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. 2017 Apr 11;114(15):E3022-E3031.
doi: 10.1073/pnas.1617451114. Epub 2017 Mar 27.

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

Helena Vazão  1 , Susana Rosa  1 , Tânia Barata  1   2 , Ricardo Costa  3 , Patrícia R Pitrez  1 , Inês Honório  1 , Margreet R de Vries  4 , Dimitri Papatsenko  5 , Rui Benedito  3 , Daniel Saris  2 , Ali Khademhosseini  6   7   8   9   10 , Paul H A Quax  4 , Carlos F Pereira  1 , Nadia Mercader  11 , Hugo Fernandes  1   2 , Lino Ferreira  12
Affiliations

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

Helena Vazão et al. Proc Natl Acad Sci U S A. .

Abstract

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Keywords: embryonic endothelial markers; endothelial cells; high-throughput screening; pluripotent stem cells; vascular toxicity.

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Conflict of interest statement

Conflict of interest statement: A patent has been filed for this work (“Differentiated Cell Population of Endothelial Cells Derived from Human Pluripotent Stem Cells, Composition System, Kit and Uses Therefore”; PCT/IB2013/061110).

Figures

Fig. 1.
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 corresponding gene expression in undifferentiated hESCs. Results are mean ± SEM (n = 4). (C) Expression of EC proteins 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 activation 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).
Fig. 2.
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 fluphenazine. Values are normalized against nontreated cells (control). Results are mean ± SEM (n = 4).
Fig. 3.
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) and sprouts (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. 4.
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 modulation (eNOS), and vascular injury sensing (DDAH1 and DDAH2) in hESC-derived ECs and HUAECs after 24 h of incubation with 0 or 1 μM 7-Cyclo. Results are 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.
Fig. 5.
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.
Fig. 6.
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 protein 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) and ERK (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.
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