David G. Harrison, M.D.

Current Research Interests

The major direction of the Harrison laboratory is to understand factors that regulate the balance between production of nitric oxide (NO·) and reactive oxygen species that inactivate NO·. In the mid-1980s, Dr. Harrison and his co-workers were among the first to demonstrate that endothelium-dependent vasodilation is dramatically reduced in atherosclerosis. Since this phenomenon is mediated by release of NO· from the endothelium, Dr. Harrison began to study production of NO· by endothelial cells from normal and atherosclerotic vessels. Surprisingly, in the setting of early atherosclerosis, it was found that the production of nitrogen oxides (NO· and oxidation products of NO·) was paradoxically increased. This led to the hypothesis that in early atherosclerosis, NO· is normally produced, but that it is degraded to vasoinactive products of NO·, such as nitrate and nitrite. The most likely "suspect" for degradation of NO· is the superoxide anion (O2·-). In subsequent studies, Dr. Harrison's group showed that O2·- production was dramatically increased in atherosclerotic vessels and that treatment of either intact animals or vessels with membrane-targeted forms of superoxide dismutase dramatically improves endothelium-dependent vasodilation.

These studies led to the concept that an imbalance between levels of NO· and O2·- is a major mechanism whereby endothelium-dependent vasodilation and NO· "bioavailability" may be lost. This phenomenon has subsequently been shown to occur not only in atherosclerosis, but also in hypertension, diabetes, heart failure, cigarette smoking and aging.

 

As shown in the figure above, the reaction between O2·- and NO· not only leads to a loss of eNOS, but formation of the storng oxidant peroxynitrite (OONO-).

In the past ten years, Dr. Harrison's laboratory has made several contributions to understanding factors that modulate the balance between O2·- and NO·. Dr. Kathy Griendling found in the early 1990s that the NADPH oxidase is a major source of O2·- in vascular tissues, and together with Dr. Kathy Griendling, Dr. Harrison's group has shown that the vascular NADPH oxidase is activated in vivo both in atherosclerosis and hypertension. Interestingly, this enzyme system is also activated in nitrate tolerance, and may contribute tolerance by causing inactivation of nitric oxide released from nitroglycerin.

Studies from other groups have shown that eNOS, when derived of its critical co-factor H2O2, begins to produce O2·- rather than NO·. This phenomenon has been referred to as eNOS uncoupling. Recent studies from Dr. Harrison's laboratory have elaborated a mechanism for eNOS uncoupling. In these studies, it has been demonstrated that peroxynitrite can potently oxidize tetrahydrobiopterin and that this occurs in both atherosclerosis and hypertension. Studies of mice lacking p47phox, a subunit of the NADPH oxidase, show that this enzyme is likely the source of O2·- leading to peroxynitrite formation and eventual oxidation of tetrahydrobiopterin. We have coined the terms "kindling radicals" to refer to moderately low levels of O2·- produced by the NADPH oxidase, and "bonfire radicals" to refer to the large amounts of O2·- produced by the uncoupled eNOS. A schematic depicting interactions between these enzyme systems is shown in the figure below.

In addition to the above studies examining mechanisms whereby O2·- is produced, other studies have examined regulation of antioxidant defense mechanisms in the vessel wall. One such antioxidant is nitric oxide. In the early 1990s Dr. Harrison's laboratory, in collaboration with Dr. T.J. Murphy, cloned the cDNA for the endothelial isoform of NO synthase. In their first paper describing this enzyme, it was shown that its expression was rather markedly increased by shear stress. Subsequently, several laboratories have repeated this and have shown that exercise training (which increases cardiac output and consequently shear forces over the endothelium) increases eNOS expression. Dr. Harrison's laboratory has continued to study factors that regulate eNOS expressoion. A recent study has examined in detail the signaling pathways involved in increasing eNOS expression in response to shear. This pathway involves activation of the tyrosine kinase c-Src as an early event. After this, the signaling events seem to diverge. One pathway, dependent on Ras/Raf/ERK1/2 leads to a transient increase in eNOS transcription. A second pathway, also dependent on c-Src, but independent of Ras/Raf or ERK1/2, leads to a prolonged stabilization of eNOS mRNA. These two pathways seem complimentary, in that one allows for short-term modulation of eNOS mRNA, while the second controls eNOS expression in response to shear over a longer term.

 

In a related recent study, Dr. Harrison's group, together with Dr. Tohru Fukai, have shown that exercise training in mice dramatically increases expression of the extracellular SOD. This was found to be dependent on the increase in eNOS that occurs with exercise training, in that it did not occur in eNOS deficient mice.

Recently, methods have been developed to assay extracellular SOD in humans, and in preliminary studies, we have found that exercise training also causes an increase in ecSOD expression in humans.