Lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1, also known as OLR-1), is a class E scavenger receptor that mediates the uptake of oxLDL by vascular cells. LOX-1 is involved in endothelial dysfunction, monocyte adhesion, the proliferation, migration, and apoptosis of smooth muscle cells, foam cell formation, platelet activation, as well as plaque instability; all of these events are critical in the pathogenesis of atherosclerosis. These LOX-1-dependent biological processes contribute to plaque instability and the ultimate clinical sequelae of plaque rupture and life-threatening tissue ischemia. Administration of anti-LOX-1 antibodies inhibits atherosclerosis by decreasing these cellular events. Over the past decade, multiple drugs including naturally occurring antioxidants, statins, antiinflammatory agents, antihypertensive and antihyperglycemic drugs have been demonstrated to inhibit vascular LOX-1 expression and activity. Therefore, LOX-1 represents an attractive therapeutic target for the treatment of human atherosclerotic diseases. This review aims to integrate the current understanding of LOX-1 signaling, regulation of LOX-1 by vasculoprotective drugs, and the importance of LOX-1 in the pathogenesis of atherosclerosis.
Atherosclerosis; Oxidized LDL; LOX-1; Soluble LOX-1; Review
Rapamycin, also known as sirolimus, is an immunosuppressant drug used to prevent rejection organ (especially kidney) transplantation. However, little is known about the role of Rapa in cardiac hypertrophy induced by isoproterenol and its underlying mechanism. In this study, Rapa was administrated intraperitoneally for one week after the rat model of cardiac hypertrophy induced by isoproterenol established. Rapa was demonstrated to attenuate isoproterenol-induced cardiac hypertrophy, maintain the structure integrity and functional performance of mitochondria, and upregulate genes related to fatty acid metabolism in hypertrophied hearts. To further study the implication of NF-κB in the protective role of Rapa, cardiomyocytes were pretreated with TNF-α or transfected with siRNA against NF-κB/p65 subunit. It was revealed that the upregulation of extracellular circulating proinflammatory cytokines induced by isoproterenol was able to be reversed by Rapa, which was dependent on NF-κB pathway. Furthermore, the regression of cardiac hypertrophy and maintaining energy homeostasis by Rapa in cardiomyocytes may be attributed to the inactivation of NF-κB. Our results shed new light on mechanisms underlying the protective role of Rapa against cardiac hypertrophy induced by isoproterenol, suggesting that blocking proinflammatory response by Rapa might contribute to the maintenance of energy homeostasis during the progression of cardiac hypertrophy.
Cell surface localization and intracellular trafficking of ATP-binding cassette transporter A-1 (ABCA1) are essential for its function. However, regulation of these activities is still largely unknown. Brefeldin A (BFA), a uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism.
Methods and Results
By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells and inhibited by 60% cholesterol release. In contrast, BIG1 over-expression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through over-expression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 siRNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1 (ARF1).
BIG1, through its ability to activate ARF1, regulates cell surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action.
BIG1; ABCA1; trafficking; cholesterol efflux
Nuclear factor kappa-B (NF-κB) signalling plays an important role in diabetic nephropathy. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. The aim of the current study was to investigate the role of Cx43 in the activation of NF-κB induced by high glucose in glomerular mesangial cells (GMCs) and to determine whether c-Src is involved in this process.
We found that downregulation of Cx43 expression induced by high glucose activated NF-κB in GMCs. Orverexpression of Cx43 attenuated NF-κB p65 nuclear translocation induced by high glucose. High glucose inhibited the interaction between Cx43 and c-Src, and enhanced the interaction between c-Src and IκB-α. PP2, a c-Src inhibitor, also inhibited the tyrosine phosphorylation of IκB-α and NF-κB p65 nuclear translocation induced by high glucose. Furthermore, overexpression of Cx43 or inhibition of c-Src attenuated the upregulation of intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1) and fibronectin (FN) expression induced by high glucose.
In conclusion, downregulation of Cx43 in GMCs induced by high glucose activates c-Src, which in turn promotes interaction between c-Src and IκB-α and contributes to NF-κB activation in GMCs, leading to renal inflammation.
Connexin43; NF-κB signalling; c-Src; Diabetic nephropathy; Inflammation; Fibronectin
Peroxisome proliferator-activated receptor alpha (PPARα) has been implicated in the pathogenesis of cardiac hypertrophy, although its mechanism of action remains largely unknown. To determine the effect of PPARα activation on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore its molecular mechanisms, we evaluated the interaction of PPARα with nuclear factor of activated T-cells c4 (NFATc4) in nuclei of cardiomyocytes from neonatal rats in primary culture. In ET-1-stimulated cardiomyocytes, data from electrophoretic mobility-shift assays (EMSA) and co-immunoprecipitation (co-IP) revealed that fenofibrate (Fen), a PPARα activator, in a concentration-dependent manner, enhanced the association of NFATc4 with PPARα and decreased its interaction with GATA-4, in promoter complexes involved in activation of the rat brain natriuretic peptide (rBNP) gene. Effects of PPARα overexpression were similar to those of its activation by Fen. PPARα depletion by small interfering RNA abolished inhibitory effects of Fen on NFATc4 binding to GATA-4 and the rBNP DNA. Quantitative RT-PCR and confocal microscopy confirmed inhibitory effects of PPARα activation on elevation of rBNP mRNA levels and ET-1-induced cardiomyocyte hypertrophy. Our results suggest that activated PPARα can compete with GATA-4 binding to NFATc4, thereby decreasing transactivation of NFATc4, and interfering with ET-1 induced cardiomyocyte hypertrophy.
NFATc4; PPARα; Cardiac hypertrophy; GATA-4; Fenofibrate
A new series of phosphodiesterase-9 (PDE9) inhibitors that contain a scaffold of 6-amino-pyrazolopyrimidinone have been discovered by a combination of structure-based design and computational docking. This procedure significantly saved load of chemical synthesis and is an effective method for the discovery of inhibitors. The best compound 28 has an IC50 of 21 nM and 3.3 µM respectively for PDE9 and PDE5, and about three orders of magnitude of selectivity against other PDE families. The crystal structure of the PDE9 catalytic domain in complex with 28 has been determined and shows a hydrogen bond between 28 and Tyr424. This hydrogen bond may account for the 860-fold selectivity of 28 against PDE1B, in comparison with about 30-fold selectivity of BAY73-6691. Thus, our studies suggest that Tyr424, a unique residue of PDE8 and PDE9, is a potential target for improvement of selectivity of PDE9 inhibitors.
The accumulation of glomerular extracellular matrix (ECM) is one of the critical pathological characteristics of diabetic renal fibrosis. Fibronectin (FN) is an important constituent of ECM. Our previous studies indicate that the activation of the sphingosine kinase 1 (SphK1)-sphingosine 1- phosphate (S1P) signaling pathway plays a key regulatory role in FN production in glomerular mesangial cells (GMCs) under diabetic condition. Among the five S1P receptors, the activation of S1P2 receptor is the most abundant. Berberine (BBR) treatment also effectively inhibits SphK1 activity and S1P production in the kidneys of diabetic models, thus improving renal injury. Based on these data, we further explored whether BBR could prevent FN production in GMCs under diabetic condition via the S1P2 receptor. Here, we showed that BBR significantly down-regulated the expression of S1P2 receptor in diabetic rat kidneys and GMCs exposed to high glucose (HG) and simultaneously inhibited S1P2 receptor-mediated FN overproduction. Further, BBR also obviously suppressed the activation of NF-κB induced by HG, which was accompanied by reduced S1P2 receptor and FN expression. Taken together, our findings suggest that BBR reduces FN expression by acting on the S1P2 receptor in the mesangium under diabetic condition. The role of BBR in S1P2 receptor expression regulation could closely associate with its inhibitory effect on NF-κB activation.
We have previously synthesized a series of hybrid compounds by linking ferulic acid to tacrine as multifunctional agents based on the hypotheses that Alzheimer's disease (AD) generates cholinergic deficiency and oxidative stress. Interestingly, we found that they may have potential pharmacological activities for treating AD. Here we report for the first time that tacrine-6-ferulic acid (T6FA), one of these compounds, can prevent amyloid-β peptide (Aβ)-induced AD-associated pathological changes in vitro and in vivo. Our results showed that T6FA significantly inhibited auto- and acetylcholinesterase (AChE)-induced aggregation of Aβ1–40
in vitro and blocked the cell death induced by Aβ1–40 in PC12 cells. In an AD mouse model by the intracerebroventricular injection of Aβ1–40, T6FA significantly improved the cognitive ability along with increasing choline acetyltransferase and superoxide dismutase activity, decreasing AChE activity and malondialdehyde level. Based on our findings, we conclude that T6FA may be a promising multifunctional drug candidate for AD.
Malignant gliomas represent one of the most aggressive types of cancers and their recurrence is closely linked to acquired therapeutic resistance. A combination of chemotherapy is considered a promising therapeutic model in overcoming therapeutic resistance and enhancing treatment efficacy. Herein, we show by colony formation, Hochest 33342 and TUNEL staining, as well as by flow cytometric analysis, that LY294002, a specific phosphatidylinositide-3-kinase (PI3K) inhibitor, enhanced significantly the sensitization of a traditional cytotoxic chemotherapeutic agent, tamoxifen-induced apoptosis in C6 glioma cells. Activation of PI3K signaling pathway by IGF-1 protected U251 cells from apoptosis induced by combination treatment of LY294002 and tamoxifen. Interference of PI3K signaling pathway by PI3K subunit P85 siRNA enhanced the sensitization of U251 glioma cells to tamoxifen -induced apoptosis. By Western blotting, we found that combination treatment showed lower levels of phosphorylated AktSer473 and GSK-3βSer9 than a single treatment of LY294002. Further, we showed a significant decrease of nuclear β-catenin by combination treatment. In response to the inhibition of β-catenin signaling, mRNA and protein levels of Survivin and the other three antiapoptotic genes Bcl-2, Bcl-xL, and Mcl-1 were significantly decreased by combination treatment. Our results indicated that the synergistic cytotoxic effect of LY294002 and tamoxifen is achieved by the inhibition of GSK-3β/β-catenin signaling pathway.
Macrophage-derived foam cell formation elicited by oxidized low-density lipoprotein (oxLDL) is the hallmark of early atherogenesis. Detection of foam cell formation is conventionally practiced by Oil Red O (ORO) staining of lipid-laden macrophages. Other methods include 1,1′-dioctadecyl-3,3,3′3′-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL) uptake and Nile Red staining. The purpose of the present study is to report an optimized method for assessing foam cell formation in cultured macrophages by ORO staining and DiI-oxLDL uptake. After incubation with oxLDL (50 μg/ml) for 24 h, the macrophages were fixed, stained with ORO for just 1 min, pronounced lipid droplets were clearly observed in more than 90% of the macrophages. To test the in vivo applicability of this method, lesions (or foam cells) of cryosections of aortic sinus or primary mouse peritoneal macrophages from ApoE deficient mice fed a high cholesterol diet were successfully stained. In another set of experiments, treatment of macrophages with DiI-oxLDL (10 μg/ml) for 4 h resulted in significant increase in oxLDL uptake in macrophages as demonstrated by confocol microscopy and flow cytometry. We conclude that the optimized ORO staining and fluorescent labeled oxLDL uptake techniques are very useful for assessing intracellular lipid accumulation in macrophages that are simpler and more rapid than currently used methods.
Atherosclerosis; Macrophages; Foam cell; Oil Red O; DiI-oxLDL
Primary culture of smooth muscle cells has been widely used as a valuable tool to study the molecular mechanisms underlying atherosclerosis and restenosis. Currently, tissue explants and enzymatic digestion methods are frequently applied to produce smooth muscle cells. Explants method is time consuming, usually taking several weeks. The enzymatic digestion method requires large amounts of proteolytic enzymes to generate enough cells for cardiovascular research. The present study reports an optimized method by combining both techniques to obtain high purity smooth muscle cells. The cultured cells exhibited the characteristic “hills and valleys” growth pattern as observed by phase contrast microscopy and showed α-SM-actin positive staining by indirect immunocytochemistry and immunofluorescence. Purity of the cells is guaranteed by the lack of von Willebrand Factor immunoreactivity. Finally, the cultured cells well proliferate on oxidized-LDL stimulation, suggesting the practical utility of this new method.
Smooth muscle cell; Cell culture; Thoracic aorta; Protocol
Primary culture of cardiomyocytes has been widely used as a valuable tool for pharmacological and toxicological studies. However, the fact that heart is a solid organ and cardiomyocytes do not proliferate after birth makes the primary myocardial culture a tedious job. The present study reports an improved method for rapid isolation of cardiomyocytes, as well as the culture maintenance and quality assurance. The whole culture process can be shortened to 3.5 h by reducing enzyme digestion period. Moreover, the new protocol guarantees cell yield and viability, and produces more than 95% cardiomyocytes in culture. The cardiomyocytes can respond to Angiotension II stimulation with increased protein synthesis, suggesting the practical value of this new culture method.
Cardiomyocytes; Culture; Neonatal; Precondition; Protocol