Fukai Lab Research
Our lab has been investigating a role of oxidative stress and copper (Cu) transport system in cardiovascular disease. In particular, we are focusing on role of extracellular SOD (ecSOD, SOD3, a major vascular extracellular antioxidant Cu enzyme) and its regulator Cu transport system in pathophysiology of cardiovascular disease (hypertension, inflammatory angiogenesis, atherosclerosis, diabetes). To achieve this, our laboratory employ variety of state-of-art experimental tools and strategies that include the use of genetic mouse models, highly innovative synchrotron-based x-ray fluorescence microscopy (XFM) to examine quantitative spatial resolution of cellular metals at sub-micrometer resolution, 64Cu metabolic labeling analysis, inductively coupled plasma mass spectrometry (ICP-MS), imaging protein-protein interaction at caveolin-enriched lipid rafts using Total Internal Reflection Fluorescence (TIRFM), and real time monitoring ATP7A in live cell image.
Extracellular Superoxide Dismutase (ecSOD, SOD3) and Cardiovascular disease
1. Extracellular SOD and vascular pathophysiology
Role in hypertension and atherosclerosis:
Due to its extracellular location, ecSOD plays a critical role in regulating endothelium-derived nitric oxide (NO) bioavailability by preventing the reaction between NO and O2•- and peroxynitrite (ONOO-) formation. During the past several years, our lab has demonstrated that expression of ecSOD is subject to a substantial degree of regulation at the transcriptional and posttranscriptional levels in cardiovascular diseases and by various stimulants, including exercise training. Of note, there is little to no detectable expression of ecSOD in endothelial cells (ECs), but it is synthesized and secreted by vascular smooth muscle cells (VSMCs), and binds to the heparan sulfate on the EC surface and extracellular matrix through its C-terminal heparin binding domain. Using ecSOD deficient mice, we demonstrated that ecSOD plays an important role in regulating blood pressure and endothelial function by modulating the levels of O2•- in the extracellular space. Recently, we also found that Cu transport proteins, key regulator of ecSOD activity, plays a role in regulating Angiotensin II-induced hypertension, as shown below. In atherosclerosis, we demonstrated that ecSOD is highly expressed in lipid-laden macrophage as well as atherosclerotic lesions. We also found that peroxidase activity of ecSOD may play a role in atherosclerosis, because ecSOD activity is partially restored by elevating endogenous levels of uric acid, a potent inhibitor of peroxidase activity of Cu/ZnSODs in vivo and in vitro. Moreover, we recently found that both ecSOD and its regulator Cu transport protein Atox1 are markedly increased in atherosclerotic intimal lesions. Given these findings, we are studying how ecSOD plays a role in cardiovascular diseases such as hypertension, atherosclerosis as well as physiological status such as exercise training.
2. ExtracellularSOD and angiogenesis
Neovascularization is an important physiological repair mechanism in response to ischemic injury, and its process is dependent on reactive oxygen species (ROS). Overproduction of O2•- rather contributes to various cardiovascular diseases. We have demonstrated that ecSOD in bone marrow and tissues induced by hindlimb ischemia may represent an important compensatory mechanism that blunts the overproduction of O2•- , which may contribute to reparative neovascularization in response to ischemic injury. In collaboration with Dr. Ushio-Fukai’s lab, we examined molecular mechanisms by which ecSOD regulates post-ischemic angiogenesis, and found that ecSOD-derived H2O2 in caveolae/lipid rafts promotes VEGF receptor2-mediaed signaling linked to angiogenesis in endothelial cells.
3. Extracellular SOD and extracellular matrix
Although heparan sulfate proteoglycan is an important ligand for ecSOD in the extracellular matrix, little is known about other biological partners of ecSOD. Using yeast two-hybrid system, we recently discovered fibulin-5 as a novel binding protein for ecSOD. We have demonstrated that: ecSOD-fibulin-5 interaction is required for ecSOD binding to vascular tissues, thereby regulating vascular O2•- levels. This interaction may represent a novel mechanism for controlling vascular redox state in the extracellular space in various cardiovascular diseases such as atherosclerosis and hypertension in which oxidative stress is increased.
Cu transport proteins and Cardiovascular disease
1. Cu transport proteins, Extracellular SOD, and Hypertension
physiological and pathological processes in eukaryocytes; however, it is also toxic in excess. Thus, Cu homeostasis is tightly controlled by various Cu transport proteins. Our laboratory provided the first evidence that ecSOD requires Cu transport proteins (i.e. Atox1 and ATP7A) to obtain its full activity. Furthermore, we demonstrated that Cu transporters play an important role in regulating oxidative stress-dependent hypertension and endothelial function by regulating ecSOD activity. Recently, we showed that decrease in ATP7A protein expression in blood vessels of type1 diabetes contributes to decrease in ecSOD activity, resulting in O2•- overproduction and endothelial dysfunction.
Cu, an essential trace element, serves as a cofactor of key metabolic and redox enzymes that regulate
2. Cu transport proteins, vascular remodeling, and other vascular diseases
lipid rafts at the leading edge as well as regulating Cu-containing lysyl oxidase (LOX) activity. This may contribute to neointimal formation after vascular injury. Using mice lacking Atox1, we found that Atox1 is involved in neointimal formation after vascular injury through promoting VSMC migration and inflammatory cell recruitment in injured vessels. We also demonstrated that Atox1 functions as a Cu-dependent transcription factor for Cyclin D1 to promote proliferation. Based on these findings, our lab extensively investigates a role of Cu transport proteins in oxidative stress- and inflammation-dependent cardiovascular disease.
Cu has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. We recently found that ATP7A plays an important role in Cu-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to