Lawrence James Rizzolo PhD, FARVO
Associate Professor of Surgery (Gross Anatomy) and of Ophthalmology and Visual Science; Director of Medical Studies
Retinal disease; Tight junctions; Outer blood-retinal barrier; Epithelia; Human embryonic stem cells; Bio/tissue engineering; Medical education
- We use fetal and stem cell-derived culture models of human retinal pigment epithelium RPE to study mechanisms of retinal degeneration and examine putative therapeutic agents.
- We are investigating the role of autophagy in young and aged RPE, and how changes in autophagy might relate to age-related macular degeneration.
- We use embryonic stem cells to study the interactions of retinal and RPE progenetors with the goal of developing superior engineered tissues for drug testing and transplantation.
- We are exploring how medical students web based learning tools with the aim of develop interactive tools that foster the habits-of-mind needed for clinical practice and that foster integration of the various basic and clinical science disciplines.
Retinal degenerations are major causes of visual impairment, especially in aging and diabetic populations. My laboratory uses human embryonic and induced pluripotent stem cells to study and treat retinal disease. The retinal pigment epithelium (RPE) separates photoreceptors from their blood supply in the choroid, and therefore, is responsible for their health. We demonstrated how RPE functions are regulated by interactions with the retina and the choroid. Our goal is to bioengineer a three-dimensional culture model that includes RPE and retinal neurons derived from human stem cells. We will use this platform to screen potential therapeutic agents and determine their mechanism of action. In mouse models, early transplantation of RPE or retinal progenitors slows retinal degeneration, but later transplantation does not reverse degeneration that has already occurred. We are examining whether the engineered tissue restores vision in late stage disease. In a second approach, we are examining how the process of autophagy may be exploited to reverse degeneration of the RPE.
Extensive Research Description
The retinal pigment epithelium (RPE) plays a central role in retinal physiology by forming the outer blood-retinal barrier and supporting the function of the photoreceptors. Many retinopathies involve a disruption of the epithelium's interactions with the neural retina or its uncontrolled proliferation. Surgical interventions limit the progression of disease, but fail to restore function. Although encouraging progress has been made with RPE transplantation, its effectiveness is limited to the earliest stages of disease when patients would be reluctant to have surgery. Our goal is to expand that window of opportunity by understanding the interactions of the RPE with its neighbors, the choroid and the neural portion of the retina. Our early studies with chick RPE demonstrated that: 1) As the neural retina matures, it secretes factors that induce the RPE to form the outer blood-retinal barrier by decreasing the permeability of RPE junctions. 2) At the RPE/neural retina interface, extracellular matrix or cell-cell interactions regulate the distribution of certain integrins. These integrins are redistributed when the neural retina and its extracellular matrix mature. 3) Initially, diffusible factors produced by the neural retina maintain the apical polarity of the Na,K-ATPase. These retinal factors differ from those that decrease the permeability of the monolayer, and may act indirectly through effects on the structure of the apical microvilli. 4) Gene array studies demonstrate that 40% of the RPE transcriptome changes in parallel with retinal development. Retinal secretions regulate many of these changes.
Our current research asks whether the chick studies are relevant to human biology. Surprisingly, we found that the tight junctions of human RPE differ significantly from those of non-primate vertebrates -- surprising because tight junctions serve a conserved function. Tight junctions are an integral part of any blood-tissue barrier, because they regulate diffusion across the paracellular spaces of an epithelial monolayer. Tight junctions form a network of anastomosing strands that encircles each cell and binds it to its neighbors in the monolayer. They regulate the permeability and selectivity of the paracellular path and are matched with the ion channels and transporters that regulate the transcellular movement of solutes. Claudins are a family of at least 24 proteins that determine the properties of tight junctions and each epithelium expresses a subset that reflects the physiology of the organ. Human RPE expresses a different set of claudins that non-primate vertebrates which implies differences in retinal physiology.
In both cultures of human fetal RPE (hfRPE) and human embryonic stem cell (hESC)-derived RPE, we found that we could make the barrier function more in vivo-like by using a serum free medium that we call SFM-1. Studies of the transcriptome demonstrate that SFM-1 affects many genes and that adaptation of the cultures to SFM-1 furthers the maturation of hESC-RPE. This was demonstrated in two cells, H1 and H9. Examining function and the transcriptome leads us to believe that even after SFM-1, hESC-RPE remains less mature than hfRPE isolated from 16 week-gestation fetal eyes. The study identifies 25 marker genes that can be used to monitor the maturation of RPE that we predict will occur when hESC-RPE is co-cultured with hESC-derived retinal precursors (RPC).
To test this hypothesis, RPE and RPC need to be culture in the same media. It appears that SFM-1 furthers the maturation of RPC so that co-culture is feasible. We have found that a woven scaffold of polycaprolactone provides a way to culture RPC as a flat sheet that can be layered atop a sheet of RPE. Preliminary data indicate that gene expression is altered in both the RPE and RPC layers following co-culture.
I believe this co-culture model will provide a superior platform to explore the efficacy of drugs that might treat retinal degenerations or improve the efficacy of transplantation. Further, the model itself may prove to be a suitable tissue for transplantation into patients with advanced retinal degeneration.
The international community of medical educators struggles with how to decompress an overcrowded curriculum. The questions have become what to teach, when to teach it and how to teach it in less time. The problem is especially acute for anatomy. Even though the classical anatomy course is a large component of medical school, residency programs believe their residents come ill-prepared. Further, the pool of qualified instructors is shrinking. To address these issues of content, efficiency and instructors, I investigated what students need, how they learn and how instructors teach. I call the resulting method “Clinically-Engaged Anatomy”. Clinically-engaged anatomy engages students in professional behaviors to learn the anatomy that prepares them for clerkships. Students learn how to draw inferences from skillful observation to form testable hypotheses, test them and teach others about the process. The coursework requires students to develop the teamwork skills that characterize modern medical practice. The clinical cases that drive the curriculum are cases commonly encountered in Yale affiliated hospitals. Students study the anatomy that underlies the patient’s history, physical exam, imaging studies and medical or surgical resolution. The cadaver becomes a simulated patient whereby anatomy is explored by performing surgical procedures. This approach fosters integration of anatomy with clinical training and has attracted large numbers of clinic faculty to participate. Despite a 30% reduction in course hours, we demonstrated that students recall more when they enter clerkships. Clinically-engaged anatomy merges advanced web resources with laboratory dissection. My “Anatomy Clinic” website attracts more than 17,000 non-Yale page viewings per year from around the world and is used in Great Britain for their “Basic Training Programme for Anatomy Professionals”. The anesthesiology and otolaryngology residency training programs adapted these methods to their laboratory sessions. Therefore, clinically engaged anatomy has identified important anatomy to teach, conveys that knowledge effectively in less time, and attracts a large number of faculty who would not participate in the old course.
Current research asks how to train the next generation of anatomy instructors. Very few training programs remain for anatomists. Most institutions require instructors to maintain competitive biomedical research programs, which means mastering a discipline that is unrelated to anatomy. The goal is to develop a training program for upper-tier postdoctoral fellows that has minimal impact on their biomedical research training.
A second project investigates how clinical students incorporate interactive computer activities into their daily work and how that information might guide curricular development.