Robert D. Koos, PhD

Molecular and Cellular Aspects of Angiogenesis and other Developmental Processes in the Ovary and Uterus:

The goal of my research is to understand the mechanisms that control developmental events, particularly angiogenesis (the growth of new blood vessels), in the follicle and corpus luteum (CL) of the ovary and the endometrium of the uterus. Blood vessels repeatedly grow and regress during the rapid, hormonally regulated development of these tissues, unlike in other tissues, during the reproductive cycle and pregnancy, making them excellent systems for the study of this fundamental developmental process. Understanding the factors that regulate normal vascular development is not only crucial to understanding these key reproductive events, but also the abnormal angiogenesis that contributes to the progression of many diseases, including cancer and atherosclerosis. In recent years, we have concentrated our efforts on examining the roles of fibroblast growth factors (FGFs) and vascular endothelial growth factor/vascular permeability factor (VEGF) in this process. Both FGFs and VEGF are endothelial cell mitogens. VEGF is also a potent stimulator of capillary permeability. The latter property is especially relevant to the reproductive system because increased vascular permeability and edema occur in the ovary prior to ovulation and in the uterus in response to estrogen, and is believed to be essential for those changes. Using the sensitive and specific technique of quantitative reverse transcription-polymerase chain reaction (RT-PCR), we have recently shown that VEGF expression increases markedly following the ovulatory signal, and immediately before the onset of follicular edema, suggesting that VEGF plays a role in follicle rupture as well as vascularization of the CL. In vitro, VEGF expression by granulosa cells is strongly stimulated by hypoxia, a fundamental stimulus for angiogenesis, and the normal state in the developing follicle and early CL. We have also shown that VEGF expression is rapidly stimulated by estrogen in the uterus, and that the increase immediately precedes uterine edema, suggesting that VEGF induces this response. Having identified two physiological stimuli that regulate VEGF expression, we are now exploring the molecular mechanisms involved. We are currently using the powerful technique of formaldehyde cross-linking/chromatin immunoprecipitation (ChIP) to map the sites in the promoter region of the VEGF gene through which estrogen acts.

FGF-10 and Follicle Development:

FGF-10 is a growth factor that plays essential roles in the development of many tissues and organs including the limbs and lungs. Mice that lack a functional FGF-10 gene are born without legs or lungs. FGF-10 is also expressed in the ovary, and expression correlates with the formation of mature follicles. To understand the role that FGF-10 plays in this process we are transplanting the ovaries from newborn FGF-10 knockout mice to host animals, in which the ovary can mature. The preliminary results of these studies show that the absence of FGF-10 completely disrupts normal follicle growth. The mechanism of this effect is now being examined.

Estrogen Action:

Estrogen regulates the growth and differentiation of the uterus and other target tissues via binding to estrogen receptors (ERs), which are members of the nuclear receptor family of transcription factors. Two forms of ER are now known to exist: ERa and ERb. ERa is a well-characterized mediator of estrogen-induced transcriptional activity in target cells, but the function of the more recently discovered ERb is unclear. Recent in vitro studies suggest that both of the splicing forms of ERb expressed in rat tissues, b1 and b2, may function in part as inhibitors of ER transcriptional activity. To gain further insight into the actions of estrogen in target tissues and into the role of ERb in those actions, we recently examined its effects and those of relaxin, a ligand-independent activator of ERs, on the expression of ERb1 and ERb2 mRNA in the uterus in vivo. Estrogen markedly decreased the steady-state levels of the mRNAs for both ERb1 and ERb2 at 3, 6, and 24 h after treatment. Likewise, relaxin induced a similar rapid down-regulation. Neither hormone had any significant effect on ERa mRNA levels. In marked contrast to the effects on ERb, estrogen rapidly increased VEGF mRNA levels. To determine if estrogen from the ovary exerts a similar effect, we examined the pattern of expression of ERbs in the uterus during the normal reproductive cycle. Levels of both isoforms were highest at diestrus (low estrogen), were significantly lower at early proestrus (rising estrogen), reached a nadir during late proestrus (peak estrogen), and rebounded at estrus (declining estrogen). Thus, ERb expression is lowest when estrogen levels are highest. These data support the proposal that ERbs may function primarily as negative modulators of estrogen action and that rapid down-regulation of ERb expression may be essential for estrogen to exert, via ERb, its full trophic effects on the uterus. They are also relevant to the mounting evidence that decreased ERb may contribute to the development of cancer, in that they demonstrate that estrogen itself induces such a decline. To pursue this possibility, we have produced human breast cancer cells that express high levels of ERb. The effect of this on the estrogen-induced growth of the cells both in vitro and in vivo is now being evaluated.

Relaxin Activation of Estrogen Receptors:

Relaxin is a peptide hormone produced by the ovary that belongs to the same family as insulin and insulin-like growth factors. It plays important roles in the growth and remodeling of the reproductive tract during pregnancy and in preparation for birth. Interestingly, many of relaxin’s effects closely resemble those of estrogen. We hypothesized that this was due to relaxin activating ERs. We have now demonstrated that this is true by blocking relaxin’s ability to stimulate uterine edema with a drug that blocks ER action. We have also demonstrated that relaxin can induce the expression of a reporter gene (luciferase) linked to the DNA sequence that recognizes ER, the estrogen response element. These novel studies have opened up new possibilities for understanding the function of relaxin. They also suggest that relaxin may play a role in the growth of so-called “estrogen-dependent” cancers, such as breast cancer. The effect of relaxin on the reproductive system of mice that lack ERs (ER knockouts) on the growth of human breast cancer cells in mice is now being tested.

• 1990-95, Member, Editorial Board, Biology of Reproduction
• 1997-00, Member, Editorial Board, Endocrinology
• 1992-95, Member, Physiological Sciences Study Section, NIH
• 1993- , Ad Hoc Reviewer, Cancer Research Manpower Review Committee
• 1998-01, Review Panel, Specialized Cooperative Centers Program in Reproduction Research
• 1999, Special Review Committee, Scientific Programs of Research Excellence (SPORE) in Ovarian Cancer, NCI
• 1999, Reproductive Biology Study Section, temporary member
• 1995-96, Chair, Publications Committee, Society for the Study of Reproduction
• 1997-00, Chair, Public Affairs Committee, Society for the Study of Reproduction
• 1998-02, Public Affairs Executive Committee, Federation of American Societies for Experimental Biology (FASEB)
• 1998-02, FASEB Board Representative for the Society for the Study of Reproduction
• 1999-02, Chair, Local Arrangements Comm., 35th Annual Meeting of the Society for the Study of Reproduction, Baltimore, MD, 2002

• 1998, Invited speaker, 14th Annual Meeting of the European Society of Human Reproduction and Embryology, Göteborg, Sweden
• 2000, Invited speaker, Relaxin 2000: 3rd International Conference on Relaxin and Related Peptides, Broome, Australia
• 2001, Invited speaker, 34th Annual Meeting of the Society for the Study of Reproduction, Ottowa, Canada

In addition to obtaining in-depth knowledge of basic ovarian and uterine physiology, trainees can learn a wide range of cellular and molecular techniques, including cell culture, RNA/DNA isolation, quantitative RT-PCR, Western blotting and immunohistochemistry, DNA cloning, transfection, transgenic methodologies, and formaldehyde cross-linking/chromatin immunoprecipitation (ChIP).

• Armina Kazi (Goucher College)
• Christine Campo (Dartmouth College)
• Suresh Pillai (2001), just began a postdoctoral fellowship with Dr. Larry Jameson at Northwestern University.
• Karina Marciani (2001), recently became a Technical Specialist at Human Genome Sciences, Inc., a leading biotechnology company in Rockville, MD.
• Jo Ann Forsythe (1996), is a Clinical Research Scientist at GlaxoSmithKline Pharmaceuticals near Philadelphia. Jo’s work involves the development of new drugs for the treatment of diabetes.
• Kathleen Cullinan-Bove (1994), is currently a Staff Scientist at the Veterans Administration Hospital in Albany, NY.