Rio Shiraki

Possible Role of Brain-Derived Neurotrophic Factor in the Pathogenesis of Coronary Artery Disease

Background— The neurotrophin (NT) family, including nerve growth factor NT-3 and brain-derived neurotrophic factor (BDNF), has a critical role in the survival, growth, maintenance, and death of central and peripheral neurons. NTs and their receptors are expressed in atherosclerotic lesions; however, their significance in cardiovascular disease remains unclear. Methods and Results— To clarify the role of NTs in the pathogenesis of coronary artery disease, NT plasma levels in the aorta, coronary sinus, and peripheral veins of patients with unstable angina (n=38), stable effort angina (n=45), and non–coronary artery disease (n=24) were examined. In addition, regional expression of BDNF in coronary arteries was examined in autopsy cases and patients with angina pectoris by directional coronary atherectomy. The difference in BDNF levels, but not NT-3, between the coronary sinus and aorta was significantly greater in the unstable angina group compared with the stable effort angina and non–coronary artery disease groups. Immunohistochemical investigations demonstrated BDNF expression in the atheromatous intima and adventitia in atherosclerotic coronary arteries. BDNF expression was enhanced in macrophages and smooth muscle cells in atherosclerotic coronary arteries. Stimulation with recombinant BDNF significantly enhanced NAD(P)H oxidase activity and the generation of reactive oxygen species in cultured human coronary artery smooth muscle cells. Conclusions— BDNF has an important role in atherogenesis and plaque instability via the activation of NAD(P)H oxidase.

Enhanced expression of TLR4 in smooth muscle cells in human atherosclerotic coronary arteries

Toll-like receptors (TLRs) play an essential role in innate immunity as components of the primary defense system against microbial infections. It has become evident that TLRs are also involved in the pathogenesis of various cardiovascular diseases. However, the expression patterns of TLRs in the human coronary arteries of coronary artery disease (CAD) patients and the regulatory mechanisms of their expression remain unknown. The TLR4 expression patterns were invstigated by immunohistochemical analysis of coronary specimens obtained from autopsy cases or CAD patients by using directional coronary atherectomy. In atherosclerotic coronary arteries (n = 8), TLR4 immunoreactivity was colocalized with infiltrating inflammatory cells. Interestingly, vascular smooth muscle cells of atherosclerotic coronary arteries intensely expressed TLR4 even in the regions that had few inflammatory cells. In contrast, TLR4 expression was barely detected in the vascular smooth muscle cells of nonatherosclerotic coronary arteries (n = 4). Furthermore, intense expression of smooth muscle TLR4 was observed in the coronary arteries of CAD patients (n = 52). Stimulation with tumor necrosis factor α and angiotensin II increased the expression of TLR4 mRNA in cultured human vascular smooth muscle cells. Candesartan, an antagonist of the angiotensin II type 1 receptor (AT1), and N-acetylcystine inhibited angiotensin II-induced TLR4 mRNA expression in these cells. These findings suggest that the vascular smooth muscle cells of atherosclerotic coronary arteries may be activated to express TLR4. Furthermore, proinflammatory cytokines and oxidative stress in the inflammatory lesions might contribute to the enhanced expression of TLR4 in vascular smooth muscle cells of atherosclerotic arteries.

Local Overexpression of Toll-Like Receptors at the Vessel Wall Induces Atherosclerotic Lesion Formation Synergism of TLR2 and TLR4

Objective—Atherosclerosis is now considered as a chronic inflammatory disease, and inflammation is closely related to immune systems, which consist of innate-immunity and adaptive-immunity. Recently, toll-like receptors (TLRs) have been identified as key components of innate-immunity. We examined the role of local expressions of TLRs at the vessel wall in atherosclerosis. Methods and Results—We transfected cDNA encoding human TLR2 and TLR4 into the carotid arterial vessel wall of rabbits fed high-cholesterol diets with the use of HVJ-liposome. The rabbits were transfected with (1) pCMV-&bgr;-gal, (2) empty vector, (3) TLR2, (4) TLR4, (5) TLR2+4. X-gal staining and immunohistochemical analysis showed that the transfected plasmids were mainly expressed in the media. Neither TLR2 nor TLR4 transfection induced significant augmentation of atherosclerosis. Transfection of TLR2- and TLR4-containing HVJ synergistically accelerated atherosclerosis and increased expressions of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and MCP-1. Moreover, transfection of TLR2 and TLR4 resulted in synergistic activation of NF-&kgr;B at the vessel wall in vivo, and in vascular smooth muscle cells in vitro. Conclusions—Expressions of both TLR2 and TLR4 at the vessel wall synergistically accelerated atherosclerosis. The present study revealed the role of TLRs expressed locally at the vessel wall in the early stage of atherosclerosis.

Endothelial Urocortin Has Potent Antioxidative Properties and Is Upregulated by Inflammatory Cytokines and Pitavastatin

Background: Urocortin, a neuropeptide discovered in the midbrain, is a member of the corticotropin-releasing factor family and is expressed in heart tissues. Urocortin exerts potent cardioprotective effects under various pathological conditions including ischemia/reperfusion. However, the regulation and function of vascular urocortin are unknown. Methods and Results: Immunohistochemistry showed definitive expression of urocortin in endothelial cells of coronary large arteries and microvessels from autopsied hearts. RT-PCR confirmed the expression of urocortin in human umbilical vein endothelial cells (HUVECs). Urocortin (10–8M) potently suppressed the generation of angiotensin II-induced reactive oxygen species (ROS) in HUVECs. Tumor necrosis factor-α and interferon-γ increased the urocortin mRNA levels and its release from HUVECs. Incubation with pitavastatin (0.1–3.0 µM) significantly increased the urocortin mRNA levels and its release from HUVECs. Furthermore, treatment with pitavastatin (2 mg/day) for 4 weeks increased the serum urocortin level from 11.0 ± 6.5 to 16.4 ± 7.3 ng/ml in healthy volunteers. Conclusion: Endothelial urocortin was upregulated by inflammatory cytokines and pitavastatin and suppressed ROS production in endothelial cells. Treatment with pitavastatin increased the serum urocortin level in human subjects. Thus, endothelial urocortin might protect cardiomyocytes in inflammatory lesions. Urocortin might partly explain the mechanisms of various pleiotropic effects of statins.

Augmentation of Vascular Remodeling by Uncoupled Endothelial Nitric Oxide Synthase in a Mouse Model of Diabetes Mellitus

Objective—Diabetes mellitus is associated with increased oxidative stress, which induces oxidation of tetrahydrobiopterin (BH4) in vessel wall. Without enough BH4, eNOS is uncoupled to L-arginine and produces superoxide rather than NO. We examined the role of uncoupled eNOS in vascular remodeling in diabetes. Methods and Results—Diabetes mellitus was produced by streptozotocin in C57BL/6J mice. Under stable hyperglycemia, the common carotid artery was ligated, and neointimal formation was examined 4 weeks later. In diabetic mice, the neointimal area was dramatically augmented. This augmentation was associated with increased aortic superoxide formation, reduced aortic BH4/dihydrobiopterin (BH2) ratio, and decreased plasma nitrite and nitrate (NOx) levels compared with nondiabetic mice. Chronic BH4 treatment (10 mg/kg/d) reduced the neointimal area in association with suppressed superoxide production and inflammatory changes in vessels. BH4/BH2 ratio in vessel wall was preserved, and plasma NOx levels increased. Furthermore, in the presence of diabetes, overexpression of bovine eNOS resulted in augmentation of neointimal area, accompanied by increased superoxide production in the endothelium. Conclusions—In diabetes, increased oxidative stress by uncoupled NOSs, particularly eNOS, causes augmentation of vascular remodeling. These findings indicate restoration of eNOS coupling has an atheroprotective benefit in diabetes.

Essential Role of NOXA1 in Generation of Reactive Oxygen Species Induced by Oxidized Low-Density Lipoprotein in Human Vascular Endothelial Cells

Oxidative stress induced by superoxide plays an important role in pathogenesis of cardiovascular diseases. NAD(P)H oxidase is a principal enzymatic origin for superoxide in vasculature. Recently, novel homologues of cytosolic components of NAD(P)H oxidase, Nox organizer 1 (NOXO1) and Nox activator 1 (NOXA1), are identified. On the other hand, oxidized low-density lipoprotein (ox-LDL) generates reactive oxygen species (ROS) in endothelial cells via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). In the present investigation, the authors examined the expression, the regulation, and the role of NOXA1 in the generation of ROS in endothelial cells. The expression of NOXA1 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). Dihydroethidium method showed that ox-LDL and angiotensin II increased the generation of intracellular ROS. Once the expression of p22(phox) or NOXA1 was suppressed by siRNA, the generation of ROS induced by ox-LDL and angiotensin II were potently decreased. Moreover, the expression of NOXA1 was increased by ox-LDL in a time-and dose-dependent manner. In conclusion, endothelial NOXA1 plays an essential role in generation of ROS. Ox-LDL not only increased the generation of ROS via LOX-1, but also enhanced the expression of NOXA1 in endothelial cells. NOXA1 is likely a key player that links ox-LDL with the activation of endothelial NAD(P)H oxidase.