Preeclampsia is a gestational disorder characterized by maternal hypertension, often with proteinuria, affecting up to 10% of all pregnancies worldwide leading to maternal mortality in extreme cases. In the United States, PE affects roughly 3-5% of pregnancies and has increased 25% over the last 20 years. Fetal complications often include intrauterine growth restriction and increased incidence of cardiovascular disorders throughout life. At the endothelial level, the loss of vasodilator production and further ongoing decline in monolayer integrity largely account for the observed symptoms of hypertension and edema/proteinuria, respectively. Work from our own lab suggests that loss of Cx43 function by direct phosphorylation underlies the immediate loss of vasodilation response while others have shown longer term progressive breakdown of cell-cell junctions may be responsible for further edema/proteinurea. Of note, both/all these responses are known to be mediated by Src family kinases sensitive to the drug PP2. Treatment of preeclampsia is difficult in part because of a limited understanding of the origin of the disease and the failure to identify a single consistentl responsible endocrine factor. We have recently pointed out a critical convergence point of the many potential negative growth factors and cytokines at a post receptor level is co-regulation of Src. Limited preliminary data showing PP2 can rescue endothelial cell dysfunction in vitro in response to multiple ligands supports the hypothesis. We now propose to develop high throughput methods to 1) verify our hypothesis that the molecular co-regulation of endothelial dysfunction at the level of Cx43 and cell-cell junctions by growth factors and cytokines is through a common Src (PP2 sensitive) pathway, and 2) screen a novel family of nutraceutical compounds which are safe for human use and may have therapeutic relevance in preeclampsia.
Aim 2 in particular follows from preliminary experiments showing the natural food product conjugated linoleic acid (CLA) in the 10,12 isoform, a Src inhibitor, can rescue inhibited Ca2+ signaling function in our ovine model. We further hypothesize that 10,12 and other CLA isoforms will recover both Ca2+ responses and monolayer integrity in HUVEC otherwise inhibited by growth factors and cytokines. A major limitation to progress is the Ca2+ imaging method used to date is time consuming, so screening of multiple hormones and drugs is impractical. The electrical cell substrate impedance sensing (ECIS) system measures electrical resistance across a cell monolayer in real time, and provides a high resolution temporal view of cell-cell junction breakdown in multiwell format. Thus, the proposed aims will provide a two- pronged, high throughput screening approach for both initiation and recovery of endothelial dysfunction at the levels of vasodilatory function and monolayer integrity. To that end:
Specific Aim 1 screens growth factors and cytokines for the ability to inhibit Cx43 mediated Ca2+ signaling in HUVEC and monolayer junctional integrity.
Specific Aim 2 screens CLA isomers for their ability to rescue Ca2+ signaling and monolayer integrity function in HUVEC.
Preeclampsia (PE) is a potentially devastating disease of pregnancy that can threaten the life of both mother and the unborn child. It affects up to 10% of all pregnancies worldwide and roughly 3-5% of the U.S. population. Even mild PE is dangerous and can cause preterm (early) birth and this then gives the baby an increased risk to develop several diseases such as blood pressure, diabetes and obesity as an adult. We have shown in mothers with PE that the cells lining the umbilical cord blood vessels have reduced function, and we think we know why. The aim of this project is to now use that information to further develop high throughput methods to screen for novel drugs that could provide therapy to treat this otherwise devastating disease.
| Boeldt, D S; Bird, I M (2017) Vascular adaptation in pregnancy and endothelial dysfunction in preeclampsia. J Endocrinol 232:R27-R44 |
