Optimizing Cellular Metabolism to Improve Chronic Skin Wound Healing James J. Slade Honors Thesis Luis Felipe Ramirez Biomedical Engineering Rutgers University, New Brunswick Under the direction of Dr. Francois Berthiaume and Dr. Gabriel Yarmush Abstract—Despite significant advances, chronic skin wounds Wound healing is a complex process with four identifiable remain a large problem both in terms of morbidity and cost. It stages: hemostasis, inflammation, proliferation, and remodel- is estimated that in the United States, this problem afflicts 6.5 ing [6]. Hemostasis involves the formation of a blood clot that million people a year and costs more than 30 billion dollars for di- abetic foot ulcers alone [4,11]. Currently approved treatments are stops the loss of blood at the site of injury [6]. Growth factors often ineffective. This thesis seeks to leverage the large amount of released by activated platelets during the hemostasis stage information that has accumulated about metabolism in the hu- recruit immune cells such as neutrophils and macrophages [6]. man body, and to mine that information with computational mod- The infiltration of the injured tissue with these immune cells eling. It seeks to uncover whether metabolites commonly available leads to the inflammatory phase [6]. The role played by inflam- in the human body can be used to bolster the metabolism of wounded cells such as keratinocytes (the cells that form the mation is classified as both positive and negative [5]. On the epidermis), and therefore improve the natural process of wound one hand the inflammatory response clears the wound site of healing. This would offer treatment options with fewer side effects pathogens and dead cells, on the other hand the inflammatory than what is currently offered to improve wound healing. The response is thought to prevent the wound from healing and author first reduced the global set of reactions and metabolites to be responsible for scarring [5]. In the proliferation stage, in a publicly available database of cellular metabolism (the recon Database), to 357 reactions and 339 metabolites that are most keratinocytes repopulate the injury and angiogenesis leads to applicable to keratinocytes. Then, metabolic flux through a key revascularization of the tissue [6]. Keratinocytes migrate from set of 25 reactions was defined for mass balance analysis. Lastly, the edge of the injury and from hair follicles towards the the impact of variations in metabolic inputs on the metabolic flux deeper portions of the injury in part due to stimulation from distribution was investigated. By analyzing the variability of the transforming growth factor (TGF)-β [6]. Hypoxic conditions flux through each reaction in Monte Carlo simulations, reactions that are likely to be amenable to manipulation were identified. within the injury site lead to release of vascular endothelial This analysis points to the citric acid cycle as the pathway growth factor, which results in angiogenesis [6]. Fibroblasts most amenable to manipulation. Other key reaction categories are also important in the proliferation stage. In particular, they identified were glycolysis, the pentose phosphate pathway, amino regenerate the extracellular matrix by releasing proteins such acid metabolism, transport reactions, and energy production and as collagen, elastin, and matrix metalloproteinase [6]. The homeostasis. remodeling stage lasts the longest, and involves remodeling of the extracellular matrix [6]. Of particular importance is I. INTRODUCTION the conversion of type III collagen to type I collagen, which strengthens the injured tissue [6]. A. The Medical Problem Chronic wounds are characterized by a sustained inflam- Chronic skin wounds can be defined as wounds that do not matory response, increased presence of microbes, and fewer heal within three months, and include three types of wounds: and functionally deficient fibroblasts [5]. The sustained in- vascular ulcers, diabetic ulcers, and pressure ulcers [5, 2]. In flammatory response presents a problem for wound healing the United States alone, this problem is said to afflict 6.5 as it taxes oxygen and nutrient availability. The immune cells million patients [11]. The burden of diabetic foot ulcers alone that infiltrate chronic wounds also appear to have diminished on the health system in the United States is estimated to be phagocytic ability, leading to a buildup of necrotic debris [5]. $30 billion annually [4]. The prevalence of diabetic foot ulcers A common problem with chronic wounds is also the presence alone was estimated to be 68.4/1000 person-years [3]. All of of bacteria and fungi that invade the wound [5]. The interplay this information points to a set of conditions that cause a severe between the sustained inflammatory response and the presence burden on individuals and the health system, and that merits of microbes is unclear [5]. Fibroblasts found in chronic sustained research efforts. wounds have also been found to lack a normal response to growth factor and to lack normal migratory capacities [5]. TABLE I Chronic wound fluid has also been found to have an inhibitory SUMMARY OF THE METABOLISM OF WOUNDS effect on the proliferation of fibroblasts and keratinocytes, indicating problems with the factors present in chronic wounds Type of Condition Condition Sub-type Positive/ [6]. Matrix Metalloproteinases have also been found to be Negative Effect overactive in chronic wounds, which leads to a degradation Acute Hypoxia- Has positive of the extracellular matrix [6]. leads to platelet effects aggregation and the release of B. Metabolism of Wounds Hypoxia cytokines Chronic Hypoxia- Has large Many processes of wound healing demand large amounts leads to impaired negative energy production, effects of energy [13]. An especially important factor in the ability a prolonged of cells to obtain energy is the oxygen availability. Shortly inflammatory phase, after the onset of the wound, hypoxic conditions occur in and too much ROS the wound area as a result of vessel constriction and platelet accumulation Xanthine - Negative effect aggregation [8]. Acute hypoxic conditions can have benefi- Purine Catabolism Oxidoreductase- cial impacts on wound healing, whereas chronic hypoxia is Excessive ROS believed to have large deleterious effects on wound healing production L-Arginine Negative effect [10]. Chronic hypoxia impairs cellular function by limiting Deaminase- the production of ATP through aerobic glycolysis, the citric Arginine Metabolism ROS produced acid cycle, and fatty acid oxidation [10]. This ATP production Arginase - No Positive effect ROS produced is critical for sustaining all of the steps of wound healing, Decreased flux Negative effect including cell proliferation, bacterial defense, and collagen through glycolysis, synthesis [10]. Furthermore, the oxygen dependent NADPH the citric acid cycle, nucleotide synthesis, oxygenase produces reactive oxygen species (ROS) that are and fatty acid crucial for bacterial control, cytokine release, cell proliferation, catabolism- leads and angiogenesis [10]. As mentioned above, acute hypoxia is to reduced energy in the cells available also believed to play a beneficial role in wound healing [10]. for wound healing The initial hypoxia, for example, leads to platelet aggregation Impaired Glucose Uptake Increased catabolism Negative effect and the stimulation of cytokine release through the production of amino acids and carboxylic acids- of ROS [10]. Moreover, it is believed that leukocytes use the leads to breakdown of ROS gradient to infiltrate the wound tissue [10]. proteins and a limited ability to construct new Chronic wounds are characterized by local hypoxia that proteins needed for is caused by alterations of the blood vessels and reduced wound healing vascular perfusion [10]. These hypoxic conditions lead to Reduced flux through Negative effect the Pentose Phosphate reduced energy for cells, as well as to signaling cascades Pathway- less that lead to inflammatory responses [10]. In addition, hypoxia reducing equivalents leads to increased production of cytokines that promote the produced movement of neutrophils and macrophages into the wound, and the subsequent production of pro-inflammatory molecules [10]. Prolonged hypoxic conditions also prevent the production of nitric oxide through the nitric oxide synthase enzyme [10]. produces reactive oxygen species [1]. Conversely, arginine Nitric oxide normally prevents the accumulation of large quan- metabolized by arginase does not generate ROS [1]. tities of ROS, and its absence can lead to harmful accumulation of ROS [10]. Due to increased energy demand, central carbon metabolism Another key metabolic pathway involved in wound healing is very important in wound healing. Defective glucose uptake, is purine metabolism. In particular, it has been found that for example, has been found to negatively impact wound due to increased energy demand, cells engage in substantial healing [14]. The researchers found that the decline in glucose purine catabolism during wound healing [13]. Weinstein et al. uptake led to significant reductions in flux through glycol- found that xanthine oxidoreductase leads to excessive ROS ysis, the citric acid cycle, nucleotide synthesis, and fatty formation in diabetic patients, thus contributing to impaired acid catabolism [14]. The cells compensated by increasing wound healing [13]. A similar pathway involving arginine catabolism of amino acids and carboxylic acids [14]. Also of metabolism has also been studied in wound healing [1].