High-throughput identification of small molecules that PNAS PLUS affect human embryonic vascular development

Helena Vazãoa,1, Susana Rosaa,1, Tânia Barataa,b, Ricardo Costac, Patrícia R. Pitreza, Inês Honórioa, Margreet R. de Vriesd, Dimitri Papatsenkoe, Rui Beneditoc, Daniel Sarisb, Ali Khademhosseinif,g,h,i,j, Paul H. A. Quaxd, Carlos F. Pereiraa, Nadia Mercaderk, Hugo Fernandesa,b, and Lino Ferreiraa,l,2

aCenter for Neurosciences and Cell Biology, University of Coimbra, 3000 Coimbra, Portugal; bMIRA Institute for Biomedical Engineering and Technical Medicine, University Twente, Enschede, 7500AE, The Netherlands; cDepartment of Cardiovascular Development and Repair, Centro Nacional de Investigaciones Cardiovasculares Carlos III, 28029 Madrid, Spain; dEinthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; eBlack Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; fCenter for Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; gHarvard-Massachusetts Institute of Technology Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139; hWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; iDepartment of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea; jDepartment of Physics, King Abdulaziz University, Jeddah 21569, Saudi Arabia; kInstitute of Anatomy, University of Bern, 3012 Bern, Switzerland; and lFaculty of Medicine, University of Coimbra, 3000 Coimbra, Portugal

Edited by Michael A. Gimbrone, Brigham and Women’s Hospital, Boston, MA, and approved March 2, 2017 (received for review October 27, 2016) Birth defects, which are in part caused by exposure to environmen- computational systems modeling have been used for the identifi- tal chemicals and pharmaceutical drugs, affect 1 in every 33 babies cation of vascular-disruptive developmental drugs (3, 4); however, born in the United States each year. The current standard to screen they do not account for differences between species. drugs that affect embryonic development is based on prenatal Human pluripotent stem cells (hPSCs) represent a potential animal testing; however, this approach yields low-throughput and source of embryonic endothelial cells (ECs) (7). hPSC-derived limited mechanistic information regarding the biological pathways ECs have not been used for the identification of molecules that and potential adverse consequences in humans. To develop a screen- disrupt vascular development, in part because it is relatively un-

ing platform for molecules that affect human embryonic development known whether hPSC-derived ECs exhibit embryonic features, SCIENCES based on endothelial cells (ECs) derived from human pluripotent stem because a set of markers to distinguish embryonic ECs from cells, we differentiated human pluripotent stem cells into embryonic postnatal ECs has to be identified. In addition, it requires the APPLIED BIOLOGICAL ECs and induced their maturation under arterial flow conditions. These validation of the hits identified in static screening conditions under cells were then used to screen compounds that specifically affect flow conditions to replicate the hemodynamics of blood vessels. embryonic vasculature. Using this platform, we have identified two Approaches to mimic the hemodynamic forces experienced by compounds that have higher inhibitory effect in embryonic than vessels in vivo require the development of microfluidic platforms. postnatal ECs. One of them was fluphenazine (an antipsychotic), Recently, researchers have replicated the circular cross-section of which inhibits calmodulin kinase II. The other compound was blood vessels in microfluidic devices (8); however, these tools have pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular not been used in the context of drug screening. Moreover, it re- endothelial 2 (VEGFR2), decreases EC viability, quires a final validation of the hits in animal embryos. induces an inflammatory response, and disrupts preformed vascular Here, we report a platform suitable for the high-throughput networks. The vascular effect of the pyrrolopyrimidine was further screening of compounds that affect embryonic vascular development. validated in prenatal vs. adult mouseECsandinembryonicandadult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of Significance research for the study and modulation of embryonic vasculature. It is well recognized that several chemicals and/or drugs are high-throughput screening | endothelial cells | vascular toxicity | potentially harmful if used during pregnancy. Unfortunately, pluripotent stem cells | embryonic endothelial markers systems capable of predicting which drugs affect embryonic development rely almost exclusively on prenatal animal test- he development of platforms for the rapid profiling of chem- ing, with all the associated limitations. Using human pluripo- Tical/pharmaceutical substances that have an effect on embry- tent stem cells, we developed a fully humanized system onic development is of great interest to reduce human embryo capable of predicting which drugs affect, specifically, vascular lethality and birth defects (1). In the United States, ∼3% of all embryonic development. The system was used to screen a li- brary of chemicals (1,280 drugs), and two compounds were babies born each year have birth defects (https://www.cdc.gov/ identified as specific inhibitors of human embryonic vascula- ncbddd/birthdefects/data.html). The majority of birth defects were ture. The platform described here is a valid alternative to ani- the result of multiple environmental and/or genetic effects that mal testing and can be used to screen existing and newly acted in concert. Environmental causes included pesticides, developed drugs. pharmaceuticals, solvents, metals, and air pollutants (2). The cardiovascular system is the first functional organ to develop in the Author contributions: H.V., S.R., R.B., D.S., A.K., P.H.A.Q., H.F., and L.F. designed research; mammalian embryo, and thus, the disruption of the vascular sys- H.V., S.R., T.B., R.C., P.R.P., I.H., M.R.d.V., D.P., R.B., C.F.P., N.M., H.F., and L.F. performed tem is important for the identification of compounds with de- research; H.V., S.R., T.B., R.C., P.R.P., I.H., M.R.d.V., D.P., R.B., D.S., A.K., P.H.A.Q., C.F.P., N.M., H.F., and L.F. analyzed data; and H.V., H.F., and L.F. wrote the paper. velopmental toxicity (3, 4). Disruption of vascular development Conflict of interest statement: A patent has been filed for this work (“Differentiated Cell has been correlated with fetal loss, human malformations, and Population of Endothelial Cells Derived from Human Pluripotent Stem Cells, Composition cognitive impairment (5, 6). Standard protocols for assessing the System, Kit and Uses Therefore”; PCT/IB2013/061110). effect of chemicals on vascular development involve testing on This article is a PNAS Direct Submission. animals. Unfortunately, these tests are low-throughput, expensive, 1H.V. and S.R. contributed equally to this work. yield limited mechanistic information, and do not account for 2To whom correspondence should be addressed. Email: [email protected]. differences between species. Recent approaches combining high- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. throughput screening and high-content screening platforms with 1073/pnas.1617451114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1617451114 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 We established the conditions for the differentiation of hPSCs derived ECs responded to the vasoactive agonists, similar to into embryonic ECs followed by their maturation under flow HUVECs or HUAECs, by increasing the intracellular levels of + conditions for more accurate toxicological assessment. Using a Ca2 (Fig. 1G). hESC-derived ECs did not respond to thrombin as high-throughput assay, we identified fluphenazine (an antipsy- HUAECs, and they had different response profiles to VEGF165, chotic) and 7-cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2, prostaglandin H2-analog, and histamine. No similarity was found in 3-d]pyrimidin-4-ylamine (7-Cyclo; an antiinflammatory agent) as the response profiles of hESC-derived ECs and HUVECs. Fur- compounds that interfere with cell viability and disrupt in vitro thermore, hESC-derived ECs responded to proinflammatory stim- embryonic vascular networks. Our results further show that 7-Cyclo’s uli, such as tumor necrosis factor alpha (TNF-α), by increasing the effect is mediated by the inhibition of vascular endothelial growth expression of ICAM1, CD40, and VCAM1 (Fig. 1H). Together, our factor receptor 2 (VEGFR2) highly expressed in the embryonic results show that hESC-derived ECs are functional; however, they ECs. These findings were validated in vitro, where we demonstrated show differences in their activity compared with HUAECs and that 0.001 μM 7-Cyclo interferes with hPSC-derived EC cord-like HUVECs, which is likely due to their embryonic properties. structure in Matrigel and cell viability. Additionally, in vivo, we To induce the maturation of hESC-derived ECs, we cultured the showed that 0.1 μM 7-Cyclo specifically blocks the motility and cells under flow conditions (20 dyne/cm2) for 7 d. Knowledge about sprouting of arterial ECs during intersomitic vessel develop- flow conditions during human embryo development is scarce, and ment in zebrafish embryos. thus we selected arterial flow conditions (14) to culture the hESC- derived ECs that have a expression profile more related to Results arterial ECs (SI Appendix,Fig.S1B). Previous studies have shown Derivation of ECs from hPSCs. To differentiate human embryonic that a mechanosensory complex formed by CD31, VECAD, and stem cells (hESCs) into ECs, we used a protocol that combined VEGFR2 mediates the responsiveness of ECs to flow shear stress VEGF165 (9), thymosin β4(Tβ4) (10), and TGF-β inhibitor (15). Indeed, the expression of CD31 and VEGFR2 was up- (SB431542) (11) as inductive agents of EC differentiation (Fig. regulated in flow conditions (SI Appendix,Fig.S1D and E),as + 1A). We obtained ∼5% CD31 cells at 18 d of differentiation. To previously shown in adult ECs (15). The maturation of the hESC- + determine whether CD31 cells after 18 d of differentiation could derived ECs was also evaluated by their capacity to express hep- + differentiate into ECs, CD31 cells were isolated by using magnetic aran sulfate proteoglycan (HSPG), a component of the glycocalyx activated cell sorting (MACS) and cultured in EGM-2 medium layer(16)(SI Appendix,Fig.S1F). HSPG is absent in ECs cultured supplemented with SB431542. Gene expression analysis in cells in static conditions, as shown in previous studies (16). However, differentiated for three passages (between 18 and 22 d after cell both hESC-derived ECs and control HUAECs cultured under flow seeding) indicated that the cells expressed CD34,vascularendo- conditions were abundantly decorated with HSPGs. HSPGs are thelial cadherin (VECAD), and VEGFR2 at the same or at a higher detected in the apical region of ECs (XZ view) exposed to flow. level compared with human umbilical vein ECs (HUVECs), albeit These results showed that hESC-derived ECs responded to flow by they exhibited a lower expression of VWF and CD31,whichmay producing HSPG, as observed in vivo. Overall, our results indicated indicate different levels of maturation (Fig. 1B). Flow cytometry that hESC-derived ECs mature under flow conditions, as shown by + and immunocytochemistry analyses showed that CD31 cells cul- the up-regulation of the mechanosensory complex and their ca- tured for three passages expressed high levels of EC markers (Fig. pacity to express HSPG. 1 C and D), but not other mesoderm-derived cell lineages, such as the smooth muscle cell marker α-SMA (SI Appendix,Fig.S1A). High-Throughput Identification of Compounds That Interfere with hESC- Similar results were obtained for ECs derived from human induced Derived EC Activity Followed by Hit Validation in Flow Conditions. To pluripotent stem cells (hiPSCs) generated from cord blood (12) (SI investigate whether hESC-derived ECs cultured under static and Appendix,Fig.S2). Microarray data from hESC-derived ECs, hu- flow (20 dyne/cm2) conditions can respond to compounds that man umbilical artery ECs (HUAECs), human arterial ECs (HAECs), interfere with EC activity, we cultured cells for 7 d in each con- and HUVECs were integrated. Clustering analysis showed that dition, after which the culture medium was supplemented or not hESC-derived ECs are more related to an arterial than a ve- with terbinafine (0.1 and 1 μM), an antiangiogenic drug that sup- nous gene expression profile (SI Appendix,Fig.S1B). Overall, presses EC proliferation and activates EC (17, 18), for an our results showed that we obtained a significantly pure EC additional day. Our results indicated that the hESC-derived ECs population from hPSCs. are highly sensitive to terbinafine because the expression of in- Next, we performed gene microarray for hESC-derived ECs, flammation (ICAM-1; E-SELECTIN), oxidative stress sensing HUAECs (fetal cells), and HAECs (adult cells) and compared (HO-1), and vasculature modulation (eNOS)geneswasup- the global gene expression with data from ECs isolated from regulated in cells cultured in flow conditions with terbinafine (SI embryonic day 8.5 (E8.5) mouse embryos (13). Interestingly, the Appendix,Fig.S3). In addition, the expression of dimethylarginine- hESC-derived ECs showed a robust clustering to embryonic ECs dimethyl-amino- (DDAH) , a family of that (Fig. 1E). We then used k-means clustering to extract the set of metabolizes asymmetric dimethylarginine (ADMA) (19), a marker genes enriched in both embryonic and hES-derived ECs (Fig. 1E of EC dysfunction, was significantly down-regulated in hESC- and SI Appendix, Table S2). Thirteen genes were selected and derived ECs cultured under flow conditions (P < 0.05 or 0.01; confirmed by quantitative RT-PCR (qRT-PCR) (DLL1, EPHB2, n = 4), but not in static conditions. Furthermore, the secretion of LYN, TEK, ID1, NRP2, CAST, FLT1, IGF1, DKK3, NIN, LEF1, ADMA and the ratio of the von Willebrand factor propeptide and SORBS3; Fig. 1F and SI Appendix, Fig. S1C). ECs isolated (vWFpp):von Willebrand factor (vWF) (20), both indicators of EC from embryonic mouse aorta at day E12.5 (mAECs E12.5) and activation/injury, were higher in hESC-derived ECs cultured in flow postnatal day 1 (mAECs p1) were used to confirm the embryonic conditions in the presence of terbinafine than in static conditions. identity. qRT-PCR results validated the microarray analysis and Overall, these studies demonstrate that hESC-derived ECs can be further showed that the 13 genes were up-regulated in mAECs used to test inhibitory molecules, and cells cultured under physi- E12.5 compared with mAECs p1 (Fig. 1F and SI Appendix, Fig. ologic shear stress have a higher sensitivity to terbinafine than cells S1C). Together, the results indicated that hESC-derived ECs cultured in static conditions. have embryonic-like properties. Having demonstrated the drug sensitivity of hESC-derived ECs, Next, we asked whether hESC-derived ECs are functional. we next asked whether we could identify compounds that interfered hESC-derived ECs are able to take up Dil-labeled acetylated low- with embryonic-like ECs using high-throughput screening. Thus, we density lipoprotein and formed cord-like structures when cultured exposed hESC-derived ECs in static conditions to a Library of in the basement membrane Matrigel (Fig. 1C). In addition, hESC- Pharmacologically Active Compounds (LOPAC) consisting of

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1617451114 Vazão et al. Downloaded by guest on September 29, 2021 A B PNAS PLUS

CD

E SCIENCES APPLIED BIOLOGICAL

F

G H

Fig. 1. Differentiation and properties of hESC-derived ECs. (A) Scheme illustrating the differentiation protocol. (B) Gene expression on hESC-derived ECs. hESC- derived ECs were obtained from CD31+ cells isolated by MACS and differentiated for three passages (∼22 d after cell seeding). Gene expression was evaluated by qRT-PCR, and the values were normalized by the corresponding gene expression observed in HUVECs, except for OCT-4, which was normalized by the corre- sponding gene expression in undifferentiated hESCs. Results are mean ± SEM (n = 4). (C) Expression of EC and functionality of hESC-derived ECs. (Scale bars: 50 μm.) (D) Flow cytometry analysis of hESC-derived ECs. Percentages of positive cells were calculated based on the isotype controls (gray plot) and are shown in each histogram plot. Results are mean ± SEM (n = 3). (E) Hierarchical clustering showing the integration of gene expression data from hESC-derived ECs (in blue) and mouse embryonic ECs (data from ref. 13). Our results show that hESC-derived ECs cluster with embryonic ECs more than fetal or adult arterial ECs. The heatmap displays 10 clusters of genes. The one highlighted in green is a cluster of genes enriched in embryonic and hESC-derived ECs (presented in SI Appendix). Red designates increased expression, and blue designates decreased expression relative to the mean. (F) qRT-PCR analysis for genes more highly expressed in embryonic ECs than in fetal or adult ECs. hESC-derived ECs at passage 4, HUAECs, embryonic mouse aortic ECs at day 12.5 (mAEC E12.5), and postnatal day 1 (mAEC p1) have been characterized. Gene expression was normalized by the expression of GAPDH. Results are mean ± SEM (n = 4). Statistical analyses were performed by an unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (G) Variation of intracellular Ca2+ in FURA-2–loaded cultured hESC-derived ECs, HUAECs, or HUVECs in response to several agonists. Traces are representative of six independent experiments for each condition. (H) hESC-derived EC ac- tivation by exposure to TNF-α (10 ng/mL) for 24 h. The shift in each plot indicates the percentage of cells that express a specific marker after exposure to TNF-α, subtracted by the percentage of cells that express the corresponding marker in the absence of TNF-α. Results are mean ± SEM (n = 4).

Vazão et al. PNAS Early Edition | 3of10 Downloaded by guest on September 29, 2021 1,280 bioactive compounds, and we assessed cell viability after 4 d extensive to 0.001 μM(SI Appendix,Fig.S7A). A similar trend was using a PrestoBlue assay (resazurin-based solution that is reduced observed for cells treated with fluphenazine, although less pro- by viable cells) (Fig. 2A). The library was screened at a single nounced than 7-Cyclo (Fig. 3C and SI Appendix,Fig.S6B). concentration (4.5 μM) according to previous studies using ECs We complemented these results by evaluating cell metabolism (21–24) and other cells (25, 26), in a volume of 200 μLperwellof as well as cell viability by annexin V/propidium iodide (PI) staining EGM-2 medium containing 0.25% DMSO (vol/vol). To identify in hESC-derived ECs and HUAECs cultured on top of Matrigel. compounds that selectively target ECs, we screened the same li- Our results show that hESC-derived ECs reduce significantly ATP brary against human anterior cruciate ligament (ACL) cells. These production and have significant apoptosis/necrosis when cultured cells were chosen because they are nonvascular cells isolated from a with 7-Cyclo in concentrations up to 0.001 μM for 3 h (Fig. 3 D poorly vascularized tissue, and thus their survival does not heavily and E and SI Appendix,Fig.S7B). This effect was less pronounced rely on blood supply (27). Of the 1,280 compounds, 99 compounds in HUAECs cultured with 7-Cyclo. In addition, the higher toxicity induced differences in cell viability (hESC-derived ECs vs. ACL of 7-Cyclo (1 μM) against human embryonic ECs compared with cells) of >50% and were considered for further analyses (SI Ap- fetal ECs was also confirmed in mouse ECs, specifically mAECs pendix,TableS3). To identify compounds that were selective to E12.5 against mAECs p1 (SI Appendix,Fig.S8). Moreover, hESC- embryonic ECs, but not fetal ECs, we screened the library against derived ECs were more sensitive to the toxicity effects of flu- HUAECs (Fig. 2A). Six compounds (danazol, chlorpromazine hy- phenazine than HUAECs, although the effect in both cells was drochloride, ellipticine, 3′,4′-dichlorobenzamil, fluphenazine dihy- relatively lower than that observed for 7-Cyclo (Fig. 3 F and G). drochloride, and 7-Cyclo) affected cell viability in both cells by a Fluphenazine has been described as an antipsychotic agent that difference of 20% (Fig. 2B). The compounds selected from the inhibits calmodulin in ECs and increases intracellular concentra- + primary screen were then tested against hESC-derived ECs and tion of Ca2 (28). The inhibition of calmodulin leads to the in- HUAECs at different concentrations to obtain a dose–response hibition of calmodulin kinase II, which, in turn, inhibits the curve (Fig. 2C and SI Appendix,Fig.S4). Compounds 7-Cyclo and phosphorylation of extracellular signal-regulated kinase (ERK) fluphenazine dihydrochloride were selected for further testing (29) and finally affects cell survival. Our results indicate that due to the significant differenceintheeffectsonhESC-derived embryonic ECs cultured with fluphenazine showed higher mobi- + ECs vs. HUAECs. lization of intracellular levels of Ca2 (SI Appendix,Fig.S9), lower To test the properties of 7-Cyclo and fluphenazine hydrochlo- calmodulin kinase II activity, and lower levels of ERK and AKT ride in the disruption of vascular networks, microvessels of hESC- phosphorylation than postnatal ECs (SI Appendix,Fig.S10). For derived ECs and HUAECs were formed on top of Matrigel to further testing, we selected 7-Cyclo because hESC-derived ECs have a patent lumen (SI Appendix,Fig.S5) and subsequently ex- were more susceptible to this compound than fluphenazine. posed to the drug between 3 and 20 h (depending on the assay). To evaluate the effects of 7-Cyclo in flow conditions, hESC- Our results showed that there was a statistically significant re- derived ECs were cultured in a poly(dimethylsiloxane) (PDMS) duction in the network length and number of sprouts in micro- microfluidic system with cylindrical channels for 7 d at 20 dyne/cm2 vessels formed by hESC-derived ECs after incubation with 1 μM (Fig. 4A). ECs were able to form a confluent monolayer on the 7-Cyclo, whereas a negligible effect was observed in microvessels entire inner surface of the channel after 48 h. At day 7, cells were formed by HUAECs (Fig. 3 A and B and SI Appendix,Fig.S6A). exposed to EGM-2 medium supplemented with 1 μM7-Cyclo Importantly, the toxicity of 7-Cyclo against hESC-derived ECs was for 24 h, and their gene expression and secretome were analyzed

AB

C

Fig. 2. High-throughput screening (HTS) to identify compounds that interfere with hESC-derived ECs. (A) Schematic representation of the HTS assay. (B) Small molecules identified after the analysis of the primary screen. The hits have preferential cytotoxicity

against hESC-derived ECs. IC50 values are for hESC- derived ECs. (C)Dose–response curve for HUAECs and hESC-derived ECs exposed to 7-Cyclo and flu- phenazine. Values are normalized against nontreated cells (control). Results are mean ± SEM (n = 4).

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1617451114 Vazão et al. Downloaded by guest on September 29, 2021 A PNAS PLUS

B.1 B.2

C.2 C.1 SCIENCES

DEFG APPLIED BIOLOGICAL

Fig. 3. Effect of 7-Cyclo and fluphenazine in angiogenesis, cell survival, and metabolism. (A) Secondary assays to show the preferential effect of 7-Cyclo in hESC- derived ECs than HUAECs. (B and C) Quantification of length (B1 and C1)andsprouts(B2 and C2) of cord-like structures in hESC-derived ECs, HUAECs, or HUAECs overexpressing VEGFR2 cultured on top of Matrigel for 12 h and then exposed for 0, 3, and 20 h to 7-Cyclo (B) or fluphenazine (C). Results are mean ± SEM (n = 4; two phase-contrast images per well and time). In B and C, statistical analyses between experimental groups and no treatment (0 μM 7-Cyclo) for the same time were performed by a one-way ANOVA test followed by a Newman–Keuls multiple comparisons test. (D and F) ATP analyses on hESC-derived ECs or HUAECs cultured on top of Matrigel for 12 h and then exposed for 3 h to 7-Cyclo (D) or fluphenazine (F). Results are mean ± SEM (n = 4). Statistical analyses were performed by one-way ANOVA test followed by a Newman–Keuls multiple comparisons test. (E and G) Quantification by flow cytometry of cell viability (annexin−/PI−), necrosis (annexin−/PI+), early (annexin+/PI−), and late (annexin+/PI+) apoptosis by using annexin V/PI staining, in cells cultured on top of Matrigel for 12 h and then exposed for 3 h to 7-Cyclo (E) or fluphenazine (G). Results are mean ± SEM, n = 4. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

(Fig. 4B). Our results revealed that hESC-derived ECs showed observed compared with HUAECs. This result was likely due to significant cell death (SI Appendix,Fig.S11A). In addition, hESC- differences in the expression profile of 7-Cyclo molecular targets derived ECs exposed to 7-Cyclo expressed significantly higher (i.e., tyrosine kinases; see below) either in static or flow conditions levels of inflammatory genes, such as ICAM-1, E-SELECTIN, (SI Appendix,Fig.S12). Overall, our results indicated that hESC- HO-1,andeNOS (P < 0.0001, n = 4), and expressed lower levels of derived ECs were more sensitive to the effects of 7-Cyclo com- DDAH1 and DDAH2 (P < 0.05 or 0.0001, n = 4), which are en- paredwithHUAECs. zymes that metabolize ADMA, compared with cells cultured under To further confirm the effects of 7-Cyclo in the embryonic vas- static conditions (Fig. 4C). Interestingly, EC inflammation and culature, we incubated mAECs E12.5 and mAECs p1 with 7-Cyclo injury occurred upstream of cell apoptosis/necrosis (SI Appendix, (1 μM) for 24 h under static conditions. Inflammation, oxidative Fig. S11B). In the case of HUAECs cultured under the same stress sensing, vascular modulation, and vascular injury-sensing conditions, the effect was less pronounced. No down-regulation of genes were statistically up-regulatedinmAECsE12.5compared DDAH-1 and -2 was observed. We complemented these gene with cells without treatment (SI Appendix,Fig.S13). In contrast, analyses with analyses of ADMA and the ratio of vWFpp:von vWF 7-Cyclo had no effect on mAECs p1. The degree of action of secreted by these cells (Fig. 4D). hESC-derived ECs or HUAECs 7-Cyclo in mAECs E12.5 was similar to the effect identified in cultured in static conditions in the presence of the drug demon- hESC-derived ECs (Fig. 4C). strated similar secretion of ADMA or vWFpp:vWF as control We further validated these findings in vivo using zebrafish conditions (i.e., without the drug). Importantly, hESC-derived ECs Tg(fli1a:eGFP)y1 embryos (30) by evaluating the effect of 7-Cyclo cultured under flow conditions in the presence of 7-Cyclo secreted on the development of intersegmental blood vessels (ISVs). The higher levels of ADMA (2.5-fold) and vWFpp:vWF (1.6-fold) than 7-Cyclo was added to the water of 22–23 h postfertilization (hpf) without the drug, and significantly higher levels of ADMA were embryos at a concentration of 0, 0.1, 1, and 10 μM for 8 h, and

Vazão et al. PNAS Early Edition | 5of10 Downloaded by guest on September 29, 2021 A B Microfluidic system; Inflammation (ICAM-1; flow: 20 dyne/cm2 E-SELECTIN) Oxidative stress (HO-1) Vascular modulation HUAECs 0 (eNOS) hESC-ECs VECAD Nuclei VECAD Nuclei 24 h Vascular injury (DDAH1, DDAH2)

Vascular injury Microfluidic system; (ADMA; flow: 0 dyne/cm2 propeptide:vWF ratio) (static)

C 1 hESC-ECs 1 HUAECs hESC-ECs HUAECs 10 10 100 100

* -1 0 **** 10 **** * 10 **** *** 100 * 10-1 **** ** 10-2 10-1 * ** 10-3 10-1 * 10-2 10-2 Gene expression Gene expression Gene expression -4 Gene expression 10

10-2 10-3 10-5 10-3

HO-1eNOS HO-1eNOS ICAM-1 ICAM-1 DDAH1 DDAH2 DDAH1 DDAH2

E-SELECTIN E-SELECTIN D hESC-ECs HUAECs hESC-ECs HUAECs

Fig. 4. Effect of 7-Cyclo in flow conditions. (A) Macroscopic view of the PDMS microfluidic system (the microchannels have a diameter of 900 μm and an average length of 0.5 cm) and fluorescent images of microchannel cross-sections showing that ECs can grow in the inner surface of the microfluidic channel after 48 h and be stable for at least 7 d at 20 dyne/cm2. (Scale bars: 50 μm.) (B) Schematic representation of the experiments performed to evaluate the effect of 7-Cyclo in ECs cultured under flow or static conditions. (C) Expression of genes involved in inflammation (ICAM-1; E-SELECTIN), oxidative stress sensing (HO-1), vascular mod- ulation (eNOS), and vascular injury sensing (DDAH1 and DDAH2) in hESC-derived ECs and HUAECs after 24 h of incubation with 0 or 1 μM7-Cyclo.Resultsare mean ± SEM (n = 4). Statistical analyses between groups at static or flow conditions were performed by an unpaired t test. (D) Quantification of ADMA and vWFpp:vWF by ELISA in hESC-derived ECs and HUAECs after 24 h incubation with 1 μM 7-Cyclo. Results are mean ± SEM (n = 6). Statistical analyses were performed by one-way ANOVA test followed by a Newman–Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

(i) the number of ISVs, (ii) number of ISVs reaching the dorsal nases (32). To understand the distinct effect of 7-Cyclo in em- longitudinal anastomotic vessel (DLAV), and (iii)percentageof bryonic vs. fetal/adult ECs, we mined the microarray data and caudal plexus sprouts was quantified (Fig. 5 A and B). Our results compared the expression levels of different kinases. Of the show that 7-Cyclo is toxic to zebrafish embryos at a concentration 38 genes that encode for tyrosine kinases (Fig. 5D and SI Ap- of 0.1–1 μM. At concentrations of 1 μM, 7-Cyclo blocked the pendix, Table S4), 13 of the genes (EFS, VEGFR2, LYN, EGFR, motility and sprouting behavior of arterial ECs during intersomitic ZAP70, FLT1, FLT4, LTK, MERTK, NRP1, NTRK2, TEK, and vessel development in zebrafish embryos. We also evaluated the y1 TYRO3) were expressed at higher levels in hESC-derived ECs effect of 7-Cyclo in adult zebrafish Tg(fli1a:eGFP) . Because drug compared to HUAECs or HEACs. The expression of EFS, accessibility and pharmacokinetics is likely different in both models VEGFR2, LYN, EGFR, ZAP70, NRP1, and TEK was further (31), the concentration and time of exposure of 7-Cyclo will be confirmed by using qRT-PCR (Fig. 5C). different, making a direct comparison difficult. To overcome this Next, we evaluated the expression of genes on issue,weevaluatedtheeffectof7-Cycloonthezebrafishcaudalfin mAECs E12.5 and p1 to validate the results obtained in hESC- regeneration model (SI Appendix,Fig.S14). In this model, we derived ECs. The same results were observed—that is, tyrosine could monitor simultaneously the effect of 7-Cyclo in the pre- kinases were expressed more in embryonic ECs (in human and formed vasculature (mature ECs) and in the forming vasculature [immature ECs; more dependent in VEGF signaling than pre- mouse) compared with fetal/adult tissues [except for EGFR in formed vasculature (31)]. The zebrafish tail fin is very thin and mouse (Egfr)]. The kinase activity of hESC-derived ECs and optically transparent, which facilitates the vascular toxicity moni- HUAECs was assessed by using luminescence (signal is inversely toring. Our results clearly show a more dramatic effect of the drug correlated with the level of kinase activity) in the absence or on newly formed vessels than on the preexistent vessels in the adult. presence of compound 7-Cyclo (Fig. 5E). After 24 h of in- cubation with 7-Cyclo (0.1 or 1 μM), the kinase activity of hESC- Effect of 7-Cyclo in Embryonic ECs. The 7-Cyclo is a cell-permeable derived ECs decreased significantly from time 0 (P < 0.01), pyrrolopyrimidine that acts as a potent inhibitor of tyrosine ki- whereas no significant decrease was observed in HUAECs.

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1617451114 Vazão et al. Downloaded by guest on September 29, 2021 AB PNAS PLUS

C SCIENCES APPLIED BIOLOGICAL D E

Fig. 5. Effect of 7-Cyclo in zebrafish embryos and molecular targets. (A) Effect of 7-Cyclo on zebrafish embryos. Tg(fli1a:EGFP)y1 Danio rerio were incubated for 8 h at the concentrations shown (A1–A4) and starting at 22–23 hpf. Insets show the effect of 7-Cyclo in ISVs reaching the DLAV (arrowheads). (Scale bars: 100 μm.) (B) Embryos were scored for the number of ISVs along the anterior–posterior axis, the number of ISV’s that reach the DLAV, and for the presence or absence of sprouts at the caudal plexus. Ten or more embryos were tested per experimental group per independent experiment (total of three independent experiments). The data shown are representative of one of three independent experiments. Statistical analyses were performed by one-way ANOVA test followed by a Bonferroni multiple comparisons test. (C) Expression of tyrosine kinases by qRT-PCR. Gene expression was normalized by the expression of GAPDH. Results are mean ± SEM (n = 4). Statistical analyses were performed by a Mann–Whitney test. (D) Microarray analysis showing the expression of tyrosine kinases in hESC-derived ECs, HUAECs, and HAECs. The list of genes is linked to the heatmap. Some of the tyrosine kinases are more highly expressed in hESC-derived ECs than in HUAECs or HAECs (displayed in the zoom of the microarray). (E) Kinase activity on hESC-derived ECs and HUAECs after incubation with variable concentrations of 7-Cyclo. Luminescence is inversely related to kinase activity. Results are mean ± SEM (n = 6). Statistical analyses were performed by one-way ANOVA test followed by a Newman–Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

Together, our results indicate that 7-Cyclo affects hESC-derived as well as in the length and sprouts of cord-like vessels formed on ECs, which likely inhibits tyrosine kinases that are highly Matrigel (Fig. 3B). Like in the human system, in the mouse system expressed in the embryonic state. the phosphorylation decreased in mAEC E12.5, but not in VEGFR2 is an important target of 7-Cyclo because the IC50 of mAECs p1, indicating that embryonic ECs are more sensitive to the drug for this tyrosine kinase is 1.57 μM (33). Therefore, we 7-Cyclo than postnatal ECs (Fig. 6A). Our results further showed evaluated the effect of the drug in the phosphorylation of that the increased sensitivity of embryonic ECs to 7-Cyclo is likely VEGFR2. The phosphorylation decreased significantly in hESC- due to their higher expression of VEGFR2 (hESC-ECs: 76.5 ± derived cells, but not in HUAECs (Fig. 6A). However, if we 2.9; mAEC E12.5: 67.3 ± 3.1) compared with postnatal ECs overexpressed VEGFR2 in HUAECs (SI Appendix,Fig.S15), we (HUAECs: 70.4 ± 2.1; mAECs p1: 49.6 ± 2.6) (Fig. 6B). The had a significant decrease in VEGFR2 phosphorylation (Fig. 6A), increased sensitivity of embryonic ECs to 7-Cyclo is not due to

Vazão et al. PNAS Early Edition | 7of10 Downloaded by guest on September 29, 2021 AB

CD E

F

Fig. 6. Effect of 7-Cyclo in VEGFR2. (A) Phosphorylation of VEGR2 in human and mouse embryonic and postnatal cells treated with 0 or 0.1 μM 7-Cyclo for 72 h (by ELISA). Results were normalized by the total form of and indicate mean ± SEM (n = 4). HUAECs-VEGFR2 cells are HUAECs overexpressing VEGFR2. Statistical analyses between groups were performed by an unpaired t test. (B) VEGFR2 is more highly expressed in embryonic cells (hESC-ECs or mAECs E12.5) than in postnatal cells (HUAECs or mAECs p1), either in human or mouse cells. Percent of positive cells was calculated based on the isotype controls (gray plot) and is shown in the histogram plots. Values in histogram plots indicate mean ± SEM (n = 3). (C and D) Effect of 7-Cyclo (0.1 M) and ZM323881 (1 M; VEGFR2-specific inhibitor) in the phosphorylation of AKT (C)andERK(D) in hESC-derived ECs and HUAECs for 15 min (by ELISA). Results were normalized by the total form of protein and indicate mean ± SEM (n = 4). Statistical analyses between experimental group “no-treatment” and the other two groups was performed by one-way ANOVA test followed by a Newman–Keuls multiple comparisons test. (E) Correlation between the inhibition of VEGFR2 phosphorylation and the inhibition of AKT (P = 0.914) or ERK (P = 0.987) phosphorylation. Correlations indicate a strong relationship between both events. Values indicate mean ± SEM (n = 4). (F) Schematic representation of the impact of 7-Cyclo in embryonic ECs. The 7-Cyclo inhibits VEGFR2 phosphorylation, leading to the inhibition of downstream pathways involved in cell proliferation and survival (ERK and AKT pathways). The 7-Cyclo also increases the expression of the molecules involved in vascular injury such as ADMA, propeptide vWF, eNOS, ICAM-1, eSelectin, and HO-1. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

changes in the level of VEGF expression after drug exposure (SI highly selective inhibitor of VEGFR2 (34), 7-Cyclo reduced Appendix,Fig.S16). significantly the phosphorylation of both AKT and ERK in Downstream effectors of VEGFR2 are ERK and AKT sig- hESC-ECs (Fig. 6 C and D). This effect was significantly lower in naling pathways (34). Phosphorylation of ERK activates cell HUAECs. To determine whether the inhibition of AKT and proliferation, while phosphorylation of AKT activates cell pro- ERK was synchronized with VEGFR2 inhibition, we performed liferation, migration, and survival (34, 35). Like ZM323881, a dose-effect analyses and calculated the correlation between the

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1617451114 Vazão et al. Downloaded by guest on September 29, 2021 slopes of both graphs (Fig. 6E). The correlations found (AKT: cell viability after 96 h of exposure. This endpoint may reflect PNAS PLUS 0.914; ERK: 0.987) indicate that there is a strong relationship multiple effects of the selected drug, and thus we further con- between AKT and ERK inhibition and VEGFR2 blocking by firmed its toxicity in multiple secondary assays for low concen- 7-Cyclo. Together, our results indicate that 7-Cyclo inhibits the trations of the drug (up to 0.001 μM). Although not explored, the kinase activity of VEGFR2, which further inhibits AKT and effect of the chemical library in specific properties of ECs, such as ERK signaling pathways, inducing cell death (Fig. 6F). The migration, capillary tube formation, vascular inflammation, among higher sensitivity of embryonic ECs to 7-Cyclo than postnatal others (24), may also be important for the identification of drugs ECs is likely due to high expression of VEGFR2. affecting embryonic vascular development. The concentration selected for the screening was based on previous screenings (21, Discussion 22, 24) and was below the toxicity of thalidomide (>5 μM) (44). In this study, we described a platform for the high-throughput In recent years, several drugs have been identified to disrupt screening of chemicals that affect embryonic vascular develop- vascular development in zebrafish and Xenopus embryos (36, 37, ment. This platform includes (i) the differentiation of hPSCs into 45); however, in many cases, the drugs have not been validated in embryonic-like ECs, followed by their maturation under flow human cells, and most of the drugs likely affect both the adult shear stress; (ii) high-throughput identification of small mole- vascular system and embryonic vascular development. In this study, cules that interfered with embryonic-like EC survival in a two- we identified two embryonic EC inhibitors, namely, fluphen- step protocol; (iii) confirmation of the hits in embryonic-like ECs azine and 7-Cyclo, using high-throughput screening. Fluphenazine cultured under flow shear stress; and (iv) final validation in medication is not recommended during pregnancy; however, for a mouse embryonic ECs and the zebrafish animal model. woman who is taking this medication at the time of conception, by Previous studies have performed very useful in vivo screens for the time the pregnancy is confirmed, most or all of the organo- inhibitors of vascular development in zebrafish embryos (36) and genesis has already occurred. An international epidemiological Xenopus embryos (37). Although in vivo screening systems in- survey of drug use in pregnancy showed that 3.5% of the women tegrate all physiological processes, they have some limitations that took psychotropic drugs during pregnancy (46). It was found that can be addressed by high-throughput cellular platforms. First, the the human fetus exposed to fluphenazine had severe rhinorrhea, identified compounds might affect embryonic as well as fetal/adult respiratory distress, and delayed extrapyramidal symptoms (47). vasculature. In the present work, we identified drugs that more Our study shows that fluphenazine has a higher effect in the dis-

specifically target embryonic ECs using a two-step protocol in- ruption of embryonic vascular networks than postnatal ones. SCIENCES volving (i) a primary screen against a nonvascular cell (ligament Previous studies have demonstrated that 7-Cyclo (20 μM) dis- cells) and (ii) a primary screen against fetal ECs (HUAECs). rupts blood vessel formation in Xenopus embryos and is an inhibitor APPLIED BIOLOGICAL Second, in the in vivo testing, the test compounds did not have of in vitro lymphangiogenesis (21, 37). Our results indicated that direct access to their target, but were metabolized or resorbed. In hESC-derived ECs exposed to medium supplemented with 7-Cyclo addition, other cell types or the immune system can alter the drug (1 μM) for 24 h under flow conditions showed an up-regulation of effects, which makes difficult to attribute the primary target for inflammatory genes and secretion of vascular injury markers. This the drug (21). Third, studies in animal embryos (zebrafish, Xen- effect was higher on hESC-derived ECs compared with HUAECs. opus, rats, and rabbits) do not account for differences in the vas- Similar results were also obtained for mouse embryonic ECs and cular development between humans and small animals (4). postnatal ECs (i.e., mouse embryonic ECs were sensitive to the Recent studies have shown the derivation of ECs from hPSCs toxicity of 7-Cyclo, whereas postnatal ECs showed no measurable with high efficiency in chemical defined medium (38, 39) and effect against the same compound). We also confirmed in vivo the exhibiting features of arterial-like ECs (39–42); however, it was effect of 7-Cyclo on ISV development of zebrafish embryos and unclear what their maturation stage was under flow conditions showed that it specifically blocked the motility and sprouting be- and whether they exhibited embryonic or adult features. Both havior of arterial ECs. features have significant impact on drug screening, as confirmed The effect of 7-Cyclo against embryonic-like ECs involved the by results of the present work. In this study, we derived inhibition of VEGFR2, which is highly expressed in embryonic ECs embryonic-like ECs. At the gene level, clustering analyses compared with fetal or adult ECs. This inhibition (at 0.1 μM) occurred showed that hESC-derived ECs were more related to embryonic in embryonic ECs (hESC-ECs), but not in fetal ECs (HUAECs), due than adult ECs. Although a defined set of embryonic EC gene to the high level of VEGFR2 expression in the former. However, the markers has not yet been identified, in this study, we identified overexpression of VEGFR2 in HUAECs makes these cells sensitive 328 embryonic EC genes, some of which were validated by qRT- to 0.1 μM 7-Cyclo. The inhibition of VEGFR2 leads to a significant PCR (DLL1, EPHB2, LYN, TEK, ID1, NRP2, CAST, FLT1, inhibition of AKT and ERK pathways with higher efficiency than a IGF1, DKK3, NIN, LEF1, and SORBS3). We further showed that commercial inhibitor. This inhibition, in turn, leads to cell necrosis. In embryonic ECs have a high expression of tyrosine kinases, in- conclusion, the platform described in this study is promising for the cluding EFS, VEGFR2, LYN, EGFR, ZAP70, NRP1, and TEK. identification of compounds that affect vascular development, as well Importantly, flow shear stress enhanced the maturation of ECs as to study embryonic vascular biology. as shown by an up-regulation of EC mechanosensory complex proteins VECAD and VEGFR2, cell alignment in the direction Methods of the flow, and production of HSPGs. Detailed methods are available in SI Appendix, SI Materials and Methods. There is currently a limited set of chemical compounds that have been described to affect human embryonic vascular devel- hPSC Culture and Differentiation. Undifferentiated hESCs (passages 33–36; H9, opment. Thalidomide, which was prescribed in the 1960s, is the WiCell) or hiPSCs K2 (passages 32–35; cord blood-derived iPSCs kindly donated most well-known embryonic vascular disruptive drug. Thalido- by Ulrich Martin; Leibniz Research Laboratories for Biotechnology and Artifi- mide inhibits angiogenesis via the prevention of filopodial ex- cial Organs, Department of Cardiothoracic, Transplantation and Vascular Sur- tensions from the endothelial tip cell (43). Thalidomide is one of gery, Hannover Medical School, Hannover, Germany) were grown on an inactivated mouse embryonic fibroblast feeder layer, as described (9, 48). Cells the drugs of the LOPAC; however, the drug was not a hit in our were induced to differentiate into ECs by using different protocols (SI Ap- screening. This finding was likely due to the high concentration > μ pendix, SI Materials and Methods). hESCs were used with the approval of the of the drug ( 5 M) that is required to affect cell viability, as ethics commitee from University of Coimbra. previously shown (44). + + In the present study, the screening of the chemical library was Isolation of CD31 Cells. CD31 cells were isolated from differentiated hESCs performed at one concentration (4.5 μM), and the endpoint was or hiPSCs at day 18 by using MACS (Miltenyi Biotec). Isolated cells were grown

Vazão et al. PNAS Early Edition | 9of10 Downloaded by guest on September 29, 2021 on Petri dishes (1.5 × 104 cells per cm2) coated with 0.1% gelatin and containing High-Throughput Screening. The LOPAC (Sigma-Aldrich) was used to screen EGM-2 (Lonza) supplemented with SB431542 (10 μM). The methodolo- compounds that interfere with embryonic ECs. The compounds were solubilized gies for cell culture under flow conditions and cell characterization at in DMSO, and the final concentration used in the screen was 4.5 μM in a final gene, protein, and functional levels are provided in SI Appendix, SI Ma- volume of 200 μL per well (96-well plate). After 4 d of incubation, HUAEC or terials and Methods. hESC-derived EC viability was assessed by using a PrestoBlue (Invitrogen) assay.

Human and Mouse Primary Cells. HUAECs and HUVECs were acquired from Zebrafish Line and Treatment with 7-Cyclo. Zebrafish Tg(fli1a:eGFP)y1 embryos Lonza. mAECs E12.5 and mAECs p1 were isolated from mice cultured for 2– (30) were used to evaluate the effect of 7-Cyclo on ISV development. The 3 d in vitro in EC medium and immediately used. The cells were obtained protocol is described in SI Appendix, SI Materials and Methods. from Innoprot. Statistical Analysis. An unpaired t test or one-way ANOVA with Newman– Gene Expression Analyses (Microarray and qRT-PCR), Kinase Activity Quantification, Keuls posttest was performed for statistical tests using GraphPad Prism Phosphorylation of AKT/ERK/VEGFR2, Matrigel Assays, Cell Viability Assays, and software. Results were considered significant when P < 0.05. + Intracellular Ca2 Analyses. The methods are found in SI Appendix, SI Materials and Methods. ACKNOWLEDGMENTS. This work was supported by FEDER through the Pro- gram COMPETE and by Portuguese funding through FCT in context of the projects PTDC/SA-BEB/098468/2008, PTDC/SAU-TOX/121887/2010, and MITP- Evaluation of the Levels of Vascular Injury by Specific Markers. ELISA kits for TB/ECE/0013/2013. The work was also supported by COMPETE in the context vWF and vWFpp (Gen-Probe GTI Diagnostic) and ADMA (Enzo Life Sciences) of Project “Stem-Cell Based Platforms for Regenerative and Therapeutic were used to analyze supernatants collected from the shear stress experi- Medicine” (Centro-07-ST24-FEDER-002008). H.V. was supported by FCT for ments, according to manufacturer’s recommendations. BD and BPD Fellowships SFRH/BD/40077/2007 and SFRH/BPD/87039/2012.

1. Makris SL, et al. (2011) Current and future needs for developmental toxicity testing. 26. Alves H, Dechering K, Van Blitterswijk C, De Boer J (2011) High-throughput assay for Birth Defects Res B Dev Reprod Toxicol 92(5):384–394. the identification of compounds regulating osteogenic differentiation of human 2. Vasvari G, Dyckhoff G, Herold-Mende C (2005) Thalidomide protects endothelial cells mesenchymal stromal cells. PLoS One 6(10):e26678. from doxorubicin-induced apoptosis but alters cell morphology—a rebuttal. J Thromb 27. Laurencin CT, Freeman JW (2005) Ligament tissue engineering: An evolutionary Haemost 3(4):816–817, author reply 817–818. materials science approach. Biomaterials 26(36):7530–7536. 3. Knudsen TB, Kleinstreuer NC (2011) Disruption of embryonic vascular development in 28. Blanc A, Pandey NR, Srivastava AK (2004) Distinct roles of Ca2+, calmodulin, and predictive toxicology. Birth Defects Res C Embryo Today 93(4):312–323. C in H2O2-induced activation of ERK1/2, p38 MAPK, and protein kinase 4. Kleinstreuer NC, et al. (2011) Environmental impact on vascular development pre- B signaling in vascular smooth muscle cells. Antioxid Redox Signal 6(2):353–366. dicted by high-throughput screening. Environ Health Perspect 119(11):1596–1603. 29. Borbiev T, et al. (2003) Role of CaM kinase II and ERK activation in thrombin-induced 5. Mellin GW, Katzenstein M (1962) The saga of thalidomide. Neuropathy to embry- endothelial cell barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 285(1): opathy, with case reports of congenital anomalies. N Engl J Med 267:1184–92. L43–L54. 6. Tideman E, Marsál K, Ley D (2007) Cognitive function in young adults following in- 30. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development – trauterine growth restriction with abnormal fetal aortic blood flow. Ultrasound using transgenic zebrafish. Dev Biol 248(2):307 318. Obstet Gynecol 29(6):614–618. 31. Bayliss PE, et al. (2006) Chemical modulation of receptor signaling inhibits re- – 7. Levenberg S, Zoldan J, Basevitch Y, Langer R (2007) Endothelial potential of human generative angiogenesis in adult zebrafish. Nat Chem Biol 2(5):265 273. embryonic stem cells. Blood 110(3):806–814. 32. Hirst G, et al. (2006) US Patent 7071199 B1. 8. Fiddes LK, et al. (2010) A circular cross-section PDMS microfluidics system for repli- 33. Arnold LD, et al. (2000) Pyrrolo[2,3-d]pyrimidines containing an extended 5-sub- cation of cardiovascular flow conditions. Biomaterials 31(13):3459–3464. stituent as potent and selective inhibitors of I. Bioorg Med Chem Lett 10(19): – 9. Ferreira LS, et al. (2007) Vascular progenitor cells isolated from human embryonic 2167 2170. stem cells give rise to endothelial and smooth muscle like cells and form vascular 34. Endo A, Fukuhara S, Masuda M, Ohmori T, Mochizuki N (2003) Selective inhibition of networks in vivo. Circ Res 101(3):286–294. vascular endothelial growth factor receptor-2 (VEGFR-2) identifies a central role for 10. Smart N, et al. (2007) Thymosin beta4 induces adult epicardial progenitor mobiliza- VEGFR-2 in human aortic endothelial cell responses to VEGF. J Recept Signal Transduct Res 23(2-3):239–254. tion and neovascularization. Nature 445(7124):177–182. 35. Ferrara N, Gerber H-P, LeCouter J (2003) The biology of VEGF and its receptors. Nat 11. James D, et al. (2010) Expansion and maintenance of human embryonic stem cell- Med 9(6):669–676. derived endothelial cells by TGFbeta inhibition is Id1 dependent. Nat Biotechnol 36. Tran TC, et al. (2007) Automated, quantitative screening assay for antiangiogenic 28(2):161–166. compounds using transgenic zebrafish. Cancer Res 67(23):11386–11392. 12. Haase A, et al. (2009) Generation of induced pluripotent stem cells from human cord 37. Kälin RE, Bänziger-Tobler NE, Detmar M, Brändli AW (2009) An in vivo chemical li- blood. Cell Stem Cell 5(4):434–441. brary screen in Xenopus tadpoles reveals novel pathways involved in angiogenesis 13. Swiers G, et al. (2013) Early dynamic fate changes in haemogenic endothelium and lymphangiogenesis. Blood 114(5):1110–1122. characterized at the single-cell level. Nat Commun 4:2924. 38. Patsch C, et al. (2015) Generation of vascular endothelial and smooth muscle cells 14. Malek AM, Alper SL, Izumo S (1999) Hemodynamic shear stress and its role in ath- from human pluripotent stem cells. Nat Cell Biol 17(8):994–1003. erosclerosis. JAMA 282(21):2035–2042. 39. Orlova VV, et al. (2014) Functionality of endothelial cells and pericytes from human 15. Tzima E, et al. (2005) A mechanosensory complex that mediates the endothelial cell pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xeno- response to fluid shear stress. Nature 437(7057):426–431. grafts. Arterioscler Thromb Vasc Biol 34(1):177–186. 16. Potter DR, Damiano ER (2008) The hydrodynamically relevant endothelial cell glyco- 40. Rufaihah AJ, et al. (2013) Human induced pluripotent stem cell-derived endothelial calyx observed in vivo is absent in vitro. Circ Res 102(7):770–776. cells exhibit functional heterogeneity. Am J Transl Res 5(1):21–35. 17. Ho PY, Zhong WB, Ho YS, Lee WS (2006) Terbinafine inhibits endothelial cell mi- 41. White MP, et al. (2013) Limited gene expression variation in human embryonic stem cell gration through suppression of the Rho-mediated pathway. Mol Cancer Ther 5(12): and induced pluripotent stem cell-derived endothelial cells. Stem Cells 31(1):92–103. – 3130 3138. 42. Ditadi A, et al. (2015) Human definitive haemogenic endothelium and arterial vas- 18. Ho PY, Liang YC, Ho YS, Chen CT, Lee WS (2004) Inhibition of human vascular en- cular endothelium represent distinct lineages. Nat Cell Biol 17(5):580–591. – dothelial cells proliferation by terbinafine. Int J Cancer 111(1):51 59. 43. Therapontos C, Erskine L, Gardner ER, Figg WD, Vargesson N (2009) Thalidomide 19. Pullamsetti S, et al. (2005) Increased levels and reduced catabolism of asymmetric and induces limb defects by preventing angiogenic outgrowth during early limb forma- – symmetric dimethylarginines in pulmonary hypertension. FASEB J 19(9):1175 1177. tion. Proc Natl Acad Sci USA 106(21):8573–8578. 20. Louden C, et al. (2006) Biomarkers and mechanisms of drug-induced vascular injury in 44. Kaushal V, Kaushal GP, Melkaveri SN, Mehta P (2004) Thalidomide protects endo- – non-rodents. Toxicol Pathol 34(1):19 26. thelial cells from doxorubicin-induced apoptosis but alters cell morphology. J Thromb 21. Schulz MMP, et al. (2012) Phenotype-based high-content chemical library screening Haemost 2(2):327–334. identifies statins as inhibitors of in vivo lymphangiogenesis. Proc Natl Acad Sci USA 45. Wang C, et al. (2010) Rosuvastatin, identified from a zebrafish chemical genetic 109(40):E2665–E2674. screen for antiangiogenic compounds, suppresses the growth of prostate cancer. Eur 22. Kitami T, et al. (2012) A chemical screen probing the relationship between mito- Urol 58(3):418–426. chondrial content and cell size. PLoS One 7(3):e33755. 46. Marchetti F, Romero M, Bonati M, Tognoni G; Collaborative Group on Drug Use in 23. Qosa H, et al. (2016) High-throughput screening for identification of blood-brain Pregnancy (CGDUP) (1993) Use of psychotropic drugs during pregnancy. A report of barrier integrity enhancers: A drug repurposing opportunity to rectify vascular am- the international co-operative drug use in pregnancy (DUP) study. Eur J Clin yloid toxicity. J Alzheimers Dis 53(4):1499–1516. Pharmacol 45(6):495–501. 24. Kleinstreuer NC, et al. (2014) Phenotypic screening of the ToxCast chemical library to 47. Iqbal MM, et al. (2005) The potential risks of commonly prescribed antipsychotics: classify toxic and therapeutic mechanisms. Nat Biotechnol 32(6):583–591. during pregnancy and lactation. Psychiatry (Edgmont) 2(8):36–44. 25. Ghebes CA, van Lente J, Post JN, Saris DB, Fernandes H (2017) High-throughput 48. Vazão H, das Neves RP, Grãos M, Ferreira L (2011) Towards the maturation and screening assay identifies small molecules capable of modulating the BMP-2 and characterization of smooth muscle cells derived from human embryonic stem cells. TGF-β1 signaling pathway. J Biomol Screen 22(1):40–50. PLoS One 6(3):e17771.

10 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1617451114 Vazão et al. Downloaded by guest on September 29, 2021