Summary:The Shestopalov laboratory is focused on studying the molecular and cellular mechanisms underlying pathophysiology of the two most blinding human diseases: cataract and glaucoma. The complex multifactorial nature of the both disorders, slow progression and strong influence of age require comprehensive systems approach to study them. We utilize the vast array of modern molecular biology, functional genomics and bioinformatics methods to elucidate these mechanisms at the levels of gene regulation, protein interaction and activity of pathways and protein networks.

Valery I. Shestopalov, Ph.D.
Assistant Professor of Ophthalmology

View published research articles by this doctor in the National Library of Medicine.

Current Research Summary: Cell-cell communication in the lens
Ocular lens is an avascular tissue, which sustains the metabolism of the mature lens fiber cells by "metabolic cooperation" with cells at the periphery. This is a physiological pre-requisite for tissue transparency throughout the lifetime of the organism. Electrophysiological studies supported this view of the lens demonstrating that this tissue is organized as a metabolic syncytium, where all cells are coupled via abundant gap junctions. However, as Dr. Shestopalov demonstrated recently, in addition to gap junctions, fiber cells in the core of the lens become coupled by a novel cell-cell communication pathway. The novel pathway couples fiber cells into a true syncytium. This study revealed that lens fibers become interconnected via the network of membrane fusion pores, which allow proteins and macromolecules to diffuse between cells.

Dr. Shestopalov's research is focused on the novel pathway. He has been using confocal microscopy, real-time imaging and three-dimensional image reconstruction to study developmental dynamics of this unique pathway formation in living lenses. Currently, these studies focus on the molecular mechanism underlying formation of intercellular fusion pores. The physiological significance of the lens core syncytium is being studied using a transgenic approach. The transgenic experimental model is being constructed which will allow functional inactivation of syncytium formation in the lens. This model will provide a powerful system for the dissection of the molecular mechanism, identification of major protein players and for testing the physiological significance of this novel cell-cell communication pathway. We are currently testing potential contribution of ADAM12, connexin 46, CD9 and MP19/Lim2 proteins to the lens syncytium formation using molecular and genetic (transgenic) approaches. This project has won the prestigious US government Presidential (PECASE) award in 2004 and is funded by NIH/NEI R01 grant.

The role of pannexins in cell-cell communication and signaling

Panx-1 and -2 are the newly discovered members of pannexin family of gap junction-forming proteins functionally analogous to connexins related to invertebrate innexins. Panx-1 has been demonstrated to form gap-junction-like molecular channels when expressed in frog oocytes. In addition, it has been shown to form a gated hemichannel capable of transporting ATP and Ca ions through by-lipid layers of plasma membrane and endoplasmic reticulum. In collaboration with Dr. Gerhard Dahl, we demonstrated that Panx-1 is abundantly expressed in the ER and plasma membranes in many cell types of epithelial origin including lens fibers. At the same time Panx-1 showed no specific association with abundant lens fiber cell gap junction, suggesting function(s) that are alternative to the intercellular coupling. Based on our preliminary data and suggested molecular properties we hypothesize that Panx-1 forms hemichannels (half-channels) in cell membranes. These abundant hemichannels may mediate ATP and Ca2+ release and, theoretically, contribute to propagation of Ca2+ waves. We will examine the role of Panx-1 in cell communication in the lens.

Systems biology of glaucoma
Glaucoma is an ocular neuropathy, a complex multifactorial disease most commonly associated with high intraocular pressure (IOP), aging and other neurological disorders like diabetes and Alzheimer's. Retinal ganglion cells (RGCs) and their axons are the major target of this neutopathy, but their survival is dependent on support from glial cells, particularly astrocytes. In pathological conditions in CNS astrocytes have been shown to exert neurotoxicity and threaten the stressed neurons even further. We seek to elucidate cellular changes in RGCs as well as in glial cells, which contribute to development of the glaucoma pathology. We utilize latest generation systems biology tools to analyze neural tissue homeostasis in the retina and optic nerve regions tested for global gene expression profile by microarray analysis. In our preliminary work we detected several glial cellular pathways potentially relevant to glaucoma in humansGlaucoma is an ocular neuropathy, a complex multifactorial disease most commonly associated with high intraocular pressure (IOP), aging and other neurological disorders like diabetes and Alzheimer's. Retinal ganglion cells (RGCs) and their axons are the major target of this neutopathy, but their survival is dependent on support from glial cells, particularly astrocytes. In pathological conditions in CNS astrocytes have been shown to exert neurotoxicity and threaten the stressed neurons even further. We seek to elucidate cellular changes in RGCs as well as in glial cells, which contribute to development of the glaucoma pathology. We utilize latest generation systems biology tools to analyze neural tissue homeostasis in the retina and optic nerve regions tested for global gene expression profile by microarray analysis. In our preliminary work we detected several glial cellular pathways potentially relevant to glaucoma in humans.

The rat ocular hypertension model will be used to test our hypothesis. In this study we are using Systems Reconstructions Platform aims to explore a data-mining potential of a systems biology approach as applied to in vivo rat ocular hypertension model of glaucoma. This project implies an in silico functional reconstruction of astrocyte- and RGC-specific metabolic and regulatory pathways implicated in the onset of optic nerve head pathology in glaucoma. Three recently discovered genes, implicated in primary open-angle glaucoma, provide the evidence that alterations in metabolic (TIGR/MYOC encoding myocillin and CYP1B1 gene, encoding human cytochrome P4501B1) and regulatory (OPTN, encoding optineurin, a protein involved in TNF a signaling) networks are essential in the onset of the pathology.

The critical advantage of Systems Reconstructions Platform is the algorithm, which utilizes a multi-level verification of existing data by integrating all kinds of functional data, high-throughput proteomics and expression data, and imposing the requirement of system-level self-consistency. This project is a cooperative effort of Dr. Shestopalov's laboratory with Washington University School of Medicine Department of Ophthalmology (Dr. Rosario Hernandez) and GeneGo Inc. GeneGo is Michigan-based bioinformatics and systems biology company, specializing on post-genomic data-mining technologies. GeneGo has developed the Systems Reconstructions TM technology and created a proprietary database (GeneGo DB), which summarizes in a graphical format the present day knowledge of human metabolic and regulatory pathways and networks.