Energy metabolic failure within the arterial wall in atherosclerosis

Abstract

Atherosclerosis is a disease that affects large and medium sized arteries. The majority of deaths in a Western population can be directly attributed to complications of this disease. Although research efforts have been vast, the pathophysiological mechanisms underlying atherosclerosis are not fully understood. According to the anoxemia theory of atherosclerosis, an imbalance between the demand for and supply of oxygen and nutrients at depth of the arterial wall is a key factor in the development of atherosclerotic lesions. In vitro evidence indicates that zones of hypoxia occur at depth of the atherosclerotic plaque. However, the energy metabolic characteristics of the arterial wall in vivo are not fully described.The focus of this study was to study different aspects of arterial wall energy metabolism in vivo. The hypoxia marker 7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline (NITP) was used to study arterial wall hypoxia. The technique of hypoxia markers have been developed in cancer research and have not previously been used on arterial tissue in vivo. In short, hypoxia markers are trapped in hypoxic, but viable, cells and can be detected in tissue sections with immunohistochemistry. NITP was injected in atherosclerotic rabbits in vivo and the aorta was removed after an accumulation period of 4-6.5 hours. In paraffin sections of the aorta, zones of hypoxia were demonstrated at depth of atherosclerotic plaques that were more than 4-500 µm thick. To analyze local concentrations of ATP (adenosine triphosphate), glucose and lactate in arteries a bioluminescence technique, bioluminescence imaging, was used. Bioluminescence imaging is used to study energy metabolic heterogeneities in cryosections of snap frozen tissue and have not previously been used on arteries. In vitro experiments were performed on arterial segments from pigs (normal vessels) and rabbits (normal and atherosclerotic vessels) to test and optimize the methodology. Bioluminescence imaging allowed for quantification of local concentrations of ATP, glucose and lactate at a spatial resolution of 1-200 µm. To study the in vivo situation, a technique to freeze the aorta in situ in anaesthetized rabbits was used. With this technique, the delay between cessation of normal blood flow and freezing was kept at an absolute minimium (3-4 seconds), thus the in vivo situation was reflected. Energy metabolic failure (ATP depletion) was demonstrated at a distance of 3-500 µm from the lumen in rabbit lesions. Glucose concentrations were low, and lactate concentrations were high in lesion, probably reflecting an increase in both aerobic and anaerobic metabolism. Furthermore, ATP concentrations were reduced and lactate concentrations increased in the media underlying lesions compared to the undiseased media.In conclusion, the results presented in this thesis suggest that hypoxia and energy metabolic failure occurs at depth of the atherosclerotic plaque in vivo. Furthermore, energy metabolic predicaments are also seen in the underlying media. We suggest that the strained energy metabolic situation is the result of a local shortage of oxygen and glucose due to increased diffusion distances in combination with an increased metabolic demand and lactate accumulation within the lesions.