Animal models of preeclampsia have been difficult to establish, primarily due to the fact that preeclampsia does not spontaneously occur in most species other than humans and the presence of inter-species variations [34]. The most reliable animal model of preeclampsia is the surgically-induced RUPP model in rats or mice. This model has been shown to induce hypertension[35], proteinuria[35], renal dysfunction[35, 36], an anti-angiogenic state[37], abnormal immune responses[38–40], vasoconstriction[41], oxidative stress[42], cardiac dysfunction[43] and IUGR [44, 45] like that of preeclampsia in humans. However, the cardiovascular dysfunction particularly in terms of heart health has not been extensively studied in this model of preeclampsia before[46]. Although there is substantial evidence published exploring this model’s reflection of human features of preeclampsia, we aimed to confirm the cardiovascular phenotype in RUPP rats and to determine the presence of altered FKBPL expression. This is the first study that explores the role of this novel anti-angiogenic protein in cardiovascular features of preeclampsia.
In this study, surgically reduced perfusion to the uterus in pregnant rats resulted in anatomical and physiological alterations to maternal hearts, placentae and kidneys with a number of features of preeclampsia being demonstrated. Given the well-established association between preeclampsia and increased risk of developing future CVD[47], this is an important aspect in modelling the manifestation of preeclampsia. In fact, recently published results describe the overlapping mechanisms between preeclampsia and future cardiovascular disease with angiogenesis- and inflammatory-related pathways playing a key role[15].
A significant increase in systolic, diastolic and mean arterial blood pressure confirmed that the RUPP procedure had been successful and increased heart rate with LV hypertrophy were evident, as determined by echocardiography. In our model, we did not apply too much pressure on the uterine clips and hence did not observe significant changes in the embryo weight or number in the RUPP rats, suggesting that it may be more representative of a LOPE phenotype. LOPE is diagnosed from 34 weeks of gestation, it is a less understood phenotype of preeclampsia, likely occurring secondary to maternal microvascular diseases, reflective of underlying vascular dysfunction. It seems to develop due to maternal inability to meet metabolic and cardiovascular demands of the growing foetus. On the other hand, EOPE is usually diagnosed before 34 weeks’ of gestation and associated with foetal growth restriction as well as inadequate or incomplete trophoblast invasion of spiral uterine arteries, often implicating placenta as the root cause[17]. Nevertheless, our analysis of placental collagen deposition found RUPP placenta to be more fibrotic compared to sham controls. Placental fibrosis has been found to occur in placentae of women with preeclampsia compared to normotensive controls[48]. Additional quantification of fibrotic factors including connective tissue growth factor (CTGF) or fibronectin in these placentae could aid in determining levels of fibrosis. Of note, placental fibrosis is a prominent feature of preeclamptic placentae and has been shown to be associated with activation of stromal fibroblasts via the TGF-β1 signalling pathway[48]. Reduced angiogenesis as depicted by decreased Flt1 expression further support this finding.
Further, given that preeclampsia is frequently associated with altered renal function and histology, RUPP and sham kidneys were inspected for altered tissue morphology showing that glomeruli in RUPP samples had a significantly larger surface area. While the RUPP model has not yet been described as displaying glomerular endotheliosis, a characteristic feature of preeclampsia[49, 50], some changes to kidney tissue were previously noted. Nevertheless, this measurement is limited to 2-dimensional analysis rather than a 3-dimensional measurement of each glomerular structure and the characteristic endothelial swelling was not observed, our data suggests glomerular endotheliosis may be present in RUPP rats, which has previously been observed in an sFlt-1 administration model of preeclampsia in rats[51]. Electron microscopy or glomerular filtration rate would be important tools to measure this in future experiments. Moreover, previous studies have demonstrated an increase in proteinuria and a decrease in glomerular filtration rate following the RUPP procedure, with the latter lasting up to 8 weeks postpartum[52, 53].
In relation to cardiovascular health in the RUPP model, picrosirius red staining of RUPP hearts suggests the presence of cardiac fibrosis compared to sham controls, which is consistent with the preeclampsia phenotype and increased risk of CVD including cardiac fibrosis, later in life[54–56]. These results are also consistent with another study characterising the cardiac effects of RUPP in rats that showed a significant increase in collagen I/III fibrotic markers in RUPP hearts [57]; these effects have been shown to be reversed post a RUPP pregnancy[53]. BNP is released from the cardiac ventricles in response to diastolic and systolic dysfunction, which places additional stress on the heart walls[58, 59]. We have noted a significant increase in cardiac levels of BNP, which is supported by other studies’ findings in preeclampsia models [57, 60].
In addition to cardiac fibrosis, FKBPL was significantly increased at the mRNA level as a result of RUPP procedure. However, FKBPL is a chaperone protein and prone to post-transcriptional modification so quantification of its expression at the protein level is more informative[61]. Therefore, ELISA was performed on rat heart protein lysates and indeed confirmed an increase in FKBPL protein levels in RUPP hearts, suggesting that FKBPL could be a novel mechanism of cardiovascular damage in preeclampsia. Further, recent data from our group has shown that FKBPL is increased as a result of cardiac fibrosis and TGF-β stimulation in cardiac fibroblasts[62]. FKBPL is also increased in plasma and placentae from women with preeclampsia[19], and in people with CVD[20].
The use of an appropriate 3D cell culture model to study the effects of secreted placental factors on cardiac heart cells would enable a deeper exploration of the cardiac effects of preeclampsia. While traditional 2D cell culture methods are limited in their ability to recapitulate the cellular structure and function within a tissue, three-dimensional (3D) cell culture methods with polymer-containing extracellular matrix (ECM), provide a 3D cell architecture allowing interaction in all spatial dimensions with both other cells and their environment. This allows for fine-tuning of the microenvironment by modifying properties including elasticity, stiffness, conductivity and porosity[63].
FKBPL in the cardiac spheroid model was evaluated to determine its role in a human models of cardiac spheroids in the presence of plasma from patients with EOPE, LOPE and healthy controls. CD31 is a marker of endothelial cells and as such evaluation of this marker was used to interpret FKBPL expression in the context of endothelial cell health within cardiac spheroids. Analysis determined that FKBPL expression was elevated in LOPE-treated spheroids when compared to healthy controls. We also noted a trend of increased FKBPL expression in EOPE-treated spheroids compared to control, though this was not significant likely due to small sample size. This is in alignment with studies performed by Gonzalez-Quinero et al. showing that endothelial microparticles were over expressed in women with preeclampsia compared to women with healthy pregnancies and those presenting with gestational hypertension[64]. Therefore, it appears that circulating factors in maternal plasma during preeclampsia may induce an overexpression of FKBPL in cardiac fibroblast and/or endothelial cells. While this did not induce a notable change in CD31 expression, this model could be used to further study the mechanism of this effect. Adding cardiomyocytes to the co-culture in a 2:1:1 ratio with the fibroblasts and endothelial cells used has been shown to produce a vascularised cardiac spheroid suspension, which have the potential to produce a more representative model of cardiac health in preeclampsia[65].
Perspectives and Significance
This is the first study that implicates FKBPL as a novel anti-angiogenic mechanism in cardiovascular dysfunction as a result of preeclampsia that could be explored in the future for better understanding of the association between preeclampsia and future CVD risk. Furthermore, our study demonstrated that preeclampsia could induce cardiac fibrosis and diastolic dysfunction in association with increased FKBPL levels. This knowledge is important for developing future monitoring and treatment strategies to prevent heart disease in women during and post preeclampsia.