Research Overview

Cell Injury, Repair and Death

Disruption of the cell membrane’s lipid bilayer is fundamental feature of trauma linked acute cellular necrosis. Structural integrity of the lipid bilayer is essential for cell viability. Clinically, loss of cell membrane integrity can result from exposure to high temperatures (>42 ^oC) associated with thermal burns; ice propagation in cryopreservation; free-radical mediated membrane lipid peroxidation that is associated with tissue reoxygenation injuries (i.e. myocardial infarction and stroke); acoustic shock waves in vehicular or military trauma; electroporation in electrical shock victims; crush injury, and other common injuries.

In the United States, tissue reoxygenation injury (ischemia-reperfusion) is probably the most common clinical cause of membrane disruption and is a critical aspect of many common medical illnesses such as cerebral palsy, myocardial infarction, and cerebrovascular stroke.

Regenerating Cell Membranes

Investigators in the Institute for Regenerative Medicine (IRM) collaborate to bring the broad interdisciplinary capabilities required to develop molecular regeneration therapeutics have focused on therapeutic strategies which promise to augment natural cell wound healing mechanisms and improve tissue survival after injury.

The ability to augment molecular regeneration of punctured cell membranes using synthetic surfactants was initially demonstrated more than a decade ago at The University of Chicago. It strategy is now the subject of research at various labs and pharmaceutical companies. The exact physicochemical mechanism of surfactant facilitated membrane regeneration is an essential area of investigation for the IRM. IRM scientista discovered most of basic dynamics. Effective sealing agents include surface-active multi-block copolymers, such as poloxamer surfactants.

Poloxamer surfactants were first shown to regenerate electroporated membranes. Subsequent efforts have been shown to P188 can also seal membrane defects following reperfusion injury, heat-shock, detergent lysis, and ultra-high dose ionizing radiation. Very recently, P188 has been shown to protect embryonic neurons against death following exposure to excitotoxic drugs. In each instance, membrane sealing resulted in enhanced cell survival.

Thus far thee surfactants used for membrane repair have been designed for other pharmaceutical purposes. A major goal of the CMR is to bring together experts to optimize the design of membranes sealing polymers and cell resuscitation cofactors that are effective and safe for intravascular administration.

Chaperoned Protein Folding

Protein function is critically dependent on its conformational structure. Altered conformation can be produced by non-physiological physical and chemical mechanisms similar to those associated with cell membrane damage. Some molecular chaperones, also known as heat-shock proteins (HSP), which help newly synthesized proteins to achieve their proper conformation, also restore denatured proteins to native conformation and prevent them from aggregating within the cell.

CMR investigators recently reported that surfactant copolymers exhibit capabilities to disaggregate and refold denatured proteins similar fashion as natural chaperones. For the first time, blood compatible surfactants have been used successfully to recover the conformation and function of heat denatured enzymes. Investigators in the CMR program are comparing natural and synthetic chaperones with goal of understanding what structural features are important for chaperone function.

In addition, investigators in the CMR have recently shown that neurons can import HSP from the external medium, protecting cell function from heat damage. Recent reports indicate that small molecular weight fragments of HSP GroEL can renature some proteins. These minichaperones have great potential use in medical therapy.

Principal Research Initiatives

• Molecular interactions between surfactant copolymers and disordered membrane lipids.
• Copolymer surfactants for brain ischemia and excitotoxic injury
• Copolymer engineering design for optimum lipid immobilization;
• Structural characterization of damaged cell membrane lipid bilayers using X-ray and neutron scattering in conjunction with AFM imaging;
• Multi-focal ESR spin probe labeling of biopolymer domains to determine water-membrane interaction energies;
• Therapeutics for reperfusion injury following vascular surgery;
• Biomimetic biopolymer design based on chaparone and chaparonin structures;
• Development of therapies for brain trauma, spinal cord injury and cerebral palsy;
• Development of enhanced tissue preservation media for transplantation to reduce rate of late apoptosis;
• Surfactant refolding of denatured proteins.
• Stabilizing membranes in Muscular dystrophy

Selected Publications

Lee, R.C., Pan, Fu-S., River, L.P., Li Ji and Wollmann, R.L. "Surfactant-Induced Sealing of Electropermeabilized Skeletal Muscle Membranes In Vivo" Proc. Natl. Acad. Sci. 89(10): 4524-28, 1992 (PubMed)

Lee, R.C., and Astumian, R.D. "The Physicochemical Basis for Thermal and Non-thermal Burn Injury "Burns J. 22(7): 509-19, 1996 (PubMed)

Merchant, FA, Holmes, W.H., Capelli-Schellpfeffer, M., Lee, R.C., and Toner, M. “Poloxamer 188 Enhances Functional Recovery of Lethally Heat-Shocked Fibroblasts”, J Surg Res. 74:1031-40, 1998 (PubMed)

Feder ME, Hofmann GE. “Heat-shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology.” Ann. Review Physiology 61: 243-82, 1999 (PubMed)

Maskarinec SA, Hannig J., Lee RC, Lee K-Y "Direct Observation of P188 Insertion into Lipid Monolayers" Biophysical Journal, 78: 328A, 2000. (PubMed)

Marks JD, Pan C-Y, Bushell T, Cromie W, and Lee RC. “Amphiphilic, tri-block Copolymers provide potent, membrane-targeted Neuroprotection” FASEB 10.1096/fj.00-0547 Feb 20, 2001.

Despa, F, Orgill, DP, Neuwalder, J. and Lee RC “Effects of Crowding on the Thermal Stability of Heterogeneous Protein Solutions” J. Biomed Engineering 33(8): 1125-1131, 2005. (PubMed)

Walsh, AM, Mustafi, D, Makinen, M, and Lee, RC, “A Copolymer Surfactant Facilitates Functional Recovery of Heat Denatured Lysozyme” Annuals NYAS Vol. 1006 : 321-327, 2006.

Research Funding Sources

Research is supported by the National Institutes of Health, the National Science Foundation, the Department of Defense and other public and private agencies. Applications for Center and Training grants from NIH and DOD will be submitted.

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Raphael Lee, MD