Wood smoke extract induces oxidative stress-mediated caspase-independent apoptosis in human lung endothelial cells: role of AIF and EndoG

Although a link between toxic smoke and oxidant lung vascular injury has been indicated, the cellular mechanisms of smoke-induced injury to lung endothelial cells are unknown. We investigated oxidative stress and apoptosis induced by wood smoke extract (SE) in human pulmonary artery endothelial cells (HPAECs) and delineated their relationship. We found that SE increased intracellular reactive oxygen species (ROS), depleted intracellular glutathione, and upregulated Cu/Zn superoxide dismutase and heme oxygenase-1 (2 antioxidant enzymes), but it failed to alter the expression of catalase and glutathione peroxidase. In addition, SE promoted apoptosis as indicated by the external exposure of membrane phosphatidylserine, the loss of mitochondrial membrane potential, an increase in the level of Bax (a proapoptotic protein), and enhanced DNA fragmentation. This apoptosis was associated with mitochondrial-to-nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) (2 apoptogenic proteins) but was independent of caspase cascade activation. Whereas N-acetylcysteine (an antioxidant) effectively reversed the SE-induced increase in ROS and depletion of glutathione, it also suppressed SE-induced nuclear translocation of either AIF or EndoG and prevented the enhanced DNA fragmentation that would have resulted from this. We conclude that 1) although SE upregulates Cu/Zn superoxide dismutase and heme oxygenase-1, it nevertheless increases intracellular oxidative stress in HPAECs, and 2) SE promotes oxidative stress-mediated caspase-independent HPAEC apoptosis that involves mitochondrial-to-nuclear translocation of AIF and EndoG. Thus modulations of the expression of antioxidant enzymes and the caspase-independent apoptotic pathway are possible target choices for potential therapeutic regimes to treat smoke-induced lung injury.     

Contribution of HDL-apolipoproteins to the inhibition of low density lipoprotein oxidation and lipid accumulation in macrophages

High-density lipoprotein (HDL) is known as a protective factor against atherosclerosis. However, whether HDL-apolipoproteins (apo-HDL) contribute to the protection in arterial cells remains unclear. The localization patterns of human apolipoproteins in atherosclerotic arteries were determined using immunohistochemical examination. The results indicate that several apolipoproteins are retained in component cells of the coronary artery walls. To elucidate the possible roles of apo-HDL in the protection of atherosclerotic lesion formation, we investigated the effects of apo-HDL on the formation of conjugated diene (CD) in a cell-free system and thiobarbituric acid-reactive substances (TBARS) in the medium of a macrophage-mediated LDL oxidation system. The results showed that apo-HDL significantly exerted an inhibitory effect on LDL lipid oxidation in vitro. In addition, apo-HDL decreased cholesterol influx but enhanced cholesterol efflux from J774 macrophages in a dose-dependent manner. These results are consistent with the notion that there is reduced intracellular lipid accumulation in apo-HDL treated macrophages. These data provide a direct evidence for apo-HDL in protecting LDL from oxidative modification and in reducing the accumulation of cholesterol and lipid droplets by J774 macrophages.     

Effects of Ginkgo biloba extract on the proliferation of vascular smooth muscle cells in vitro and on intimal thickening and interleukin-1beta expression after balloon injury in cholesterol-fed rabbits in vivo

Restenosis may develop in response to cytokine activation and smooth muscle cell proliferation. Ginkgo biloba extract (EGb) has been used to treat cardiovascular and cerebrovascular diseases. In the present study, the effects of EGb on the growth of cultured vascular smooth muscle cells (VSMC), as well as on the expression of interleukin-1beta (IL-1beta) and the intimal response in balloon-injured arteries of cholesterol-fed rabbits, were investigated. Using bromodeoxyuridine incorporation as an index of cell proliferation, EGb was found to inhibit serum-induced mitogenesis of cultured rat aorta VSMC in a dose-dependent manner. In vivo, EGb and probucol ( positive control) reduced the atheroma area in thoracic aortas of male New Zealand white rabbits fed a 2% cholesterol diet for 6 weeks with balloon denudation of the abdominal aorta being performed at the end of the third week. Intimal hyperplasia, expressed as the intimal/medial area ratio, in the abdominal aortas was significantly inhibited in the both the EGb group (0.61 +/- 0.06) and the probucol group (0.55 +/- 0.03) compared to the C group (0.87 +/- 0.02). In the balloon-injured abdominal aorta, both EGb and probucol significantly reduced IL-1beta mRNA and protein expression and the percentage of proliferating cells. The inhibitory effects of EGb on the intimal response might be attributed to its antioxidant capacity. EGb may have therapeutic potential for the prevention of restenosis after angioplasty.     

miR-26a attenuates cardiac apoptosis and fibrosis by targeting ataxia–telangiectasia mutated in myocardial infarction

Apoptosis and fibrosis play a vital role in myocardial infarction (MI) induced tissue injury. Although microRNAs have been the focus of many studies on cardiac apoptosis and fibrosis in MI, the detailed effects of miR-26a is needed to further understood. The present study demonstrated that miR-26a was downregulated in ST-elevation MI (STEMI) patients and oxygen-glucose deprivation (OGD)-treated H9c2 cells. Downregulation of miR-26a was closely correlated with the increased expression of creatine kinase, creatine kinase-MB and troponin I in STEMI patients. Further analysis identified that ataxia–telangiectasia mutated (ATM) was a target gene for miR-26a based on a bioinformatics analysis. miR-26a overexpression effectively reduced ATM expression, apoptosis, and apoptosis-related proteins in OGD-treated H9c2 cells. In a mouse model of MI, the expression of miR-26a was significantly decreased in the infarct zone of the heart, whereas apoptosis and ATM expression were increased. miR-26a overexpression effectively reduced ATM expression and cardiac apoptosis at Day 1 after MI. Furthermore, we demonstrated that overexpression of miR-26a improved cardiac function and reduced cardiac fibrosis by the reduced expression of collagen type I and connective tissue growth factor (CTGF) in mice at Day 14 after MI. Overexpression of miR-26a or ATM knockdown decreased collagen I and CTGF expression in cultured OGD-treated cardiomyocytes. Taken together, these data demonstrate a prominent role for miR-26a in linking ATM expression to ischemia-induced apoptosis and fibrosis, key features of MI progression. miR-26a reduced MI development by affecting ATM expression and could be targeted in the treatment of MI.     

Viscolin reduces VCAM-1 expression in TNF-α-treated endothelial cells via the JNK/NF-κB and ROS pathway

Viscolin, a major active component in a chloroform extract of Viscum coloratum, has antioxidative and anti-inflammatory properties. We focused on its effects on the expression of vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor-α (TNF-α)-treated human umbilical vein endothelial cells (HUVECs). The TNF-α-induced expression of VCAM-1 was significantly reduced by respectively 38 ± 7 or 34 ± 16% when HUVECs were pretreated with 10 or 30 μM viscolin, as shown by Western blotting, and was also significantly reduced by pretreatment with the antioxidants N-acetylcysteine, diphenylene iodonium chloride, and apocynin. Viscolin also reduced TNF-α-induced VCAM-1 mRNA expression and promoter activity, decreased reactive oxygen species (ROS) production, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and significantly reduced the binding of monocytes to TNF-α-stimulated HUVECs. The attenuation of TNF-α-induced VCAM-1 expression and cell adhesion was partly mediated by a decrease in JNK phosphorylation. Furthermore, viscolin reduced VCAM-1 expression in the aorta of TNF-α-treated mice in vivo. Taken together, these data show that viscolin inhibits TNF-α-induced JNK phosphorylation, nuclear translocation of NF-κB p65, and ROS generation and thereby suppresses VCAM-1 expression, resulting in reduced adhesion of leukocytes. These results also suggest that viscolin may prevent the development of atherosclerosis and inflammatory responses.     

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo-E-deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP-2 and MMP-9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo-E-deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP-2 and MMP-9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP-2 and barely detectable levels for MMP-9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS-induced cell migration through the inactivation of MMP-2 and MMP-9 protein synthesis as well as the downregulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These results demonstrate that Sal B has anti-migration properties on smooth muscle cells and may explain its anti-atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.     

18beta-glycyrrhetinic acid promotes src interaction with connexin43 in rat cardiomyocytes

The mechanism by which 18beta-glycyrrhetinic acid regulates gap junction intercellular communication (GJIC) remains poorly understood. In this study, treatment of cultured rat neonatal cardiomyocytes with 18beta-glycyrrhetinic acid resulted in dose-dependent inhibition of GJIC as assessed by fluorescent dye transfer analysis. 18beta-Glycyrrhetinic acid induced time-dependent serine/threonine dephosphorylation and redistribution of connexin43 (Cx43) in cardiomyocytes and the induced Cx43 dephosphorylation was prevented by the protein phosphatase inhibitor, calyculin A. However, functional analyses showed that the inhibitory effect of 18beta-glycyrrhetinic acid on dye spreading among cardiomyocytes was not blocked by calyculin A, but was blocked by the Src-selective tyrosine kinase inhibitor, PP2. 18beta-Glycyrrhetinic acid also induced an increase in the levels of phosphorylated Src, and this effect was prevented by PP2. Immunoprecipitation using anti-Cx43 and anti-p-Src antibodies showed that 18beta-glycyrrhetinic acid increased the association between p-Src and Cx43 and induced tyrosine phosphorylation of Cx43. We conclude that the inhibitory effect of 18beta-glycyrrhetinic acid on GJIC in cardiomyocytes involves Src-mediated tyrosine phosphorylation of Cx43.     

Ganoderma lucidum polysaccharides prevent platelet-derived growth factor-stimulated smooth muscle cell proliferation in vitro and neointimal hyperplasia in the endothelial-denuded artery in vivo

Ganoderma lucidum is used in traditional Chinese medicine to prevent or treat a variety of diseases, including cardiovascular disorders. We previously demonstrated that a glucan‐containing extract of Reishi polysaccharides (EORP) has the potent anti‐inflammatory action of reducing ICAM‐1 expression in lipopolysaccharide (LPS)‐treated human aortic smooth muscle cells (HASMCs) and LPS‐treated mice. In the present study, we examined whether EORP inhibited platelet‐derived growth factor‐BB (PDGF)‐stimulated HASMC proliferation and the mechanism involved. EORP dose‐dependently reduced cell numbers and DNA synthesis of PDGF‐treated HASMCs in vitro. EORP also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21^Cip1 and upregulation of the cyclin‐dependent kinase inhibitor p27^Kip1. The anti‐proliferative effect of EORP was partly mediated by downregulation of PDGF‐induced JNK phosphorylation. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial‐denuded and the mice were fed a diet containing 100 mg/kg/day of EORP. On day 14, both cell proliferation (proliferating cell nuclear antigen‐positive cells) in the neointima and the neointima/media area ratio (0.67 ± 0.03 vs. 1.46 ± 0.30) were significantly reduced. Our data show that EORP interferes with the mitogenic activation of JNK, preventing entry of HASMCs into the cell cycle in vitro and reducing cell proliferation in the neointima and decreasing the neointimal area in vivo. Thus, EORP may represent a safe and effective novel approach to the prevention and treatment of vascular proliferative diseases. J. Cell. Physiol. 227: 3063–3071, 2012. © 2011 Wiley Periodicals, Inc.