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. 2022 Oct 31;14(19):1139–1155. doi: 10.2217/epi-2022-0110

Alteration of the N6-methyladenosine epitranscriptomic profile in synthetic phthalate-treated human induced pluripotent stem cell-derived endothelial cells

Jordan Jousma 1,‡, Zhenbo Han 1,‡, Gege Yan 1,‡, Sarath Babu Nukala 1, Youjeong Kwon 1, Hoai Huong Thi Le 2, Ya Li 1, Sang-Bing Ong 3,4,5,6, Won Hee Lee 2,*, Sang-Ging Ong 1,3,7,**
PMCID: PMC9710528  PMID: 36314267

Abstract

Background

This study aimed to characterize the N6-methyladenosine epitranscriptomic profile induced by mono(2-ethylhexyl) phthalate (MEHP) exposure using a human-induced pluripotent stem cell-derived endothelial cell model.

Methods

A multiomic approach was employed by performing RNA sequencing in parallel with an N6-methyladenosine-specific microarray to identify mRNAs, lncRNAs, and miRNAs affected by MEHP exposure.

Results

An integrative multiomic analysis identified relevant biological features affected by MEHP, while functional assays provided a phenotypic characterization of these effects. Transcripts regulated by the epitranscriptome were validated with quantitative PCR and methylated RNA immunoprecipitation.

Conclusion

The authors' profiling of the epitranscriptome expands the scope of toxicological insights into known environmental toxins to under surveyed cellular contexts and emerging domains of regulation and is, therefore, a valuable resource to human health.

Keywords: : epitranscriptomic, iPSCs, mono(2-ethylhexyl) phthalate, N6-methyladenosine, synthetic phthalate, toxicology screening

Plain language summary

Synthetic phthalates, such as mono(2-ethyhexyl) phthalate, have long been recognized as environmental toxins. What effect these compounds have on endothelial cells remains poorly understood. To address this, the authors utilized a human-induced pluripotent stem cell-derived endothelial cell model to screen for an environmental toxin. They then obtained a profile of the epitranscriptomic changes involving the N6-methyladensosine modification and performed biochemical and functional assays. Overall, this study demonstrated how stem cell-based approaches can be used for toxicological screening and provided a valuable resource that profiles the epitranscriptomic response, which was complemented with RNA sequencing and functional and biochemical assays. This study provides relevant toxicological insights into the context of human health.

The global production of phthalates for plasticizers is more than 2 million tons per year [1]. Synthetic phthalates are most often used as plasticizers to promote flexibility in plastic products. The inability to form covalent bonds within the polymerized lattice in plastics makes them ideal for these industrial applications; however, this principle also makes them particularly susceptible to leaching. As a result, exposure to phthalates is generally considered widespread and has been implicated in the development of numerous health problems. The most-used synthetic phthalate is di(2-ethylhexyl) phthalate. Exposure to di(2-ethylhexyl) phthalate under physiological conditions results in the production of the biologically active metabolite mono(2-ethylhexyl) phthalate (MEHP) [2,3]. Phthalates are found in a wide range of plastic products, including packaged foods, furniture, toys, and medical devices [4]. Exposure to synthetic phthalates such as MEHP is generally considered to be widespread within a wide range of serum concentrations [5,6]. Despite their pervasive use in society, plasticizers such as MEHP have been recognized as an environmental toxin, known to cause harm to humans, most often in reproductive health contexts [7–11]. Specific environmental conditions play an important role in determining the net exposure to which an individual might be subjected. Among those subjected to the highest exposure levels are individuals receiving intravenous therapies such as hemodialysis [12–14]. This is believed because medical tubing contains an extraordinarily high concentration of synthetic phthalates, sometimes as much as 80% di(2-ethylhexyl) phthalate by material weights [15]. Recent investigations have recognized this route of exposure and have begun to address adverse effects believed to be imparted to the cardiovascular system as a result [16,17]. Others have also implicated the involvement of synthetic phthalate exposure in establishing vascular dysfunction [18–21].

While such observations have expanded the toxicological insights regarding the effects of synthetic phthalates into new physiological contexts, there remains an overall lack of understanding of the precise molecular mechanisms responsible for mediating dysfunction. The epitranscriptome comprises a series of chemical modifications made post-transcriptionally to RNAs and has recently been appreciated as a mechanism responsible for upholding a myriad of molecular mechanisms involved in health and diseases. The most abundant epitranscriptomic modification utilized by mammalian cells is the reversible N6-methyladenosine (m6A) modification, which affects many aspects of RNA biology, including stability, splicing, localization and the establishment of structural configurations, which are crucial for the proper function of ncRNAs [22–25]. The presence of m6A modification is determined by the activity of m6A ‘writer’ enzymes such as METTL3/14, ‘eraser’ enzymes such as FTO and ALKBH5 and ‘reader’ enzymes such as YTHDF1/2/3 [24]. By recognizing the m6A modification, reader enzymes can regulate RNA. These modifications have crucial roles in mediating cellular stress responses to changes in environmental conditions, since they allow for a rapid change in the activity of a transcript [26,27]. Additionally, these modifications have been shown to have an essential role in development, health and disease [28–30]. The effects that synthetic phthalates have on the epitranscriptome have thus far been largely unexplored.

The use of human induced pluripotent stem cell (hiPSC)-based approaches has presented a tremendous opportunity over the last decade to investigate human health in more relevant contexts and screen for toxicological consequences induced by drugs or environmental toxins [31–35]. Experimentally hiPSC-derived endothelial cells (hiPSC-ECs) have been shown to be highly accurate in recapitulating the biological functions of primary ECs [36]. Therefore, this study sought to leverage advances in hiPSC-based approaches to evaluate the potential toxicological consequences of synthetic phthalate exposure in hiPSC-ECs. Furthermore, it brings attention to the epitranscriptome, which is largely unexplored in the context of synthetic phthalate exposure. This epitranscriptomic approach was then complemented with RNA sequencing performed in parallel as well as in vitro functional assessments.

Materials & methods

EC differentiation & cell culture

ECs were differentiated from three different lines of human iPSCs as previously described [37,38]. All procedures conformed to the University of Illinois Chicago institutional review board-approved protocol. To initiate differentiation, cells were cultured in N2B27 media supplemented with 8 μmol CHIR and 25 ng/ml BMP4 for 72 h. Media were then replaced with advanced Dulbecco's modified Eagle medium/F12 supplemented with 100 ng/ml of VEGF and 2 μmol forskolin and replenished every 24 h for 4 days. Affinity purification using magnetic beads conjugated to CD144 was used to isolate the ECs. Immunofluorescence staining for endothelial markers, CD31, CD144, and acetylated low-density lipoprotein were used to validate endothelial identity. Differentiated hiPSC-ECs were cultured in EGM-2™ Endothelial Cell Growth Media (Lonza) and incubated at 37°C with 5% CO2 in plates precoated with fibronectin. Cells were treated with 120 μmol MEHP (Wako Chemicals) for 72 h. MEHP was dissolved in dimethyl sulfoxide (DMSO), and the concentration of DMSO was maintained at 0.1% for MEHP treatment and control.

RNA sequencing preparation & analysis

Total RNA extractions were obtained from three lines of hiPSC-ECs and submitted for RNA sequencing. Raw Fastq files were then uploaded to a cloud-based analysis suite, BioJupies [39], for mapping and quality control, while EnrichR was used for ontological analysis [40,41]. The set of differentially expressed genes (DEGs) with average expression values and Log2FC were supplied to Ingenuity Pathway Analysis (Qiagen) and assessed for downstream toxicological effects and disease states associated with the set of DEGs. Canonical pathways were identified from the set of DEGs and were then assigned p-values and ranked in order to assemble a network map. Quantile normalized read counts were used for gene set enrichment analysis. Heat maps were constructed in Morpheus (Broad Institute). Raw FASTQ files can be accessed through the Gene Expression Omnibus database at the accession number GSE214420.

Microarray hybridization

Total RNA extractions were obtained from three lines of hiPSC-ECs after treatment with MEHP and prepared according to the Arraystar standard protocol. Total RNAs were labeled with an anti-m6A antibody and immunoprecipitated with magnetic beads. Immunoprecipitated RNAs were labeled with Cy5 (IPCy5), while the remaining unmodified, supernatant portion was labeled with Cy3 (SupCy3). Labeled RNAs were then hybridized onto Arraystar Human mRNA & lncRNA Epitranscriptomic Microarray (8 × 60 K, Arraystar). Raw intensities of immunoprecipitated RNAs were normalized with an average Log2-scaled spike-in RNA control. Hybridized slides were scanned and analyzed with Agilent Scanner G2505C and Agilent Feature Extraction software. The probes used within this microarray were designed within unique regions of gene isoforms, allowing for efficient detection of all isoforms within a gene family. Labeling both m6A-modified and -unmodified transcripts provided a read for both the m6A methylation quantity and the m6A methylation level. The m6A methylation level was calculated as the percentage of m6A-modified transcripts (Cy5-labeled) relative to the total amount of transcripts detected.

Fold changes and p-values were calculated from raw intensities. A significance threshold of p ≤ 0.05 and Log2FC ± 1 was used to identify differentially methylated transcripts across conditions. In addition to the transcript ID, each probe used in the array was supplied with feature annotations to assign a subcellular location to the transcript ID, which was curated by Arraystar Human mRNA & lncRNA Epitranscriptomic Microarray database. Raw data files be accessed through the Gene Expression Omnibus database at the accession number GSE214420.

Quantitative reverse transcription PCR

Total RNA was isolated with the RNeasy (Qiagen) extraction kit using the gDNA eliminator column. RNA concentration and purity were measured using BioDrop (BioDrop). RNA was reverse transcribed using a high-capacity RNA to cDNA kit (Applied Biosystems). Gene expression assays of m6A readers (YTHDF1/2/3), writers (METTL3/14) and erasers (FTO and ALKBH5) were evaluated using TaqMan Real-Time PCR master mix, while all other targets were assayed using standard PCR and SYBR Green. All primer sequences are listed in the Supplementary Materials Table. Reactions were analyzed by QuantStudio™ 7 Flex using the ∆∆Ct method to determine expression levels.

RNA methylation quantification

Quantification of m6A was achieved using the EpiQuick m6A RNA methylation quantification kit (Fluorometric, EpiGentek) from total RNA isolates. RNA concentrations were measured using BioDrop. As per the manufacturer's instructions, 200 ng of total RNA was used in each reaction and incubated with m6A capture and detection antibodies. The FlexStation III plate reader was set to 530Ex/590EM to measure fluorescent intensity.

m6A RNA dot blot

A total of 400 ng of RNA was serially diluted to 200 and 100 ng. Each 3 µl dot was placed onto Hybond-N+ (nylon transfer membrane), 0.45 µm (GE Amersham, RPN 203B), and crosslinked to the membrane using a UV crosslinker (Analytik Jena). The blocking solution contained 5% skim milk (BD, 232100) in phosphate-buffered saline with 0.1% Tween® 20. Total RNA blots were visualized using 0.2% methylene blue solution (Sigma, M4159-25G), and m6A blots were visualized using an m6A antibody (Millipore, ABE572).

Western blotting

Protein samples were lysed in RIPA buffer and measured using the Pierce Rapid Gold BCA protein assay kit (ThermoScientific, A53225). Absorbance was measured at 480 nm using FlexStation III. Samples were separated with polyacrylamide gel and blocked with a blocking solution (Thermo, 37515). Antibodies used are listed in the Supplementary Materials Table. Images were analyzed using the iBright western Blot Imaging System. The resulting images were quantified for relative intensity using ImageJ.

Wound healing assay

hiPSC-ECs or mouse cardiac ECs were seeded onto either fibronectin or gelatin-coated 24-well plates and grown to confluence. Cells were then exposed to 120 μmol MEHP. Scratches were made using a sterile pipette tip. Images were recorded at time zero and time 18 h. Images were then analyzed using the MRI wound healing tool plug-in for ImageJ, and the resulting areas calculated were used to determine percent closure.

Tube formation assay

hiPSC-ECs were cultured for 72 h in 120 μmol MEHP or DMSO and then seeded into wells of μ-Slide Angiogenesis (Ibidi #81506). The μ-Slide Angiogenesis wells were precoated with 10 μl of Matrigel and incubated at 37°C for 1 h, then seeded with 1 × 104 cells in 50 μl. Images were then recorded after 14 h on the Echo Revolution microscope and analyzed using the angiogenesis plugin for ImageJ.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 15 min, washed in phosphate-buffered saline, permeabilized in 0.5% TritonX100/Dulbecco's phosphate-buffered saline at room temperature for 10 min and then blocked in 5% donkey serum for 1 h at room temperature. Primary antibodies were then diluted in 5% donkey serum and incubated overnight at 4°C. Samples were then washed in phosphate-buffered saline with 0.1% Tween 20 before secondary antibody incubation for 2 h at room temperature, washed and incubated in 4′,6-diamidino-2-phenylindole and sealed with PermaFluor (Thermo Scientific).

Results

A hiPSC-EC model of phthalate exposure

Three healthy hiPSC lines were differentiated into ECs. After differentiation, magnetic CD144 beads were used to isolate ECs. hiPSC-ECs were then validated for endothelial identity. This was achieved by immunofluorescence staining for endothelial-specific markers CD31 (PECAM-1) and CD144 (VE-Cadherin), as well as uptake of acetylated-low density lipoprotein, a hallmark feature of ECs. The isolated ECs were also observed to have a cobblestone morphology (Figure 1A). After confirming endothelial identity, hiPSC-ECs were treated with 120 μmol MEHP, which is within the observed range of synthetic phthalates observed in stored blood products and recognized as eliciting inflammatory responses. Furthermore, the selected concentration is consistent with that used in previous in vitro models, where it has been shown to induce dysfunction at levels that are below the threshold of inducing cytotoxicity [12,42–44]. The effects of MEHP treatment were then characterized using a multiomic approach to understand how changes in epitranscriptomic regulation involving the m6A modification might contribute to alterations in gene expression and endothelial function (Figure 1B & C).

Figure 1. . Validation of human induced pluripotent stem cell-derived endothelial cell identity and experimental design.

Figure 1. 

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(A) Endothelial cell (EC) identity was validated with immunofluorescence staining showing positive staining for CD31 and CD144. Uptake of acetylated low-density lipoprotein and cobblestone morphology provide additional features that validate EC identity. (B) Schematic differentiation protocol used to create ECs from human induced pluripotent stem cell-derived EC (hiPSC) and experimental design. hiPSC-ECs were treated with either dimethyl sulfoxide or mono(2-ethylhexyl) phthalate and subjected to multiomic evaluation and functional characterization. An epitranscriptomic N6-methyladenosine (m6A) microarray was used to evaluate m6A levels. (C) Illustration of the most common epitranscriptomic modifications with the m6A modification shown in green, the most abundant modification made to coding regions created using Biorender.

RNA sequencing of MEHP-treated hiPSC-ECs

hiPSC-ECs were treated with MEHP or DMSO for 72 h and then subjected to RNA sequencing. MEHP exposure resulted in the upregulation of 165 genes and downregulation of 384 genes (p ≤ 0.05 and Log2FC ± 1; Figure 2A). Individual z-scores for DEGs were organized by hierarchical clustering and compiled into a heat map (Figure 2B). Additional annotations describing the overall quality of the data and effects of MEHP treatment, including principal component analysis, are shown in Supplementary Figure 1A–C. To determine downstream pathways that might be affected by exposure to MEHP, gene set enrichment analysis was performed using quantile normalized read counts and assessed using the Reactome database (Figure 2C). This resulted in the identification of pathways that have previously been reported to be altered by exposure to synthetic phthalates, such as IFN-α/β signaling and pathways involving translation initiation, which is a process that can be regulated by m6A modifications [45,46]. Additional ontological analyses were performed using the top 1000 DEGs using the Gene Ontology database, which highlighted the involvement of other biological activities such as extracellular substrate-binding activities and focal adhesion binding (Supplementary Figure 1D). Ingenuity Pathway Analysis predicted downstream consequences associated with gene expression patterns. This analysis provided a list of the annotations related to potential disease outcomes and biological functions in one module. The most significant term identified in this analysis is cancer-related, but it also recognizes the involvement of cardiovascular functions and RNA post-transcriptional modifications (Figure 2D). A similar module created a toxicological profile from the Ingenuity Pathway Analysis software, highlighting several possible signatures associated with cardiovascular dysfunction (Figure 2E). Finally, a network map was constructed that identified signatures of canonical pathways contained with the set of dysregulated genes, where network edges reveal a shared set of genes contained within these annotated terms, highlighting a highly interconnected set of genes involved in the canonical pathways shown. Within this network map, there were multiple canonical signaling nodes identified that are known to be involved in endothelial functions and thus may have phenotypic consequences. These included, but were not limited to, ephrin and thrombin signaling (Figure 2F).

Figure 2. . Transcriptomic signature of mono(2-ethylhexyl) phthalate-treated human induced pluripotent stem cell-derived endothelial cells.

Figure 2. 

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(A) The effects of MEHP treatment on gene expression are shown in the volcano plot with the most differentially regulated genes annotated (p ≤ 0.05; Log2FC ± 1). (B) Hierarchical clustering organization of genes with significant changes in expression values shows how corresponding changes vary across treatment groups in three separate lines of human induced pluripotent stem cell-derived endothelial cells. (C) Gene set enrichment analysis was performed using the Reactome database to recognize pathways that might be dysregulated after treatment with MEHP. (D) Analysis of downstream effects associated with the gene expression signature was determined using Ingenuity Pathway Analysis (IPA) to assess for signatures of diseases and biological functions and (E) to create a toxicological profile. (F) The most significantly identified canonical pathways identified by IPA were compiled into a network where nodes represent the indicated pathway. The edges indicate a shared set of genes contained within the respective canonical pathway as determined by IPA.

MEHP: Mono(2-ethylhexyl) phthalate.

Epitranscriptomic signature of MEHP exposure

The epitranscriptomic profile was also obtained from hiPSC-ECs treated with DMSO or MEHP for 72 h, determined by an m6A-specific microarray (Arraystar). Overall, 628 out of 20,370 genes probed had significant differences in m6A quantity detected (p ≤ 0.05 and Log2FC ± 1). Most of the differentially methylated transcripts experienced reductions in m6A deposition (586 decreased, 42 increased). However, most array probes indicated that no significant changes had occurred (Figure 3A & B). Among the differentially regulated transcripts, there was a trend toward downregulation across all classes of transcripts interrogated by the microarray (Supplementary Figure 2A–C). Each probe within the microarray represented a unique RefSeq ID. If a single isoform within a gene family was significant, then that gene was considered to be significantly affected. The expression changes of each specific RefSeq ID within a gene family were compiled in a supplementary data file (Supplementary Table 1). To provide an overview of how the epitranscriptome is affected by MEHP exposure, an ontological analysis was performed to evaluate the entire population of differentially expressed transcripts, including mRNAs, lncRNAs and miRNAs that experienced changes in m6A status. Downregulated and upregulated transcripts were each specifically considered (Figure 3C). These ontological annotations revealed selected terms similar to those identified within the RNA sequencing (RNA-seq) dataset, including terms related to the extracellular matrix. There also were numerous other terms that bore little resemblance to ontological annotations identified within the RNA-seq set. Additional annotations were also performed on transcripts that had alterations in m6A levels, which were determined as the expression of m6A transcripts relative to the expression of non-immunoprecipitated transcripts (Supplementary Figure 2A–E). This population of transcripts was overall smaller in terms of the total number of affected transcripts and magnitude of effect.

Figure 3. . Epitranscriptomic profile of RNA with altered N6-methyladenosine quantity in response to mono(2-ethylhexyl) phthalate exposure.

Figure 3. 

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(A) Volcano plot of transcripts recognized by microarray as having differences in N6-methyladenosine abundance on a transcript. (B) Pie chart showing the relative proportions of differentially methylated transcripts in terms of the direction of effect. (C) Gene Ontology analysis was performed on the set of differentially methylated transcripts with p ≤ 0.05 and Log2FC ≥ ± 1.

M6A modifications serve as a regulatory nexus, capable of mediating a diverse set of regulatory outcomes depending on how epitranscriptomic regulators interpret the mark. For instance, the presence of m6A modification has been reported to increase transcript stability, leading to increased transcript abundance. In contrast, m6A modification can also destabilize a transcript and lead to a reduction in transcript abundance [47]. Thus, m6A profiling performed in parallel with RNA-seq has shown both positive and negative correlations with transcript abundance [48,49]. To provide a functional context to those transcripts identified as having significant differences in the quantity of m6A detected by microarray, an integrative analysis involving both the RNA-seq and microarray data was performed. In comparing the entire population of transcripts differentially regulated, there were different proportions of transcripts identified in a single dataset or both datasets (Figure 4A). All transcripts within overlapping, cis-regulatory regions were then plotted against one another in terms of Log2FC (Figure 4B). The Log2FC change in abundance of m6A on the corresponding transcript was strongly associated with Log2FC changes in expression values obtained in the RNA-seq data. Thus, the authors consider this population of transcripts to be one that may experience changes in gene expression, which might involve epitranscriptomic regulation. To gain insights into this functionally distinct class of epitranscriptomic regulated changes in gene expression, a pathway analysis was performed using the set of 170 genes contained within the overlapping regions of the Venn diagram (Figure 4C). Within this pathway analysis, the NOTCH1 regulation of the EC cell calcification pathway stood out from the rest. This set of genes was also annotated with ontological classifications using the Gene Ontology database (Figure 4D). Interestingly, among the most significant annotations identified within this Gene Ontology cellular compartment analysis was the collagen-containing extracellular matrix annotation, which was also listed in the previous analysis (Figure 3C).

Figure 4. . Epitranscriptomic profile of RNA with altered N6-methyladenosine quantity in response to mono(2-ethylhexyl) phthalate exposure.

Figure 4. 

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(A) Venn diagram showing the overlap between the microarray and RNA sequencing data of significantly dysregulated genes with p ≤ 0.05 and Log2FC ≥ ± 1. (B) The correlation matrix of transcripts with a significant difference in gene expression detected by RNA sequencing and N6-methyladenosine abundance was determined by comparing with Log2FC. (C) The resulting 170 transcripts that experienced epitranscriptomic cis-regulatory outcomes were then isolated from the combined datasets and used for downstream pathway analysis using the WikiPathway database and (D) the Gene Ontology database.

Functional consequences of MEHP exposure in hiPSC-ECs

To determine the effects of MEHP on endothelial health, immunofluorescence staining was performed to evaluate the expression of vascular inflammation markers, VCAM-1 and ICAM-1 (Figure 5A & B). There was no overt difference observed in cells treated with MEHP compared with DMSO-treated cells in the expression of either of these markers. To address possible functional impairments that might arise from exposure to MEHP, hiPSC-ECs were treated with MEHP and then evaluated in a wound healing assay and a tube formation assay (Figure 5C & D). Quantification of wound healing and tube formation assays did not reveal any significant changes in hiPSC-EC function. Wound healing capabilities were also evaluated in mouse cardiac ECs, which showed no significant difference in endothelial function (Supplementary Figure 3A). The proliferation marker KI-67 was assessed by immunofluorescence staining to determine how MEHP affects the rate of proliferation of hiPSC-ECs. After treatment with MEHP for 72 h, there was a significant increase in the expression of KI-67, indicating that MEHP appears to promote proliferation (Figure 5E). Additionally, a cell viability assay was performed (Figure 5F), which showed that there were no significant impairments to cell viability after treatment with MEHP. Overall, these results indicate that MEHP has no clear effects on inflammation; however, it was seen that MEHP induced a significant increase in endothelial proliferation.

Figure 5. . Molecular characterization of mono(2-ethylhexyl) phthalate treatment in human induced pluripotent stem cell-derived endothelial cells.

Figure 5. 

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(A) Immunofluorescence staining of human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) treated with mono(2-ethylhexyl) phthalate (MEHP) assessing for markers of vascular inflammation, such as VCAM-1 and (B) ICAM-1. (C) Functional assessments of MEHP exposure in hiPSC-ECs were determined using a wound healing assay (n = 6) and (D) tube formation assay (n = 3). (E) Immunofluorescence staining determined EC proliferation activity using the proliferation marker KI-67 with quantification of fluorescence intensity relative to controls (n = 4). (F) Cell viability assay performed in hiPSC-ECs treated with the indicated concentrations of MEHP for 72 h.

*p ≤ 0.05. Data are represented as mean ± standard error of the mean.

MEHP: Mono(2-ethylhexyl) phthalate.

Validation of m6A regulation & transcriptomic responses to MEHP

The gene and protein expression levels of m6A readers, writers and eraser enzymes were evaluated from hiPSC-ECs after treatment with MEHP for 72 h (Figure 6A & B). No significant changes were observed in either the gene or protein expression level. However, a trend toward increased gene expression of the eraser enzyme ALKBH5 could be recognized. The overall change in global m6A content appeared comparable across conditions as assessed by a colorimetric m6A quantification kit in hiPSC-ECs and an m6A RNA dot (Figure 6C & Supplementary Figure 3B). The authors then sought to validate the changes in gene expression and m6A abundance for a select number of transcripts within their defined modality of the regulation (Figure 4B). Gene expression levels were determined by quantitative PCR (qPCR), while m6A abundance was determined by methylated RNA immunoprecipitation (MeRIP)-qPCR. The gene expression levels of all five genes selected were consistent with changes observed in the RNA-seq analysis (Figure 6D). When determining the differences in m6A abundance, though, only two of these genes agreed with the microarray results, ANGPTL4 and DIO3. Thus, there was a significant increase in gene expression levels and m6A abundance on ANGPTL4, while DIO3 experienced a significant decrease in gene expression levels and m6A abundance, which was consistent with both the RNA-seq and microarray data. While the gene expression levels of all five transcripts selected for this validation process were shown to be consistent with the RNA-seq data, the authors' approach to validating data from the m6A microarray was successful in only two instances. In contrast to the findings from the microarray, the results from MeRIP-qPCR showed that POSTN, TNFSF10 and IGFBP3 were enriched in the m6A immunoprecipitated fraction, indicating that the m6A abundance on these transcripts was increased (Figure 6E).

Figure 6. . Validation of epitranscriptomic regulated transcripts dysregulated by mono(2-ethylhexyl) phthalate.

Figure 6. 

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(A) Gene expression levels of reader, writer and eraser enzymes were determined by quantitative PCR (n = 9). (B) Protein expression levels of N6-methyladenosine (m6A) reader, writer and eraser enzymes (n = 3). (C) Global m6A levels were determined using a colorimetric assay of total RNA extracts from MEHP-treated human induced pluripotent stem cell-derived endothelial cells (n = 3). (D) Gene expression levels of transcriptionally up- and down-regulated targets identified by RNA sequencing (n = 3) determined using the ∆∆Ct method. (E) m6A immunoprecipitated transcripts were isolated and used for quantitative PCR to validate the status of m6a modification. Statistical significance was determined using Student's t-test.

*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Data are represented as mean ± standard error of the mean.

MEHP: Mono(2-ethylhexyl) phthalate.

Discussion

Exposure to synthetic phthalates is widespread and is known to cause harm to human health [50–52]. Recent attention to this topic has brought into focus those possible adverse consequences that might contribute to cardiovascular dysfunction [53,54]. In the authors' studies, they showed that MEHP induces a strong proliferative response in hiPSC-ECs, as indicated by the increased expression of the proliferative marker KI67. However, they did not observe any difference in the expression of inflammatory markers or any indication that endothelial function was impaired, by wound healing assay, tube formation assay or cell viability assay. While others have reported that MEHP can induce oxidative stress or apoptosis in ECs [19,21], the findings of the present study suggest that the resulting dysfunction is below the threshold of causing functional impairments, specifically those involving angiogenic functions and wound healing capacity. ECs represent an extraordinarily diverse group of cells, and so the cellular responses elicited by exposure to synthetic phthalates may vary widely, depending on whether the endothelium is contained within a greater vessel or microvasculature. In the authors' studies though, they have made use of a hiPSC-based approach, using multiple cell lines to evaluate the effects of MEHP. In doing so, they could obtain results that have greater relevance to a broader population of humans, since they were able to incorporate some degree of genetic heterogeneity into the evaluation, like that which exists within human populations. This allowed them to account for possible confounding genetic factors that might influence susceptibility to environmental toxins. Thus, this hiPSC-based model for toxicological screening, which is simple and reproducible, demonstrates how stem cells can be used in the field of toxicology. However, these findings are limited by the fact that the authors relied primarily on a single cell type. Thus, it remains of great importance to continue to monitor how these chemicals might interfere with endothelial function, given their widespread use in society. Furthermore, vascular disease is often preceded by chronic inflammatory conditions, such as obesity, sustained mechanical stress and aging, which are conditions that are not well adapted to in vitro models. While the authors have attempted to model such chronic conditions by using an extended duration of treatment time, future studies may benefit from the use of animal models. Overall, the authors observed dysfunction, which appeared to be only subtle, yet this profiling of the epitranscriptome may still serve as a valuable resource for future studies that address the role of synthetic phthalates in cardiovascular dysfunction.

Analysis of data generated from this multiomic approach resulted in the identification of transcripts with significant alterations in m6A abundance as well as transcripts that experienced alterations in overall expression levels. The analysis focused primarily on transcripts that had changes in the overall quantity of m6A and how these changes coincided with expression determined by RNA-seq. Analysis of the transcriptome identified a negative enrichment of IFN-α/β signaling (Figure 2C). This observation is consistent with previous reports that have reported that synthetic phthalates can suppress the expression of interferons [45,55]. In addition to these findings, several ontological analyses and a network map were constructed to provide a broad overview of the transcriptomic response. Among the most significant terms identified within this analysis was an annotation implicating the involvement of cancer (Figure 2D). Given that plasticizers have been reported to induce genotoxicity and promote cancer in rodents [56] and the observed increase in proliferation, the authors performed an additional analysis to highlight the expression changes occurring in a broad range of oncogenic genes and proliferation-related genes, so that individual targets could be better recognized. This analysis resulted in the identification of an additional 12 genes related to oncogenesis and 18 proliferation-related genes (Supplementary Table 1). This is also a relevant finding, given that the formation of new blood vessels is a hallmark of cancer. Furthermore, another feature of cancer is rapid and uncontrolled cellular proliferation. This characteristic of cancer was consistent with the observed phenotype after treatment with MEHP, since there was an observed increase in proliferation. Thus, this annotation is worth noting in the sense that it may warrant general concern regarding the effects of MEHP exposure and that it fits with observed dysfunction and, as such, may contribute to aberrant vascular remodeling.

Analysis of the epitranscriptome began with a general survey of all dysregulated transcripts, followed by a targeted analysis of a specific subset of transcripts that may exist within a particular modality of epitranscriptomic regulation. The authors first showed that among those transcripts identified by the microarray that contained significant changes in m6A abundance (628 in total), there was a trend toward downregulation (586 decreased and 42 increased; Figure 3B). However, when surveyed on a broader scale, it appeared that greater than 90% of the epitranscriptome was unaffected by MEHP treatment. The microarray data were therefore consistent with the experimental data, which showed stable expression of m6A enzymes and unchanged global m6A deposition (Figure 6A–C). Given that there was an apparent trend toward downregulation, though, the authors sought to highlight the features of the RNA-seq data for a larger collection of recently defined m6A reader, writer and eraser enzymes (Supplementary Table 1) [57], which might offer an explanation for this. This analysis failed to observe any significant changes in these enzymes, though. One possible explanation for the observed trend toward downregulation might include a more nuanced regulation of the epitranscriptome, which may involve recently described a post-translational modifications which can be made to certain m6A enzymes leading to alterations in their activity in establishing m6A marks. [58–60]. Such changes in the activity of these enzymes might therefore be able to explain the directionality of regulation observed, since these effects may be mediated with stable expression of reader and writer enzymes.

To provide biological insights into the epitranscriptomic response, the authors first created an extensive ontological classification of all epitranscriptomic transcripts that were differentially regulated (Figure 3C). These data may serve as a valuable reference for those processes that might be subjected to epitranscriptomic regulation, and thus may offer mechanistic insights into how synthetic phthalates can induce endothelial dysfunction. Since the epitranscriptome can either promote or reduce the stability of the transcript that it marks, the authors sought to define and specifically address a subset of transcripts with alterations in m6A abundance, which might be involved in mediating a specific type of regulatory outcome (Figure 4A). After isolating this subset of epitranscriptomic transcripts, the authors performed an additional analysis to gain insights into the functions of this gene set. After performing a pathway analysis using the WikiPathway 2021 database (Figure 4C), they identified a strong signal for EC calcification. This specific type of endothelial dysfunction might therefore be a process regulated through the epitranscriptome acting through a specific type of regulatory modality. EC calcification has been described as a process that occurs over time with aging and can contribute to the development of numerous adverse cardiovascular consequences [61]. While it has not been clearly established that any connection exists between exposure to synthetic phthalates and EC calcification, there have been multiple reports recognizing an association between vascular stiffness and circulating levels of synthetic phthalates [62,63]. It may therefore be of interest to continue to monitor this association in the future. The present study's phenotypic characterization did not identify any overt impairments to functionality or cell viability. In the cell viability assay, though, the authors did observe a trend which suggested that potentially harmful effects may be elicited at higher concentrations. They did, however, note a stark increase in expression of the proliferative marker KI-67. Others have shown that synthetic phthalates can mediate endothelial dysfunction by causing oxidative stress, and oxidative stress is known to induce cellular proliferation. Thus, there may be multiple ways by which exposure to synthetic phthalates might induce endothelial dysfunction.

In an attempt to validate targets subjected to epitranscriptomic regulation, the authors found success with only two transcripts, ANGPTL4 and DIO3. The upregulation of ANGPTL4 has also been reported to occur in response to treatment with MEHP and similar phthalates [64,65]. By validating the gene expression levels, and m6A deposition of this gene, the authors identified a possible mechanism for this MEHP-mediated response involving the epitranscriptome. ANGPTL4 was previously implicated in the development of atherosclerotic plaques, endothelial inflammation and numerous other cardiometabolic disorders due to its role in regulating lipid homeostasis [66,67]. Future investigations may seek to determine if exposure to MEHP compromises a cell's ability to maintain homeostatic functions when faced with relevant pathological stressors such as lipotoxic stress. Interestingly, many of the targets selected for validation with MeRIP-qPCR in the present study could not be validated. The authors believe that this may be related to the fact that probe sequences used to identify differentially regulated transcripts were only 60 nt long, while it is well known that the m6A landscape varies along the length of transcripts [68]. Furthermore, it has also been shown that in pathological states, the shape of this landscape can dynamically rearrange [69]. Thus, it is reasonable to assume that the probes used in the present study's microarray may exist in a dynamic location, sensitive enough to detect significant alterations within a transcript but unable to evaluate the overall state of that transcript. The use of MeRIP exposed a limitation of the microarray platform in this way. However, in the cases of these possible rearrangements of the landscape, the authors feel that their probes were still capable of accurately identifying significant changes in m6A occurring on a transcript but that they may not necessarily reflect the status of the entire transcript.

Conclusion

In summary, we have provided a multiomic characterization to highlight previously unrecognized areas of endothelial functions affected by exposure to MEHP. As such, we have provided evidence that might aid in bridging the gap between clinical observations that associate levels of synthetic phthalates with vascular phenotypes. Therefore, the molecular profiling performed in this study will serve as a valuable resource for future research that seeks to address how synthetic phthalate exposure may contribute to endothelial and vascular dysfunction. Given the widespread use of synthetic phthalates and the large burden of cardiovascular disease, it remains of great importance to continue to survey this topic for potential threats to human harm using up-to-date advances in molecular understanding, as we have done here in our survey of the epitranscriptome. Our findings offer important insights into the alterations occurring throughout the m6A epitranscriptome and the gene expression changes that occur in hiPSC-ECs after treatment with a synthetic phthalate.

Future perspective

Our study has made use of a hiPSC-EC model for the screening of an environmental toxin. We believe that future studies conducting toxicological screens may benefit from similar approaches. In our profiling of the epitranscriptomic response that hiPSC-ECs experienced to treatment with MEHP, we have provided a resource that may be utilized for studies that seek to better understand how synthetic phthalates might affect human health.

Summary points.

  • This study makes uses of recent advances in the field of stem cell biology to demonstrate how toxicological screens for environmental toxins can be conducted.

  • The advantages of a human induced pluripotent stem cell-derived endothelial cell-based model include the opportunity to use cell lines from multiple donors to evaluate diverse genetic backgrounds and the ability to direct differentiation toward cell types that were previously difficult to obtain.

  • The profile of the epitranscriptomic effects elicited by treatment with a synthetic phthalate was, overall, rather modest and caused little change on a global scale.

  • Multiomic integration of data obtain from RNA sequencing performed in parallel with N6-methyladenosine profiling allowed for a specific subset of transcripts to be identified.

  • Analysis of dysregulated genes identified relevant pathways and biological networks that might be impaired by mono(2-ethylhexyl) phthalate (MEHP) in endothelial cells.

  • The effects of MEHP were observed to induce a strong proliferative response, as indicated by the increase in the expression of the proliferation marker KI67.

  • Individual genes of interest selected from the RNA sequencing data were validated by quantitative PCR, while those of interest obtained from N6-methyladenosine microarray were validated by methylated RNA immunoprecipitation–quantitative PCR.

  • Overall, the data contained within this study offer a comprehensive overview of the epitranscriptomic profile and corresponding gene expression changes in human induced pluripotent stem cell-derived endothelial cells treated with MEHP and therefore may serve as a valuable human health resource.

Supplementary Material

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Footnotes

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at: www.futuremedicine.com/doi/suppl/10.2217/epi-2022-0110

Author contributions

J Jousma carried out bioinformatic analysis, data visualization, cellular and molecular experimental methods as well as manuscript preparation and revisions. SB Nukala contributed to cellular experimental methods. Z Han contributed to cellular experimental methods. G Yan and HHT Le carried out validation of statistical analysis. Y Kwon and Y Li contributed cellular experimental methods. WH Lee and SG Ong carried out project conceptualization, design, supervision, resources and manuscript review. All authors read and approved the final manuscript. SB Ong made intellectual contributions during review and preparation.

Financial & competing interests disclosure

This paper was supported and funded by the National Heart, Lung, and Blood Institute (NHLBI) T32 HL007829 (JJ), (NHLBI) R00 HL130416 (S-GO) and R01 HL148756 (S-GO). The authors also received funding from the American Heart Association (AHA), Postdoctoral Fellowship 917176 (ZH), AHA Scientist Development Grant 16SDG27560003 (WHL). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Data availability

RNA sequencing and N6-methyladenosine microarray data will be made publicly accessible upon request to the corresponding authors and will be available in the Gene Expression Omnibus database at the accession number GSE214420.

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