About our work
Over the last 20 years we have been able to provide a comprehensive analysis of the plasma membrane proteins and intracellular signaling pathways that mediate human RPE cell physiology and its interactions with retinal photoreceptors (Adijanto et al., 2009; Li et al., 2009; Miller et al., 2010; Maminishkis et al., 2006; Quinn et al., 2001).
In the back of the vertebrate eye, the apical membrane of the retinal pigment epithelium (RPE) and the photoreceptor outer segments form a very tight anatomical relationship (Figure 1). This structural feature supports a whole host of mechanical, electrical, and metabolic interactions that maintain the health and integrity of the neural retina throughout the life of the organism. Like all epithelia, the RPE plasma membrane contains a wide variety of proteins, enzymes, and small molecules that are specifically segregated to the apical or basolateral sides of the epithelium, which face the neural retina and choroidal blood supply, respectively (Figure 2). The asymmetrical distribution of these functionally distinct molecules is maintained by junctional complexes that surround each cell and by the continuous synthesis and regulated traffic of these molecules to each membrane. Epithelial polarity is defined by the steady-state maintenance of this asymmetric distribution and is critical for the ongoing vectorial transport of Ions, metabolites, fluid, and waste products across the RPE. Epithelial polarity is also fundamentally important for controlling changes in the volume and chemical compositions of the extracellular spaces on either side of the RPE, following transitions between light and dark. In the distal retina, the extracellular or subretinal space (SRS) separates the photoreceptor outer segments and the RPE apical processes. The chemical composition of this space is tightly buffered by the cells which surround it (Mller cells, photoreceptors, and RPE). On the opposite side of the RPE, an extracellular space is formed between its basolateral membrane and Bruch’s membrane, which is adjacent to the choriocapillaris.
The physiological and pathophysiological states of the RPE/distal retina complex are significantly affected by changes in the chemical composition of these extracellular spaces as evidenced in disease processes such as age-related macular degeneration (AMD)or uveitis. AMD develops within the RPE/distal retina complex and eventually leads to RPE impairment and loss of photoreceptor function. The RPE’s ability to control and respond to varying levels of oxidative insult from light quanta, outer segment phagocytosis, vitamin A uptake and delivery, and oxygen consumption diminishes with age. These changes significantly affect the chemical composition of the surrounding extracellular spaces, SRS and choroid, and are a major factor in disease pathogenesis.
Miller et al., 2010 summarizes some recent experiments from our lab and others, which show that inflammation induced changes in the environment surrounding human RPE can significantly alter intracellular signaling and physiology. This study provides a basis for understanding disease progression and regression begining with a description of our development of a robust and well defined primary cell culture model of human fetal retinal pigment epithelium (Maminishkis et al., 2006).We use this model to analyze how metabolic waste products, produced in the retina following light/dark transitions, can be disposed of by CO2/HCO3 and lactate transporters located in the apical and basolateral cell membranes (Adijantor et al., 2009). We have also used this cell culture model to analyze RPE antioxidant mechanisms that are protective against disease processes, such as AMD or uveitis. Finally, we describe a series of experiments that use this model to define the impact of cytokines on human RPE function with focus on the role of interferon gamma (INFg) in controlling RPE physiology (Li et al., 2009).
Recent Lab projects include refinements of a robust and well defined primary cell culture model of human fetal retinal pigment epithelium (Figure 3), development of pre-clinical animal models of disease, including CNV in AMD (Wang et al., 2003;2007) and retinal re-attachment (Maminishkis et al., 2002) and has provided the basis for several clinical trials (eg, NIH Protocol 09-EI-0191). In the latter phase II trial we are investigating the ability of IFN to increase fluid removal from the distal retina of uveitis patients. At the molecular level, we have recently identified micro RNAs that are enriched in human RPE compared to the adjacent retina and choroid. We showed how several of them maintain tight junction integrity and epithelial phenotype (Wang et al., 2010). Recently, we have identified a signature set of 154 genes that distinguish the human RPE from other cells in the body (Strunnikova et al., 2010). Using a similar approach, we have identified several hundred additional genes that distinguish human fetal and adult RPE tissue. One of our current goals is to provide a molecular and physiological characterization of RPE tissues derived from a number of extant iPS/ES cell lines. This work is intended to determine variability in RPE cells derived from iPS cells of different genetic and epigenetic origins. This will lead to the identification of a signature set of molecular and physiological assays that can truly and reproducibly represent RPE tissue derived from genetically diverse iPS cells. This work will provide a first step toward the development of a rational basis for a consistent, successful, and safe therapeutic intervention in retinal/RPE diseases using iPSC technology (Bharti et al., 2010). In parallel, we use iPS cell-derived RPE to study the mechanisms of disease initiation in monogenic neurodegenerative disease and AMD.
Selected background reviews (retinal pigment epithelium)
Gallemore, R.P., Hughes, B.A., and S. S. Miller (1998). Light-induced responses of the retinal pigment epithelium. Retinal Pigment Epithelium: Current Aspects of Function and Disease, eds: Marmor, M.F. and Wolfensburger T.J., Oxford University Press, pp. 175-198.
Hughes, B.A., Gallemore, R.P., and S. S. Miller (1998). Transport Mechanisms in the retinal pigment epithelium. Retinal Pigment Epithelium: Current Aspects of Function and Disease, eds: Marmor, M.F. and Wolfensburger T.J, Oxford University Press, pp. 103-134.
Peterson, W.M., Quong, J.N. and S.S.Miller (1998). Mechanisms of fluid transport in retinal pigment epithelium. The Third Great Basin Visual Science Symposium on Retinal Research, Volume III: 34-42.
Maminishkis A, Miller SS (2010). Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology. JoVE. http://www.jove.com/index/Details.stp?ID=2032(link is external), doi: 10.3791/2032
Miller, S.S., Maminishkis, Li, R., and J. Adijanto (2010). Retinal Pigment Epithelium: Cytokine Modulation of Epithelial Physiology, (Encyclopedia of the Eye, Chapter 184: Phototransduction: RPE transport Retina | Phototransduction: RPE transport ELSEVIER ). Hardbound, 2540 pages, publication date: MAY-2010, ISBN-13: 978-0-12-374198-1, Imprint: ACADEMIC PRESS.
Miller, S.S., Hughes, B.A. and T.E. Machen (1982). Fluid Transport Across the Retinal Pigment epithelium is Inhibited by Cyclic AMP. Proceedings of the National Academy of Science, 79:2111-2115.
Hughes, B.A., Miller, S.S. and T.E. Machen (1984). Effects of cAMP on Fluid Absorption and Ion Transport Across Frog Retinal Pigment Epithelium: Measurement in the Open-Circuit State. Journal of General Physiology, 83:875-899.
Adorante, J.S. and S.S. Miller (1990). Potassium Dependent Volume Regulation in Retinal Pigment Epithelium is Mediated by Na, K, Cl Cotransport. Journal of General Physiology, 96:1153-1176.
Joseph, D.P. and S.S. Miller (1991). Apical and Basal Membrane Ion Transport Mechanisms in Bovine Retinal Pigment Epithelium. Journal of Physiology, 435:439-463.
Lin, H. and S.S. Miller (1991). pHi Regulation in Frog Retinal Pigment Epithelium: Two Apical Membrane Mechanisms. American Journal of Physiology 261:C132-C142.
Edelman, J.L. and S.S. Miller (1991). Epinephrine Stimulates Fluid Absorption Across Bovine Retinal Pigment Epithelium. Investigative Ophthalmology and Visual Science 32:3033-3040.
Joseph, D. and S.S. Miller (1992). Alpha-1 Adrenergic Modulation of K and Cl Transport In Bovine Retinal Pigment Epithelium. Journal of General Physiology 99:263-290.
Quinn, R., and S.S. Miller (1992). Ion Transport Mechanisms in Native Human Retinal Pigment Epithelium. Investigative Ophthalmology and Visual Science 33:3513-3527.
Lin, H., Kenyon, E. and S.S. Miller (1992) Na-Dependent pHi Regulatory Mechanisms in Native Human Retinal Pigment Epithelium. Investigative Ophthalmology and Visual Science 33:3528-3538.
Lin, H. and S.S. Miller (1994). I. pHi-dependent Cl-HCO3 Exchange at the Basolateral Membrane of Frog Retinal Pigment Epithelium. American Journal of Physiology 266:C935-C945.
Edelman, J.L., Lin, H. and S.S. Miller (1994). II. Acidification stimulates chloride and fluid absorption across frog pigment epithelium. American Journal of Physiology (cell) 266:C946-C956.
Edelman, J.L., Lin, H. and S.S. Miller (1994). III. Potassium-induced chloride secretion across the frog retinal pigment epithelium. American Journal of Physiology (cell) 266:C957-C966.
Kenyon, E., Yu, K., LaCour, M. and S.S. Miller (1994). Lactate transport mechanisms at the apical and basolateral membranes of bovine retinal pigment epithelium. American Journal of Physiology 267: C1561-C1573.
Bialek, S., Joseph, D.P. and S.S. Miller (1995). The delayed basal hyperpolarization of the bovine retinal pigment epithelium: Mechanisms of generation. Journal of Physiology (London) 484: 53-67.
Kenyon, E., Maminishkis, A. Joseph D.P. and S.S. Miller (1997). Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium. American Journal of Physiology 273: (Cell Physiol. 42) C456 - C472.
Peterson, W.M., Meggyesy, C., Yu, K., and S.S. Miller (1997). Extracellular ATP activates calcium signaling, ion and fluid transport in retinal pigment epithelium. The Journal of Neuroscience 17: 2324-2337.
Quinn, R.H., Quong, J.N., and S.S. Miller (2001). Adrenergic receptor activated ion transport in native human fetal retinal pigment epithelium. Investigative Ophthalmology & Visual Science 42:255-264.
Maminishkis, A., Jalickee, S., Rymer, J., Yerxa, B.R., Peterson, W.M. and S.S. Miller (2002). The P2Y2 receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat. Investigative Ophthalmology & Visual Science 43: 3555-3566.
Wang, F., Rendahl, K.G., Manning, W.C., Quiroz, D., Coyne, M. and S.S. Miller (2003).Adeno-Associated Virus Mediated Expression of Vascular Endothelial Growth Factor Induced Choroidal Neovascularization in Rat. Investigative Ophthalmology & Visual Science 44: 781-790.
Voloboueva, L.A., Liu, J., H. Suh, J.H., Ames, B.N., and S.S. Miller (2005). (R)–Lipoic Acid Protects Retinal Pigment Epithelial Cells from Oxidative Damage. Investigative Ophthalmology & Visual Science 47: 3612-3624.
Maminishkis, A., Chen, S., Jalickee, S., Banzon, T., Shi, G., Ehalt, T., Wang, F.E., Hammer, J.A., and S. S. Miller (2006). Confluent monolayers of cultured human fetal retinal pigment epithelium (hfRPE) exhibit morphology and physiology of native tissue. Investigative Ophthalmology & Visual Science 46: 4302-4310.
Jia, L., Liu, Z., Sun, L., Miller, S.S., Ames, B.N., Cotman, C.W., and J. Liu (2007). Acrolein, a toxin in cigarette smoke, causes oxidative damage and mitochondrial dysfunction in retinal pigment epithelial cells: protection by (R)–lipoic acid. Investigative Ophthalmology & Visual Science 48: 339-348.
F.E. Wang, G.Shi, M.R. Niesman D.A. Rewolinski, S.S. Miller (2007). Receptor Tyrosine Kinase Inhibitors AG013764 and AG013711 Reduce Choroidal Neovascularization in Rat Eye. Experimental Eye Research 84: 922-933.
Li, R., Maminishkis, A., Wang, F.E., S.S. Miller (2007). PDGF-C and -D Induced Proliferation/Migration of Human RPE is Abolished by Inflammatory Cytokines. Investigative Ophthalmology & Visual Science 48: 5722 - 5732.
Shi, G., Maminishkis, A., Banzon, T., Jalickee, S., Li, R., Hammer, J., and S.S. Miller (2008). Control of Chemokine Gradients by the Retinal Pigment Epithelium. Investigative Ophthalmology & Visual Science 49: 4620 - 4630.
Economopoulou, M., Hammer, J., Wang, F., Fariss, R., Maminishkis, A., and S.S.Miller (2009). Expression, Localization, and Function of Junctional Adhesion Molecule-C (JAM-C) in Human Retinal Pigment Epithelium. Investigative Ophthalmology & Visual Science 50:1454-1463.
Adijanto, J., Jalickee, S., Banzon, T., Wang, NS, and S.S. Miller (2009). CO2 - induced Ion and Fluid Transport in Human Retinal Pigment Epithelium. Journal of General Physiology, 133: 602-622.
Li, R., Maminishkis,A., Zahn, G.„ Vossmeyer,D., and S.S. Miller (2009). Integrin 51 Mediates Attachment, Migration, and Proliferation in Human Retinal Pigment Epithelium: Relevance for Proliferative Retinal Disease. Investigative Ophthalmology & Visual Science, 50: 5988-5996.
Li, R., Maminishkis, A., Banzon, T., Wan, Q., Jalickee, S., Chen S. and S.S. Miller (2009). IFN Regulates Retinal Pigment Epithelial Fluid Transport. Am J Physiol Cell Physiol 297: C1452-C1465, 2009.
Wang, F., Zhang, C.*, Maminishkis, A., Dong.L., Zhi, C., Rong Li, Jing Zhao, J., Majerciak, V., Gaur, AB., Chen, S, and S. S. Miller (2010). MicroRNA-204/211 Alters Epithelial Physiology 2010 May;24(5):1552-71.) Epub 2010 Jan 7.
Strunnikova, N.V., Barb, J., Chen, W., Maminishkis, A., Wang, F., Zhi, C., Sergeev, Y., Stambolian, D., Edwards, A.O., Abecasis, G. Swaroop, A., Munson, P.J., and S.S. Miller (2010). Transcriptome Analysis and Molecular Signature of Human Retinal Pigment Epithelium. Human Molecular Genetics, Jun 15;19(12):2468-86. Epub 2010 Apr 1.
Bharti, K., Miller, SS., and H. Arnheiter (2010). The new paradigm: Retinal pigment epithelium cells generated from embryonic stem cells or induced pluripotent cells. Pigment Cell Melanoma Res. 2010 Sep 15. [Epub ahead of print]