Prediction of nitric oxide concentrations during inflammation and carcinogenesis

Abstract

Nitric oxide is a biological messenger which is synthesized enzymatically throughout the body and which has numerous physiological functions, including roles in blood pressure control, regulation of clotting, and neurotransmission. It is produced also by macrophages as part of the non-specific immune response, and has been shown to be toxic to invasive microorganisms. However, sustained overproduction of NO, as observed with chronic inflammation or infection, may damage the host tissue and has been linked to increased risk for cancer. Nitric oxide may facilitate tumorigenesis or metastasis by influencing tumor-cell proliferation, angiogenesis, or lymphangiogenesis. However, the concentrations of NO in inflamed tissues or during carcinogenesis are largely unknown. The objective of the research in this thesis was to predict NO concentrations in an inflamed colon and a cutaneous metastatic melanoma, two areas where NO has been implicated in the development or progression of disease. Nitric oxide production in the colon has been linked to inflammatory bowel disease (IBD) and increased risk for colon cancer. However, measurements of NO concentration in the inflamed colon have not been available and it is not known what levels of NO are pathophysiological. A computational model, based on anatomical length scales and rates of NO production measured in cell cultures, was developed to predict spatially varying NO concentrations within a colonic crypt under inflammatory conditions. A variety of scenarios were considered, including different spatial distributions of macrophages and a range of possible macrophage and epithelial synthesis rates for NO.

(cont.) The results were used to predict the range of NO concentrations (<0.3 tM) and cumulative NO dose (560 [mu]M * min) experienced by a given epithelial cell migrating from the base to the top of the crypt. This first set of predictions was based on literature data on the cellular synthesis and consumption of NO. Knowledge of the rates at which macrophages and epithelial cells synthesize NO is critical for predicting the concentrations of NO and other reactive nitrogen species in colonic crypts during inflammation, and elucidating the linkage between inflammatory bowel disease, NO, and cancer. Macrophage-like RAW264.7 cells, primary bone marrow-derived macrophages (BMDM), and HCT116 colonic epithelial cells were subjected to simulated inflammatory conditions, and rates of formation and consumption were determined for NO, O2, and 02-. Production rates of NO were determined in either of two ways: continuous monitoring of NO concentrations in a closed chamber, with corrections for autoxidation; or NO2- accumulation measurements in an open system, with corrections for diffusional losses of NO. The results obtained using the two methods were in excellent agreement. Rates of NO synthesis (2.3 + 0.6 pmol s-1 106 cells-), NO consumption (1.3 ± 0.3 s-1), and 02 consumption (58.8 ± 17 pmol/s/10 6 cells) for activated BMDM were indistinguishable from those of activated RAW264.7 cells. NO production rates calculated from NO2- accumulation data for HCT1 16 cells infected with Helicobacter cinaedi (3.9 ± 0.1 pmol/s/106 cells) were somewhat greater than those of RAW264.7 macrophages infected under similar conditions (2.6 ± 0.1 pmol/s/106 cells).

(cont.) Thus, RAW264.7 cells have nearly identical NO kinetics to primary macrophages, and stimulated epithelial cells are capable of synthesizing NO at rates comparable to macrophages. Using this new cellular data to refine the predictions of the colon model, simulations of NO diffusion and reaction in a crypt during inflammation gave maximum NO concentrations of about 0.2 [mu]M. The presence of iNOS and nitrotyrosine in human metastatic melanoma tumors suggest that NO plays a role in the pathophysiology of metastatic melanomas. However, the concentrations of NO that melanoma cells are exposed to in vivo remains unknown. Cellular rates of NO synthesis and consumption were experimentally determined and used in a reactiondiffusion model to predict NO concentrations in a cutaneous melanoma. NO synthesis by A375 melanoma cells was undetectable using Griess assays. The rate constant for intracellular NO consumption by A375 cells (7.1 ± 1.1 s-1), was determined by monitoring NO concentrations in a closed chamber with corrections for autoxidation and consumption from media-generated 02-. Incorporating NO kinetic data from A375 cells and macrophages into a reaction-diffusion model, NO was found to be localized to the periphery of the melanoma; roughly 90% of the NO synthesized by macrophages is consumed within 30 [mu]m of the tumor edge. Several additional scenarios were modeled, including the effects of varying the volume fraction of macrophages surrounding a melanoma tumor and intratumoral NO synthesis by melanoma cells. As such, a range of NO concentrations were predicted (< 0.2 VM) for the edge of the tumor.

(cont.) Our model may offer some insight into the role NO plays in the metastasis of cutaneous melanomas, which occurs primarily through lymphatic spread. At the melanoma edge, NO may inhibit melanoma cell apoptosis, contributing to abnormal tumor growth and invasion of local lymph vessels or NO may act as a lymphangiogenic factor. The colonic crypt model and the cutaneous melanoma model developed in this work provide the first NO concentration predictions for tissues during inflammation. Despite the different geometries and different cell types involved, the maximum values estimated in both cases were - 0.2 [mu]M. The results from this work can be used for predicting intracellular levels of other reactive nitrogen species, and should help guide further studies to understand the mechanisms by which NO contributes to carcinogenesis in IBD and in cutaneous melanomas.