In the November issue of the JCI, Wang et al. provide new molecular insights into the well-known effects of maternal smoking on impairing fetal growth. The research team found elevated expression of the Wnt antagonist secreted frizzled-related protein 1 (sFRP1) in placental tissue from mothers who smoked. Using a mouse model, they then showed that adenoviral overexpression of sFRP1 in pregnant mice impaired the proliferation of trophoblast cells and induced fetal growth restriction. They further demonstrated that a carbon monoxide analog reduces Wnt activity and is associated with increased expression of sFRP-1 in a human trophoblast cell line. First author Alice Wang recently shared some of her experiences from this project and how she incorporates research with clinical work as a neonatologist.
Could you tell us about your training and what brought you into the lab as a practicing neonatologist?
I majored in biology and literature as an undergraduate student at Duke and spent several summers and semesters working in basic science labs, which exposed me to biomedical research. I stayed at Duke for medical school, which has a unique curriculum that involves a full year dedicated to basic or clinical research following a compressed schedule of preclinical and clinical rotations during the first two years. I spent my elective year, as a Sarnoff fellow, in the laboratory of Shaun Coughlin at UCSF, where I developed an interest in developmental and vascular biology.
After finishing a residency in pediatrics and subsequently a fellowship in newborn medicine, I received Flight Attendant Medical Research Institute (FAMRI) funding to work on projects related to smoking-related fetal growth restriction, a topic that had always intrigued me. I wanted to understand how maternal and extrinsic environmental factors could affect the placenta and the fetus. That funding has really made it possible for me to do this research. At the time, I had very little preliminary data, and FAMRI really took a chance on me to do this.
How did you begin the work for this particular manuscript?
We didn’t start out examining Wnt signaling in the placenta. We started with a directed focus on hepatocyte growth factor and its potential role in regulating fetal growth. This project unfortunately yielded mostly negative data, although it was based on a very reasonable hypothesis. In the process of performing this work, I learned how to prioritize my efforts and when to cut bait. We then took a more unbiased approach to identify candidate genes altered in smoking-related fetal growth restriction, and in analyzing our microarray findings from the placentas, we found huge changes in Wnt signaling. That signal pointed us in this direction.
What was the biggest obstacle you faced in completing this project?
I found that this kind of research has been much more challenging than I had originally imagined. In papers, the experiments seem to flow so linearly and so logically. However, the papers don’t reveal all the experiments that don’t work, or the process by which hypotheses develop. You work hard, spend a lot of time on experiments, and sometimes they don’t work. It’s very hard to know when to persist or when to give up on an idea, and one can struggle with self-doubt. It can be very discouraging to start over, knowing how much time you’ve invested and knowing how long it will take to answer the question. As clinician-scientists, we have so many questions we want to answer, yet we have so many demands on our time. With these kinds of demands, it’s easy to become discouraged and to think that we’re just wasting our time on research.
What clinical commitments and responsibilities did you have during this work?
As neonatologists, we work in shifts. We take a lot of mandatory in-house night call assignments, where we are frequently up much of the night to go to deliveries and to take care of sick babies. This kind of work has its advantages and disadvantages, because you don’t have months of being on service that totally takes you away from the lab, but you’re up working all night, which is also not great for your productivity.
So I would multitask a little bit. When I’m on call in the hospital, I try to collect placentas. Placentas would otherwise be extremely difficult to obtain because most babies (and placentas) are not delivered on a schedule or between the hours of 9:00 and 5:00. In order to get the placentas, we have to be there ideally within 30 minutes to an hour of delivery to get fresh tissue. The only person that’s going to be there at 3:00 am to collect the placenta, unless you have an extensive research team, is the neonatologist or the obstetrician.
Can tell any more about the difficulties you face in studying the placenta, as it pertains to this work?
I think research in placental biology is pretty challenging in itself. Although we have access to the placentas at time of delivery, we can’t get placentas at the onset of disease or to placentas throughout gestation. Our animal models are not as good as in some other diseases, in the sense that they aren’t perfectly representative of human pregnancy. I think our field also doesn’t yet have all the cellular and molecular tools that other fields like oncology or hematology or cardiology might have.
Would alternative samples, such as fresh frozen placenta or tissue from the pathology lab, be useful?
Pathology samples stored in formalin are good, but they’re not going to be helpful for RNA gene expression or if you want tissue that’s really fresh. Placentas tend to sit in the refrigerator and don’t always get immediately processed by pathologists. Having liquid nitrogen for samples would be enormously helpful, but it’s hard to get liquid nitrogen in a hospital setting. The logistics of obtaining, transporting, and storing frozen placentas can be very challenging, because we can’t really leave the hospital when we are on call. So I frequently have to bring a big bucket of liquid nitrogen or dry ice or RNAlater with me to the hospital.
What factors contributed to the success of your project, particularly as you worked across multiple institutions to get this done?
I wouldn’t have been able to do this project without all the help and support that I received, not only from my mentor, but also from the colleagues in my lab who contributed. I am very grateful for the guidance and teaching from the PhD members of the lab. My husband, Paul Yu, who’s also a physician-scientist, has also helped me tremendously. He’s been very supportive and very encouraging. He’s helped me with experimental techniques, with my writing, and provides good criticism. Institutional support at Boston Medical Center by Barry Zuckerman and now Bob Vinci and Mark Mirochnick has been very important as well. They’ve really allowed me the time and flexibility to do this research across institutions, to work at the Beth Israel Deaconess Medical Center with my mentor, Ananth Karumanchi. And of course, without my mentor, none of this work would have been possible. He’s given me the resources, freedom, and time to pursue this work. Mentorship is so critical for our development. I don’t think it would be possible for a clinician-scientist nowadays to just bloom and flourish on their own without support from many corners.
Is there anything at the institutional level that could change or that you believe is most helpful for the developing physician-scientist?
I think that institutions should definitely focus on supporting physician-scientists at the late fellowship stage and/or just after completing fellowship. That stage is a very challenging time in general. Both funding support and institutional support are critical. Even if you are fortunate to have funding and a position, some institutions don’t protect your time, which is our most limited resource. You can’t get any traction on getting the work done without sufficient time. If you have experimental failures, then you’re set back even longer. Institutions can’t support young investigators ad infinitum, but sometimes the research doesn’t fit into two years, but maybe in four years there might be something really interesting.
How do you balance the clinical and research commitments of your career path as you strive for success in both?
There’s always this tension between clinical responsibilities and research time. This is especially true in the NICU, which is staffed 24 hours a day. This tension is even more apparent for our obstetric and surgical colleagues, who need to spend a significant amount of time to maintain their surgical acumen. Balancing this tension is difficult.
However, because the patients that we take care of inspire our research questions, we’re driven by a stubborn curiosity to go into the lab to figure out the causal factors, to determine how a disease process may be driven by either inherent genetic mechanisms or by signaling pathways that are altered by external factors.
Alice Wang, MD, received her medical degree from Duke University School of Medicine. She completed her pediatrics residency in the Boston Combined Residency Program in 2005 and her neonatology fellowship in the Harvard Neonatal-Perinatal Medicine Fellowship Program in 2008. She is currently an Assistant Professor of Pediatrics at Boston University School of Medicine and an attending neonatologist at Boston Medical Center.
Freddy T. Nguyen is an MD/PhD candidate at the University of Illinois at Urbana-Champaign. He is the founder of the American Physician Scientists Association and served on the Associate Member Council of the American Association for Cancer Research. His research interests currently lie at the intersection of biomedical optics and cancer research. He received his BS in chemistry and BA in mathematics from Rice University.
James M. Pauff, MD, PhD, is a Clinical Fellow in Medical Oncology at Vanderbilt University Medical Center. He has served as President and on the Board of Directors of the American Physician Scientists Association. His research interests are in molecular pathways of cancer and targeted therapeutics, and he is currently in the laboratory of Carlos Arteaga, where he studies mechanisms of resistance to compounds targeting the estrogen receptor in hormone-positive breast cancer. Dr. Pauff completed his internal medicine residency at Vanderbilt and received his MD and PhD from the Ohio State University.
Maternal cigarette smoking during pregnancy remains one of the most common and preventable causes of fetal growth restriction (FGR), a condition in which a fetus is unable to achieve its genetically determined potential size. Even though epidemiologic evidence clearly links maternal cigarette smoking with FGR, insight into the molecular mechanisms of cigarette smoke–induced FGR is lacking. Here, we performed transcriptional profiling of placentas obtained from smoking mothers who delivered growth-restricted infants and identified secreted frizzled-related protein 1 (sFRP1), an extracellular antagonist of endogenous WNT signaling, as a candidate molecule. sFRP1 mRNA and protein levels were markedly upregulated (~10-fold) in placentas from smoking mothers compared with those from nonsmokers. In pregnant mice, adenovirus-mediated overexpression of sFRP1 led to FGR, increased karyorrhexis in the junctional zone, and decreased proliferation of labyrinthine trophoblasts. Consistent with our hypothesis that placental WNT signaling is suppressed in maternal smokers, we found that exposure to carbon monoxide analogs led to reduced WNT signaling, increased
Alice Wang, Zsuzsanna K. Zsengellér, Jonathan L. Hecht, Roberto Buccafusca, Suzanne D. Burke, Augustine Rajakumar, Emily Weingart, Paul B. Yu, Saira Salahuddin, S. Ananth Karumanchi