The mission of wound healing is to increase our basic understanding of the molecular and cellular events of the cellular repair and wound healing processes, and to use this information as the basis for developing new therapies that minimize the adverse consequences of burns injuries. Such novel therapies could enhance cellular repair, promote rapid wound closure, minimize hypertrophic scarring, or control scar contracture. To accomplish these goals, our research is focused on four principal molecular and cell processes of healing wounds: inflammatory cell infiltration and cutaneous immune function, keratinocyte activation and migration, endothelial cell function during angiogenesis, and fibroblast activation leading to scar formation. In addition to specific cell types, our investigations also involve identification and characterization of the growth factors and extracellular matrix proteins which are synthesized by the cells of the healing wound. We seek to establish how these molecules orchestrate the temporal and spatial events important for wound closure.
To understand these events, we use a variety of methods to analyze and manipulate the cells and molecules of the healing wound. These experimental approaches include: analyzing patterns of expression of extracellular matrix proteins and growth factors during wound repair; testing structure-function relationships between cytokines, extracellular matrix proteins and their receptors; employing genetic engineering of skin cells to over-express various growth factors, as well as regulation of the immune system. In the latter case, these modified cells are then transplanted and evaluated as skin equivalents, both wounded and unwounded, in animal models. Using these approaches, we expect to identify which growth factors and extracellular matrix molecules exert the greatest influence over specific facets of the wound healing process (i.e., inflammation, angiogenesis, re-epithelialization and matrix formation), determine how these molecules regulate the wound healing cascade and develop an improved understanding of the roll of the immune system in wound healing and its relationship to allograft rejection. Moreover, we will determine if dysregulation of these key regulatory molecules underlies some of the pathologies of the healing such as wound contracture and hypertrophic scarring. Such investigations may also lead to the development of much needed animal models of wound pathologies, in which new therapies can be tested.
We anticipate that the research within this area will yield future therapies that can intervene in the wound-healing process to the benefit of the burned patient. Prospective new therapies include small molecules which act as specific agonists or antagonists of selected wound healing processes, and improved skin equivalents which can aid the closure of large burned areas. There are several areas of inquiry which are relevant to the wound healing, but not yet actively investigated. These future areas include the exploitation of the new revolution in gene isolation to identify previously uncharacterized genes which participate in wound healing, the use of embryonic stem cells to prepare specific lineages of skin cells which are genetically deficient in selected functions, the underlying basis for the scarless wound healing environment of the fetus and the integrated analysis of molecular/cellular events with the biomechanical and structural/physical properties of the skin.
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Burn injury triggers a hypermetabolic state which is manifested by a negative nitrogen balance with increased protein degradation and urea excretion, hyperglycemia and hyperlactatemia. Ongoing research in the metabolism aims at characterizing these changes and elucidating the molecular mechanisms which control the onset, maintenance, and abatement of the post burn hypermetabolic state. Currently there is a lack of understanding of the quantitative relationships between the various burn-induced metabolic and physiological changes. Our efforts attempt to fill this gap by:
- probing and quantitatively analyzing the in-vivo activity of anabolic and catabolic pathways of key nutrients in burn patients, both at the whole-body level and in major organs/tissues.
- investigating the metabolic processes on a molecular level using in-vitro systems such as perfused organs and cell culture preparations.
Ultimately, this knowledge will help develop a more efficient nutritional and pharmacological regimen to accelerate the recovery process of burn patients. The key questions that are being addressed in the current studies are described below.
Conditionally essential amino acids: These studies examine the metabolism of tyrosine, cysteine, proline, glutamic acid, and arginine in burn patients and the effect of supplementation of these amino acids on the metabolic status. There is evidence that burn injury leads to an increase in the demand together with a decrease in the de-novo synthesis of these amino acids, which suggests that they may become nutritionally essential in burn patients.
Nitrogen balance and urea synthesis: These studies explore the mechanisms leading to increased amino acid nitrogen conversion into urea, the form which accounts for most of the patient's nitrogen loss after burn injury. The regulation of urea cycle activity and protein turnover after burn injury and its impact on nutritional support are being studied at the whole body level using labeled leucine and urea cycle intermediates (arginine, ornithine and citrulline) used as tracers. To better delineate the role of intrinsic (e.g., alterations in enzyme expression) vs. extrinsic (e.g., changes in substrate load coming from the portal circulation) factors which regulate urea synthesis in burns, other investigations focus on the fate of amino acids in isolated liver preparations and in cultured liver cell systems. Because one of the main sequelae of hypermetabolism and nitrogen loss is muscle wasting, extensive studies currently attempt to elucidate the roles of the various pathways (e.g., ubiquitin proteasomes) in muscle proteolysis using a rat burn model as well as stable differentiated muscle cell cultures. Similar pathways are being investigated in peripheral blood lymphocytes, which may provide a simple and effective diagnostic tool to assess proteolytic activity in burn patients. Lymphocyte protein catabolism is also being examined as a possible underlying mechanism of immunosuppression in burn patients.
Increased energy expenditure: The main goal is to determine, particularly in muscle, liver, gut, and the wound, the specific pathways which are upregulated within and around the tricarboxylic acid (TCA) cycle after burn injury and contribute to the increased metabolic rate. These studies are performed at the whole body level as well as in isolated liver preparations using different nutritional support formulations and with the aid of various stable and positron-emitting isotope tracers.
Effect of mediators on metabolism: These studies are aimed at understanding the effects of specific inflammatory mediators and stress hormones on energy and carbohydrate metabolism. The effect of altered hormone levels are investigated in vivo using isotope tracers and positron emission tomography (PET) imaging. Studies on cultured hepatocytes allow the precise characterization of the effect of inflammatory mediators on mitochondrial redox state. In addition, there is a significant effort on understanding the role of insulin and more specifically on the development of insulin resistance in burn-induced muscle catabolism.
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Inflammation and Infection
The overall goal of the inflammation and infection is to obtain a better understanding of the pathogenesis of systemic inflammation following burns and the resulting deleterious effects on patients. This information is being used to rationally design selective and safe therapeutic agents to block the most detrimental aspects of the systemic inflammatory response. Ongoing efforts can be grouped into three categories, each addressing a major aspect of the inflammatory response:
- interactions of infectious agents with the inflammatory system
- organ-specific and systemic inflammatory responses to injury
- new therapeutic agents.
Infection: Little is known about the pathogenesis of infection following burns.
The factors that make certain bacteria pathogenic or invasive ("virulence factors") are currently being studied by molecular biologists and surgeons, who are identifying genes responsible for virulence characteristics in Pseudomonas species. Furthermore, mechanisms whereby bacterial products such as the outer membrane proteins on Enterobacteria mediate an inflammatory response in the host are being elucidated. Organisms with small and well characterized genomes, such as the plant Arabidopsis and the nematode Caenorhabditis, are used to identify clinically relevant glycoproteins and genetic factors in the host which are involved in the natural defenses against microorganisms.
Inflammation: Systemic inflammation following burns involves multiple organ systems which exhibit different responses. Currently, the main efforts focus on understanding molecular mechanisms of inflammation in the lungs. The role of specific inflammatory mediators is being investigated using genetically modified mice that either do not express (i.e. knockouts) or overexpress genes related to inflammation as well as by cellular transfection techniques in vivo. The susceptibility to pneumonia following burns is being characterized in these animal models. Another important aspect of lung injury is the behavior of sequestered inflammatory cells such as neutrophils. Using animal models of burns and sepsis, current studies focus on the role of lung macrophages as well as a variety of inflammatory cytokines (e.g., GM-CSF and TNFa) and arachidonic acid metabolites (e.g., leukotrienes) in the control of neutrophil sequestration and programmed cell death (apoptosis) in the lungs. Some of these are collaborative studies with investigators in the Metabolism area, who focus on the role of these mediators in the metabolic response to burn injury.
Therapeutics: New therapeutic agents and approaches are evolving from the basic studies described above. The identification of virulence factors is allowing the development of animal-independent screening systems for the virulence of clinical isolates of Pseudomonas bacteria. Furthermore, immunotherapies targeted at pro-inflammatory bacterial surface antigens are being investigated to treat bacterial sepsis. Gene therapy using retroviral transfection techniques as well as various pharmacological agents are being used to control neutrophil trafficking and apoptosis in the lungs and prevent lung damage mediated by these cells following burn injury.
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Biotechnology & Bioengineering Research
The biotechnology and bioengineering research encompasses several distinct projects that seek to analyze, evaluate and develop therapies for a variety of clinical problems facing the severely burned patient. Each of these projects is united by the fact that they utilize either state-of-the-art biotechnology processes for the design and production of recombinant proteins/gene therapeutics and/or they use quantitative bioengineering analyses to evaluate complex physiological processes. Both approaches have a common goal of developing novel therapies that address a medical need which impacts the mortality and morbidity of the burned patient.
Smoke injury, especially when associated with surface burns, can lead to severe respiratory complications that often is a leading cause of death for burn patients. Inflammatory mediators evoked by the presence of toxic substances and debris associated with smoke injury are thought to be major contributors to acute respiratory syndrome (ARDS) in the burned patient. One project using a bioengineering approach seeks to use and evaluate oxygen carrying perfluorocarbon liquid as a means to simultaneously remove toxic debris/mediators while providing liquid ventilation of the lungs. The effects of perfluorocarbon liquid ventilation will be followed by using non-invasive functional imaging (Positron Emission Tomography) to assess the intrapulmonary distribution of lung function.
Inflammatory mediators such as IL-6, which are synthesized at high levels after burn trauma, may also play a role in the onset of multiorgan disorder system (MODS). The liver is especially sensitive to these types of inflammatory mediators and undergoes an acute phase response when exposed to high levels of IL-6. The overall goal of this research project is to specifically control the liver response to IL-6 by engineering an antisense DNA which hybridizes and down- regulates the messenger RNA encoding the receptor for IL-6. These antisense molecules are targeted to the hepatocytes of the liver by complexing the antisense to an asialoglycoprotein, which is selectively taken up by receptors found only on hepatocytes. These antisense conjugates have been shown to selectively downregulate the hepatocyte's response to IL-6 and hold promise as a means to modulate specific organ responses to inflammatory mediators after burn injury.
Cryopreservation of engineered and cadaveric skin grafts is crucial to the treatment of the burned patient. Multiple types of grafts are available to the surgeon including autografts, cadaver allografts as well as grafts of cultured keratinocytes and composite grafts of cultured keratinocytes seeded on dermal analogues. Although the structural integrity of the skin is preserved following a freeze-thaw cycle, the cellular viability of the skin immediately after freezing has never been optimized and may be critical for the optimal performance of the skin graft.
This project seeks to obtain an understanding of the cellular response to cryoprotectants and ice nucleation and to integrate these biophysical parameters into a coherent theoretical heat and mass transfer model to predict the optimal cryopreservation protocol that preserves cell viability. After testing using animal models, these optimal cryopreservation protocols will assure that valuable skin grafts will have optimal performance after transplantation to the burned patient.
In summary, Biotechnology & Bioengineering is focused on the development and testing of novel therapies and protocols to benefit the burned patient. In addition to these aforementioned efforts, future areas of interest include non-invasive methods to assess the extent of burns, therapeutics for hypertrophic scarring, copolymers to seal thermally damaged plasma membranes, and mechanically induced muscle culture systems for studies of muscle wasting.