(L-472) Pericyte Recruitment to the Endothelium at the Maternal-Fetal Interface During Preeclampsia

Introduction:: Preeclampsia impacts around 2-8% of all pregnancies globally each year, accounting for 70,000 maternal and 500,000 fetal deaths¹. Currently, the only cure is delivery of the baby, which often involves preterm delivery. Preeclampsia is the new onset of high blood pressure during the 2nd or 3rd trimester¹. It can also manifest as proteinuria, or protein in the urine¹. Failure of the spiral artery to transform into a low resistance conduit is widely accepted as the initiating event for preeclampsia². However, adaptations that occur within the placenta, specifically at the maternal-fetal interface, are not well understood and likely fuel this condition.

Long-term goals for our study include developing mouse models to observe early placenta vascular formation. Vascular development entails differentiation, proliferation, and morphological rearrangement of endothelial cells (EC) in processes known as vasculogenesis and angiogenesis. ECs line the inside of the blood vasculature as it forms³. In contrast, pericytes (PCs) are vascular mural cells in an abluminal position that assist in maintaining blood vessel integrity, among other roles. They interact with ECs to promote microvessel stability³. PC recruitment to ECs is therefore an essential step in the early phases of vascular development, specifically of capillaries.


Materials and Methods:: Tissue Collection. Human placenta samples from preeclamptic and normotensive patients (Carilion IRB #20-954) were collected after birth and fixed in paraformaldehyde. After fixation, samples were washed and transferred to a 30% sucrose solution for 24 hours, followed by embedding in OCT for cryo-sectioning. For initial comparisons, normal healthy WT (C57BL/6) mouse placenta samples were collected on embryonic day 14.5 (E14.5) and prepared similarly as human tissue for immunolabeling, high-power confocal imaging, and quantitative image analysis.

Immunofluorescence. Placenta sections were immuno-stained with primary antibodies against: (1) platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) for ECs, and (2) Neural Glial Antigen-2 (NG2/Cspg4) for PCs. Cell nuclei were labeled with DAPI. These labels were applied to allow assessment of vascular structures in the placenta, with a specific focus on fetus-associated vessels within the chorionic villi that facilitate nutrient and waste exchange.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Mouse placenta samples were harvested into Trizol, following thoracotomy and PBS perfusion, at each of the three time-points: E8.5, E13.5, E18.5. Target genes were Cspg4, Krt7, Pecam1, and Kdr/Flk1/Vegfr2. These genes were selected as initial candidates to determine the temporal expression of vascular- and trophoblast-related genes, guiding the construction of a time-line for their expression.


Results, Conclusions, and Discussions:: Failure of maternal spiral arteries to transform is widely accepted as the major initiator of preeclampsia; however, the impact this may have on developing placental vessels is not well known. To begin to understand corresponding vascular morphology changes in preeclamptic placentas, we immuno-stained human normotensive and preeclamptic placentas for PECAM-1 (ECs) and NG2 (PCs). We found PCs adjacent to ECs in both normotensive and preeclamptic human placentas. The preeclamptic placentas were further divided into preeclamptic with severe features (SF) and without SF. Quantified images showed a downward trend in PECAM-1 and NG2 signal, suggesting there may be some impairment in vessel remodeling and PC recruitment in preeclamptic patients. Similar to human placentas, immuno-stained mouse placentas showed PCs adjacent to ECs along microvessels. However, the PCs appear to be less pronounced in the mouse placentas compared to the human placentas, possibly due to the different stages of placental development. The mouse placentas imaged were at embryonic day 14.5 (E14.5), which is when the placenta is still developing before it reaches full volume. Since the human placentas samples were collected after birth, we then looked into developing mouse normotensive placentas to see how gene expression within PCs and ECs may change over time. We chose three different placental development time-points: (i) early in development (E8), (ii) after primary structures are established (E13.5), and (iii) after trophoblast invasion is complete but before birth (E18.5). Quantitative PCR data shows a decrease in Pecam1 over time, consistent with current literature. Blastocytes (early embryos) highly express Pecam1, presumably to facilitate uterine implantation. PC marker expression (Cspg4/NG2) consistently increased over time, tracking placental development. EC (Kdr/Flk1/Vegfr2) and trophoblast (Krt7) marker expression however appeared relatively unchanged throughout development, though additional biological replicates are needed to better establish these trends. Some future directions for this project include collecting more replicates of mouse placentas for imaging and qPCR, aiming to image them at the different developmental stages. Also, in vivo mouse placenta data will guide the development of an in vitro tissue engineered model of placental vascular formation utilizing murine embryonic stem cells and a fibrinogen-based hydrogel.


Acknowledgements (Optional): :

References (Optional): : [1] S. Rana, E. Lemoine, J. P. Granger, and S. A. Karumanchi, “Compendium on the Pathophysiology and Treatment of Hypertension Preeclampsia,” p. 19.

[2] C. E. Powe, R. J. Levine, and S. A. Karumanchi, “Preeclampsia, a Disease of the Maternal Endothelium,” Circulation, vol. 123, no. 24, pp. 2856–2869, Jun. 2011, doi: 10.1161/CIRCULATIONAHA.109.853127.

[3] L. B. Payne, M. Hoque, C. Houk, J. Darden, and J. C. Chappell, “Pericytes in Vascular Development,” Curr. Tissue Microenviron. Rep., vol. 1, no. 3, pp. 143–154, Sep. 2020, doi: 10.1007/s43152-020-00014-9.

[4] K. A. Pennington, J. M. Schlitt, and L. C. Schulz, “Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay,” JoVE, no. 59, p. 3202, Jan. 2012, doi: 10.3791/3202.