Characterisation of cardiac health in the reduced uterine perfusion pressure model and a 3D cardiac spheroid model, of preeclampsia

Fig. 1. RUPP surgery increases collagen deposition and BNP45 concentration in rat hearts. a, b Paraffin-embedded rat hearts were sectioned at 10μm thickness and stained with picrosirius red to visualise collagen I/III (red) and muscle cell cytoplasm (yellow) in sham and RUPP rats. Scalebar = 50 μm. Images taken at × 5 using an Axioscan microscope were analysed for percentage area stained red to quantify collagen deposition as an indicator of cardiac fibrosis. c Rat heart protein isolated by homogenisation with RIPA lysis buffer was quantified by indirect ELISA against known standards to quantify protein concentration of BNP45. Data points are expressed as mean percentage ± SEM; n ≥ 6, unpaired Student’s t-test; *p < 0.05, ***p < 0.001

Fig. 2. RUPP hearts express higher mRNA and protein levels of FKBPL. Total RNA extracted from rat hearts by TRIsure reagent was analysed by RT-qPCR to determine mRNA expression levels of Fkbpl, Flt1 and Vegfa. Relative mRNA expression of a Fkbpl, b Flt1 and c Vegfa mRNA levels comparing sham and RUPP hearts normalized to B-actin. Data presented as mean fold change ± SEM; n ≥ 6; Mann-Whitney t-test; *p < 0.05. d Fkbpl expression was verified at the protein level by enzyme-linked immunosorbent assay (ELISA), concentration calculated against a known standard and plotted against total mg of protein per sample. Data presented as mean ± SEM; n ≥ 6; unpaired Student’s t-test. e, f Cardiac FKBPL expression was further analysed by immunofluorescent staining and intensity measured using ImageJ by analysing greyscale value in six × 40 images per animal. Image brightness was adjusted equally for representative images displayed. Data plotted as mean fold change ± SEM; n ≥ 5; unpaired Student’s t-test; *p < 0.05

Fig. 3. RUPP rats have increased collagen deposition and decreased Flt1 mRNA expression in their placentae. a Paraffin-embedded rat placentae were sectioned at 10μm thickness and stained with picrosirius red to visualise collagen I and III fibres (red) in sham and RUPP hearts. Scalebar = 100μm. b Images of picrosirius red-stained tissue were taken at × 5 using an Axioscan microscope and analysed for percentage of area stained red to quantify collagen deposition as an indicator of placental fibrosis. Data points are mean ± SEM; n = 7, unpaired Student’s t-test; *p < 0.05. RNA extracted from rat placentae by TRIsure reagent was analysed by RT-qPCR to determine mRNA expression levels of c Flt1 and d Vegfa. Data presented as mean fold change ± SEM; n = 7; Mann-Whitney t-test; *p < 0.05

Fig. 4. RUPP rats present with larger glomeruli in rat kidneys. Paraffin-embedded rat kidneys were cut at 10-μm sections and stained with H&E staining to visualise tissue morphology. a Images of entire tissue sections were taken at × 4, × 10 and × 20 objective using an Axioscan microscope to produce virtual slides of sham and RUPP kidneys. Scalebar = 20 μm. b Average glomerular area was measured at a × 20 magnification using ZEN Lite 3.2 software. Data plotted as mean ± SEM; n = 7; unpaired Student’s t-test; *p < 0.05

Fig. 5. Immunofluorescent images of cardiac spheroids treated with plasma samples from healthy control, early-onset and late-onset preeclampsia patients. Cardiac spheroids were generated in hanging droplets by co-culturing primary human cardiac fibroblast cells (HCFs) and human coronary artery endothelial cells (HCAECs) in a 1:1 ratio. Following formation of 3D structures, the spheroids were treated with human plasma samples from women with or without preeclampsia. After plasma treatment, spheroids were fixated and permeabilised prior to labelling with antibodies (FKBPL, CD31) and fluorescent stain (Hoechst). a Spheroids treated with plasma from normotensive control pregnancies. b Spheroids treated early-onset preeclampsia (EOPE) patient plasma. c Spheroids treated with late-onset preeclampsia (LOPE) patient plasma

Fig. 6. Expression of FKBPL and CD31 in cardiac spheroids treated with plasma from healthy control, early-onset and late-onset preeclampsia patients. Cardiac spheroids generated by co-culturing primary human cardiac fibroblast cells (HCFs) and human coronary artery endothelial cells (HCAECs) were treated with patient plasma from normotensive controls, early-onset preeclampsia and late-onset preeclampsia. Immunofluorescent expression of a FKBPL and b CD31 in cardiac spheroids was quantified. Data plotted as mean ± SEM; n = 3; ordinary one-way ANOVA with Tukey’s multiple comparison test; *p < 0.05