Optimizing cellular metabolism to improve chronic skin wound healing
Creators: name: Ramirez, Luis
Publisher: name: No Publisher Supplied
Year: 2023

description: Despite significant advances, chronic skin wounds remain a large problem both in terms of morbidity and cost. It is estimated that in the United States, this problem afflicts 6.5 million people a year and costs more than 30 billion dollars for diabetic foot ulcers alone[1,2]. The ineffectiveness of currently approved treatments merits continued attempts at ameliorating this set of conditions. This thesis seeks to leverage the large amount of information that has accumulated about metabolism in the human body and use computational modeling to uncover whether metabolites commonly available in the human body can be used to bolster the metabolism of wound cells, and therefore improve the natural process of wound healing. This would offer treatment options with fewer side effects than what is currently offered to improve wound healing. This work focuses on keratinocytes (the cells that form the epidermis), and makes use of the Recon database, which is a public database that seeks to aggregate all of the metabolic pathways in the human body. In 2014, Quek et al. outlined a method for reducing the entirety of the Recon database to a set of 357 reactions and 339 metabolites that were more appropriate for human cells in culture[3]. The methods outlined in that paper were used to achieve a reduced set of reactions to optimize metabolic fluxes that would be most promising for the improvement of chronic skin wounds. For this work, biomass and ATP production were used as key outputs targeted for optimization. They are used as surrogate markers for cell proliferation and cell migration (an energy intensive process), respectively, both key processes during wound healing. The set of experimental measurements reported by Quek et al. was utilized as a starting point to perform a sensitivity analysis in order to determine which reactions involving extracellular metabolites, and thus most amenable to experimental manipulation, had the most impact on biomass and ATP production. Monte Carlo simulations, which involved varying inputs randomly around Quek’s average values assuming a normal distribution with standard deviation equal to 10%, were carried out for this purpose. First, it was found that &gt;1000 simulations were needed in order for simulations to converge to a value within 5% of the average output value. Subsequently, 1098 simulations were performed for each analysis. Second, both biomass and ATP production were among the reactions with the highest average flux, validating them as targets for intervention. Of note, there were numerous reactions from fatty acid metabolism that exhibited large relative variation (as determined by the standard deviation normalized to the average flux). Monte Carlo simulations were performed to identify the subset of simulations yielding the highest ATP production. In this case, the largest change from the average flux in extracellular metabolites was that of glycine. Of importance, glycine demonstrated the largest percentage change. Then, simulations with the greatest biomass production were analyzed; it became evident once again that the extracellular metabolite with the greatest percentage change was glycine. Analysis of the totality of the simulations also showed that biomass production is highly correlated with ATP production, meaning that when one is maximized the other is also likely to be maximized; thus, the same intervention would likely increase both outputs at the same time. Maximizing ATP and biomass generally involved increasing oxygen uptake; however, there is low oxygen availability in chronic wounds. Therefore, another set of simulations was carried out in which the flux of oxygen into the cell was kept constant, this constraint used as a way to account for limited oxygen supply, and also identified glycine as the most promising intervention. Meanwhile, glutamine showed consistently positive and was among the largest change in average flux in those conditions that most closely resemble chronic skin wounds, namely those with constant oxygen and those with low oxygen conditions. Thus, the model suggested that a possible intervention to improve ATP and biomass production could be an increase in the uptake of both glutamine and glycine. This hypothesis was tested in an in vitro cell culture system using immortalized human keratinocytes (HaCaT cells). Uptake of these amino acids was tentatively increased by supplementing the base culture media with additional glycine and/or glutamine. Valine supplementation was used as control. First, proliferative activity under various amino acid supplementation regimes was measured. In particular, supplementation with glycine yielded significantly greater cellular proliferation than base media. Second, wound closure rate in an in vitro wound scratch assay of confluent HaCaT monolayers was measured. Again, glycine supplementation showed significant improvement in wound closure rate. Supplementation with valine or glutamine and the combination of glycine and glutamine did not show significant improvement in either the proliferation assay or scratch assay. It is hoped that the examination of these interventions in vivo will yield improvements in the chronic skin wound healing process.