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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Hypertension. 2019 Jun 3;74(1):154–163. doi: 10.1161/HYPERTENSIONAHA.118.12569

Sexual Dimorphisms of Preeclampsia-Dysregulated Transcriptomic Profiles and Cell Function in Fetal Endothelial Cells

Chi Zhou 1, Qin Yan 1,2, Qing-yun Zou 1, Xin-qi Zhong 1,3, Chanel T Tyler 1, Ronald R Magness 4, Ian M Bird 1, Jing Zheng 1,5,*
PMCID: PMC6561818  NIHMSID: NIHMS1528523  PMID: 31154903

Abstract

Preeclampsia impairs fetoplacental vascular function and increases risks of adult-onset cardiovascular disorders in children born to preeclamptic mothers, implicating that preeclampsia programs fetal vasculature in utero. However, the underlying mechanisms remain elusive. We hypothesize that preeclampsia alters fetal endothelial gene expression and disturbs cytokines- and growth factors-induced endothelial responses. RNAseq analysis was performed on unpassaged human umbilical vein endothelial cells (HUVECs) from normotensive and preeclamptic pregnancies. Functional assays for endothelial monolayer integrity, proliferation, and migration were conducted on passage 1 HUVECs from normotensive and preeclamptic pregnancies. Compared with normotensive cells, 926 and 172 genes were dysregulated in unpassaged female and male HUVECs from preeclamptic pregnancies, respectively. Many of these preeclampsia-dysregulated genes are associated with cardiovascular diseases (e.g., heart failure) and endothelial function (e.g., cell migration, calcium signaling, and endothelial nitric oxide synthase signaling). TNFα-, TGFβ1-, FGF2-, and VEGFA-regulated gene networks were differentially disrupted in unpassaged female and male HUVECs from preeclamptic pregnancies. Moreover, preeclampsia decreased endothelial monolayer integrity in responses to TNFα in both female and male HUVECs. Preeclampsia decreased TGFβ1-strengthened monolayer integrity in female HUVECs, while it enhanced FGF2-strengthened monolayer integrity in male HUVECs. Preeclampsia promoted TNFα-, TGFβ1-, and VEGFA-induced cell proliferation in female, but not in male HUVECs. Preeclampsia inhibited TNFα-induced cell migration in female HUVECs, but had an opposite effect on male HUVECs. In conclusion, preeclampsia differentially dysregulates cardiovascular diseases- and endothelial function-associated genes/pathways in female and male fetal endothelial cells in association with the sexual dimorphisms of preeclampsia-dysregulated fetal endothelial function.

Keywords: Preeclampsia, Fetal endothelial dysfunction, Transcriptome, Sexual dimorphisms

Graphical Abstract

graphic file with name nihms-1528523-f0001.jpg

Introduction

Preeclampsia is a maternal hypertensive disorder, which is a leading cause of maternal and fetal morbidity and mortality during human pregnancy1,2. Preeclampsia also impairs fetal endothelial function including monolayer permeability, calcium responses, and nitric oxide (NO) production3-6. Moreover, children born to preeclamptic (PE) mother exhibit increased risks of adult-onset cardiovascular disorders7-9, demonstrating in utero programming of fetal vascular function in preeclampsia. Recently, we reported that preeclampsia-dysregulated fetal endothelial mircoRNAs expression could contribute to part of the mechanisms underlying preeclampsia-impaired fetal endothelial function10. Thus, further dissecting such mechanisms is critical to the development of new therapeutic strategies for this common pregnancy complication.

Endothelial function is tightly regulated by peptide growth factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2), which are key regulators of placental angiogenesis and vasodilation11. Additionally, cytokines also regulate vascular function during pregnancy. For example, tumor necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that regulates angiogenesis, endothelial apoptosis, NO production, and vascular integrity12-15. Similarly, transforming growth factor-beta1 (TGFβ1) regulates endothelial function, vascular development, and vascular barrier function16,17. Preeclampsia elevates the VEGFA18 and FGF219 levels in human placentas, as well as the sVEGFR120, TNFα21 and TGFβ122 levels in maternal circulation, all of which are associated with the endothelial dysfunction in preeclampsia.

The exact mechanisms underlying preeclampsia-dysregulated fetal endothelial function are poorly understood. Previous studies on genome-wide transcriptomic profiling have identified different sets of differentially expressed genes in placentas from preeclampsia23-26. However, as the placental tissue is highly heterogeneous in cell composition, the roles of these preeclampsia-dysregulated gene networks/pathways remain to be defined in any single cell type.

Human umbilical vein endothelial cells (HUVECs) are widely utilized to study preeclampsia-dysregulated fetal endothelial function. Indeed, fetal endothelial cells from preeclampsia exhibit impaired endothelial function including dysregulated microRNA expression, reduced endothelial migration, and decreased vasodilator production3-6, 10, 27. Sexual dimorphisms of fetal endothelial function have been reported in cultured HUVECs from normotensive pregnancies (NT). Specifically, male NT-HUVECs exhibit decreased cell proliferation, cell viability, tube formation capacity, migration, and endothelial NO synthase (eNOS) expression compared with their female counterparts28, 29. To date, the molecular mechanisms underlying these sexual dimorphisms of fetal endothelial function in NT pregnancies remain elusive. Furthermore, it is also unknown if there are sexual dimorphisms in preeclampsia-dysregulated fetal endothelial function.

In this study, we hypothesize that preeclampsia alters fetal endothelial (HUVECs) gene expression and disturbs cytokines- and growth factors-induced endothelial function in a sex-specific manner. We characterized preeclampsia-dysregulated transcriptomic profiles in unpassaged (P0) female and male HUVECs from NT and PE pregnancies using RNAseq as these P0 cells might closely mimic the in vivo condition. Bioinformatics and gene ontology analysis were performed to identify preeclampsia-dysregulated genes/pathways in female and male P0-HUVECs. To determine if transcriptomic changes corresponded to changes in phenotypic endothelial function, we also examined the effect of preeclampsia on cellular responses to cytokine and growth factors in passage 1 (P1, ~5 days culture) female and male HUVECs.

Methods

All supporting data are available within the article and its online supplementary files. RNAseq analysis data have been deposited in NCBI Gene Expression Omnibus (GEO) database (GEO accession: GSE116428).

Ethical approval

All procedures were conducted in accordance to the Declaration of Helsinki. The tissue collection protocol was approved by the Institutional Review Board of UnityPoint Health-Meriter Hospital (Madison, WI) and the Health Sciences Institutional Review Boards of the University of Wisconsin-Madison (Protocol#2004-006). All subjects gave written, informed consent. The obstetricians at the UnityPoint Health-Meriter Hospital Birth Center where the umbilical cords were collected made clinical diagnosis of NT and PE. Preeclampsia was defined according to the standard American College of Obstetricians and Gynecologists criteria30.

Isolation and characterization of primary HUVECs

Female (F) and male (M) HUVECs were isolated immediately after deliveries of NT and PE pregnancies (TableS1). All patients are Caucasian due to the local demographic distribution. Cells were purified after ~16 h of culture10,31 (Supplemental Methods). After purification, the majority of cells (referred as P0-HUVECs) were either immediately frozen in liquid nitrogen until total RNA isolation or cultured to P1 (~5 days of culture). To validate the purity of each cell preparation, DiI-Ac-LDL uptake assay10 was performed with each P0 cell preparation (Supplemental Methods). Only cell preparations with ≥96% DiI-Ac-LDL uptake were used.

RNA isolation and quality control

Total RNA were isolated from P0-HUVECs using an RNeasy Mini Kit (Qiagen, Valencia, CA). The concentration and quality of each RNA sample were assessed using NanoDrop™ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) and Agilent 2100-bioanalyzer (Agilent Technologies, Santa Clara, CA)10. Only RNA samples with high RNA integrity number (>8.5) were utilized.

RNA sequencing (RNAseq) and bioinformatics analysis

We performed RNAseq analysis of total RNA samples from P0-HUVECs (n=5~8 individual cell preparations/sex/group, TableS1) using Illumina TruSeq RNA library prep kits and an Illumina HiSeq2000 sequencer (Illumina, San Diego, CA) as described in supplemental methods. EdgeR was utilized to determine the differentially expressed (fold change (FC)>∣2∣ and false discovery rate (FDR)-adjusted P-value<0.05) genes. Gene ontology analysis was performed to determine the enriched biological processes, diseases/canonical pathways, and gene networks using Ingenuity Pathway Analysis (IPA; www.qiagenbioinformatics.com)32.

RT-qPCR verification

To validate the RNAseq data, 15 genes with different expression patterns were selected for RT-qPCR analysis10 in another set of samples (n=10 individual cell preparations/sex/group, TableS1) using commercially available mRNA Primer Assays (TableS2), miScript SYBR Green PCR Kit (Qiagen), and a StepOnePlus qPCR system (Life Technologies, Carlsbad, CA). Data were normalized to an external control (miRTC, Qiagen) along with 3 internal housekeeping genes (GAPDH, YWHAZ, and SDHA). The normalized data were then analyzed using the 2−ΔΔCT method10,32.

Cell functional assays

Cell functional assays were performed on P1-HUVECs from NT and PE pregnancies (n=5 individual cell preparations/sex/group; TableS1; Supplemental Methods). Cell monolayer integrity was determined using the ECIS Zθ+96-well array station and 96W10idf plates (Applied BioPhysics, NY)10. Cell proliferation was assessed using the CCK-8 kit (Dojindo Molecular Technologies, MD)33. Cell migration was assessed using a transwell system (Corning, NY)33,34.

Statistical analyses

SigmaPlot software (Jandel Co., San Rafael, CA) was used for statistical analyses. Data are represented as the medians±standard deviation (SD). Data analyses were performed using the Mann-Whitney Rank Sum Test or Kruskal-Wallis test when appropriate. Differences were considered significant when P<0.05. Benjamini and Hochberg FDR-adjustment was used for multiple comparison correction when appropriate.

Results

Sexual dimorphisms of preeclampsia-dysregulated transcriptomic profiles in P0-HUVECs

Compared with NT-F cells, 1561 differentially expressed genes (81 up-regulated and 1480 down-regulated) were identified in NT-M cells (Figure.1A/B; TableS3). Out of these 1561 genes, 41 and 23 are located on X- and Y-chromosomes, respectively (TableS3).

Figure 1. Preeclampsia differentially dysregulates transcriptomic profiles of P0-HUVECs.

Figure 1.

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(A) Circos plot illustrating the chromosomal position of differentially expressed genes between NT-M vs. NT-F (grey dots, 1561 genes), PE-F vs. NT-F (pink dots, 927 genes), and PE-M vs. NT-M (blue dots, 172 genes). Each dot represents one gene. The numbers and letters in the outer ring indicate the chromosomal location. For each scatter plot track, dots outside and inside of the centerline are up- and down-regulated genes, respectively. (B,C,D) Volcano plots showing differentially expressed genes between (B) NT-M vs. NT-F, (C) PE-F vs. NT-F, and (D) PE-M vs. NT-M in RNAseq analysis. Grey dots: no significant difference; Red and green dots: >2-fold up- and down-regulation, respectively (FDR-adjusted P<0.05) in PE vs. NT; n=5~8/group. (E,F) RT-qPCR validation of preeclampisa-dysregulated genes in F and M cells. *Differ (P<0.05) from NT, n=10/group.

Compared with NT within the same sex, preeclampsia dysregulated 926 and 172 genes in F and M cells, respectively (Figure.1A/C/D; TableS4). Compared to NT-F cells, 255 and 671 gens were up- and down-regulated in PE-F cells, respectively, among which 24 are located on X-chromosome (TableS4). Compared to NT-M cells, 155 and 17 genes were up- and down-regulated in PE-M cells, respectively, among which 3 are located on X-chromosome; none of these genes is located on Y-chromosome (TableS4). Thirty-nine genes were commonly dysregulated in PE-F and PE-M cells. Out of these 39 genes, 35 were down-regulated in PE-F but up-regulated in PE-M, including 6 Heat Shock Protein family members and 4 Zinc Finger Protein family members (TableS4). Additionally, only 1 gene (GCH1) was commonly upregulated and 3 (MLIP, PALMD, and LINC00704) were commonly down-regulated in F and M cells. Only one (PLCXD1) is located on X-chromosome.

RT-qPCR data are highly correlated (correlation coefficient value of 0.897) to RNAseq analysis (Figure.1E/F). Specifically, VCAM1 and GCH1 were up-regulated, whereas MAOA was down-regulated in PE-F and PE-M cells. CDCA2, HSPH1, and CDKN1C were down-regulated in PE-F, but up-regulated in PE-M cells. COL1A1 was up-regulated in PE-M but not in PE-F cells. COL18A1, COL8A2, and FASN were up-regulated, while down-regulated LAMA2 was down-regulated in PE-F but not in PE-M cells. Preeclampsia did not alter FGF2, ICAM2, AKT1, and KDR expression in F and M cells.

PE dysregulates cardiovascular diseases- and endothelial function-associated genes in P0-HUVECs

Canonical pathways enrichment analysis indicated that 9 pathways including eNOS signaling, calcium signaling, Gαq signaling, and cardiac β-adrenergic signaling pathways were enriched in differentially expressed genes between NT-F and NT-M cells (Figure.S1).

Biological processes enrichment analysis on preeclampsia-dysregulated genes in F and M cells revealed that 6 biological processes including seizures, diabetes mellitus, and cell viability were enriched in PE-F and M cells (Figure.2A). Genes associated with growth failure and blood pressure were only enriched in PE-M cells, whereas genes associated with other 13 biological processes including adhesion of immune cells, angiogenesis, and chemotaxis were only enriched in PE-F cells.

Figure 2. Preeclampsia differentially dysregulates (A) biological functions-, (B) cardiovascular diseases-, and (C) canonical pathways-associated genes in P0-HUVECs.

Figure 2.

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Significant enrichments were determined using IPA software (P<0.05, Fisher’s exact test).

Diseases and Bio-function enrichment analysis on preeclampsia-dysregulated genes in F and M cells (Figure.2B, TableS5) further showed that genes associated with heart failure, coronary artery disease, congenital heart disease, pulmonary hypertension, and cardiac dysfunction were enriched in PE-F, but not in PE-M cells. Moreover, genes associated with chronic heart failure were only enriched in PE-M cells (Figure.2B).

Canonical pathways enrichment analysis on preeclampsia-dysregulated genes in F and M cells (Figure.2C) indicated that eNOS signaling, dendritic cell maturation, and LXR/RXR activation pathways were enriched in PE-F and PE-M cells. Five canonical pathways including calcium signaling, TNFR2 signaling, and PI3K/AKT signaling pathways were enriched only in PE-F cells.

Upstream regulator analysis (Table1, TableS6) on preeclampsia-dysregulated genes in F and M cells revealed that TNF-, FGF2-, FGFR2-, and VEGFA-regulated genes were enriched in PE-F and PE-M cells, with PE-F cells exhibiting more preeclampsia-dysregulated genes. In addition, NFκB-, TGFB1-, pro-inflammatory cytokines-, ICAM1-, and TGFB1-regulated genes were uniquely enriched in PE-F cells.

Table 1.

PE dysregulated gene network in female and male P0-HUVECs

Gene network Female P0-HUVECs
 Male P0-HUVECs
p-value Differentially expressed in
PE vs. NT		 p-value Differentially expressed in
PE vs. NT
TNF-regulated genes 8.59E-27 144 1.70E-02 9
NFkB-regulated genes 9.35E-27 79    
TGFB1-regulated genes 5.94E-17 121    
Pro-inflammatory Cytokine- regulated genes 5.02E-05 10    
ICAM1-regulated genes 1.08E-04 8
TGFBR1-regulated genes 3.94E-02 6 2.07E-06 9
FGF2-regulated genes 4.86E-06 29 8.59E-03 7
FGFR2-regulated genes 2.16E-03 11 8.62E-03 4
VEGFA-regulated genes 5.16E-05 23 1.17E-05 6
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PE dysregulates endothelial monolayer integrity in responses to TNFα, TGFβ, and FGF2 in P1-HUVECs

After 5h of treatment, TNFα decreased electrical resistance (weakening cell monolayer integrity) of NT-F and PE-F cells by 20% and 35%, respectively (Figure.3A). This TNFα-decreased electrical resistance was recovered after 12h in NT-F cells; however, this decrease was sustained for up to 25h in PE-F cells. TNFα did not affect monolayer integrity of NT-M cells, whereas decreased monolayer integrity of PE-M cells by 25% at 7h.

Figure 3. Preeclampsia differentially dysregulates endothelial monolayer integrity of P1-HUVECs.

Figure 3.

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After serum starvation, confluent cells were treated with ECM-b (control), (A) TNFα (10ng/ml), (B) TGFβ1 (10ng/ml), (C) VEGFA (10ng/ml), (D) FGF2 (10ng/ml), or (E) ECM for 25h. Electrical resistance at 4000Hz was constantly recorded. Data are expressed as medians ± SD fold of control at corresponding time (n=5). #Different (P<0.05, Kruskal-Wallis test) between PE and NT groups within each corresponding treatment.

TGFβ1 time-dependently increased electrical resistance (strengthening cell monolayer integrity) of NT-F cells, starting at 10h and reaching its maximum effect at 25h (by 23%; Figure.3B). TGFβ1 did not significantly affect monolayer integrity of PE-F, NT-M and PE-M cells (Figure.3B).

FGF2 time-dependently strengthened cell monolayer integrity of NT-F (by 91% at 25 hr), PE-F (by 119%), NT-M (by 46%), and PE-M (by 93%) cells (Figure.3C). This FGF2-strengthened monolayer integrity was higher in PE-M vs. NT-M cells.

VEGFA did not significantly alter monolayer integrity of all four groups of cells studied (Figure.3D). Endothelial complete growth medium (ECM) time-dependently increased monolayer integrity of all cells treated (Figure.3E).

PE dysregulates endothelial proliferation in responses to TNFα and TGFβ in P1-HUVECs

Cell proliferation data are shown in Figure.4A. Compared with control media, TNFα stimulated cell proliferation in PE-F (117%), NT-M (140%), and PE-M (127%), but not in NT-F cells. TGFβ1 promoted cell proliferation in PE-F, but not in NT-F, NT-M, and PE-M cells. FGF2 stimulated cell proliferation in all four groups of cells studied, with F cells exhibiting a more profound response than M cells. However, no difference was observed in FGF2-stimulated cell proliferation between NT and PE cells within the same sex. VEGFA stimulated cell proliferation in PE-F, NT-M, and PE-M, but not in NT-F cells. ECM stimulated cell proliferation of all four groups of cells studied. This ECM-induced cell proliferation in PE-F cells was lower than that in NT-F cells.

Figure 4. Preeclampsia differentially dysregulates the endothelial (A) proliferation and (B) migration of P1-HUVECs.

Figure 4.

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After serum starved, sub-confluent cells were treated with ECM-b (control), TNFα (10ng/ml), TGFβ1 (10ng/ml), VEGFA (10ng/ml), FGF2 (10ng/ml), or ECM for (A) 48h (cell proliferation) or (B) 16h (cell migration). Data are expressed as medians ± SD fold of control (n=5). *Differ from corresponding control. #Differ in PE vs. NT in F or M HUVECs within each treatment. ΔDiffer between female and male in PE or NT groups within each treatment. (P<0.05, Kruskal-Wallis test).

PE dysregulates endothelial migration in responses to TNFα in P1-HUVECs

Cell migration data are shown in Figure.4B. Compared with control media, TNFα stimulated cell migration in NT-F (215%), PE-F (177%), NT-M (176%), and PE-M (237%) cells. However, compared with NT, TNFα-stimulated cell migration was attenuated in PE-F cells, but enhanced in PE-M cells. TGFβ inhibited cell migration in NT-F (by 25%), PE-F (by 27%), NT-M (by 24%), and PE-M (by 16%) cells. FGF2 increased cell migration in NT-F (149%), PE-F (172%), NT-M (167%), and PE-M (176%) cells. VEGFA also promoted cell migration in NT-F (271%), PE-F (267%), NT-M (222%), and PE-M (204%) cells. VEGFA-stimulated cell migration was greater in F than M cells; but there were no differences between PE and NT cells within the same sex. ECM stimulated cell migration in all cells treated. Compared with NT cells, ECM-induced cell migration was enhanced in PE-F, but not in PE-M cells. ECM-induced cell migration was significantly higher in PE-F vs. PE-M cells.

Discussion

In this study, we have defined for the first time the transcriptomic profiles of female and male fetal endothelial cells from NT and PE pregnancies utilizing high-purity, unpassaged HUVECs from Caucasian pregnancies. We have further demonstrated that preeclampsia-dysregulates many cardiovascular diseases- and fetal endothelial function-associated gene networks in a fetal sex-dependent manner. Such dysregulation is associated with defective fetal endothelial responses to cytokines and growth factors. Overall, female fetal endothelial cells are much more impacted by preeclampsia than male cells. These data clearly indicate sexual dimorphisms of preeclampsia-associated transcriptomics and endothelial function dysregulation in fetal endothelial cells.

Transcriptomic differences between female and male endothelial cells have been reported in passage 2 (P2)-HUVECs from NT using microarray (GEO accession: GSE52212), where 21 differentially expressed genes were identified28. Our RNAseq analysis in P0-HUVECs extensively expanded the list of these genes between male and female fetal endothelial cells (TableS3). This is not surprising as P0-HUVECs may more closely mimic the in vivo phenotype and RNAseq is more sensitive in detection of differential expression and even discover new genes. Additionally, the current finding that only ~4% (64 genes) of these differentially expressed genes are located on X- and Y-chromosomes suggests that sex chromosome-specific genes may not be the major contributor to the transcriptomic differences between female and male fetal endothelial cells.

To date, it is unknown if the differential expression of eNOS and calcium signaling pathways between female and male HUVECs (Figure.S1) contributes to the sexual dimorphisms of fetal endothelial function observed in NT cells28,29. It is also unknown if the differential expression of the cardiovascular diseases-associated (including Gαq35,36 and cardiac β-adrenergic37 signaling) pathways (Figure.S1) is associated with different risks of cardiovascular diseases and outcomes in men and women in general38.

We further show that the transcriptomic profiles were differentially dysregulated between female and male fetal endothelial cells from preeclampsia. It is noteworthy that female HUVECs have significantly more (~5-fold) preeclampsia-dysregulated genes than male cells. Another important finding is that the majority (72%) of preeclampsia-dysregulated genes are down-regulated in female cells, while most (90%) are up-regulated in male cells. Similarly, the majority (90%) of the 39 commonly dysregulated genes are down-regulated in female, but are up-regulated in male cells from preeclampsia. These differentially dysregulated genes include heat shock proteins and zinc finger proteins, both of which are key stress responses-associated proteins. These data agree with the previous report that female HUVECs have more pronounced transcriptional responses to shear stress than male cells28, and imply that female fetal endothelial cells are transcriptionally more responsive to preeclampsia stress than their male counterparts are.

Mechanisms underlying these sexual dimorphisms of preeclampsia-dysregulated transcriptomic profiles in fetal endothelial cells remain elusive. There are only 24 (2.6%) and 3 (1.7%) of the preeclampsia-dysregulated genes in female and male P0-HUVECs, respectively, are located on X-chromosome (none on Y-chromosome, TableS3). Hence, sex chromosome-specific genes appear to have a limited role in these sexual dimorphisms. Sex hormones may contribute to transcriptional sexual dimorphisms, as there are small differences in levels of gonadal hormones in the blood of umbilical veins from female and male fetueses39. However, in the current study, we do not detect any significant differential expression of estrogen and androgen receptors in HUVECs as reported29.

Preeclampsia differentially dysregulates many critical biological processes and pathways-associated genes between female and male HUVECs. Many of these processes (e.g., viability, organization of cytoskeleton, and eNOS signaling pathway-associated genes) are closely associated with preeclampsia-impaired fetal endothelial function (e.g., reduced cell migration, impaired calcium signaling, decreased NO production, and increased endothelial permeability)3-5,40. Strikingly, “seizures” is the most significant dysregulated biological process in both female and male PE-HUVECs, implicating that preeclampsia might increase fetal seizure susceptibility. Preeclampsia also uniquely dysregulates a number of cardiovascular diseases- and endothelial function (e.g. migration of endothelial cells, vascularization, and angiogenesis)-related biological processes in female cells, and growth failure, blood pressure and chronic heart failure-associated genes in male cells. We also observe that preeclampsia dysregulates more cardiovascular diseases-associated gene networks and canonical pathways in female than in male cells.

The current finding that preeclampsia significantly dysregulates the LXR/RXR activation pathway (a lipid metabolism and inflammation regulation-associated pathway41), Glucose metabolism disorder, and Diabetes mellitus-associated genes in both female and male HUVECs. These data are consistent with previous reports that PE-offspring are at increased risks of metabolic disorders7,42. Additionally, the differential regulation of GPCR-mediated nutrient sensing in enteroendocrine cells pathway- and growth failure-associated genes in female and male cells may be associated with the different weight growth trends between male and female children from PE pregnancies43.

It is not surprising to see that preeclampsia dysregulates inflammation-associated gene networks in HUVECs since excessive inflammation is a hallmark of preeclampsia44-46. Elevated circulating pro-inflammatory cytokines and adhesion molecules can up-regulate inducible NO synthase (iNOS), contributing to the pathogenesis of preeclampsia47-49. However, sexual dimorphisms of such dysregulation is a novel observation in HUVECs. Specifically, our data suggest that preeclampsia dysregulates nuclear transcription factor kappa B (NFκB)-regulated gene networks in both female and male HUVECs (Table1; Figure.1D/E), with PE female cells having more dysregulated genes than male cells. Additionally, preeclampsia dysregulates iNOS signaling pathway-, adhesion of immune cells-, and maturation of antigen presenting cells-associated as well as pro-inflammatory cytokines-regulated genes only in female P0-HUVECs (Figure.2A/C, Table1). Collectively, compared with male cells, PE female fetal endothelial cells exhibit higher inflammatory responses implicating a more severely impaired endothelial function as supported our current data discussed below.

Preeclampsia up-regulated TNFα21 could damage endothelial monolayer integrity14. The current study provides novel evidence of sexually dimorphic regulation of TNFα in fetal endothelial cell monolayer integrity, proliferation and migration of fetal endothelial cells. Notably, preeclampsia further decreases and sustains TNFα-weakened monolayer integrity in female HUVECs and only briefly induces TNFα-weakened monolayer integrity in male cells. Additionally, preeclampsia moderately promotes TNFα-stimulated endothelial proliferation but decreases TNFα-stimulated endothelial migration in female cells. In female HUVECs, preeclampsia specifically dysregulates the TNF receptor 2 (TNFR2) signaling pathway. Given the TNFα-TNFR2 pathway is important in regulating cell migration15, the impairment of this pathway could participate in preeclampsia-dysregulated endothelial responses to TNFα in female HUVECs.

TGFβ1 regulates diverse endothelial function including monolayer integrity, proliferation, and migration17,50. Similar to TNFα, TGFβ1 may contribute to preeclampsia-induced endothelial dysfunction, as preeclampsia elevates TGFβ1 levels in the maternal circulation22. In this study, we demonstrate that TGFβ1 decreases the permeability of NT-F cells and inhibits cell migration in all cells examined. Moreover, preeclampsia abolishes TGFβ1-decreased cell permeability and increases cell proliferation in female cells, which is consistent with our observation that preeclampsia dysregulates TGFβ1-regulated genes in female HUVECs. Together with the report that inhibition of TGFβ signaling disrupts endothelial monolayer integrity51, the present data suggest that preeclampsia impairs the female fetal endothelial monolayer integrity and promotes proliferation partially via the TGFβ1 pathway.

The current observation that FGF2 strengthens fetal endothelial monolayer integrity concurs with our previous report10. However, preeclampsia further promotes FGF2-strengthened monolayer integrity only in male cells. Unexpectedly, VEGFA does not significantly alter monolayer integrity of NT or PE P1-HUVECs, which is contradictory to our recent observation in NT P4-HUVECs10. The exact cause of this discrepancy is unclear, but different passages of cells used may be a contributing factor. It is also noteworthy that FGF2-stimulated cell proliferation is much higher in female than male cells, and preeclampsia actually enhances cell proliferative response to VEGFA in female cells. Further studies are needed to determine if preeclampsia differentially-dysregulated FGF2-, FGFR2-, and VEGFA-related gene networks in female and male HUVECs (Table1) are contributing to the sexual dimorphisms of growth factors-induced fetal endothelial function in preeclampsia.

To date, it is unclear what causes these transcriptomic differences in PE female and male fetal endothelial cells and if these differences will lead to sexual disparities in cardiovascular function of adults born from PE pregnancies. Many risk factors, including maternal and/or paternal genetics and cadre of genes underling preexisting maternal vascular dysfunction may contribute to the development of PE preeclampsia, leading to fetal endothelial dysfunction and cardiovascular disorders in children born to preeclampsia. Additionally, as women in general have lower risks of cardiovascular diseases but with worse outcomes than men do38, the more profound transcriptional and cellular responses to preeclampsia in female vs. male fetal endothelial cells might be associated with preeclampsia-induced vascular dysfunction in the fetus and preeclampsia-increased cardiovascular risks in adult offspring.

In conclusion, preeclampsia differentially dysregulates cardiovascular diseases- and endothelial function-associated genes in female and male fetal endothelial cells in association with sexual dimorphisms of fetal endothelial dysfunction. These fetal sex-specific preeclampsia-dysregulated gene networks could contribute to early fetal programming, which may be associated with the developmental origins of cardiovascular diseases of children of PE pregnancies later in life, as endothelial dysfunction is a hallmark of various cardiovascular diseases. As all data are derived from Caucasian subjects, the conclusion we drawn here may not be fully applicable to other racial groups.

Perspectives

Preeclampsia dysregulates the fetal endothelial transcriptome and endothelial function in cells in a fetal sex-specific manner, with female fetal endothelial cells are more dramatically impacted by preeclampsia than male cells. These sexual dimorphisms of preeclampsia-dysregulated fetal endothelial responses may help us identify therapeutic targets and risk predictors for adult-onset cardiovascular diseases in children born to PE mothers.

Supplementary Material

Supplemental Material

Novelty and Significance:

What Is New?

  • There are sexual dimorphisms of preeclampsia-dysregulated cardiovascular diseases and endothelial function-associated gene networks in unpassaged HUVECs using RNAseq

  • Preeclampsia differentially-dysregulated female and male fetal endothelial function in responses to cytokines and growth factors.

What Is Relevant?

  • We have identified fetal sex-specific gene networks that are associated with preeclampsia-induced fetal endothelial dysfunction. Such dysfunction may contribute to the increased risks of cardiovascular diseases in children born to preeclamptic mothers.

  • Our findings advance our understanding of preeclampsia-associated fetal endothelial dysfunction and may open new prospects for developing precision medicine for cardiovascular risk prediction and treatment of preeclampsia-associated cardiovascular diseases in children born to preeclamptic mothers.

Summary

Preeclampsia differentially dysregulates cardiovascular diseases- and endothelial function-associated genes in female and male fetal endothelial cells which may lead to sexual dimorphisms of preeclampsia-associated fetal endothelial dysfunction and contribute to the increased risks of cardiovascular diseases in children born to preeclamptic mothers.

Acknowledgments

The authors thank the University of Wisconsin Biotechnology Center DNA Sequencing Facility and Bioinformatics Resource Center for providing RNAseq and bioinformatics facilities and services. The authors thank Lori Uttech-Hanson, a grant writer from the Office of Research Administration and Proposal Development, UW-Madison for English editing.

Sources of Funding

This study is supported by the American Heart Association award 17POST33670283 (CZ) and National Institutes of Health grants P01HD38843 (JZ, RRM, IMB), R01HL117341 (JZ, RRM).

Footnotes

Disclosures

None.

References

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