3D in vitro modelling of post-partum cardiovascular health reveals unique characteristics and signatures following hypertensive disorders in pregnancy

Abstract

Background: Hypertensive disorders of pregnancy (HDP) affect 2-8% of pregnancies and are associated postpartum with increased cardiovascular disease (CVD) risk, although mechanisms are poorly understood.

Methods: Human induced pluripotent stem cells (iPSC)-derived cardiomyocytes, cardiac fibroblasts and coronary artery endothelial cells were cocultured to form cardiac spheroids (CSs) in collagen type-1 hydrogels containing 10% patient plasma collected five years postpartum [n = 5 per group: normotensive control, gestational hypertension (GH) and preeclampsia (PE)]. Plasma-treated CSs were assessed for cell viability and contractile function and subjected to immunofluorescence staining and imaging. A quantitative proteomic analysis of plasma samples was conducted (controls n = 21; GH n = 5; PE n = 12).

Results: Contraction frequency (CF) was increased in PE-treated CSs (CF: 45.5 ± 3.4 contractions/minute, p < 0.001) and GH-treated CSs (CF: 45.7 ± 4.0 contractions/minute, p < 0.001), compared to controls (CF = 21.8 ± 2.6 contractions/min). Only PE-treated CSs presented significantly increased fractional shortening (FS) % (9.95 ± 1.8%, p < 0.05) compared to controls (3.7 ± 1.1%). GH-treated CSs showed a reduction in cell viability (p < 0.05) and an increase in α-SMA expression (p < 0.05). Proteomics analyses identified twenty differentially abundant proteins, with hemoglobin A2 being the only protein perturbed in both GH and PE versus control plasma (p < 0.05).

Conclusions: The innovative patient-relevant CS platforms led to the discovery of biomarkers/targets linked to cell death signaling and cardiac remodeling in GH-induced CVD and vascular/endothelial cell dysfunction in PE-induced CVD.

Keywords: Biomarkers; Cardiovascular disease; Hypertensive disorders of pregnancy; Post-partum; Pregnancy; Proteomics.

Fig. 1

Plasma-derived from women with HDP reduces the cell viability of CSs. A-C Representative collapsed Z-stacks images of all and dead cells within CSs. The percentage of live cells remaining following exposure to human plasma from women with HDP was quantified for A) Control (normotensive plasma). B GH plasma. C PE plasma. 10% of normotensive, GH or PE plasma are added to CSs plated in collagen hydrogel, respectively. After 96 h, CSs were stained with Hoechst for nuclei stain, and ethidium homodimer for dead cells. Scale bar equals 200 µM. D Statistical analysis of the fold change in live versus dead cells comparing all three sample groups: controls, GH and PE. Data represented as Mean ± SD; n = 5 patients per group; P-value was calculated using one-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05

Fig. 2

HDP-derived plasma impairs contractile function in CSs. A-C representative images from videos of contracting control- (A), GH- (B) and PE- (C) treated CSs, where yellow outlines the area of the cardiac spheroid during the phase of contraction in each spheroid. Scale bar 500 μm. (D-E) Statistical analyses of contraction frequency (D) and fractional shortening % (E) in CS. 4–8 CSs per group from 1–3 patients per group; The number of CSs per group: control (n = 8), GH (n = 9), PE (n = 9–11); P-value was calculated using one-way ANOVA post-hoc analysis. Data represented as Mean ± SD; n = 1–3 patients per group; n ≥ 8; P-value was determined using one-way post-hoc analysis; * p < 0.05, *** p < 0.001

Fig. 3

Treatment with HDP plasma does not change the numbers of cardiomyocytes, cardiac endothelial cells and cardiac fibroblasts in CSs. A-C Representative maximum-intensity projection confocal images of CSs stained for the three cell markers. CSs were exposed to 10% plasma (A) normotensive (control), (B) GH, and (C) PE, for 96 h, fixed and stained with antibodies against cardiomyocytes (cTNT, magenta), endothelial cells (CD31, yellow) and fibroblasts (vimentin, green). Nuclei are stained with DAPI stain (cyan). Confocal images are shown for representation only; widefield images were used for quantification of protein expression. Scale bar 100 μm. D-F Statistical analysis of the fold change in protein expression of vimentin (D), cTNT (E) and CD31 (F) for normotensive, GH and PE CSs, normalized to control. Data represented as Mean ± SD; n = 5 patients per group; The number of CSs per group: control (n = 23), GH (n = 31–32) and PE (n = 20–21); Kruskal-Wallis test witht Dunn’s multiple comparison test

Fig. 4

HDP plasma treatment increases α-SMA in CSs. A-C Maximum-intensity projection images from confocal Z-stacks of CSs exposed to normotensive (control) (A), GH (B) and PE (C) plasma for 96 h. Samples were stained with DAPI (cyan) for nuclei stain, as well as antibodies against FKBPL (magenta) and α-SMA (yellow). Confocal images are presented for visual representation only, while widefield images were utilized for the quantification of protein expression. Scale bar 100 μm. D Statistical analysis of the fold change of protein expression of FKBPL for control, GH and PE groups, normalized to control. E Statistical analysis of the fold change of protein expression of α-SMA for control, GH and PE groups, normalized to control. The number of CSs per group: control (n=31), GH (n=28-29) and PE (n=36). Data represented as Mean ± SD; n = 5 patients per group; P-value was determined using Kruskal-Wallis test with Dunn’s multiple comparison test; *p < 0.05

Fig. 5

Gal-3 is increased in PE-treated CSs, and no changes in VE-Cadherin among groups were shown. A-C Maximum-intensity projection images from confocal Z-stacks of CSs treated with normotensive (control) (A), GH (B) or PE (C) plasma for 96 h. Samples were stained with DAPI (cyan) for nuclei stain, as well as antibodies against Gal-3 (magenta) and VE-Cadherin (yellow). Confocal images are shown for illustration purposes only, while widefield images were used for quantitative analysis of protein expression. Scale bar 100 μm. D Statistical analysis of the fold change of protein expression of Gal-3 for control, GH and PE groups, normalized to control. E Statistical analysis of the fold change of protein expression of VE-Cadherin for control, GH and PE groups, normalized to control. Data represented as Mean ± SD; n = 5 patients per group; The number of CSs per group: control (n = 26), GH (n = 31) and PE (n = 30); P-value was calculated using Kruskal-Wallis test with Dunn’s multiple comparison test; *p<0.05

Fig. 6

Differential expression of grouped patient plasma following untargeted proteomics analyses. Grouped samples were measured in triplicates to account for anomalies. (A) Principal component analysis (PCA) plot of grouped proteomic data for control (purple dots), GH (blue dots) and PE (orange dots) generated using Progenesis. (B) Multigroup heatmap with hierarchical clustering dendrogram of proteomic data levels across control, GH and PE groups. Volcano plots of proteomic data for (C) PE vs. normotensive (D) GH versus normotensive (E) PE vs. GH. Significant proteins were defined as Benjamini–Hochberg adjusted p-value < 0.05