Can the effects of physical trauma to the human body be undone? Research on how individual cells become injured and repair themselves suggests it may even be possible now.The study of cell injury and repair crosses the boundary between the physical and biological sciences: the people who study the inner workings of cells tend to be chemists, rather than biologists. Several years ago, researchers from the University of Chicago and the U.S. Department of Energy's Argonne National Lab joined together to create the Program for Research in Molecular Repair to understand what happens when cells are injured, and to find ways to repair them. Raphael Lee, a surgeon, biomedical engineer, and pioneer in the field, directs the program.
In 2004, the University of Chicago began a year-long seminar series that featured experts on cell injury and repair. The speakers were drawn from disciplines ranging from physics to plastic surgery. Many described how cells naturally repair themselves, discussing vesicle fusion, sealing damaged cell membranes, DNA repair strategies, and how proteins involved in the stress response refold or remove damaged proteins. Others discussed their discoveries about how cells respond to injuries such as burns, electric shock, and freezing. Some used a broad definition of cell injury to discuss the damage that accrues during diseases such as diabetes. The seminars also covered modes of cell failure after injury including paths leading to cell death.. The series has led to a new, more rule-based approach to teaching students about the pathogenesis of cell injury, repair, and death. The proceedings of these seminars are published in Cell Injury: Mechanisms, Responses, and Repair, Volume 1066 of the Annals of the New York Academy of Sciences, edited by Lee and two University of Chicago colleagues, Florin Despa and Kimm J. Hamann.
As Tom Hunt, the founder of modern wound healing and professor emeritus at the University of California, San Francisco, writes in the book's foreword, the volume is a major accomplishment: it truly is "the first compendium on injured cells," the first to get the concept of cell healing "on paper and in one place."
Here is a glimpse of some of the research, touching on cell structure, injury, repair, and therapeutic approaches.
Before researchers could discuss repairing injured cells, they had to take a good look at what happens when cells are injured. While there are many roads to cell injury—burn, frostbite, temporary oxygen deprivation, and electrical shock, to name a few—they all lead to one common event: the rupture of the cell membrane. The membrane's importance to cell health cannot be overstated. It is a lipid bilayer that acts as a barrier, maintaining a delicate balance of ions (negatively or positively charged atoms) on either side of the cell wall. The cell exerts considerable energy controlling that traffic. When the cell wall is ruptured, intracellular fluid rushes out, calcium rushes in, and the cell will die within minutes if the membrane is not resealed.
Many cells are capable of repairing a membrane rupture. As Dennis Orgill and his colleagues at Harvard Medical School explain, when the cell wall is breached a two-part rescue ensues. First, an influx of calcium signals helpers called vesicles to fuse together with the damaged membrane to form a patch. This causes the surface tension of the membrane to decrease, allowing it to reseal. Next, F-actin and myosin 2, the same pair of proteins that allow muscles to contract and relax, form a circle around the damaged area that pulls the sides together.
While holes in the cell membrane must be sealed if the cell is to survive, damaged cellular proteins need fixing as well. When the body is exposed to stresses such as temperature extremes, a process of protein unfolding called denaturation occurs. Between 20% and 30% of the cell membrane is made up of protein, so another crucial aspect of cell-based therapy is addressing protein damage.
When proteins become unfolded or misfolded, they cannot perform their crucial functions. In a protein's correctly folded state, its hydrophobic (water-averse) sections face inward, while hydrophilic (water-attracted) sections are exposed at the molecule's surface. As Stephen Meredith of the University of Chicago describes eloquently in his paper, unfolding results in the sudden exposure of hydrophobic areas, and these exposed domains are attracted to the hydrophobic sites of their misfolded neighbors. If left to their own devices, the wayward proteins will link up to form aggregates which interfere with cell function and lead to cell death.
When a cell is injured and its proteins undergo denaturation, the crowded cell environment makes it very likely that misfolded proteins will stick together. Protein misfolding and aggregation are associated with many diseases, including Alzheimer's Disease and Type II diabetes.
Clearly, someone needs to clean up this tangled mess. Cells respond to all kinds of stress by increasing their production of heat shock proteins. These act as chaperones, finding and re-folding damaged proteins. If a protein cannot be refolded, the chaperone designates it for removal. Chaperones can also attract the hydrophobic domains of misfolded proteins and bind them securely, which prevents the damaged proteins from binding to each other. In fact, they function much as human chaperones have for centuries: They prevent their confused, vulnerable charges from making ill-fated matches. Some, called chaperonins, actually hide misfolded proteins by taking them into a cavity in themselves—the cell version of a nunnery or Rapunzel tower.
The heat-shock response enables the body to resist damage from some types and severities of injuries. But some injuries are too much for cells to withstand. Once researchers understood what happened to cells during injury, they started looking for ways to enhance the body's repair systems. Could cell membranes be induced to reseal? Could artificial chaperones step in when our systems are overwhelmed?
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Bubble solution and other surfactants, like detergents, lower the surface tension of liquids. Lee looked for a surfactant that could seal cell membranes without causing harm. A few years and several experiments later, he published the first of many papers on poloxamer-188 (P-188), an agent blood banks have used safely for decades as an emulsifier. In 1992, Lee published the finding that P-188 could seal cell membranes that were damaged by electric shock. Sealing the membrane with P-188 stopped leaking intracellular fluids and prevented cells from dying, giving them a chance to repair themselves.
Throughout the 1990s, further research by Lee and others showed that the polymer could seal cell membranes after a variety of injuries. Lee received FDA approval in 1995 to test P-188 in clinical trials for victims of electric shock. But the trials did not go forward: he had trouble both in securing funding and in convincing doctors to refer patients to the study. Perhaps because the approach was so unfamiliar, or because P-188's mode of action was not yet well understood, the medical trauma community was skeptical.
It did not help that in 1996, in a series of clinical trials to help stop heart attacks, P-188 had caused kidney damage in some patients. The organizers of those trials had hypothesized that a continuous supply of the polymer was needed to keep blood vessels open, and had given the drug for days, in doses hundreds of times higher than the small amount needed for membrane sealing.
Scientists now understand that P-188 inserts itself into the holes of cells that have sustained trauma, preventing their death. The three-part structure of P-188 matches that of the cell membrane. Both feature a hydrophobic middle sandwiched between two hydrophilic ends. When the cell membrane is ruptured, the broken places in the wall expose their inner hydrophobic regions. P-188's hydrophobic middle is drawn to these sites. When the polymer inserts itself into a hole in the membrane, surface tension is reduced, which facilitates sealing.
Today, Both P-188 and the concept of treating injury by healing cell membranes are receiving new attention. In 2004, researchers at Purdue University's Center for Paralysis Research tested P-188 on a group of 16 paralyzed dogs that would otherwise have been put down. In addition to being given standard treatments—steroids to reduce inflammation and surgery to relieve spinal cord pressure and take out bone fragments, the dogs were injected with P-188 within 72 hours of their spinal injury. They were compared with animals treated with the pure hydrophilic polymer polyethylene glycol (PEG), and a set of past cases of dogs with similar injuries that received only standard treatment, so that no injured dogs received a placebo. Six to eight weeks after treatment, the dogs that received P-188 and PEG injections had healed much better than dogs in past cases. More than half of the dogs treated with P-188 (9 of 16) recovered their ability to walk, compared to 25% (6 of 24) of the dogs in the historical control group. The Purdue research drew attention to the use of polymers to heal damaged cells.
P-188 also offers promise for injuries to the brain and central nervous system. Jeremy Marks, a pediatrician and neurologist at the University of Chicago, has demonstrated that P-188 can reduce damage after a simulated stroke or reduction of oxygen to the brain. In his recent work, adding P-188 to neurons in a Petri dish up to eight hours after injury allowed the neurons to fully recover. Marks is interested in learning how quickly the drug might be able to cross the placenta to prevent injury to babies during distressed labor. Lee believes that with polymer therapy, "we will soon be able to reduce the consequences of diseases like Cerebral Palsy," commonly caused when the umbilical cord is compressed and prevents oxygen from reaching a baby's brain. Polymers could potentially reverse the damage that results and prevent the disease from developing.
Some of the most recent work on P-188 also suggests that it functions as an artificial chaperone, taking on the job of disentangling and refolding damaged proteins. Studies at Argonne National Lab's Advance Photon Source , which provides brilliant X-ray beams, in the laboratory of Pappannan Thiyagarajan, have looked at how surfactants affect molecules as they experience heating and cooling. In tests using small-angle X-ray scattering, which involves focusing an X-ray on a sample and studying the way the beam scatters, poloxamers broke apart aggregates of denatured proteins. Other work shows that P-188 can actually re-fold denatured proteins. [Read the article.]
How does P-188 know where to go in the body, and what happens to it after cell membranes are sealed? Using a lipid monolayer to mimic the cell wall, University of Chicago chemist Ka Yee Lee, Argonne National Laboratory chemist Millicent Firestone, and colleagues found that the poloxamer, attracted to areas of lower "lipid packing density," went directly to the holes in the film in need of repair, and nowhere else. When the density of the model membrane returned to normal, P-188 was pushed out. This suggests cells could accept the drug's help when needed, but release it once cell membranes were restored.
To date, most of the work on P-188 has been done in isolated human cells, animal models, and model cell membrane systems. But that will soon change. In 2003, Lee and a group of other researchers formed Maroon Biotech to conduct the clinical trials necessary to bring polymer therapies to market. The drug's safety has already been established in clinical trials, and by the fact that it has been in medical use for many years, but it must still be proven effective for various specific conditions. Treating brain and spinal cord trauma is the company's first priority. Another priority is treating military injuries, with the goal of reducing lifelong disability among young people. Lee explains that gunshot wounds from high-velocity bullets create a shockwave that causes extensive tissue damage. But that damage can be reduced by applying polymers, he says.
Maroon will also develop polymer therapies to treat burns. A group of researchers at Massachusetts General Hospital including Mehmet Toner, one of Lee's first postdoctoral collaborators, has shown that injection of P-188 after burn injury reduced the thickness of burns by 40% in an animal model. "If you can reduce the thickness of a burn wound by 40%, that would eliminate most of the need for skin grafts," Lee says. "And these burns would heal themselves, just like a bad sunburn."
Scientists at Maroon Biotech are working on these conditions and others. They are also tinkering with the structure of P-188 to arrive at the best possible design. Lee is anxious to see the drug make it through its required clinical trials, and given all the model systems it has been used in, he fully expects it to work. The potential impact the drug could have on people's lives is enormous, making it difficult for Lee and others at Maroon Biotech to accept the slowness of the process.
Yet, the process is moving forward. Will we see a day when EMTs arrive on the scene with an IV fluid that will repair molecular damage? When an injection given within hours of spinal cord injury will prevent lifelong paralysis? The idea may still sound like science fiction, but some trauma doctors are intrigued by it. Jonathan Wenk, an emergency room physician with MEP, a group that serves hospitals in the Washington, DC area, says, "Often, the body is better at healing and repairing itself than doctors are.A compound that can facilitate the healing process at the cellular level would certainly hold a great deal of promise." Time and clinical trials will tell.
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