Impact of NAD(P)H oxidase-derived reactive oxygen species on coronary arterial remodeling: a comparative intravascular ultrasound and histochemical analysis of atherosclerotic lesions.

BACKGROUND: Coronary arterial remodeling, which is a response to the growth of atherosclerotic plaques, is associated with plaque vulnerability. Oxidative stress induced by reactive oxygen species (ROS) via NAD(P)H oxidase in the vasculature also plays a crucial role in the pathogenesis of atherosclerosis-based cardiovascular disease. In this study, the relationship between coronary arterial remodeling and ROS generation was examined by comparing preinterventional intravascular ultrasound findings of atherosclerotic lesions to the histochemical findings of corresponding specimens obtained by directional coronary atherectomy. METHODS AND RESULTS: Predirectional coronary atherectomy intravascular ultrasound images of 49 patients were analyzed. The remodeling index was calculated by dividing the target-lesion external elastic membrane cross-sectional area by the reference-segment external elastic membrane cross-sectional area. Expansive remodeling was defined as a remodeling index of >1.0. ROS generation and NAD(P)H oxidase p22(phox) expression in directional coronary atherectomy specimens were evaluated using the dihydroethidium staining method and immunohistochemistry as the ratio of the positive area to the total surface area in each specimen, respectively. ROS generation and p22(phox) expression were significantly greater in lesions with expansive remodeling than in lesions without remodeling (0.18+/-0.12 versus 0.03+/-0.02, P<0.0001, 0.10+/-0.08 versus 0.04+/-0.05, P=0.0039, respectively). Both ROS generation and p22(phox) expression significantly correlated with the intravascular ultrasound-derived remodeling index (r=0.77, P<0.0001, r=0.53, P<0.0001, respectively). CONCLUSIONS: Simultaneous examination with intravascular ultrasound and immunohistochemistry analyses suggests that NAD(P)H oxidase-derived ROS is related to the coronary arterial remodeling process associated with plaque vulnerability.

Immunohistochemical localization of endothelial cell-derived lipase in atherosclerotic human coronary arteries.

OBJECTIVE: A novel lipoprotein lipase (LPL)-like gene, endothelial cell-derived lipase (EDL), was recently cloned from vascular endothelial cells. The presence of LPL in the vascular wall has been implicated in the progression of atherosclerosis through the bridging function between lipoprotein particles and matrix proteoglycans to enhance lipoprotein uptake into the vascular wall. The aim of this study was to investigate the local expression of EDL in human coronary arteries. METHODS AND RESULTS: Human coronary arterial specimens from 10 autopsied cases were examined by immunohistochemistry with polyclonal antibodies against specific synthetic EDL peptides. Immunohistochemical analysis revealed that EDL was expressed in endothelial cells and medial smooth muscle cells in non-atherosclerotic coronary arteries. In addition, EDL was expressed in infiltrating cells within atheromatous plaques as well as endothelial and smooth muscle cells. Double labeling immunofluorescence confirmed EDL positive-cells were endothelial cells, smooth muscle cells and macrophages. EDL immunoreactivity was also detected in neovasculature within atheromatous plaques in atherosclerotic coronary arteries. CONCLUSIONS: These results suggest that EDL may have unique functional roles in the pathogenesis of coronary artery diseases such as atherosclerosis as well as in lipid metabolism in the vessel wall.

Interaction of oxidative stress and inflammatory response in coronary plaque instability: important role of C-reactive protein.

OBJECTIVE: C-reactive protein (CRP), a predictor of cardiovascular events, localizes in atherosclerotic arteries and exerts proinflammatory effects on vascular cells. Reactive oxygen species (ROS) have been implicated in atherogenesis and plaque instability. METHODS AND RESULTS: Expressional pattern of CRP in directional coronary atherectomy specimens from 39 patients was examined. Characteristics of histological plaque instability and higher levels of serum CRP and fibrinogen were associated with the CRP immunoreactivity. In situ hybridization revealed the presence of CRP mRNA in coronary vasculature. Furthermore, the expression of CRP mRNA and protein was detected in cultured human coronary artery smooth muscle cells (CASMCs) by reverse transcriptase-polymerase chain reaction and Western blotting. In addition, CRP was frequently colocalized with p22phox, an essential component of NADH/NADPH oxidase, which is an important source of ROS in vasculature. Moreover, the incubation of cultured CASMCs with CRP resulted in the enhanced p22phox protein expression and in the generation of intracellular ROS. CONCLUSIONS: The expression of CRP in coronary arteries was associated with histological and clinical features of vulnerable plaque, and it had a prooxidative effect on cultured CASMCs, suggesting that it might play a crucial role in plaque instability and in the pathogenesis of acute coronary syndrome via its prooxidative effect.

Superoxide generation in directional coronary atherectomy specimens of patients with angina pectoris: important role of NAD(P)H oxidase.

OBJECTIVE: NADH/NADPH oxidase is an important source of reactive oxygen species (ROS) in the vasculature. Recently, we demonstrated that p22(phox), an essential component of this oxidase, was expressed in human coronary arteries and that its expression was enhanced with the progression of atherosclerosis. The present study was undertaken to investigate its functional importance in the pathogenesis of coronary artery disease. For this aim, the expression of p22(phox), the distribution of oxidized low density lipoprotein (LDL), and the generation of ROS in directional coronary atherectomy (DCA) specimens were examined. METHODS AND RESULTS: DCA specimens were obtained from patients with stable or unstable angina pectoris. The distribution of p22(phox) and of oxidized LDL was examined by immunohistochemistry. The generation of superoxide in DCA specimens was assessed by the dihydroethidium method and lucigenin-enhanced chemiluminescence. ROS were closely associated with the distribution of p22(phox) and oxidized LDL. Not only inflammatory cells but also smooth muscle cells and fibroblasts generated ROS. There was a correlation between ROS and the expression of p22(phox) or oxidized LDL. The generation of ROS was significantly higher in unstable angina pectoris compared with stable angina pectoris. CONCLUSIONS: ROS generated by p22(phox)-based NADH/NADPH oxidase likely mediate the oxidative modification of LDL and might play a major role in pathogenesis of atherosclerotic coronary artery disease.

Heterogeneous loss of connexin43 protein in nonischemic dilated cardiomyopathy with ventricular tachycardia.

INTRODUCTION: Gap junction alterations recently have been implicated in chronic heart failure, but direct evidence between gap junction manifestation in nonischemic dilated cardiomyopathy (DCM) is lacking. The current study examines whether qualitative changes or altered distribution of gap junctional connexin43 (Cx43) are related to global ventricular function and ventricular arrhythmia in DCM. METHODS AND RESULTS: We investigated 31 DCM patients (52 +/- 15 years) and 5 control subjects (55 +/- 10 years). Expression of Cx43 proteins was qualitatively and quantitatively determined using immunoconfocal microscopy in right ventricular biopsy specimens from each patient. The expression level of Cx43 protein was defined as the proportion of tissue area occupied by Cx43 (percent tissue area) in each test area. Cx43 immunoreactive signal expressed as percent tissue area was not correlated with the change in left ventricular ejection fraction (P = 0.17). Of 31 DCM patients, 23% subsequently developed sustained ventricular tachycardia (VT), which allowed retrospective division of the samples into two groups: non-VT and VT. Left ventricular ejection fraction was comparable in both groups, but the percent tissue area in the VT groups was significantly decreased compared with that of the non-VT groups (P = 0.03). Furthermore, Cx43 protein was distributed heterogeneously in the VT groups (P < 0.0001). CONCLUSION: Heterogeneous reduction of Cx43 protein may result in development of malignant ventricular arrhythmia in DCM.

Expressional changes of the vascular antioxidant system in atherosclerotic coronary arteries.

Oxidative stress induced by reactive oxygen species (ROS) plays an important role in atherogenesis, and the redox state is determined by the balance between antioxidants and the ROS generating system. To defend against enhanced ROS, mammalian cells have a complex network of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase. To clarify the role of the vascular antioxidant system, we investigated by immunohistochemistry the expressional changes of antioxidative enzymes in coronary arteries obtained from autopsied cases. In nonatherosclerotic coronary arteries, Cu/Zn SOD and Mn SOD were expressed in medial smooth muscle cells (SMC), whereas cytosolic GPx (GPx-1) was expressed mainly in endothelium and weakly in medial SMC. Catalase was expressed in medial SMC and endothelium. Progression of atherosclerosis did not result in an additional increase in the expression of antioxidative enzymes in SMC in the media or endothelium. However, migrating SMC and macrophages in atheromatous plaques expressed these four antioxidative enzymes intensively. Double staining with cell markers confirmed the cell-specific expression of the antioxidative enzymes. Thus, the expressional pattern showed regional heterogeneity. In response to oxidative stress, the vascular antioxidant system was upregulated in atherosclerotic lesions. The imbalance between vascular antioxidant and oxidant systems might play an important role in coronary atherogenesis.

Expression of tissue transglutaminase and elafin in human coronary artery: implication for plaque instability.

BACKGROUND: the extracellular matrix (ECM) is an important determinant of plaque instability. Since tissue transglutaminase (tTG) and elafin act as stabilizing factors, they might play a crucial role in the pathogenesis of acute coronary syndrome. We examined their expression in human coronary arteries and the regulation of tTG expression in cultured vascular smooth muscle cells (SMCs). METHODS AND RESULTS: immunohistochemical studies on autopsy samples of human coronary arteries revealed the expression of tTG and elafin in the endothelium, medial SMCs, and the ECM in non-atherosclerotic coronary arteries. Their expression in SMCs, endothelium, and ECM was enhanced in atherosclerotic coronary arteries. In contrast, they were hardly detectable in accumulating macrophages or at the lipid core. Double staining demonstrated that elafin was co-localized with tTG. Moreover, some tTG-expressing cells were positive for TNF-alpha, suggesting that this cytokine might play an important role in the regulation of tTG. Treatment of cultured rat aortic SMCs with TNF-alpha increased their tTG mRNA, protein expression and enzyme activity. CONCLUSIONS: the expression of tTG and elafin increased in atherosclerotic coronary arteries. The investigation with cultured SMCs suggested that TNF-alpha might mediate the upregulation of tTG. Our findings may provide new insights into the mechanism of plaque instability and the pathogenesis of acute coronary syndrome.

Cardiac myocytes are recruited by bone marrow-derived cells in intact murine heart.

It is generally believed that the cardiac myocytes withdraw from the cell cycle shortly after birth and thereafter any loss of myocardial tissue cannot be repaired. However, recent reports indicate that cardiac myocytes can be regenerated by stem cells derived from bone marrow in the damaged hearts. In this study, we investigated whether bone marrow-derived cells can differentiate into cardiac myocytes in the intact hearts. We performed bone marrow transplantation from syngenic male mice to female c57/B6 mice. In female mice's hearts, the presence of cells from male mice was examined by FISH method that detects Y chromosome. Using the same samples, we also performed immunohistochemical staining with muscle specific antibodies. In the heart sections of female mice, there were some cells that were considered as differentiated myocytes derived from male bone marrow (0.01~0.09% of total myocytes) and the proportion of the cells increased as the period after bone marrow transplantation became longer (3 months after vs. 8 months after). These results suggest that, not only in the damaged heart but also in the intact heart, a portion of cardiac myocytes is recruited by bone marrow-derived cells.