Title: Roles of Macrophage Mitochondrial Oxidative Stress and Mitochondrial Fission in Atherosclerosis
Description: Electron transportation (ET) coupled with oxidative phosphorylation (OXPHOS) in the mitochondria produces limited, physiologic levels of reactive oxygen species (ROS). While this process is adaptive under normal conditions, excessive mitochondrial oxidative stress (mitoOS) has been correlated with a number of diseases, including atherosclerotic vascular disease in humans. However, definitive evidence of causation and cell-specific pro-atherogenic mechanisms of mitoOS require further investigation. The high level of interest in this topic, the human relevance, and the potential therapeutic implications prompted us to explore causation and mechanism with a focus on the key inflammatory cell type in atherosclerosis, the macrophage (Chapter 2). For this purpose, we used a recently described model, the mitochondrial catalase (mCAT) transgenic mouse, that decreases mitoOS in vivo. Normally, glutathione perioxidase is the endogenous mitochondrial enzyme that catalyzes the reduction of H2O2 and prevents its conversion into the most detrimental ROS hydroxyl nitrites.

Catalase can carry out this role in peroxisomes, where it is exclusively located. The mCAT transgenic mouse expresses human catalase with a mitochondrial matrix-targeting motif, which quenches mitoOS and protects against mitoOS-induced damage. To focus on myeloid-derived cells in atherosclerosis, we used two strategies: transplantation of mCAT transgenic bone marrow cells into atheroprone Ldlr-/- mice and crossing Ldlr-/- mice with an mCATfl/-LysMCre model that expresses mCAT only in lysozyme M-expressing cells, notably differentiated macrophages. After 8 wk western type diet (WD) feeding, both models demonstrated evidence of decreased mitoOS in lesional macrophages, decreased atherosclerosis, suppression of Ly6chi monocyte infiltration, and lower levels of the monocyte chemotactic protein-1 (MCP-1). The decrease in lesional MCP-1 was associated with suppression of other markers of inflammation (iNOS and TNF-α) and with decreased phosphorylation of the critical transcription factor RelA (NF-κB p65), indicating decreased activation of the pro-inflammatory NF-κB pathway.

Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed MCP-1 expression by decreasing activation of the Iκ-kinase (IKK) - NF-κB (RelA) pathway. Taken together, we conclude that MitoOS in lesional macrophages amplifies early atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the early progression of atherosclerosis. The mitochondrial dynamic processes of fission and fusion influence and integrate with multiple physiologic and pathophysiologic processes. Mitochondrial fusion/fission dysregulation has been implicated in atherosclerosis, but little is known about the role of myeloid cell specific mitochondrial dynamics in the progression of atherosclerosis. Dynamin related protein 1(DRP1), a cytosolic GTPase family member, is one of the molecules that mediate mitochondrial fission.

In the second part of this thesis (Chapter 3), we used western diet-fed Drp1fl/fl LysmCre+/-Ldlr-/-mice to determine the role of Mφ mitochondrial fission in both early atherogenesis and advanced atherosclerosis. Our data thus far show that: (1) Mitochondria in lesional Mφs are elongated in Drp1fl/fl LysmCre+/-Ldlr-/ mice by transmission electron microscopy (TEM) analysis; (2) Suppression of Mφ mitochondrial fission does not affect early atherogenesis; (3) Inhibition of Mφ mitochondrial fission leads to a striking increase of necrotic core area and the accumulation of apoptotic cells, which are likely due to the defective phagocytic clearance of apoptotic cells (efferocytosis) in the advanced stage of atherosclerosis in vivo; (4) DRP1-deficient Mφs are defective in efferocytosis in vitro and in vivo. (5) The phagocytic deficiency in DRP1-deficient Mφs is associated with a reduced level of uncoupling protein 2 (UCP2), a mitochondria protein required for continuous uptake and clearance of dead cells in phagocytes.

We conclude that DRP1-mediated mitochondrial fission in Mφs promotes the clearance of apoptotic cells and thereby blocks necrotic core formation in advanced atherosclerosis. This study indicates that mitochondrial fusion/fission could be a new therapeutic target to stabilize the advanced plaques and prevent acute atherothrombosis in humans. In terms of mechanism, we hypothesize that mitochondrial fission stabilizes UCP2 in the inner membrane of mitochondria. Further studies are required to elucidate how DRP1-UCP2 pathway maintains the efferocytosis capability in phagocytosis. In summary, my thesis studies have revealed the pathological significance of macrophage mitoOS in early atherogenesis and a novel link of mitochondria dynamics to macrophage phagocytosis in the setting of advanced atherosclerosis.