Purnima Narasimhan, Ph.D.
Lab Manager/Senior Research Associate
Nadine Martinez, Ph.D.
Frauke von Versen-Höynck, M.D., Ph.D., MSc.
Visiting Assistant Professor, Division of Reproductive Endocrinology & Infertility
Obstetrics & Gynecology, Stanford University School of Medicine
Associate Professor, Obstetrics & Gynecology, Hannover Medical School, Germany
Cynthia Klein, B.S.
Senior Lab Manager
Overall Question of Interest
The Winn Laboratory research program revolves around understanding the process of normal and abnormal human placentation. Acquiring knowledge about human placentation will provide important insights about pregnancy complications such as preeclampsia, intrauterine growth restriction (IUGR), placenta accreta and gestational trophoblastic disease. As a physician scientist, my ultimate goal is to see this knowledge translate into improved clinical care resulting in healthier mothers and infants. Current projects include:
Dissecting the role of preeclampsia related molecules on cytotrophoblast differentiation and invasion.
The ultimate goal of this project is to shed light on the pathophysiology of preeclampsia, a pregnancy complication characterized by maternal hypertension, proteinuria and other end organ damage, with the hope of providing better diagnostic markers and improved therapeutic approaches. The underlying pathophysiology of preeclampsia, while still not fully understood, clearly involves alterations in the maternal endothelium and immune system. One clearly established principle is that the placenta plays a pivotal role. Preeclampsia only occurs in the presence of trophoblastic tissue and the clinical condition typically resolves with the delivery of the placenta. The placental basal plate is the site of known anatomical abnormalities in preeclampsia. Shallow cytotrophoblast (CTB) invasion and limited remodeling of the uterine spiral arteries is noted in the basal plate region of many preeclamptic placentas. Hence, this region has been the focus of our studies.
We have determined the gene expression profile of this region from normal second trimester and term samples as well as from preterm preeclamptic placentas and preterm labor placentas. Analysis of the resulting gene profiles has identified potential pathways and molecules altered within the basal plate of preeclampsia. Our hypothesis is that those differentially expressed genes in preeclampsia contribute to the abnormal CTB invasion seen in preeclampsia.
Ongoing projects in the lab are confirming the expression pattern of the identified molecules determining which cell-types express the molecules of interest (i.e. CTBs, decidua or immune cells) in the basal plate region. This is being done by using immunohistochemistry or by in situ hybridization on banked basal plate biopsies from normal, preterm labor and preeclamptic placentas. Importantly, then the functional significance of these molecules in regards to CTB invasion is explored employing functional perturbation studies using our in vitro model systems (as discussed above). Perturbations are performed by using either blocking/stimulating antibodies where appropriate or by transfection using adeno/lentiviral systems to either over-express or knockdown expression of the target gene. Ultimately the results of these studies should identify key molecules and molecular pathways that potentially serve as therapeutic targets for the prevention and/or treatment of preeclampsia.
Endothelial Progenitor Cell Biology and Preeclampsia: Implications for Maternal and Infant Health
A central feature of PE pathogenesis is maternal endothelial cell dysfunction. While much has been accomplished in characterizing the systemic endothelial dysfunction of preeclampsia, the role of endothelial progenitor cells (EPCs) in the mechanism is not fully understood and may provide a potential cellular target for novel therapies. The association of long-term health consequences of preeclampsia on cardiovascular health may also be mediated through the biology of EPCs. We are examining the types and laternation in EPC during and following preeclampsia and the correlation with endothelial function as assessed through non-invasive techniques such a brachial artery flow mediated dilation (FMD) or EndoPAT.
Placental Extracellular Vesicles Role in Pregnancy and Preeclampsia
The syncytial lining of the placenta that is in direct contact with maternal blood sheds grams of extracellular vesicles into maternal circulation. These vesicles contain not only surface molecules but internal cargo that can direct or alter the recipient cell that they encounter. We are exploring novel methods to assess these vesicles such that placental health can be assessed from a maternal blood draw during on ongoing pregnancy. In addition studies are directed at understand how these “placental packages” are used to communicate and regulate maternal and fetal physiology during pregnancy, particularly focusing on the endothelium and immune system.
The Winn Laboratory has procured placental biopsies, cord blood and maternal blood and urine from over 200 preeclamptic and normotensive pregnancies. This valuable resource supports our translational studies to understand pathophysiology as well as identify predictive and diagnostic biomarkers.
The Remarkable Process of Human Placental Development
We study the remarkable process of human placental development. Interestingly, while the placenta is one of the defining characteristics of mammals, the architecture and development of the mammalian placenta is not highly conserved. The human placenta is one of the most invasive placentas and results in the formation of an organ which comprises cells both from the fetus and the mother. How this chimeric organ forms and is not rejected is truly remarkable. The fetal cells dramatically remodel the maternal vasculature to bring large volumes of maternal blood to the placenta. The mechanisms that regulate both interstitial and endovascular invasion are not fully understood.
Our focus is on the invasive component of the placenta, the region known as the basal plate or decidua basalis.
Diagram of the human maternal-fetal interface. A: Representation of the human placenta after delivery. The placental surface that was adjacent to the uterine wall is termed the basal platedenoted ny the boxed area. B: View of the basal plate at the cellular level. This chimeric region of the placenta is composed of both maternal and fetal components: extravillous (invasive) cytotrophoblasts (dark gray), decidual cells (light gray), remodeled vasculature (both invasive CTBs and maternal endothelium) and maternal immune cells (white).
Image Credit: Winn, et. al. Endocrinology. 2007 Mar.
We utilize several in vitro models to study cytotrophoblast differentiation and invasion:
1. Primary Cytotrophoblast in vitro Differentiation - Cytotrophoblasts isolated from placenta and plated on an extracellular matrix which initiates differentiation down the invasive pathway (red).
2. Villous Explant Model - Anchoring chorionic villi are dissected and plated on an extracellular matrix. After one round of proliferation, the trophoblasts from the cell column, differentiate and invade into the extracellular matrix (blue).
Illustration of two in vitro models for studying human cytotrophoblast invasion: (A) When human cytotrophoblasts (light green cells encircled in red) are isolated from early-gestation placentas and plated on an extracellular matrix (ECM) substrate (Matrigel), they differentiate along the pathway that leads to uterine invasion. By 12 hours in culture these cells form aggregates that resemble cell columns of anchoring villi, and by 48 hours they switch on expression of a repertoire of stage-specific antigens that are expressed in cytotrophoblasts within the uterine wall in situ (dark green cells). These molecules facilitate uterine invasion, vascular mimicry, and evasion of the maternal immune response. (B) When anchoring villi are dissected from the surfaces of early-gestation human placentas (blue box) and plated on an ECM substrate, cytotrophoblasts in cell columns continue to differentiate. By 48 hours many cytotrophoblasts have left the columns and invaded the substrate (green box). During this process they execute the same phenotypic switch that isolated cells carry out.
Image credit: Red-Horse K, et. al. Trophoblast differentiation during embryo implantation and formation of the maternal-fetal interface. J Clin Invest. 2004 Sep;114(6):744-54.