(L-478) Shear Stress Modulates Transgelin Expression in Preeclampsia Affected Endothelial Colony Forming Cells

Introduction:: Preeclampsia, a hypertensive disorder of pregnancy, is one of the leading causes of infant and maternal death worldwide. It is characterized by the onset of hypertension, proteinuria, and/or other severe complications including elevation of liver enzymes and decreased platelet counts after 20 weeks of gestation, causing increased risk for hypertension, stroke, and cardiovascular disease for both the mother and the child1. Despite this, there is little known about the root cause of preeclampsia, due in part to it being a complex, multisystem pathology. While many mechanisms including oxidative stress and endothelial dysfunction are known to play a major role, there is no unifying hypothesis. One cell type that is shown to be downregulated in preeclampsia is endothelial colony forming cells (ECFCs), a type of endothelial progenitor cell that maintains vascular homeostasis and supports vessel formation. The decrease of these cells may play a role in endothelial dysfunction seen in preeclampsia. Here, ECFCs isolated from umbilical cord blood were used to understand the phenotypic difference between healthy ECFCs and their preeclamptic counterparts (PRECs). Previous studies on cord blood derived ECFCs identified transgelin (TAGLN) as one of the genes significantly increased in disease affected ECFCs with lower rates of migration and tube formation, similar to what was seen in the PRECs². Since transgelin is an F-actin binding protein that regulates the organization of the actin cytoskeleton, cellular contractility, and motility, we investigated the effect of preeclampsia on cell alignment to shear stress, cell migration, and tube formation.

Materials and Methods:: Human ECFCs and PRECs were isolated from cord blood samples from pregnancies with a variety of gestational ages. These cells were provided through clinical trials by members of the Indiana University School of Medicine, Dr. Laura Haneline and Dr. David Haas. ECFCs and PRECs were characterized using immunostaining and FACS for their endothelial surface markers, including Ulex Lectin, CD31, CD144, and CD34. The angiogenic potential of ECFCs and PRECs were characterized using in vitro angiogenesis assays. Cell proliferation and migration assays were used to compare the functionality of ECFCs and PRECs. ECFCs and PRECs were seeded into flow chambers, allowed to grow for 2-6 hours, then subjected to 7 to 48 hours of flow at a shear stress of 10 dynes/cm2. Following treatment, they were fixed and stained for Transgelin, F-actin, and DAPI. All the stained samples were imaged on the Nikon AX-R. The F-Actin fibers’ alignment to flow was quantified using the ImageJ plugin Directionality.


Results, Conclusions, and Discussions:: Noticeable differences in cell proliferation, migration, and tube formation between ECFCs and PRECs were observed. Angiogenic potential (Fig. 1A-C) and migration (Fig. 1F) were decreased in PRECs, while cell proliferation was increased (Fig. 1D-E). PRECs had more dysregulated networks (Fig. 1 B) and a lower tube-to-node ratio (Fig. 1C), indicating a decreased tube forming ability. Compared to healthy ECFCs, there was increased transgelin alignment with F-Actin in PRECs, as indicated by the blue arrows (Fig. 1G). This suggests that transgelin may more severely affect cytoskeletal rearrangement in PRECs than ECFCs. Without flow, the F-Actin fibers do not align in a particular direction (Fig. 1H). However, after 7 hours of flow treatment, the F-Actin fibers in the PRECs partially align with flow. While most of the fibers align within ± 20 degrees from the direction of flow, a group of fibers is aligning around -60 degrees (Fig. 1I), which is not seen in ECFCs. These findings indicate that preeclampsia decreases F-Actin alignment with the flow, which may play a role in the endothelial dysfunction seen in this disorder. After 48 hours of flow treatment, transgelin alignment with F-Actin decreases in both ECFCs and PRECs (Fig. 1G). The F-Actin fibers align more closely with the direction of flow in PRECs than previously seen (Fig. 1K). Though compared to the ECFCs, there are still fewer fibers aligned within ± 20 degrees of the direction of flow (Fig. 1J-K). Based on these findings, longer shear stress exposure causes increased alignment despite transgelin overexpression. Shear stress perturbations caused by a hypertension-induced decrease in placental flow could disrupt this effect. Therefore, alternative molecular mechanisms through the TGF-beta pathway must be examined to improve misalignment sooner and more efficiently. Increased transgelin expression and alignment with F-actin in response to shear stress plays a role in the decreased cell migration and impaired angiogenic potential in PRECs. This novel finding provides further insight into preeclampsia at the cellular level and provides insight into potential markers, which can be used to restore the angiogenic potential of PRECs and to improve the health of children born from preeclamptic pregnancies.

Acknowledgements (Optional): : ECFCs were kindly provided by Dr. Laura Haneline and Dr. David Haas at IUSM. We are grateful for funding from the University of Notre Dame, Indiana CTSI, The American Heart Association, and NSF.

References (Optional): : 1. Ayoubi. Pre-eclampsia: pathophysiology, diagnosis, and management. VHRM 467 (2011) doi:10.2147/VHRM.S20181.

2. Varberg, K. M. et al. Transgelin induces dysfunction of fetal endothelial colony-forming cells from gestational diabetic pregnancies. American Journal of Physiology-Cell Physiology 315, C502–C515 (2018).