase at passage 3–5. Lentivirus green fluorescent protein (JNK) [6]. A potential link between Fzd5 and angiogenesis (GFP)-transduced HUVECs and lentivirus discosoma was previously demonstrated in Fzd5 full knockout mice sp. red fluorescent protein (dsRED)-transduced pericytes [5]. Fzd5 silencing induced in utero death at approximately were used at passages 5–7. HUVECs and GFP-labeled E10.5, which was associated with vascular defects in the HUVECs were used from six different batches derived placenta and yolk sac. Furthermore, isolated ECs from from pooled donors. Pericytes and dsRED-labeled peri- Fzd5-dec fi ient mice showed a reduction in cell proliferation, cytes were used from eight different batches derived which is crucial for neovessel formation. These findings sug- from single donors. Fzd5, Ets1, and Wnt5a knockdown gest that Fzd5 can be an important regulator of angiogenesis. in HUVECs was achieved by cell transfection of a pool However, the exact type of endothelial Fzd5/Wnt signaling containing four targeting short interference RNA (siRNA) and the downstream molecular mechanism causal to the poor sequences, whereas PKC isoforms were knocked down vascular phenotype in the absence of this receptor requires with individual siRNA strands (Dharmacon), all in a further in-depth evaluation. final concentration of 100 nM. Control cells were either Here, we studied the angiogenic potential of Fzd5 and untreated or transfected with a pool of four non-targeting investigated the signaling pathways that are mediated by siRNA sequences (Dharmacon) in a final concentration of Fzd5/Wnt signaling in human ECs. Our findings indicate 100 nM. Target sequences are listed in Table 1. Inhibition that Wnt5a, which is endogenously expressed in ECs, binds of GSK3β, NFAT, JNK, and PKC activation was achieved and signals via Fzd5, but in the absence of this receptor with 20 µM LiCl (Sigma), 1 µM Cyclosporine A (CsA; triggers a poor angiogenic phenotype via an alternative Sigma), 20 µM SP600125 (Sigma), and 5, 10, and 20 nM signaling route. We demonstrated that Fzd5 is essential for staurosporine (CST), respectively. Phosphatase activity 2+ neovessel formation in vitro in a collagen matrix-based 3D was inhibited with 50 nM Calyculin A. Free Ca -induced co-culture of primary human vascular cells. Silencing of activation of NFAT-mediated transcription was achieved Fzd5 reduced EC proliferation as a result of G /G cell cycle with 10 µM A23187. In experiments involving a serum 0 1 arrest and decreased cell migration capacity. Furthermore, starvation step, the cells were cultured for 24 h in EBM2. Fzd5 knockdown resulted in enhanced expression of the factors Angiopoietin 2 (Angpt2) and Fms-Related Tyrosine Kinase 1 (Flt1), which are mainly known for their destabiliz- Table 1 siRNA sequences used in cell culture ing effects on the vasculature [ 9–11]. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced Pro- Target gene Target sequence tein Kinase C (PKC) signaling, without the involvement of Non-targetingUGG UUU ACA UGU CGA CUA A 2+ canonical Wnt signaling, non-canonical Wnt/Ca -mediated UGG UUU ACA UGU UGU GUG A activation of NFAT, and non-canonical Wnt/PCP-mediated UGG UUU ACA UGU UUU CUG A activation of JNK. Further downstream, PKC-induced tran- UGG UUU ACA UGU UUU CCU A scription of Angpt2 and Flt1 involved the transcription factor Fzd5GCA UUG UGG UGG CCU GCU A Protein C-Ets-1 (Ets1), as knockdown of both Fzd5 and Ets1 GCA CAU GCC CAA CCA GUU C resulted in a marked repression of Angpt2 and Flt1 expres- AAA UCA CGG UGC CCA UGU G sion levels. In addition, silencing of Ets1 partially restored GAU CCG CAU CGG CAU CUU C the impaired endothelial tubule formation capacity of Fzd5- Ets1AUA GAG AGC UAC GAU AGU U silenced ECs. GAA AUG AUG UCU CAA GCA U GUG AAA CCA UAU CAA GUU A CAG AAU GAC UAC UUU GCU A Methods Wnt5aGCC AAG GGC UCC UAC GAG A GUU CAG AUG UCA GAA GUA U Cell culture CAU CAA AGA AUG CCA GUA U GAA ACU GUG CCA CUU GUA U Human umbilical vein endothelial cells (HUVECs; Lonza) PKCαUAA GGA ACC ACA AGC AGU A and human brain vascular pericytes (Sciencell) were cul- PKCδCCA UGU AUC CUG AGU GGA A tured on gelatin-coated plates in EGM2 medium (EBM2 PKCεGUG GAG ACC UCA UGU UUC A medium supplemented with EGM2 bullet kit; Lonza, and PKCηGCA CCU GUG UCG UCC AUA A 100  U/ml penicillin/streptomycin; Lonza) and DMEM 1 3 Angiogenesis imaged by f luorescence microscopy, followed by analysis Quantitative PCR and Western blot analysis of the number of junctions, the number of tubules, and the tubule length using AngioSys. At least three technical Total RNA was isolated using RNA mini kit (Bioline) and reversed transcribed into cDNA using iScript cDNA syn- replicates were averaged per condition per independent replicate. thesis kit (Bioline). Gene expression was assessed by qPCR using SensiFast SYBR & Fluorescein kit (Bioline) and prim- Migration assay ers as listed in Table  2. Expression levels are relative to the housekeeping gene β-actin. For assessment of protein Twenty-four hours post siRNA transfection, HUVECs levels, cells were lysed in cold NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris, pH 8.0) supplemented with were plated at a density of 0.5 × 10 cells/well in an Oris™ Universal Cell migration Assembly Kit (Platypus Tech- 1 mM β-glycerophosphate, 1 mM PMSF, 10 mM NaF, 1 mM NaOV, and protease inhibitor cocktail (Roche). Total pro- nologies) derived 96-well plate with cell seeding stoppers. Twenty-four hours post sub-culturing, the cell stoppers were tein concentration was quantified by Pierce® BCA Protein Assay Kit (Thermo Scientific) as a loading control. Lysates removed and cells were allowed to migrate into the cell free region for 16 h in 5% C O at 37 °C. Subsequently, the cells were denaturated in Laemmli buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromo- were washed in PBS and stained by Calcein-AM followed by visualization using fluorescence microscopy. Wells in phenol blue) at 90 °C for 5 min followed by electrophoresis on a 10% SDS-page gel (Biorad). Subsequently, proteins which cell seeding stoppers were not removed were used as a negative control. Results were analyzed by Clemex. At were transferred to a nitrocellulose membrane (Pierce) and incubated for 1 h in PBS with 5% non-fat milk, followed least three technical replicates were averaged per condition per independent replicate. by incubation with rabbit anti-Fzd5 (Milipore), goat anti- β-actin (Abcam), rabbit anti-β-catenin, anti-non-phospho Intracellular immunofluorescent staining β-catenin and phospho-β-catenin (CST, validated in Sup- plemental Fig. 3A), rabbit anti-Angpt2 (Abcam), rabbit anti- Forty-eight hours post siRNA transfection, HUVECs were JNK and phospho-JNK (CST, validated in Supplemental Fig. 4C), rabbit anti-JUN and phospho-JUN (CST, validated seeded on gelatin-coated glass coverslips in 12-well plates at 5 5 a density of 0.5 × 10 cells/well (sub-confluent) and 3.5 × 10 in Supplemental Fig. 4C), rabbit anti-Wnt5a (CST) rabbit anti-Dvl2 (CST) according to the manufacturer’s descrip- cells/well (confluent). Subsequently, cells adhered for 24 h followed by fixation for 15 min in 4% paraformaldehyde tion. Protein bands were visualized with the Li-Cor detec- tion system (Westburg). Levels of secreted Flt1 in cultured and blocking for 60 min in PBS with 5% bovine serum albu- min (Sigma) and 0.3% Triton X-100 (Sigma). After block- medium were assessed 72 h post-transfection using a Flt1 ELISA kit (R&D systems). ing, coverslips were placed on droplets PBS containing 1% BSA, 0.3% Triton X-100, and rabbit anti-β-catenin antibody 3D analysis of endothelial tubule formation (CST), followed by incubation for 16 h in a humidified envi- ronment at 4 °C. Thereafter, coverslips were incubated on Twenty-four hours post siRNA transfection, GFP-labeled PBS with 1% BSA and 0.3% Triton X-100 containing an Alexa Fluor 594-labeled secondary antibody (Invitrogen) HUVECs were harvested and suspended with non-trans- fected dsRED-labeled pericytes in collagen as previously and phalloidin-rhodamine (Invitrogen) for 1 h at room tem- perature, finally followed by mounting the stained coverslips described by Stratman [12]. In summary, HUVECs and pericytes were mixed in a 5:1 ratio in EBM2 supple- on vectashield with DAPI (Brunschwig). Coverslips were imaged by confocal microscopy. mented with Ascorbic Acid, Fibroblast Growth Factor, and 2% FCS from the EGM2 bullet kit. Additionally, Proliferation, cell cycle assay, and apoptosis C-X-C motif chemokine 12, Interleukin 3, and Stem Cell Factor were added in a concentration of 800 ng/ml (R&D Twenty-four hours post siRNA transfection, HUVECs were systems). The cell mixture was suspended in bovine col- lagen (Gibco) with a final concentration of 2 mg/ml and seeded in six-well plates at a density of 0.5 × 10 cells/well. To study the effect of Fzd5 knockdown on proliferation, pipetted in a 96-well plate. One hour of incubation in 5% CO at 37 °C was followed by the addition of 100 µl of the HUVECs were harvested 24, 48, and 72 h post sub-culturing and counted by flow cytometry. For analysis of cell cycle adjusted EBM2 medium on the collagen gels. The addi- tion of recombinant human Angpt2 and Flt1 (R&D sys- progression, cells were harvested 48 h post sub-culturing and fixated in 70% ethanol for 60 min on ice. Subsequently, cells tems) was done 24 h post seeding in the collagen matrix, both in a final concentration of 1000 ng/ml. Forty-eight were stained with PI and treated with RNAse (Sigma) for 30 min at 37 °C and analyzed by flow cytometry. Apoptosis hours and 120  h post seeding, these co-cultures were 1 3 Angiogenesis Table 2 Primer sequences used Gene Sense primer sequence Antisense primer sequence for (q)PCR Fzd1GCC CTC CTA CCT CAA CTA CCA ACT GAC CAA ATG CCA ATC CA Fzd2GCT TCC ACC TTC TTC ACT GTC GCA GCC CTC CTT CTT GGT Fzd3CTT CCC TGT CGT AGG CTG TGT GGG CTC CTT CAG TTG GTT CT Fzd4ATG AAC TGA CTG GCT TGT GCT TGT CTT TGT CCC ATC CTT TTG Fzd5TAC CCA GCC TGT CGC TAA ACAAA ACC GTC CAA AGA TAA ACTGC Fzd6GCG GAG TGA AGG AAG GAT TAG TGA ACA AGC AGA GAT GTG GAA Fzd7CGC CTC TGT TCG TCT ACC TCT CTT GGT GCC GTC GTG TTT Fzd8GCC TAT GGT GAG CGT GTC CCTG GCT GAA AAA GGG GTT GT Fzd9CTG GTG CTG GGC AGT AGT TTGCC AGA AGT CCA TGT TGA GG Fzd10CCT TCA TCC TCT CGG GCT TCAGG CGT TCG TAA AAG TAG CAG Wnt1CAA CAG CAG TGG CCG ATG GTGG CGG CCT GCC TCG TTG TTG TGAAG Wnt2GTC ATG AAC CAG GAT GGC ACA TGT GTG CAC ATC CAG AGC TTC Wnt2bAAG ATG GTG CCA ACT TCA CCG CTG CCT TCT TGG GGG CTT TGC Wnt3GAG AGC CTC CCC GTC CAC AGCTG CCA GGA GTG TAT TCG CATC Wnt3aCAG GAA CTA CGT GGA GAT CATG CCA TCC CAC CAA ACT CGA TGTC Wnt4GCT CTG ACA ACA TCG CCT ACCTT CTC TCC CGC ACA TCC Wnt5aGAC CTG GTC TAC ATC GAC CCC GCA GCA CCA GTG GAA CTT GCA Wnt5bTGA AGG AGA AGT ACG ACA GCCTC TTG AAC TGG TTG TAG CC Wnt6TTA TGG ACC CTA CCA GCA TATG TCC TGT TGC AGG ATG Wnt7aGCC GTT CAC GTG GAG CCT GTG CGT GCAGC ATC CTG CCA GGG AGC CCG CAG CT Wnt7bGAT TCG GCC GCT GGA ACT GCTC TGG CCC ACC TCG CGG AAC TTAG Wnt8aCTG GTC AGT GAA CAA TTT CCGTA GCA CTT CTC AGC CTG TT Wnt8bGTC TTT TCA CCT GTG TCC TCAGG CTG CAG TTT CTA GTC AG Wnt10aCTG TTC TTC CTA CTG CTG CTACA CAC ACC TCC ATC TGC Wnt10bGCA CCA CAG CGC CAT CCT CAAG GGG GTC TCG CTC ACA GAA GTC AGG A Wnt11CAC TGA ACC AGA CGC AAC ACCCT CTC TCC AGG TCA AGC AAA Wnt14ACA AGT ATG AGA CGG CAC TCAGA AGC TAG GCG AGT CAT C Wnt15TGA AAC TGC GCT ATG ACT CGTG AGT CCT CCA TGT ACA CC Wnt16GAG AGA TGG AAC TGC ATG ATGAT GGG GAA ATC TAG GAA CT Axin2TTG AAT GAA GAA GAG GAG TGGA TCG GGA AAT GAG GTA GAG ACA Ccnd1GTC CAT GCG GAA GAT CGT CGTCT CCT TCA TCT TAG AGG CCACG C-MycCAC AGC AAA CCT CCT CAC AGCGC CTC TTG ACA TTC TCC TC Angpt1GCT GAA CGG TCA CAC AGA GACTT TCC CCC TCA AAG AAA GC Angpt2TTA TCA CAG CAC CAG CAA GCTTC GCG AGA ACA AAT GTG AG VEGFaAAG GAG GAG GGC AGA ATC ATATC TGC ATG GTG ATG TTG GA VEGFr2AGC GAT GGC CTC TTC TGT AAACA CGA CTC CAT GTT GGT CA Flt1TGT CAA TGT GAA ACC CCA GAGTC ACA CCT TGC TCC GGA AT DSCR1GAG GAC GCA TTC CAA ATC ATAGT CCC AAA TGT CCT TGT GC TFTAC TTG GCA CGG GTC TTC TCTGT CCG AGG TTT GTC TCC A Ets1GGA GCA GCC AGT CAT CTT TCGGT CCC GCA CAT AGT CCT T PKCαCGA CTG GGA AAA ACT GGA GAACT GGG GGT TGA CAT ACG AG PKCδATT GCC GAC TTT GGG ATG TTGA AGA AGG GGT GGA TTT TG PKCεAAG CCA CCC TTC AAA CCA CGGC ATC AGG TCT TCA CCA AA PKCηTCC CAC ACA AGT TCA GCA TCCCC AAT CCC ATT TCC TTC TT MMP1GAT TCG GGG AGA AGT GAT GTT CGG GTA GAA GGG ATT TGT G Β-actinTCC CTG GAG AAG AGC TAC GAAGC ACT GTG TTG GCG TAC AG 1 3 Angiogenesis was studied 72 h after transfection using an in situ cell death Results detection kit (Roche) as described by the manufacturer on 4% PFA fixated cells. Fzd5 siRNA induces a specific knockdown of endothelial Fzd5 Wnt5a adenovirus preparation, transduction, and stimulation The function of Fzd5 was studied in vitro using siRNA- mediated silencing in HUVECs, which were shown to Recombinant adenoviruses were produced using the Gate- express all Fzd receptors other than Fzd10 (Supplemen- way pAd/CMV/V5DEST vector and ViraPowerTM Ade- tal Fig. 1A), and Wnt2b, 3, 4, 5a, and 11 (Supplemental noviral Expression System (Invitrogen), according to the Fig. 1B). Both qPCR and Western blot analysis confirmed manufacturer’s instructions. Briefly, the Wnt5a expression a significant loss of Fzd5 expression in cells treated with an cassette was cloned from the pENTR™ 221 Wnt5a entry siRNA pool specific for Fzd5, compared to untreated control vector (Invitrogen) into pAd/CMV/V5-DEST expression cells and cells treated with a pool of non-targeting siRNA, vector (Invitrogen) via the LR-reaction II (invitrogen). After referred to as siSHAM (Supplemental Fig. 1C,D). Although verification by DNA sequencing, the pAd/CMV plasmids Fzd receptors share highly similar domains, knockdown of were linearized by Pac1 restriction and subsequently trans- Fzd5 was specific. None of the other Fzd receptors were fected with Lipofectamine 2000 (Invitrogen) in HEK293A differentially expressed after treatment with Fzd5 siRNA, cells. Infected cells were harvested by the time 80% of the other than Fzd5 (Supplemental Fig. 1C). cells detached from plates followed by isolation of viral particles from crude viral lysate. HeLa cells were used to Wnt5a signals via endothelial Fzd5 produce Wnt5a (or dsRED, referred to as adSHAM) by transduction with a calculated 5 viral particles per cell. Previous studies listed Wnt5a and Secreted Frizzled- Forty-eight hours post-transduction, HeLa cells were cul- Related Protein 2 (SFRP2) as most likely candidates to tured for 24 h on EBM2, which eventually was used to stim- activate Fzd5-mediated signaling in ECs [13–15]. In con- ulate serum-starved endothelium for 3 h. trast to SFRP2 [16], Wnt5a is endogenously expressed by HUVECs (Supplemental Fig. 1B). To address the potential Statistical analysis signal capacities of this endogenously expressed Wnt5a as ligand for Fzd5, HeLa cells were transduced with an adeno- For each experiment, N represents the number of independ- viral overexpression plasmid for Wnt5a to produce cultured ent replicates. Statistical analysis was performed by Graph- medium containing high levels of this Wnt ligand. HeLa Pad Prism using one-way ANOVA followed by post hoc cells were selected for this purpose over HUVECs as these Tukey’s test, unless stated otherwise. Results are expressed cells were shown to have a more refined machinery to pro- as mean ± SEM. Significance was assigned when P < 0.05 duce and secrete functional Wnt5a than HUVECs, as illus- (two-tailed). trated by enhanced mRNA expression of Wntless (WLS) and Porcupine (PORCN) (data not shown). Transduction with this overexpression vector (adWnt5a) led to a significant Fig. 1 Wnt5a induced Fzd5-mediated Dvl activation in HUVECs. a stimulation with cultured medium (CM) from HeLa cells overex- Representative Western  blot of adenoviral-based Wnt5a overexpres- pressing dsRED (adSHAM) or Wnt5a, 72 h post siRNA transfection sion in HeLa cells, 72  h post-transduction. N = 4. b Representative in HUVECs. N = 6 Western blot of Dvl and phosphorylated Dvl in HUVECs after 3  h 1 3 Angiogenesis 1 3 Angiogenesis ◂Fig. 2 Fzd5 expression is crucial for vascular formation in  vitro. cycle progression was analyzed in a cell cycle assay in a Representative fluorescent microscope images of GFP-labeled which total DNA was stained with PI, followed by flow HUVECs (green) in co-culture with dsRED-labeled pericytes (red) cytometry. A strong increase of cells in the G /G phase 0 1 in a 3D collagen matrix during vascular formation. Shown are the of the cell cycle was observed after knockdown of Fzd5, results at day 2 and 5 of non-transfected control, siSHAM, and siFzd5 conditions. Scale bar in the left columns represents 1  mm. Scale indicative of a cell cycle arrest (Fig. 3d, e). For apoptosis bar in the right columns represents 350  µm. b Bar graphs show the analysis, a terminal deoxynucleotidyl transferase dUTP quantified results of the co-culture assay. Shown are the total tubule nick end labeling (TUNEL)-based detection staining was length, and the number of endothelial junctions and tubules relative to used. Although seeded in similar densities, Fzd5 knock- the control conditions, both after 2 and 5 days. N = 4, *P < 0.05 com- pared to control and siSHAM condition down led to a significant reduction of nuclei per image field. However, the relative number of TUNEL positive nuclei in the Fzd5 knockdown condition was similar when upregulation of Wnt5a compared to dsRED control trans- compared to control and siSHAM condition, showing that duced cells (adSHAM) (Fig. 1a). To assess whether Fzd5 the reduction of ECs in the Fzd5 knockdown condition is was involved in transducing the signal of Wnt5a, cultured not related to increased apoptosis (Fig. 3f, g). medium from transduced HeLa cells was applied to serum- starved HUVECs after which Dvl activation was monitored. Western blot analysis showed that Wnt5a strongly induced Loss of Fzd5 does not interfere with endogenous Dvl phosphorylation in untreated or non-targeting siRNA- canonical Wnt signaling treated HUVECs, however, this effect was blocked in the absence of Fzd5 (Fig.  1b), confirming the importance of To further dissect the molecular mechanism of endothelial endothelial Fzd5 in transducing Wnt5a signaling. Fzd5 signaling in angiogenesis, known Fzd/Wnt signaling pathways were studied. Downstream Fzd signaling occurs Fzd5 expression is essential for endothelial via the canonical Wnt signaling pathway, also known as the proliferation, migration, and tubule formation Wnt/β-catenin pathway, or by the less well described non- canonical Wnt signaling pathways. Activation of canonical The angiogenic capacities of these Fzd5-silenced HUVECs Wnt signaling is characterized by an accumulation of cyto- were evaluated in a well-validated in vitro 3D angiogenesis plasmic β-catenin, eventually resulting in nuclear transloca- assay developed for studying formation of micro-capillary tion and subsequent expression of β-catenin-dependent tar- structures [12]. In this assay, HUVECs with GFP marker get genes. To evaluate the effect of Fzd5 knockdown on the expression and dsRED-labeled pericytes directly interact canonical Wnt signaling pathway, total levels of β-catenin, in a collagen type I matrix environment, resulting in EC as well as phospho-β-catenin (ser33/37/thr41) and non- sprouting, tubule formation, and neovessel stabilization as phospho-β-catenin (active) were examined 24, 48 and 72 h a result of perivascular recruitment of pericytes. At day 5 post-transfection by Western blot. Ser33/37/thr41 phospho- post-seeding, well-defined, micro-capillaries with pericyte rylation is induced by GSK3β and primes β-catenin for sub- coverage can be observed. Imaging and quantification of the sequent degradation, and could be indicative for a reduced vascular structures were conducted at days 2 and 5. Endothe- activity of canonical Wnt signaling. Total β-catenin, as well lial knockdown of Fzd5 strongly impaired endothelial tubule as non-phospho-β-catenin (active) levels were unaffected by formation (Fig.  2a). Quantification revealed a significant Fzd5 silencing, and non-phospho-β-catenin (ser33/37/thr41) reduction in the total tubule length, the number of endothe- was observed in all conditions (Fig. 4a, b), even though the lial junctions, and the number of endothelial tubules, both antibody was capable of detecting GSK3β-induced β-catenin after 2 and 5 days (Fig. 2b). phosphorylation (Fig. 4c). Furthermore, expression levels of To get a better insight in the causative factor for this previously described endothelial target genes of β-catenin poor vascular phenotype, the migration and prolifera- were studied using qPCR, but no differences were observed tion capacities of Fzd5-silenced ECs were studied. A in the expression of Axin2, Ccnd1, and C-myc after knock- plug-stopper-based migration assay was performed to down of Fzd5 (Fig. 4d). An immunofluorescent staining, analyze the effects of Fzd5 knockdown on endothelial validated to detect cellular distribution of β-catenin (Supple- mobility. Knockdown of Fzd5 significantly inhibited the mental Fig. 3B), was also performed on transfected ECs, as migration of ECs towards the open cell-devoid area com- stable total levels of β-catenin found by Western blot did not pared to untreated and non-targeting siRNA-treated ECs deviate between cytoplasmic or nuclear localized β-catenin. (Fig. 3a, b). In addition, knockdown of Fzd5 significantly In line with the other experiments focusing on β-catenin- reduced cell numbers compared to control and siSHAM mediated signaling, no differences in β-catenin localization condition (Fig. 3c). To clarify whether this was a result were observed after knockdown of Fzd5, both in confluent of impaired cell proliferation or increased apoptosis, cell and sub-confluent cells (Fig.  4e, f, respectively). 1 3 Angiogenesis Fig. 3 Endothelial knockdown of Fzd5 significantly inhibited EC condition (two-way ANOVA followed by Bonferroni post hoc test). migration and proliferation, but had no effect on apoptosis. a Rep - d Representative histogram of flow cytometric analysis of PI-based resentative fluorescent microscope images of Calcein-AM-labeled DNA staining showing the distribution of cells over the cell cycle HUVECs (green) in a plug-stopper-based migration assay. Shown are in the different groups at 48  h post-transfection. e Quantified results the results of 16 h of migration of non-transfected control, siSHAM, of cell cycle analysis. Percentage of cells in G /G phase is shown. 0 1 and siFzd5 conditions. Scale bar represents 500 µm. Open migration N = 3, *P < 0.05 compared to control and siSHAM condition. f Quan- areas produced by the plug-stopper before initiation of the assay are tified results of TUNEL staining. Percentage TUNEL-positive cells indicated by dotted lines. b Bar graph shows the quantified results of total number of cell is shown, 72  h post-transfection of control, of migration assay. Shown are the percentages of surface area within siSHAM, and siFzd5 conditions. N = 3, no significance. g Representa- the dotted circle covered by HUVECs after 16 h of migration. N = 4, tive fluorescent microscope images of DAPI-based nuclei staining in *P < 0.05 compared to control and siSHAM condition. c ECs expan- HUVECs (blue, upper row) and TUNEL staining of the same cells sion at 24, 48, and 72 h post seeding in similar densities, as quantified (red, lower row). Positive control was treated with DNAse solution. by flow cytometry. N = 3, *P < 0.05 compared to control and siSHAM Scale bar represents 200 µm cells, yet was statistically equal to non-targeting siRNA- Fzd5 knockdown induces the expression of several treated HUVECs (Supplemental Fig. 2). Interestingly, vas- (anti‑) angiogenic factors cular endothelial growth Factor A (VEGFa) decoy recep- tor Flt1, and the vascular destabilizing factor Angpt2 were To further elucidate the anti-angiogenic phenotype observed significantly upregulated at both mRNA and protein level after Fzd5 knockdown, expression levels of several impor- in HUVECs treated with Fzd5 siRNA when compared to tant regulators of angiogenesis were analyzed. In contrast to untreated or non-targeting siRNA-treated HUVECs (Fig. 5a, what was previously reported [17], our findings in HUVECs c). Expression levels of VEGF receptor 2, VEGFa, as well as indicate that expression of tissue factor (TF) is not positively Angpt1 remained unaffected in the absence of Fzd5 (Supple- regulated by Fzd5 signaling, as Fzd5 knockdown did not mental Fig. 2). In line with previous findings of Lobov et al., attenuate TF expression. In fact, TF was slightly upregulated combined addition of Flt1 and Angpt2 in the 3D co-culture in Fzd5-silenced HUVECs compared to untreated control 1 3 Angiogenesis Fig. 4 Fzd5 knockdown did not affect the canonical Wnt signaling Calyculin A (50 nM) with and without a 30 min pretreatment of the pathway in ECs. a Representative Western blot result of total levels GSK3β inhibitor LiCl (20  mM). d QPCR analysis of the mRNA of β-catenin, non-phospho-β-catenin, phospho-β-catenin (ser33/37/ expression levels of β-catenin target genes Axin2, Cyclin D1 (Ccnd1), thr41), and β-actin loading control, at different time points post-trans- and C-myc in different conditions 72  h post-transfection. N = 4, no fection. b Quantified results of β-catenin Western blot. Shown are significance. e Immunofluorescent staining β-catenin (green), F-actin β-catenin levels relative to β-actin loading control. N = 3, no signifi- (red), and DAPI (blue) in confluent and sub-confluent f HUVECs cance. c Western blot result of total levels of β-catenin and phospho- after knockdown of Fzd5. N = 3 β-catenin in response to treatment with the phosphatase inhibitor 2+ system completely attenuated endothelial tubule formation Wnt/Ca and PCP pathways were studied for their potential (Fig. 5d, e) [9]. role in the upregulation of Angpt2 and Flt1. Activation of the 2+ Knockdown of a Fzd receptor can not only attenuate sig- Wnt/Ca pathway could induce Flt1 and Angpt2 transcrip- 2+ nal transduction, but due to impaired inhibitory crosstalk tion, as stimulation of the Wnt/Ca pathway leads to free 2+ between the individual pathways, or via alternative receptor Ca -induced activation of Calcineurin, which in turn could binding by the Wnt ligand can also have a stimulatory effect promote NFAT-mediated transcription by dephosphorylating [18, 19]. Since Fzd5 knockdown had no effect on the canoni- NFAT [6]. The mRNA expression level of Down Syndrome cal Wnt signaling pathway, the described non-canonical Critical Region 1 (DSCR1) was evaluated to assess the 1 3 Angiogenesis Fig. 5 Fzd5 knockdown led to increased expression of vascular (green) in co-culture with dsRED-labeled pericytes (red) in a 3D col- regression-associated genes Flt1 and Angpt2. a QPCR results of lagen matrix during vascular formation. Shown are the results at day expression levels of Angpt2 and Flt1 in different conditions 72  h 5 of an untreated control, and after stimulation with PBS, Angpt2 post-transfection. N = 11, *P < 0.05 compared to control and siSHAM (1000  ng/ml), Flt1 (1000  ng/ml), and Angpt2 + Flt1 (1000  ng/ml condition. b Representative Western blot results of Angpt2 expres- both). Scale bar in the upper row represent 1 mm, in the bottom row sion levels in the different conditions 72 h post-transfection. N = 3. c 350  µm. e Bar graphs show the quantified results of the co-culture Enzyme-linked immunosorbent assay-based quantification of secreted assay. Shown are the total tubule length, and the number of endothe- Flt1 levels in cultured endothelial medium 72  h post-transfection. lial junctions and tubules after 5  days. N = 4, *P < 0.05 compared to N = 8, *P < 0.05 compared to control and siSHAM condition. d Rep- control and siSHAM condition resentative fluorescent microscope images of GFP-labeled HUVECs potential link between Fzd5 knockdown and NFAT activa- (A23187)-induced transcription of DSCR1 as a result of free 2+ tion, as DSCR1 is a profound target gene of NFAT, involved Ca -mediated NFAT activation in ECs (Fig.  6a). In line in a feedback loop to fine-tune NFAT-mediated transcrip- with the absence of DSCR1 upregulation in the Fzd5 knock- tion [20, 21]. However, no correlation between endothelial down condition, the upregulation of Flt1 and Angpt2 could knockdown of Fzd5 and DSCR1 upregulation was observed not be linked to an increase of NFAT-mediated transcription (Fig. 6a). The involvement of NFAT-mediated transcription in the Fzd5 knockdown condition, as CsA stimulation failed was also evaluated by pharmacological inhibition of the to reduce Angpt2 and Flt1 upregulation in Fzd5-silenced 2+ Wnt/Ca signaling cascade using the Calcineurin inhibitor cells (Fig. 6b). 2+ Cyclosporine A (CsA). The effectiveness of CsA (1 µM) Besides activation of the Wnt/Ca pathway, the PCP was confirmed by its ability to inhibit calcium ionophore pathway could also stimulate the expression of Flt1 and 1 3 Angiogenesis Fig. 6 Fzd5 knockdown led to increased expression of vascular tive Western blot of total JNK, phospho-JNK, and β-actin levels at regression-associated genes Flt1 and Angpt2, independent of the different time points post-transfection. d Quantified results of JNK 2+ non-canonical Wnt/Ca and PCP pathways. a QPCR results of and phospho-JNK Western blot. Shown are individual (phospho) expression levels of NFAT target gene Dscr1 in the different condi- JNK isoform (p46 and p54) levels relative to β-actin loading control. tions 72  h post-transfection and in response to ionophore A23187 N = 6, *P < 0.05 compared to control and siSHAM condition within 2+ (10 µM)-induced Ca flux with and without NFAT inhibitor Cyclo- one time comparison (24, 48 or 72  h). e Western blot of total JNK, sporin A (CsA) (1  µM). N = 5, *P < 0.05 compared to control and phospho-JNK, and β-actin levels in response to different concentra- siSHAM condition, and DMSO-treated and CsA + A23187-treated tions of JNK inhibitor SP600125 after 1 h. f QPCR analysis showing ECs, respectively. b Angpt2 and Flt1 mRNA expression levels in the effect of SP600125, supplemented 48 h post-transfection, on Flt1 HUVECs in response to CsA, supplemented 48  h post-transfection. and Angpt2 mRNA levels in the different conditions. N = 4, *P < 0.05 N = 4, *P < 0.05 compared to control and siSHAM condition (two- compared to control and siSHAM condition, P < 0.05 as indicated in way ANOVA followed by Bonferroni post hoc test). c Representa- graph (two-way ANOVA followed by Bonferroni post hoc test) Angpt2 via activation of the Wnt/PCP signaling cascade slightly increased both 48 and 72 h post-transfection (Fig. 6c, linked to downstream JNK-induced transcriptional activa- d). JNK-mediated phosphorylation of c-JUN, however, was tion of c-JUN [22, 23]. Activation of JNK/c-JUN-mediated not observed (Supplemental Fig. 4A, B). Since JNK is a transcription involves phosphorylation of JNK, which was kinase with a broad spectrum of downstream substrates [24], 1 3 Angiogenesis the JNK inhibitor SP600125 was used to block activation The effectiveness of SP600125 (20 µM) was confirmed by of JNK to define whether the enhanced phosphorylation of its ability to inhibit JNK phosphorylation in ECs (Fig. 6e). JNK played a role in the upregulation of Flt1 and Angpt2. Treatment of HUVECs with SP600125 did not diminish 1 3 Angiogenesis ◂Fig. 7 Fzd5 knockdown-induced upregulation of Angpt2 and (Supplemental Fig. 5C). Involvement of Ets1 in transcrip- Flt1 expression is mediated via enhanced PKC and Ets1 signal- tional regulation of Angpt2 and Flt1 was evaluated in the ing. a QPCR results showing expression levels of Angpt2 and Flt1 Fzd5 knockdown condition using a double knockdown in HUVECs after knockdown of Fzd5 alone, in combination with of both Fzd5 and Ets1. Knockdown of Ets1 alone had no knockdown of different PKC isoforms, and in combination with knockdown of all novel PKC isoforms (PKCδ,ε,η), 48  h post-trans- effect on the expression of Flt1 and Angpt2 compared to fection. N = 4, *P < 0.05 compared to control and siSHAM condi- control and siSHAM condition, indicating no active tran- tion, P < 0.05 as indicated in graph. b QPCR results of Angpt2 and scription regulation of these two genes by Ets1 in control Flt1 expression in HUVECs, 72  h post-transfection, with and with- conditions. However, knockdown of Ets1 in Fzd5-silenced out knockdown of transcription factor Ets1, a downstream target of PKC. N = 4, *P < 0.05 compared to control and siSHAM condition, HUVECs fully inhibited upregulation of Angpt2 and par- P < 0.05 as indicated in graph. c Representative fluorescent micro- tially inhibited the upregulation of Flt1 when compared to scope images of GFP-labeled HUVECs (green) in co-culture with Fzd5-silenced controls (Fig. 7b). The involvement of Ets1- dsRED-labeled pericytes (red) in a 3D collagen matrix during vas- induced transcription was further substantiated by a similar cular formation. Shown are the results at day 5 of non-transfected control, siSHAM, siFzd5, siEts1 and the combined knockdown of Ets1-dependent upregulation of Matrix metalloproteinase 1 Fzd5 and Ets1. Scale bar in the upper row represents 1  mm, in the (MMP1), a verified endothelial target gene of Ets1 (Supple - bottom row 350 µm. d Bar graphs show the quantified results of the mental Fig. 6A, B) [34]. To evaluate if the anti-angiogenic co-culture assay. Shown are the total tubule length, and the number phenotype of Fzd5 silencing observed in the 3D angiogen- of endothelial junctions and tubules after 5 days. N = 6, *P < 0.05 compared to control and siSHAM condition, P < 0.05 as indicated in esis co-culture assay was mediated via this pathway, Ets1 graph was silenced in GFP-labeled HUVECs. Analysis of the 3D co-culture results demonstrated that inhibition of Ets1 in the Fzd5 silencing-induced upregulation of Flt1 and Angpt2 Fzd5 knockdown condition partly rescued the Fzd5 knock- (Fig. 6f). In contrast, SP600125 treatment rather induced a down-mediated reduction of endothelial tubule formation general upregulation of Angpt2, indicating that activation (Fig. 7c, d). of JNK was not causally related to the Fzd5 knockdown- mediated upregulation of both genes. Discussion Angpt2 and Flt1 upregulation is mediated via PKC and Ets1 The main findings of the current study are (1) endothelial Fzd5 expression is essential for vascular formation, as shown Previously, it was demonstrated that Wnt signal transduction in a 3D co-culture assay. (2) Fzd5 silencing inhibits EC pro- also involves PKC [25–27]. PKCs are part of a kinase fam- liferation and migration. (3) Endothelial loss of Fzd5 expres- ily with a diverse range of potential downstream targets. To sion does not interfere with endogenous canonical Wnt sign- verify whether Fzd5 knockdown-induced upregulation of aling. (4) Fzd5 knockdown leads to increased expression Flt1 and Angpt2 depended on activation of PKC, HUVECs of vascular regression-associated factors Flt1 and Angpt2, 2+ were treated with the PKC inhibitor Staurosporine in the independent of both the non-canonical Wnt/Ca -mediated concentration range of 5–20 nM, as not all different PKC activation of NFAT and PCP-mediated activation JNK. (5) family members are equally inhibited at similar concentra- Inhibition of nPKC signaling, as well as knockdown of the tions. Interestingly, both Angpt2 and Flt1 overexpression PKC target Ets1 suppressed the upregulation of Flt1 and induced by Fzd5 knockdown were dose-dependently reduced Angpt2 in the absence of Fzd5. The Ets1 knockdown inter- by PKC inhibition compared to control and siSHAM condi- vention also partially rescued the Fzd5 knockdown-induced tion (Supplemental Fig. 5A). Since HUVECs express mul- inhibitory effect on new vessel formation. tiple PKC isoforms [28, 29], PKC expression was knocked Previously, it was reported that Fzd5 is indispensable for down by siRNA to interrogate which isoform mediated the murine embryogenesis [5]. Fzd5 knockout embryos died in observed upregulation of Angpt2 and Flt1. Individual PKC utero from severe defects in yolk sac and placenta vasculari- isoform knockdown only had a minor effect on the Fzd5 zation. Using trophoblast-specific Fzd5 knockout mice, Lu knockdown-induced overexpression of the anti-angiogenic et al. reported that the observed phenotype in the Fzd5 full factors, whereas combined knockdown of the novel PKCs knockout placenta was partly initiated by a defect in chori- (nPKCs) completely attenuated the upregulation of Angpt2 onic branching morphogenesis [35]. As defective branch- and Flt1 (Fig. 7a, Supplemental Fig. 5B). ing morphogenesis of the chorion of these mice resulted in PKC signaling can induce elevated synthesis of the a smaller placental labyrinth layer compared to wild-type transcription factor Protein C-ets1 (Ets1) [30, 31], which littermates, it remained difficult to distinguish whether the has binding sites in the promoter regions of both Angpt2 placental defects observed in the Fzd5 full knockout mice and Flt1 [32, 33]. Ets1 was significantly upregulated in were indeed vascular related, or the outcome of propor- the absence of Fzd5, which was orchestrated by PKC tional growth limitations resulting from the reduced villous 1 3 Angiogenesis volume. In our study, we demonstrated that endothelial DSCR1 is also a verified target of the transcription factor knockdown of Fzd5 in vitro leads to a severe reduction in NFAT [20, 21], yet our data showed that the expression level vascular tubule formation in a 3D co-culture model, thereby of DSCR1 remained stable after knockdown of Fzd5. More providing evidence for the direct role of Fzd5 in new vessel important, our experiments demonstrated that inhibition of growth. NFAT activation with CsA after endothelial knockdown of The most detailed described Fzd/Wnt signaling cascade Fzd5 did not inhibit the upregulation of Angpt2 and Flt1, is the canonical or β-catenin-dependent pathway. Without suggesting that the enhanced transcription of these anti- stimulation of the canonical pathway, β-catenin is degraded angiogenic factors was not mediated by enhanced activity by a destruction complex consisting of Axin, Glycogen Syn- of NFAT. Alternatively, stimulation of the Fzd/Wnt/PCP thase Kinase 3ß, Adenomatous Polyposis Coli, and Casein pathway can also induce the transcription of Flt1 and Angpt2 Kinase 1α. Upon binding of Wnt ligands to a Fzd receptor via GTPase-mediated activation of JNK, which eventually in the presence of the co-receptor Lrp5 or Lrp6, a conforma- activates c-JUN-based transcription [6]. Multiple studies tion change in Lrp extracts Axin away from the destruction provided evidence for transcriptional regulation of Flt1 and complex, leading to an increase in intracellular β-catenin Angpt2 either by c-JUN alone, or by the transcription com- levels. When translocated into the nucleus, β-catenin binds plex AP-1 involving c-JUN [22, 23]. Our data indicated that to the TCF/Lef complex and promotes the expression of Fzd5 knockdown led to an increase in JNK phosphorylation, β-catenin target genes [6–8]. Knockdown of a Fzd receptor but no increase in c-JUN phosphorylation was observed. In could both have an inhibiting effect on this pathway, due addition, inhibition of JNK activity with SP600125 ruled to a reduction in receptors capable of transducing a signal out the involvement of the PCP-JNK signal transduction for downstream signaling cascade activation, and an acti- axis as causal factor for the enhanced expression of vascular vating effect, either due to impaired inhibitory crosstalk regression-associated factors Angpt2 and Flt1 in ECs with between the individual pathways or via alternative receptor Fzd5 knockdown, as upregulation of these factors remained binding by the Wnt ligand [18, 19]. Involvement of Fzd5 evident. In future studies, however, it remains of interest to in this canonical pathway appears to be tissue dependent. further dissect the relevance of this altered JNK signaling Steinhart et al. recently demonstrated that canonical Wnt in the absence of Fzd5. signaling via Fzd5 was involved in pancreatic tumor growth Multiple reports have previously suggested a role for PKC and Caricasole et al. reported enhanced β-catenin-mediated involvement in Fzd/Wnt signaling [25–27]. Staurosporine, signaling upon Wnt7a interaction with both Fzd5 and Lrp6 as well as siRNA-mediated knockdown of nPKCs inhib- in the rat pheochromocytoma cell line PC12 [36, 37]. In ited the upregulation of Angpt2 and Flt1 in HUVECs with the mouse optic vesicle, however, no evidence suggests that suppressed expression of Fzd5, indicating the involvement Fzd5 activates or suppresses canonical Wnt signaling [38, of PKC signaling in the transcriptional regulation of these 39]. Our analysis of endogenous canonical Fzd/Wnt sign- genes in Fzd5-silenced ECs. The promoter regions of both aling suggests that Fzd5 is not involved in Wnt β-catenin Angpt2 and Flt1 contain binding sites of the transcription signaling in ECs. factor Ets1 [32, 33], which was shown by our data to be PKC In contrast to the β-catenin target genes, expression lev- dependently upregulated in the absence of Fzd5. Our results els of Angpt2 and Flt1 were significantly upregulated in demonstrate the involvement of enhanced Ets1-mediated HUVECs with suppressed Fzd5 expression. Angpt2 by itself transcription of these two genes in Fzd5-silenced ECs, as is known to have a positive effect on neovessel formation, as Ets1 knockdown resulted in a marked repression of Angpt2 it is involved in pericyte detachment and destabilization of and Flt1 expression levels. Another validated endothelial the endothelium to potentiate the actions of pro-angiogenic target of PKC/Ets1-mediated transcription, MMP1, which factors [40, 41]. However, in the absence of VEGFa, or in like Angpt2 and Flt1 was previously shown to be involved the presence of an increased expression of Flt1, a decoy in vascular regression [34], was also upregulated via Ets1 receptor for VEGFa, Angpt2 is known to induce vascular in the absence of Fzd5. The involvement of Ets1 was fur- regression [9–11]. Both Angpt2 and Flt1 are potential down- ther validated using the 3D co-culture model, in which Ets1 stream target genes of the non-canonical Fzd/Wnt signal- knockdown in Fzd5-silenced ECs partially rescued the 2+ ing pathways. Upon stimulation of the Fzd/Wnt/Ca path- inhibitory effect on new vessel formation that was observed way, activation of phospholipase C leads to cleavage of the in Fzd5-silenced conditions. These results indicate a repress- membrane component PIP2 into DAG and IP3. When IP3 ing function on PKC/Ets1 signaling by Fzd5 in ECs, leading 2+ binds to its receptor on the endoplasmic reticulum, Ca is to reduced expression of vascular regression-associated fac- released in the cytosol, activating the transcription factor tors Angpt2 and Flt1. NFAT via Calcineurin [6]. In recent studies, Flt1 and Angpt2 In this study, the effect of Fzd5 knockdown on the differ - were shown to be transcriptional targets of NFAT [42, 43]. ent Fzd/Wnt signaling routes was studied without the addi- Like Angpt2 and Flt1, the endogenous NFAT inhibitor tion of exogenous Wnt factors. HUVECs secrete Wnt factors 1 3 Angiogenesis should also aim to identify the unknown alternative Wnt5a receptor. The aim of this study was to explore the involvement of Fzd5 in vascular and perivascular biology, which might eventually serve as a foundation for future therapeutic strategies, e.g., in modulating tumor vasculature. A recent genome-wide CRISPR-Cas9 study demonstrated that Fzd5 is a potential druggable target in specific subtypes of pan- creatic tumors [36]. Signaling via Fzd5 in these tumor cells was shown to be crucial in β-catenin-mediated proliferation and treatment of these pancreatic adenocarcinoma cells with Fzd5 antibodies led to inhibited cell growth, both in vitro and in xenograft models in vivo. Although these pancreatic adenocarcinoma tumors are not excessively vascularized, they were previously shown to depend on angiogenesis for growth [45, 46]. Our data demonstrate the importance of Fzd5 in ECs during angiogenesis and might imply that tar- geting the Fzd5 in these types of tumors not only affects the pancreatic adenocarcinoma cells, but could in addi- tion potentially result in beneficial suppression of tumor vascularization. In conclusion, the current study provides evidence for an important role of endothelial Fzd5 in angiogenesis, thereby Fig. 8 Schematic representation of the proposed model of signal- providing novel insights in the molecular mechanism causal ing via Fzd5 in ECs. Our data provide evidence for a new proposed to the poor angiogenic phenotype in the absence of this model of signaling in ECs in the absence of Fzd5. Knockdown of this receptor. receptor provokes its ligand Wnt5a to signal via an alternative recep- tor, thereby triggering the activation of nPKC/Ets1-mediated tran- scription of vascular regression-associated factors, among which Flt1 Acknowledgements The authors would like to thank Dr. O. G. de and Angpt2 Jong for donating the lentiviral GFP and dsRED constructs, and L. A. Blonden and E. H. van de Kamp for their technical support. themselves, among which the typical canonical factor Wnt3 Funding This work was supported by Netherlands Foundation for and non-canonical factor Wnt5a. Knockdown of endothelial Cardiovascular Excellence [to C.C.], Netherlands Organization for Scientific Research Vidi Grant [No. 91714302 to C.C.], the Erasmus Fzd5 led to functional defects, as well as differential expres- MC fellowship Grant [to C.C.], the Regenerative Medicine Fellow- sion of important genes in the angiogenic process, indicat- ship grant of the University Medical Center Utrecht [to C.C.], and ing that lack of Fzd5 interferes with endogenous Fzd/Wnt the Netherlands Cardiovascular Research Initiative: An initiative with signaling. The nature of this endogenous signaling in the support of the Dutch Heart Foundation [CVON2014-11 RECONNECT to C.C., D.D., and M.V.]. absence of Fzd5 was shaped by the finding that combined knockdown of Fzd5 and endogenous Wnt5a significantly Compliance with ethical standards suppressed Angpt2 and Flt1 upregulation (Supplemental Fig. 7). It was previously demonstrated that Wnt factors Conflict of interest The authors declared that they have no conflict of induce signaling to a variety of Fzd and non-Fzd receptors, interest. and that binding selectivity is receptor context dependent [13, 44]. As suppression of endogenous Wnt5a signaling Open Access This article is distributed under the terms of the Crea- partially rescued the Fzd5 knockdown-induced upregula- tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion of Angpt2 and Flt1, our data suggest that endothelial tion, and reproduction in any medium, provided you give appropriate knockdown of Fzd5 provokes its ligand Wnt5a to signal via credit to the original author(s) and the source, provide a link to the an alternative receptor, thereby triggering the activation Creative Commons license, and indicate if changes were made. of the observed PKC/Ets1-mediated transcription (Fig. 8). 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Endothelial loss of Fzd5 stimulates PKC/Ets1-mediated transcription of Angpt2 and Flt1

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Biomedicine; Cancer Research; Biomedicine, general; Cell Biology; Cardiology; Ophthalmology; Oncology
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Abstract

Aims Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. Methods and results Using short interference RNA-mediated loss-of-function assays, the function and mechanism of sign- aling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neoves - sel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G /G cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in 0 1 enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of 2+ canonical Wnt signaling, non-canonical Wnt/Ca -mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. Conclusions The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2. Keywords Endothelial cells · Angiogenesis · Fzd5 · Wnt signaling Introduction New formation of blood vessels from pre-existing vessels, a process called angiogenesis, is a critical step in embryogen- esis and continues to play important roles throughout life in Electronic supplementary material The online version of this both health and disease [1]. It is a dynamic process that is article (https ://doi.org/10.1007/s1045 6-018-9625-6) contains tightly regulated by a diverse range of signal transduction supplementary material, which is available to authorized users. cascades, and imbalances in these pathways can be a causa- * Caroline Cheng tive or a progressive factor in many diseases [2]. K.L.Cheng-2@umcutrecht.nl Multiple studies suggest an important role for endothelial signal transduction via Frizzled (Fzd) receptors in angio- Experimental Cardiology, Department of Cardiology, genesis [3–5]. The Fzd receptors belong to a family of 10 Thoraxcenter, Erasmus University Medical Center, Rotterdam, The Netherlands transmembrane receptors (Fzd1–10), which can initiate Fzd/ Wnt canonical and non-canonical signaling upon binding Department of Nephrology and Hypertension, Division of Internal Medicine and Dermatology, University Medical with one of the 19 soluble Wnt ligands. Canonical Wnt sign- Center Utrecht, Utrecht, The Netherlands aling depends on Fzd receptor and LRP 5/6 co-activation, Department of Pediatric Surgery of the Erasmus initiating Disheveled (Dvl) to stabilize β-catenin, followed Medical Center, Sophia Children’s Hospital, Rotterdam, by β-catenin-mediated transcriptional regulation [6–8]. In The Netherlands Vol.:(0123456789) 1 3 Angiogenesis contrast, non-canonical Wnt signaling also involves Dvl, (supplemented with 100  U/ml penicillin/streptomycin; 2+ but proceeds via Wnt/Ca -mediated activation of nuclear Lonza, and 10% FCS; Lonza), respectively, in 5% C O factor of activated T-cells (NFAT) or Wnt/planar cell polar- at 37  °C. The experiments were performed with cells ity (PCP)-mediated activation of c-JUN N-terminal Kinase at passage 3–5. Lentivirus green fluorescent protein (JNK) [6]. A potential link between Fzd5 and angiogenesis (GFP)-transduced HUVECs and lentivirus discosoma was previously demonstrated in Fzd5 full knockout mice sp. red fluorescent protein (dsRED)-transduced pericytes [5]. Fzd5 silencing induced in utero death at approximately were used at passages 5–7. HUVECs and GFP-labeled E10.5, which was associated with vascular defects in the HUVECs were used from six different batches derived placenta and yolk sac. Furthermore, isolated ECs from from pooled donors. Pericytes and dsRED-labeled peri- Fzd5-dec fi ient mice showed a reduction in cell proliferation, cytes were used from eight different batches derived which is crucial for neovessel formation. These findings sug- from single donors. Fzd5, Ets1, and Wnt5a knockdown gest that Fzd5 can be an important regulator of angiogenesis. in HUVECs was achieved by cell transfection of a pool However, the exact type of endothelial Fzd5/Wnt signaling containing four targeting short interference RNA (siRNA) and the downstream molecular mechanism causal to the poor sequences, whereas PKC isoforms were knocked down vascular phenotype in the absence of this receptor requires with individual siRNA strands (Dharmacon), all in a further in-depth evaluation. final concentration of 100 nM. Control cells were either Here, we studied the angiogenic potential of Fzd5 and untreated or transfected with a pool of four non-targeting investigated the signaling pathways that are mediated by siRNA sequences (Dharmacon) in a final concentration of Fzd5/Wnt signaling in human ECs. Our findings indicate 100 nM. Target sequences are listed in Table 1. Inhibition that Wnt5a, which is endogenously expressed in ECs, binds of GSK3β, NFAT, JNK, and PKC activation was achieved and signals via Fzd5, but in the absence of this receptor with 20 µM LiCl (Sigma), 1 µM Cyclosporine A (CsA; triggers a poor angiogenic phenotype via an alternative Sigma), 20 µM SP600125 (Sigma), and 5, 10, and 20 nM signaling route. We demonstrated that Fzd5 is essential for staurosporine (CST), respectively. Phosphatase activity 2+ neovessel formation in vitro in a collagen matrix-based 3D was inhibited with 50 nM Calyculin A. Free Ca -induced co-culture of primary human vascular cells. Silencing of activation of NFAT-mediated transcription was achieved Fzd5 reduced EC proliferation as a result of G /G cell cycle with 10 µM A23187. In experiments involving a serum 0 1 arrest and decreased cell migration capacity. Furthermore, starvation step, the cells were cultured for 24 h in EBM2. Fzd5 knockdown resulted in enhanced expression of the factors Angiopoietin 2 (Angpt2) and Fms-Related Tyrosine Kinase 1 (Flt1), which are mainly known for their destabiliz- Table 1 siRNA sequences used in cell culture ing effects on the vasculature [ 9–11]. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced Pro- Target gene Target sequence tein Kinase C (PKC) signaling, without the involvement of Non-targetingUGG UUU ACA UGU CGA CUA A 2+ canonical Wnt signaling, non-canonical Wnt/Ca -mediated UGG UUU ACA UGU UGU GUG A activation of NFAT, and non-canonical Wnt/PCP-mediated UGG UUU ACA UGU UUU CUG A activation of JNK. Further downstream, PKC-induced tran- UGG UUU ACA UGU UUU CCU A scription of Angpt2 and Flt1 involved the transcription factor Fzd5GCA UUG UGG UGG CCU GCU A Protein C-Ets-1 (Ets1), as knockdown of both Fzd5 and Ets1 GCA CAU GCC CAA CCA GUU C resulted in a marked repression of Angpt2 and Flt1 expres- AAA UCA CGG UGC CCA UGU G sion levels. In addition, silencing of Ets1 partially restored GAU CCG CAU CGG CAU CUU C the impaired endothelial tubule formation capacity of Fzd5- Ets1AUA GAG AGC UAC GAU AGU U silenced ECs. GAA AUG AUG UCU CAA GCA U GUG AAA CCA UAU CAA GUU A CAG AAU GAC UAC UUU GCU A Methods Wnt5aGCC AAG GGC UCC UAC GAG A GUU CAG AUG UCA GAA GUA U Cell culture CAU CAA AGA AUG CCA GUA U GAA ACU GUG CCA CUU GUA U Human umbilical vein endothelial cells (HUVECs; Lonza) PKCαUAA GGA ACC ACA AGC AGU A and human brain vascular pericytes (Sciencell) were cul- PKCδCCA UGU AUC CUG AGU GGA A tured on gelatin-coated plates in EGM2 medium (EBM2 PKCεGUG GAG ACC UCA UGU UUC A medium supplemented with EGM2 bullet kit; Lonza, and PKCηGCA CCU GUG UCG UCC AUA A 100  U/ml penicillin/streptomycin; Lonza) and DMEM 1 3 Angiogenesis imaged by f luorescence microscopy, followed by analysis Quantitative PCR and Western blot analysis of the number of junctions, the number of tubules, and the tubule length using AngioSys. At least three technical Total RNA was isolated using RNA mini kit (Bioline) and reversed transcribed into cDNA using iScript cDNA syn- replicates were averaged per condition per independent replicate. thesis kit (Bioline). Gene expression was assessed by qPCR using SensiFast SYBR & Fluorescein kit (Bioline) and prim- Migration assay ers as listed in Table  2. Expression levels are relative to the housekeeping gene β-actin. For assessment of protein Twenty-four hours post siRNA transfection, HUVECs levels, cells were lysed in cold NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris, pH 8.0) supplemented with were plated at a density of 0.5 × 10 cells/well in an Oris™ Universal Cell migration Assembly Kit (Platypus Tech- 1 mM β-glycerophosphate, 1 mM PMSF, 10 mM NaF, 1 mM NaOV, and protease inhibitor cocktail (Roche). Total pro- nologies) derived 96-well plate with cell seeding stoppers. Twenty-four hours post sub-culturing, the cell stoppers were tein concentration was quantified by Pierce® BCA Protein Assay Kit (Thermo Scientific) as a loading control. Lysates removed and cells were allowed to migrate into the cell free region for 16 h in 5% C O at 37 °C. Subsequently, the cells were denaturated in Laemmli buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromo- were washed in PBS and stained by Calcein-AM followed by visualization using fluorescence microscopy. Wells in phenol blue) at 90 °C for 5 min followed by electrophoresis on a 10% SDS-page gel (Biorad). Subsequently, proteins which cell seeding stoppers were not removed were used as a negative control. Results were analyzed by Clemex. At were transferred to a nitrocellulose membrane (Pierce) and incubated for 1 h in PBS with 5% non-fat milk, followed least three technical replicates were averaged per condition per independent replicate. by incubation with rabbit anti-Fzd5 (Milipore), goat anti- β-actin (Abcam), rabbit anti-β-catenin, anti-non-phospho Intracellular immunofluorescent staining β-catenin and phospho-β-catenin (CST, validated in Sup- plemental Fig. 3A), rabbit anti-Angpt2 (Abcam), rabbit anti- Forty-eight hours post siRNA transfection, HUVECs were JNK and phospho-JNK (CST, validated in Supplemental Fig. 4C), rabbit anti-JUN and phospho-JUN (CST, validated seeded on gelatin-coated glass coverslips in 12-well plates at 5 5 a density of 0.5 × 10 cells/well (sub-confluent) and 3.5 × 10 in Supplemental Fig. 4C), rabbit anti-Wnt5a (CST) rabbit anti-Dvl2 (CST) according to the manufacturer’s descrip- cells/well (confluent). Subsequently, cells adhered for 24 h followed by fixation for 15 min in 4% paraformaldehyde tion. Protein bands were visualized with the Li-Cor detec- tion system (Westburg). Levels of secreted Flt1 in cultured and blocking for 60 min in PBS with 5% bovine serum albu- min (Sigma) and 0.3% Triton X-100 (Sigma). After block- medium were assessed 72 h post-transfection using a Flt1 ELISA kit (R&D systems). ing, coverslips were placed on droplets PBS containing 1% BSA, 0.3% Triton X-100, and rabbit anti-β-catenin antibody 3D analysis of endothelial tubule formation (CST), followed by incubation for 16 h in a humidified envi- ronment at 4 °C. Thereafter, coverslips were incubated on Twenty-four hours post siRNA transfection, GFP-labeled PBS with 1% BSA and 0.3% Triton X-100 containing an Alexa Fluor 594-labeled secondary antibody (Invitrogen) HUVECs were harvested and suspended with non-trans- fected dsRED-labeled pericytes in collagen as previously and phalloidin-rhodamine (Invitrogen) for 1 h at room tem- perature, finally followed by mounting the stained coverslips described by Stratman [12]. In summary, HUVECs and pericytes were mixed in a 5:1 ratio in EBM2 supple- on vectashield with DAPI (Brunschwig). Coverslips were imaged by confocal microscopy. mented with Ascorbic Acid, Fibroblast Growth Factor, and 2% FCS from the EGM2 bullet kit. Additionally, Proliferation, cell cycle assay, and apoptosis C-X-C motif chemokine 12, Interleukin 3, and Stem Cell Factor were added in a concentration of 800 ng/ml (R&D Twenty-four hours post siRNA transfection, HUVECs were systems). The cell mixture was suspended in bovine col- lagen (Gibco) with a final concentration of 2 mg/ml and seeded in six-well plates at a density of 0.5 × 10 cells/well. To study the effect of Fzd5 knockdown on proliferation, pipetted in a 96-well plate. One hour of incubation in 5% CO at 37 °C was followed by the addition of 100 µl of the HUVECs were harvested 24, 48, and 72 h post sub-culturing and counted by flow cytometry. For analysis of cell cycle adjusted EBM2 medium on the collagen gels. The addi- tion of recombinant human Angpt2 and Flt1 (R&D sys- progression, cells were harvested 48 h post sub-culturing and fixated in 70% ethanol for 60 min on ice. Subsequently, cells tems) was done 24 h post seeding in the collagen matrix, both in a final concentration of 1000 ng/ml. Forty-eight were stained with PI and treated with RNAse (Sigma) for 30 min at 37 °C and analyzed by flow cytometry. Apoptosis hours and 120  h post seeding, these co-cultures were 1 3 Angiogenesis Table 2 Primer sequences used Gene Sense primer sequence Antisense primer sequence for (q)PCR Fzd1GCC CTC CTA CCT CAA CTA CCA ACT GAC CAA ATG CCA ATC CA Fzd2GCT TCC ACC TTC TTC ACT GTC GCA GCC CTC CTT CTT GGT Fzd3CTT CCC TGT CGT AGG CTG TGT GGG CTC CTT CAG TTG GTT CT Fzd4ATG AAC TGA CTG GCT TGT GCT TGT CTT TGT CCC ATC CTT TTG Fzd5TAC CCA GCC TGT CGC TAA ACAAA ACC GTC CAA AGA TAA ACTGC Fzd6GCG GAG TGA AGG AAG GAT TAG TGA ACA AGC AGA GAT GTG GAA Fzd7CGC CTC TGT TCG TCT ACC TCT CTT GGT GCC GTC GTG TTT Fzd8GCC TAT GGT GAG CGT GTC CCTG GCT GAA AAA GGG GTT GT Fzd9CTG GTG CTG GGC AGT AGT TTGCC AGA AGT CCA TGT TGA GG Fzd10CCT TCA TCC TCT CGG GCT TCAGG CGT TCG TAA AAG TAG CAG Wnt1CAA CAG CAG TGG CCG ATG GTGG CGG CCT GCC TCG TTG TTG TGAAG Wnt2GTC ATG AAC CAG GAT GGC ACA TGT GTG CAC ATC CAG AGC TTC Wnt2bAAG ATG GTG CCA ACT TCA CCG CTG CCT TCT TGG GGG CTT TGC Wnt3GAG AGC CTC CCC GTC CAC AGCTG CCA GGA GTG TAT TCG CATC Wnt3aCAG GAA CTA CGT GGA GAT CATG CCA TCC CAC CAA ACT CGA TGTC Wnt4GCT CTG ACA ACA TCG CCT ACCTT CTC TCC CGC ACA TCC Wnt5aGAC CTG GTC TAC ATC GAC CCC GCA GCA CCA GTG GAA CTT GCA Wnt5bTGA AGG AGA AGT ACG ACA GCCTC TTG AAC TGG TTG TAG CC Wnt6TTA TGG ACC CTA CCA GCA TATG TCC TGT TGC AGG ATG Wnt7aGCC GTT CAC GTG GAG CCT GTG CGT GCAGC ATC CTG CCA GGG AGC CCG CAG CT Wnt7bGAT TCG GCC GCT GGA ACT GCTC TGG CCC ACC TCG CGG AAC TTAG Wnt8aCTG GTC AGT GAA CAA TTT CCGTA GCA CTT CTC AGC CTG TT Wnt8bGTC TTT TCA CCT GTG TCC TCAGG CTG CAG TTT CTA GTC AG Wnt10aCTG TTC TTC CTA CTG CTG CTACA CAC ACC TCC ATC TGC Wnt10bGCA CCA CAG CGC CAT CCT CAAG GGG GTC TCG CTC ACA GAA GTC AGG A Wnt11CAC TGA ACC AGA CGC AAC ACCCT CTC TCC AGG TCA AGC AAA Wnt14ACA AGT ATG AGA CGG CAC TCAGA AGC TAG GCG AGT CAT C Wnt15TGA AAC TGC GCT ATG ACT CGTG AGT CCT CCA TGT ACA CC Wnt16GAG AGA TGG AAC TGC ATG ATGAT GGG GAA ATC TAG GAA CT Axin2TTG AAT GAA GAA GAG GAG TGGA TCG GGA AAT GAG GTA GAG ACA Ccnd1GTC CAT GCG GAA GAT CGT CGTCT CCT TCA TCT TAG AGG CCACG C-MycCAC AGC AAA CCT CCT CAC AGCGC CTC TTG ACA TTC TCC TC Angpt1GCT GAA CGG TCA CAC AGA GACTT TCC CCC TCA AAG AAA GC Angpt2TTA TCA CAG CAC CAG CAA GCTTC GCG AGA ACA AAT GTG AG VEGFaAAG GAG GAG GGC AGA ATC ATATC TGC ATG GTG ATG TTG GA VEGFr2AGC GAT GGC CTC TTC TGT AAACA CGA CTC CAT GTT GGT CA Flt1TGT CAA TGT GAA ACC CCA GAGTC ACA CCT TGC TCC GGA AT DSCR1GAG GAC GCA TTC CAA ATC ATAGT CCC AAA TGT CCT TGT GC TFTAC TTG GCA CGG GTC TTC TCTGT CCG AGG TTT GTC TCC A Ets1GGA GCA GCC AGT CAT CTT TCGGT CCC GCA CAT AGT CCT T PKCαCGA CTG GGA AAA ACT GGA GAACT GGG GGT TGA CAT ACG AG PKCδATT GCC GAC TTT GGG ATG TTGA AGA AGG GGT GGA TTT TG PKCεAAG CCA CCC TTC AAA CCA CGGC ATC AGG TCT TCA CCA AA PKCηTCC CAC ACA AGT TCA GCA TCCCC AAT CCC ATT TCC TTC TT MMP1GAT TCG GGG AGA AGT GAT GTT CGG GTA GAA GGG ATT TGT G Β-actinTCC CTG GAG AAG AGC TAC GAAGC ACT GTG TTG GCG TAC AG 1 3 Angiogenesis was studied 72 h after transfection using an in situ cell death Results detection kit (Roche) as described by the manufacturer on 4% PFA fixated cells. Fzd5 siRNA induces a specific knockdown of endothelial Fzd5 Wnt5a adenovirus preparation, transduction, and stimulation The function of Fzd5 was studied in vitro using siRNA- mediated silencing in HUVECs, which were shown to Recombinant adenoviruses were produced using the Gate- express all Fzd receptors other than Fzd10 (Supplemen- way pAd/CMV/V5DEST vector and ViraPowerTM Ade- tal Fig. 1A), and Wnt2b, 3, 4, 5a, and 11 (Supplemental noviral Expression System (Invitrogen), according to the Fig. 1B). Both qPCR and Western blot analysis confirmed manufacturer’s instructions. Briefly, the Wnt5a expression a significant loss of Fzd5 expression in cells treated with an cassette was cloned from the pENTR™ 221 Wnt5a entry siRNA pool specific for Fzd5, compared to untreated control vector (Invitrogen) into pAd/CMV/V5-DEST expression cells and cells treated with a pool of non-targeting siRNA, vector (Invitrogen) via the LR-reaction II (invitrogen). After referred to as siSHAM (Supplemental Fig. 1C,D). Although verification by DNA sequencing, the pAd/CMV plasmids Fzd receptors share highly similar domains, knockdown of were linearized by Pac1 restriction and subsequently trans- Fzd5 was specific. None of the other Fzd receptors were fected with Lipofectamine 2000 (Invitrogen) in HEK293A differentially expressed after treatment with Fzd5 siRNA, cells. Infected cells were harvested by the time 80% of the other than Fzd5 (Supplemental Fig. 1C). cells detached from plates followed by isolation of viral particles from crude viral lysate. HeLa cells were used to Wnt5a signals via endothelial Fzd5 produce Wnt5a (or dsRED, referred to as adSHAM) by transduction with a calculated 5 viral particles per cell. Previous studies listed Wnt5a and Secreted Frizzled- Forty-eight hours post-transduction, HeLa cells were cul- Related Protein 2 (SFRP2) as most likely candidates to tured for 24 h on EBM2, which eventually was used to stim- activate Fzd5-mediated signaling in ECs [13–15]. In con- ulate serum-starved endothelium for 3 h. trast to SFRP2 [16], Wnt5a is endogenously expressed by HUVECs (Supplemental Fig. 1B). To address the potential Statistical analysis signal capacities of this endogenously expressed Wnt5a as ligand for Fzd5, HeLa cells were transduced with an adeno- For each experiment, N represents the number of independ- viral overexpression plasmid for Wnt5a to produce cultured ent replicates. Statistical analysis was performed by Graph- medium containing high levels of this Wnt ligand. HeLa Pad Prism using one-way ANOVA followed by post hoc cells were selected for this purpose over HUVECs as these Tukey’s test, unless stated otherwise. Results are expressed cells were shown to have a more refined machinery to pro- as mean ± SEM. Significance was assigned when P < 0.05 duce and secrete functional Wnt5a than HUVECs, as illus- (two-tailed). trated by enhanced mRNA expression of Wntless (WLS) and Porcupine (PORCN) (data not shown). Transduction with this overexpression vector (adWnt5a) led to a significant Fig. 1 Wnt5a induced Fzd5-mediated Dvl activation in HUVECs. a stimulation with cultured medium (CM) from HeLa cells overex- Representative Western  blot of adenoviral-based Wnt5a overexpres- pressing dsRED (adSHAM) or Wnt5a, 72 h post siRNA transfection sion in HeLa cells, 72  h post-transduction. N = 4. b Representative in HUVECs. N = 6 Western blot of Dvl and phosphorylated Dvl in HUVECs after 3  h 1 3 Angiogenesis 1 3 Angiogenesis ◂Fig. 2 Fzd5 expression is crucial for vascular formation in  vitro. cycle progression was analyzed in a cell cycle assay in a Representative fluorescent microscope images of GFP-labeled which total DNA was stained with PI, followed by flow HUVECs (green) in co-culture with dsRED-labeled pericytes (red) cytometry. A strong increase of cells in the G /G phase 0 1 in a 3D collagen matrix during vascular formation. Shown are the of the cell cycle was observed after knockdown of Fzd5, results at day 2 and 5 of non-transfected control, siSHAM, and siFzd5 conditions. Scale bar in the left columns represents 1  mm. Scale indicative of a cell cycle arrest (Fig. 3d, e). For apoptosis bar in the right columns represents 350  µm. b Bar graphs show the analysis, a terminal deoxynucleotidyl transferase dUTP quantified results of the co-culture assay. Shown are the total tubule nick end labeling (TUNEL)-based detection staining was length, and the number of endothelial junctions and tubules relative to used. Although seeded in similar densities, Fzd5 knock- the control conditions, both after 2 and 5 days. N = 4, *P < 0.05 com- pared to control and siSHAM condition down led to a significant reduction of nuclei per image field. However, the relative number of TUNEL positive nuclei in the Fzd5 knockdown condition was similar when upregulation of Wnt5a compared to dsRED control trans- compared to control and siSHAM condition, showing that duced cells (adSHAM) (Fig. 1a). To assess whether Fzd5 the reduction of ECs in the Fzd5 knockdown condition is was involved in transducing the signal of Wnt5a, cultured not related to increased apoptosis (Fig. 3f, g). medium from transduced HeLa cells was applied to serum- starved HUVECs after which Dvl activation was monitored. Western blot analysis showed that Wnt5a strongly induced Loss of Fzd5 does not interfere with endogenous Dvl phosphorylation in untreated or non-targeting siRNA- canonical Wnt signaling treated HUVECs, however, this effect was blocked in the absence of Fzd5 (Fig.  1b), confirming the importance of To further dissect the molecular mechanism of endothelial endothelial Fzd5 in transducing Wnt5a signaling. Fzd5 signaling in angiogenesis, known Fzd/Wnt signaling pathways were studied. Downstream Fzd signaling occurs Fzd5 expression is essential for endothelial via the canonical Wnt signaling pathway, also known as the proliferation, migration, and tubule formation Wnt/β-catenin pathway, or by the less well described non- canonical Wnt signaling pathways. Activation of canonical The angiogenic capacities of these Fzd5-silenced HUVECs Wnt signaling is characterized by an accumulation of cyto- were evaluated in a well-validated in vitro 3D angiogenesis plasmic β-catenin, eventually resulting in nuclear transloca- assay developed for studying formation of micro-capillary tion and subsequent expression of β-catenin-dependent tar- structures [12]. In this assay, HUVECs with GFP marker get genes. To evaluate the effect of Fzd5 knockdown on the expression and dsRED-labeled pericytes directly interact canonical Wnt signaling pathway, total levels of β-catenin, in a collagen type I matrix environment, resulting in EC as well as phospho-β-catenin (ser33/37/thr41) and non- sprouting, tubule formation, and neovessel stabilization as phospho-β-catenin (active) were examined 24, 48 and 72 h a result of perivascular recruitment of pericytes. At day 5 post-transfection by Western blot. Ser33/37/thr41 phospho- post-seeding, well-defined, micro-capillaries with pericyte rylation is induced by GSK3β and primes β-catenin for sub- coverage can be observed. Imaging and quantification of the sequent degradation, and could be indicative for a reduced vascular structures were conducted at days 2 and 5. Endothe- activity of canonical Wnt signaling. Total β-catenin, as well lial knockdown of Fzd5 strongly impaired endothelial tubule as non-phospho-β-catenin (active) levels were unaffected by formation (Fig.  2a). Quantification revealed a significant Fzd5 silencing, and non-phospho-β-catenin (ser33/37/thr41) reduction in the total tubule length, the number of endothe- was observed in all conditions (Fig. 4a, b), even though the lial junctions, and the number of endothelial tubules, both antibody was capable of detecting GSK3β-induced β-catenin after 2 and 5 days (Fig. 2b). phosphorylation (Fig. 4c). Furthermore, expression levels of To get a better insight in the causative factor for this previously described endothelial target genes of β-catenin poor vascular phenotype, the migration and prolifera- were studied using qPCR, but no differences were observed tion capacities of Fzd5-silenced ECs were studied. A in the expression of Axin2, Ccnd1, and C-myc after knock- plug-stopper-based migration assay was performed to down of Fzd5 (Fig. 4d). An immunofluorescent staining, analyze the effects of Fzd5 knockdown on endothelial validated to detect cellular distribution of β-catenin (Supple- mobility. Knockdown of Fzd5 significantly inhibited the mental Fig. 3B), was also performed on transfected ECs, as migration of ECs towards the open cell-devoid area com- stable total levels of β-catenin found by Western blot did not pared to untreated and non-targeting siRNA-treated ECs deviate between cytoplasmic or nuclear localized β-catenin. (Fig. 3a, b). In addition, knockdown of Fzd5 significantly In line with the other experiments focusing on β-catenin- reduced cell numbers compared to control and siSHAM mediated signaling, no differences in β-catenin localization condition (Fig. 3c). To clarify whether this was a result were observed after knockdown of Fzd5, both in confluent of impaired cell proliferation or increased apoptosis, cell and sub-confluent cells (Fig.  4e, f, respectively). 1 3 Angiogenesis Fig. 3 Endothelial knockdown of Fzd5 significantly inhibited EC condition (two-way ANOVA followed by Bonferroni post hoc test). migration and proliferation, but had no effect on apoptosis. a Rep - d Representative histogram of flow cytometric analysis of PI-based resentative fluorescent microscope images of Calcein-AM-labeled DNA staining showing the distribution of cells over the cell cycle HUVECs (green) in a plug-stopper-based migration assay. Shown are in the different groups at 48  h post-transfection. e Quantified results the results of 16 h of migration of non-transfected control, siSHAM, of cell cycle analysis. Percentage of cells in G /G phase is shown. 0 1 and siFzd5 conditions. Scale bar represents 500 µm. Open migration N = 3, *P < 0.05 compared to control and siSHAM condition. f Quan- areas produced by the plug-stopper before initiation of the assay are tified results of TUNEL staining. Percentage TUNEL-positive cells indicated by dotted lines. b Bar graph shows the quantified results of total number of cell is shown, 72  h post-transfection of control, of migration assay. Shown are the percentages of surface area within siSHAM, and siFzd5 conditions. N = 3, no significance. g Representa- the dotted circle covered by HUVECs after 16 h of migration. N = 4, tive fluorescent microscope images of DAPI-based nuclei staining in *P < 0.05 compared to control and siSHAM condition. c ECs expan- HUVECs (blue, upper row) and TUNEL staining of the same cells sion at 24, 48, and 72 h post seeding in similar densities, as quantified (red, lower row). Positive control was treated with DNAse solution. by flow cytometry. N = 3, *P < 0.05 compared to control and siSHAM Scale bar represents 200 µm cells, yet was statistically equal to non-targeting siRNA- Fzd5 knockdown induces the expression of several treated HUVECs (Supplemental Fig. 2). Interestingly, vas- (anti‑) angiogenic factors cular endothelial growth Factor A (VEGFa) decoy recep- tor Flt1, and the vascular destabilizing factor Angpt2 were To further elucidate the anti-angiogenic phenotype observed significantly upregulated at both mRNA and protein level after Fzd5 knockdown, expression levels of several impor- in HUVECs treated with Fzd5 siRNA when compared to tant regulators of angiogenesis were analyzed. In contrast to untreated or non-targeting siRNA-treated HUVECs (Fig. 5a, what was previously reported [17], our findings in HUVECs c). Expression levels of VEGF receptor 2, VEGFa, as well as indicate that expression of tissue factor (TF) is not positively Angpt1 remained unaffected in the absence of Fzd5 (Supple- regulated by Fzd5 signaling, as Fzd5 knockdown did not mental Fig. 2). In line with previous findings of Lobov et al., attenuate TF expression. In fact, TF was slightly upregulated combined addition of Flt1 and Angpt2 in the 3D co-culture in Fzd5-silenced HUVECs compared to untreated control 1 3 Angiogenesis Fig. 4 Fzd5 knockdown did not affect the canonical Wnt signaling Calyculin A (50 nM) with and without a 30 min pretreatment of the pathway in ECs. a Representative Western blot result of total levels GSK3β inhibitor LiCl (20  mM). d QPCR analysis of the mRNA of β-catenin, non-phospho-β-catenin, phospho-β-catenin (ser33/37/ expression levels of β-catenin target genes Axin2, Cyclin D1 (Ccnd1), thr41), and β-actin loading control, at different time points post-trans- and C-myc in different conditions 72  h post-transfection. N = 4, no fection. b Quantified results of β-catenin Western blot. Shown are significance. e Immunofluorescent staining β-catenin (green), F-actin β-catenin levels relative to β-actin loading control. N = 3, no signifi- (red), and DAPI (blue) in confluent and sub-confluent f HUVECs cance. c Western blot result of total levels of β-catenin and phospho- after knockdown of Fzd5. N = 3 β-catenin in response to treatment with the phosphatase inhibitor 2+ system completely attenuated endothelial tubule formation Wnt/Ca and PCP pathways were studied for their potential (Fig. 5d, e) [9]. role in the upregulation of Angpt2 and Flt1. Activation of the 2+ Knockdown of a Fzd receptor can not only attenuate sig- Wnt/Ca pathway could induce Flt1 and Angpt2 transcrip- 2+ nal transduction, but due to impaired inhibitory crosstalk tion, as stimulation of the Wnt/Ca pathway leads to free 2+ between the individual pathways, or via alternative receptor Ca -induced activation of Calcineurin, which in turn could binding by the Wnt ligand can also have a stimulatory effect promote NFAT-mediated transcription by dephosphorylating [18, 19]. Since Fzd5 knockdown had no effect on the canoni- NFAT [6]. The mRNA expression level of Down Syndrome cal Wnt signaling pathway, the described non-canonical Critical Region 1 (DSCR1) was evaluated to assess the 1 3 Angiogenesis Fig. 5 Fzd5 knockdown led to increased expression of vascular (green) in co-culture with dsRED-labeled pericytes (red) in a 3D col- regression-associated genes Flt1 and Angpt2. a QPCR results of lagen matrix during vascular formation. Shown are the results at day expression levels of Angpt2 and Flt1 in different conditions 72  h 5 of an untreated control, and after stimulation with PBS, Angpt2 post-transfection. N = 11, *P < 0.05 compared to control and siSHAM (1000  ng/ml), Flt1 (1000  ng/ml), and Angpt2 + Flt1 (1000  ng/ml condition. b Representative Western blot results of Angpt2 expres- both). Scale bar in the upper row represent 1 mm, in the bottom row sion levels in the different conditions 72 h post-transfection. N = 3. c 350  µm. e Bar graphs show the quantified results of the co-culture Enzyme-linked immunosorbent assay-based quantification of secreted assay. Shown are the total tubule length, and the number of endothe- Flt1 levels in cultured endothelial medium 72  h post-transfection. lial junctions and tubules after 5  days. N = 4, *P < 0.05 compared to N = 8, *P < 0.05 compared to control and siSHAM condition. d Rep- control and siSHAM condition resentative fluorescent microscope images of GFP-labeled HUVECs potential link between Fzd5 knockdown and NFAT activa- (A23187)-induced transcription of DSCR1 as a result of free 2+ tion, as DSCR1 is a profound target gene of NFAT, involved Ca -mediated NFAT activation in ECs (Fig.  6a). In line in a feedback loop to fine-tune NFAT-mediated transcrip- with the absence of DSCR1 upregulation in the Fzd5 knock- tion [20, 21]. However, no correlation between endothelial down condition, the upregulation of Flt1 and Angpt2 could knockdown of Fzd5 and DSCR1 upregulation was observed not be linked to an increase of NFAT-mediated transcription (Fig. 6a). The involvement of NFAT-mediated transcription in the Fzd5 knockdown condition, as CsA stimulation failed was also evaluated by pharmacological inhibition of the to reduce Angpt2 and Flt1 upregulation in Fzd5-silenced 2+ Wnt/Ca signaling cascade using the Calcineurin inhibitor cells (Fig. 6b). 2+ Cyclosporine A (CsA). The effectiveness of CsA (1 µM) Besides activation of the Wnt/Ca pathway, the PCP was confirmed by its ability to inhibit calcium ionophore pathway could also stimulate the expression of Flt1 and 1 3 Angiogenesis Fig. 6 Fzd5 knockdown led to increased expression of vascular tive Western blot of total JNK, phospho-JNK, and β-actin levels at regression-associated genes Flt1 and Angpt2, independent of the different time points post-transfection. d Quantified results of JNK 2+ non-canonical Wnt/Ca and PCP pathways. a QPCR results of and phospho-JNK Western blot. Shown are individual (phospho) expression levels of NFAT target gene Dscr1 in the different condi- JNK isoform (p46 and p54) levels relative to β-actin loading control. tions 72  h post-transfection and in response to ionophore A23187 N = 6, *P < 0.05 compared to control and siSHAM condition within 2+ (10 µM)-induced Ca flux with and without NFAT inhibitor Cyclo- one time comparison (24, 48 or 72  h). e Western blot of total JNK, sporin A (CsA) (1  µM). N = 5, *P < 0.05 compared to control and phospho-JNK, and β-actin levels in response to different concentra- siSHAM condition, and DMSO-treated and CsA + A23187-treated tions of JNK inhibitor SP600125 after 1 h. f QPCR analysis showing ECs, respectively. b Angpt2 and Flt1 mRNA expression levels in the effect of SP600125, supplemented 48 h post-transfection, on Flt1 HUVECs in response to CsA, supplemented 48  h post-transfection. and Angpt2 mRNA levels in the different conditions. N = 4, *P < 0.05 N = 4, *P < 0.05 compared to control and siSHAM condition (two- compared to control and siSHAM condition, P < 0.05 as indicated in way ANOVA followed by Bonferroni post hoc test). c Representa- graph (two-way ANOVA followed by Bonferroni post hoc test) Angpt2 via activation of the Wnt/PCP signaling cascade slightly increased both 48 and 72 h post-transfection (Fig. 6c, linked to downstream JNK-induced transcriptional activa- d). JNK-mediated phosphorylation of c-JUN, however, was tion of c-JUN [22, 23]. Activation of JNK/c-JUN-mediated not observed (Supplemental Fig. 4A, B). Since JNK is a transcription involves phosphorylation of JNK, which was kinase with a broad spectrum of downstream substrates [24], 1 3 Angiogenesis the JNK inhibitor SP600125 was used to block activation The effectiveness of SP600125 (20 µM) was confirmed by of JNK to define whether the enhanced phosphorylation of its ability to inhibit JNK phosphorylation in ECs (Fig. 6e). JNK played a role in the upregulation of Flt1 and Angpt2. Treatment of HUVECs with SP600125 did not diminish 1 3 Angiogenesis ◂Fig. 7 Fzd5 knockdown-induced upregulation of Angpt2 and (Supplemental Fig. 5C). Involvement of Ets1 in transcrip- Flt1 expression is mediated via enhanced PKC and Ets1 signal- tional regulation of Angpt2 and Flt1 was evaluated in the ing. a QPCR results showing expression levels of Angpt2 and Flt1 Fzd5 knockdown condition using a double knockdown in HUVECs after knockdown of Fzd5 alone, in combination with of both Fzd5 and Ets1. Knockdown of Ets1 alone had no knockdown of different PKC isoforms, and in combination with knockdown of all novel PKC isoforms (PKCδ,ε,η), 48  h post-trans- effect on the expression of Flt1 and Angpt2 compared to fection. N = 4, *P < 0.05 compared to control and siSHAM condi- control and siSHAM condition, indicating no active tran- tion, P < 0.05 as indicated in graph. b QPCR results of Angpt2 and scription regulation of these two genes by Ets1 in control Flt1 expression in HUVECs, 72  h post-transfection, with and with- conditions. However, knockdown of Ets1 in Fzd5-silenced out knockdown of transcription factor Ets1, a downstream target of PKC. N = 4, *P < 0.05 compared to control and siSHAM condition, HUVECs fully inhibited upregulation of Angpt2 and par- P < 0.05 as indicated in graph. c Representative fluorescent micro- tially inhibited the upregulation of Flt1 when compared to scope images of GFP-labeled HUVECs (green) in co-culture with Fzd5-silenced controls (Fig. 7b). The involvement of Ets1- dsRED-labeled pericytes (red) in a 3D collagen matrix during vas- induced transcription was further substantiated by a similar cular formation. Shown are the results at day 5 of non-transfected control, siSHAM, siFzd5, siEts1 and the combined knockdown of Ets1-dependent upregulation of Matrix metalloproteinase 1 Fzd5 and Ets1. Scale bar in the upper row represents 1  mm, in the (MMP1), a verified endothelial target gene of Ets1 (Supple - bottom row 350 µm. d Bar graphs show the quantified results of the mental Fig. 6A, B) [34]. To evaluate if the anti-angiogenic co-culture assay. Shown are the total tubule length, and the number phenotype of Fzd5 silencing observed in the 3D angiogen- of endothelial junctions and tubules after 5 days. N = 6, *P < 0.05 compared to control and siSHAM condition, P < 0.05 as indicated in esis co-culture assay was mediated via this pathway, Ets1 graph was silenced in GFP-labeled HUVECs. Analysis of the 3D co-culture results demonstrated that inhibition of Ets1 in the Fzd5 silencing-induced upregulation of Flt1 and Angpt2 Fzd5 knockdown condition partly rescued the Fzd5 knock- (Fig. 6f). In contrast, SP600125 treatment rather induced a down-mediated reduction of endothelial tubule formation general upregulation of Angpt2, indicating that activation (Fig. 7c, d). of JNK was not causally related to the Fzd5 knockdown- mediated upregulation of both genes. Discussion Angpt2 and Flt1 upregulation is mediated via PKC and Ets1 The main findings of the current study are (1) endothelial Fzd5 expression is essential for vascular formation, as shown Previously, it was demonstrated that Wnt signal transduction in a 3D co-culture assay. (2) Fzd5 silencing inhibits EC pro- also involves PKC [25–27]. PKCs are part of a kinase fam- liferation and migration. (3) Endothelial loss of Fzd5 expres- ily with a diverse range of potential downstream targets. To sion does not interfere with endogenous canonical Wnt sign- verify whether Fzd5 knockdown-induced upregulation of aling. (4) Fzd5 knockdown leads to increased expression Flt1 and Angpt2 depended on activation of PKC, HUVECs of vascular regression-associated factors Flt1 and Angpt2, 2+ were treated with the PKC inhibitor Staurosporine in the independent of both the non-canonical Wnt/Ca -mediated concentration range of 5–20 nM, as not all different PKC activation of NFAT and PCP-mediated activation JNK. (5) family members are equally inhibited at similar concentra- Inhibition of nPKC signaling, as well as knockdown of the tions. Interestingly, both Angpt2 and Flt1 overexpression PKC target Ets1 suppressed the upregulation of Flt1 and induced by Fzd5 knockdown were dose-dependently reduced Angpt2 in the absence of Fzd5. The Ets1 knockdown inter- by PKC inhibition compared to control and siSHAM condi- vention also partially rescued the Fzd5 knockdown-induced tion (Supplemental Fig. 5A). Since HUVECs express mul- inhibitory effect on new vessel formation. tiple PKC isoforms [28, 29], PKC expression was knocked Previously, it was reported that Fzd5 is indispensable for down by siRNA to interrogate which isoform mediated the murine embryogenesis [5]. Fzd5 knockout embryos died in observed upregulation of Angpt2 and Flt1. Individual PKC utero from severe defects in yolk sac and placenta vasculari- isoform knockdown only had a minor effect on the Fzd5 zation. Using trophoblast-specific Fzd5 knockout mice, Lu knockdown-induced overexpression of the anti-angiogenic et al. reported that the observed phenotype in the Fzd5 full factors, whereas combined knockdown of the novel PKCs knockout placenta was partly initiated by a defect in chori- (nPKCs) completely attenuated the upregulation of Angpt2 onic branching morphogenesis [35]. As defective branch- and Flt1 (Fig. 7a, Supplemental Fig. 5B). ing morphogenesis of the chorion of these mice resulted in PKC signaling can induce elevated synthesis of the a smaller placental labyrinth layer compared to wild-type transcription factor Protein C-ets1 (Ets1) [30, 31], which littermates, it remained difficult to distinguish whether the has binding sites in the promoter regions of both Angpt2 placental defects observed in the Fzd5 full knockout mice and Flt1 [32, 33]. Ets1 was significantly upregulated in were indeed vascular related, or the outcome of propor- the absence of Fzd5, which was orchestrated by PKC tional growth limitations resulting from the reduced villous 1 3 Angiogenesis volume. In our study, we demonstrated that endothelial DSCR1 is also a verified target of the transcription factor knockdown of Fzd5 in vitro leads to a severe reduction in NFAT [20, 21], yet our data showed that the expression level vascular tubule formation in a 3D co-culture model, thereby of DSCR1 remained stable after knockdown of Fzd5. More providing evidence for the direct role of Fzd5 in new vessel important, our experiments demonstrated that inhibition of growth. NFAT activation with CsA after endothelial knockdown of The most detailed described Fzd/Wnt signaling cascade Fzd5 did not inhibit the upregulation of Angpt2 and Flt1, is the canonical or β-catenin-dependent pathway. Without suggesting that the enhanced transcription of these anti- stimulation of the canonical pathway, β-catenin is degraded angiogenic factors was not mediated by enhanced activity by a destruction complex consisting of Axin, Glycogen Syn- of NFAT. Alternatively, stimulation of the Fzd/Wnt/PCP thase Kinase 3ß, Adenomatous Polyposis Coli, and Casein pathway can also induce the transcription of Flt1 and Angpt2 Kinase 1α. Upon binding of Wnt ligands to a Fzd receptor via GTPase-mediated activation of JNK, which eventually in the presence of the co-receptor Lrp5 or Lrp6, a conforma- activates c-JUN-based transcription [6]. Multiple studies tion change in Lrp extracts Axin away from the destruction provided evidence for transcriptional regulation of Flt1 and complex, leading to an increase in intracellular β-catenin Angpt2 either by c-JUN alone, or by the transcription com- levels. When translocated into the nucleus, β-catenin binds plex AP-1 involving c-JUN [22, 23]. Our data indicated that to the TCF/Lef complex and promotes the expression of Fzd5 knockdown led to an increase in JNK phosphorylation, β-catenin target genes [6–8]. Knockdown of a Fzd receptor but no increase in c-JUN phosphorylation was observed. In could both have an inhibiting effect on this pathway, due addition, inhibition of JNK activity with SP600125 ruled to a reduction in receptors capable of transducing a signal out the involvement of the PCP-JNK signal transduction for downstream signaling cascade activation, and an acti- axis as causal factor for the enhanced expression of vascular vating effect, either due to impaired inhibitory crosstalk regression-associated factors Angpt2 and Flt1 in ECs with between the individual pathways or via alternative receptor Fzd5 knockdown, as upregulation of these factors remained binding by the Wnt ligand [18, 19]. Involvement of Fzd5 evident. In future studies, however, it remains of interest to in this canonical pathway appears to be tissue dependent. further dissect the relevance of this altered JNK signaling Steinhart et al. recently demonstrated that canonical Wnt in the absence of Fzd5. signaling via Fzd5 was involved in pancreatic tumor growth Multiple reports have previously suggested a role for PKC and Caricasole et al. reported enhanced β-catenin-mediated involvement in Fzd/Wnt signaling [25–27]. Staurosporine, signaling upon Wnt7a interaction with both Fzd5 and Lrp6 as well as siRNA-mediated knockdown of nPKCs inhib- in the rat pheochromocytoma cell line PC12 [36, 37]. In ited the upregulation of Angpt2 and Flt1 in HUVECs with the mouse optic vesicle, however, no evidence suggests that suppressed expression of Fzd5, indicating the involvement Fzd5 activates or suppresses canonical Wnt signaling [38, of PKC signaling in the transcriptional regulation of these 39]. Our analysis of endogenous canonical Fzd/Wnt sign- genes in Fzd5-silenced ECs. The promoter regions of both aling suggests that Fzd5 is not involved in Wnt β-catenin Angpt2 and Flt1 contain binding sites of the transcription signaling in ECs. factor Ets1 [32, 33], which was shown by our data to be PKC In contrast to the β-catenin target genes, expression lev- dependently upregulated in the absence of Fzd5. Our results els of Angpt2 and Flt1 were significantly upregulated in demonstrate the involvement of enhanced Ets1-mediated HUVECs with suppressed Fzd5 expression. Angpt2 by itself transcription of these two genes in Fzd5-silenced ECs, as is known to have a positive effect on neovessel formation, as Ets1 knockdown resulted in a marked repression of Angpt2 it is involved in pericyte detachment and destabilization of and Flt1 expression levels. Another validated endothelial the endothelium to potentiate the actions of pro-angiogenic target of PKC/Ets1-mediated transcription, MMP1, which factors [40, 41]. However, in the absence of VEGFa, or in like Angpt2 and Flt1 was previously shown to be involved the presence of an increased expression of Flt1, a decoy in vascular regression [34], was also upregulated via Ets1 receptor for VEGFa, Angpt2 is known to induce vascular in the absence of Fzd5. The involvement of Ets1 was fur- regression [9–11]. Both Angpt2 and Flt1 are potential down- ther validated using the 3D co-culture model, in which Ets1 stream target genes of the non-canonical Fzd/Wnt signal- knockdown in Fzd5-silenced ECs partially rescued the 2+ ing pathways. Upon stimulation of the Fzd/Wnt/Ca path- inhibitory effect on new vessel formation that was observed way, activation of phospholipase C leads to cleavage of the in Fzd5-silenced conditions. These results indicate a repress- membrane component PIP2 into DAG and IP3. When IP3 ing function on PKC/Ets1 signaling by Fzd5 in ECs, leading 2+ binds to its receptor on the endoplasmic reticulum, Ca is to reduced expression of vascular regression-associated fac- released in the cytosol, activating the transcription factor tors Angpt2 and Flt1. NFAT via Calcineurin [6]. In recent studies, Flt1 and Angpt2 In this study, the effect of Fzd5 knockdown on the differ - were shown to be transcriptional targets of NFAT [42, 43]. ent Fzd/Wnt signaling routes was studied without the addi- Like Angpt2 and Flt1, the endogenous NFAT inhibitor tion of exogenous Wnt factors. HUVECs secrete Wnt factors 1 3 Angiogenesis should also aim to identify the unknown alternative Wnt5a receptor. The aim of this study was to explore the involvement of Fzd5 in vascular and perivascular biology, which might eventually serve as a foundation for future therapeutic strategies, e.g., in modulating tumor vasculature. A recent genome-wide CRISPR-Cas9 study demonstrated that Fzd5 is a potential druggable target in specific subtypes of pan- creatic tumors [36]. Signaling via Fzd5 in these tumor cells was shown to be crucial in β-catenin-mediated proliferation and treatment of these pancreatic adenocarcinoma cells with Fzd5 antibodies led to inhibited cell growth, both in vitro and in xenograft models in vivo. Although these pancreatic adenocarcinoma tumors are not excessively vascularized, they were previously shown to depend on angiogenesis for growth [45, 46]. Our data demonstrate the importance of Fzd5 in ECs during angiogenesis and might imply that tar- geting the Fzd5 in these types of tumors not only affects the pancreatic adenocarcinoma cells, but could in addi- tion potentially result in beneficial suppression of tumor vascularization. In conclusion, the current study provides evidence for an important role of endothelial Fzd5 in angiogenesis, thereby Fig. 8 Schematic representation of the proposed model of signal- providing novel insights in the molecular mechanism causal ing via Fzd5 in ECs. Our data provide evidence for a new proposed to the poor angiogenic phenotype in the absence of this model of signaling in ECs in the absence of Fzd5. Knockdown of this receptor. receptor provokes its ligand Wnt5a to signal via an alternative recep- tor, thereby triggering the activation of nPKC/Ets1-mediated tran- scription of vascular regression-associated factors, among which Flt1 Acknowledgements The authors would like to thank Dr. O. G. de and Angpt2 Jong for donating the lentiviral GFP and dsRED constructs, and L. A. Blonden and E. H. van de Kamp for their technical support. themselves, among which the typical canonical factor Wnt3 Funding This work was supported by Netherlands Foundation for and non-canonical factor Wnt5a. Knockdown of endothelial Cardiovascular Excellence [to C.C.], Netherlands Organization for Scientific Research Vidi Grant [No. 91714302 to C.C.], the Erasmus Fzd5 led to functional defects, as well as differential expres- MC fellowship Grant [to C.C.], the Regenerative Medicine Fellow- sion of important genes in the angiogenic process, indicat- ship grant of the University Medical Center Utrecht [to C.C.], and ing that lack of Fzd5 interferes with endogenous Fzd/Wnt the Netherlands Cardiovascular Research Initiative: An initiative with signaling. The nature of this endogenous signaling in the support of the Dutch Heart Foundation [CVON2014-11 RECONNECT to C.C., D.D., and M.V.]. absence of Fzd5 was shaped by the finding that combined knockdown of Fzd5 and endogenous Wnt5a significantly Compliance with ethical standards suppressed Angpt2 and Flt1 upregulation (Supplemental Fig. 7). It was previously demonstrated that Wnt factors Conflict of interest The authors declared that they have no conflict of induce signaling to a variety of Fzd and non-Fzd receptors, interest. and that binding selectivity is receptor context dependent [13, 44]. As suppression of endogenous Wnt5a signaling Open Access This article is distributed under the terms of the Crea- partially rescued the Fzd5 knockdown-induced upregula- tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion of Angpt2 and Flt1, our data suggest that endothelial tion, and reproduction in any medium, provided you give appropriate knockdown of Fzd5 provokes its ligand Wnt5a to signal via credit to the original author(s) and the source, provide a link to the an alternative receptor, thereby triggering the activation Creative Commons license, and indicate if changes were made. of the observed PKC/Ets1-mediated transcription (Fig. 8). 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AngiogenesisSpringer Journals

Published: May 29, 2018

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