Hongjiang Chen

LOX-1 Mediates Oxidized Low-Density Lipoprotein-Induced Expression of Matrix Metalloproteinases in Human Coronary Artery Endothelial Cells

Background—Oxidized LDL (ox-LDL) accumulation in the atherosclerotic region may enhance plaque instability. Both accumulation of ox-LDL and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. This study was designed to examine the role of LOX-1 in the modulation of metalloproteinases (MMP-1 and MMP-3) in human coronary artery endothelial cells (HCAECs). Methods and Results—HCAECs were incubated with ox-LDL (10 to 80 &mgr;g/mL) for 1 to 24 hours. Ox-LDL increased the expression of MMP-1 (collagenase) and MMP-3 (stromelysin-1) in a concentration- and time-dependent manner. Ox-LDL also increased collagenase activity. Ox-LDL did not significantly affect the expression of tissue inhibitors of metalloproteinases. Native LDL had no effect on the expression of MMPs. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1, because pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92, 10 &mgr;g/mL) prevented the expression of MMPs in response to ox-LDL (P <0.01). In parallel experiments, ox-LDL caused the activation of protein kinase C (PKC), which was inhibited by LOX-1 antibody. The PKC-&bgr; isoform played a critical role in the expression of MMPs, because the PKC-&bgr; inhibitor hispidin reduced ox-LDL-induced activation of PKC and the expression of MMPs. Other PKC subunits (&agr;, &ggr;, and &egr;) did not affect the expression of MMPs. Conclusions—These findings indicate that ox-LDL, via LOX-1 activation, modulates the expression and activity of MMPs in HCAECs. In this process, activation of the PKC-&bgr; subunit plays an important signaling role.

LOX-1, an Oxidized LDL Endothelial Receptor, Induces CD40/CD40L Signaling in Human Coronary Artery Endothelial Cells

Background—Despite increasing appreciation that atherogenesis involves participation of inflammatory cells, information on mediators of communication between different constituents of atherosclerotic plaque remain incomplete. We examined the role of LOX-1, a receptor for oxidized (ox) LDL, in the expression of CD40/CD40L in cultured human coronary artery endothelial cells (HCAECs). Methods and Results—We observed that ox-LDL increased the expression of CD40 and CD40L in a concentration (10 to 80 &mgr;g/mL)- and time (1 to 24 hours)- dependent manner. These effects of ox-LDL were mediated by activation of LOX-1, because pretreatment of HCAECs with a blocking antibody to LOX-1 (JTX92) prevented the expression of CD40 and CD40L in response to ox-LDL (P <0.01). In parallel experiments, HCAECs were incubated with the protein kinase C (PKC) inhibitor bisindolylmaleimide I, and the cells were then exposed to ox-LDL. Both LOX-1 antibody and the PKC inhibitor inhibited PKC activation in response to ox-LDL (P <0.01). The PKC inhibitor also blocked the effects of ox-LDL on the expression of CD40 and CD40L (P <0.01). In additional experiments, we found that it is the PKC&agr;, but not PKC&bgr; and PKC&ggr;, isoform that mediated ox-LDL–induced CD40 and CD40L upregulation. Further experiments showed that upregulation of CD40 mediated induction of proinflammatory genes, because CD40 antibody markedly reduced ox-LDL–induced TNF-&agr; generation and P-selectin expression, whereas nonspecific mouse IgG had no effect. Conclusions—These findings indicate that ox-LDL through its receptor LOX-1 triggers the CD40/CD40L signaling pathway that activates the inflammatory reaction in HCAECs. These observations provide novel insight into ox-LDL–mediated inflammation in atherosclerosis.

Statins inhibit oxidized-LDL-mediated LOX-1 expression, uptake of oxidized-LDL and reduction in PKB phosphorylation

OBJECTIVES LOX-1, a lectin-like receptor on endothelial cells, facilitates the uptake of oxidized-LDL. Expression of LOX-1 is involved in the pathobiological effects of oxidized-LDL in endothelial cells, including apoptosis, suppression of cNOS activity and cell adhesion. Recent studies show that intracellular signal protein kinase B (PKB) is involved in the regulation of cNOS. Further, HMG CoA reductase inhibitors (statins) may affect LOX-1 expression. In this study, we examined the modulation of LOX-1 expression and PKB activity in response to oxidized-LDL by two different statins (simvastatin and atorvastatin). METHODS AND RESULTS Cultured human coronary artery endothelial cells (HCAECs) were used in this study. Oxidized-LDL (40 microg/ml) was found to upregulate the expression of LOX-1 (mRNA and protein), enhance [125I]-ox-LDL uptake and to reduce the phosphorylation of PKB (p-PKB). Two different statins, simvastatin and atorvastatin (each 1 and 10 microM), upregulated the activity of PKB and decreased LOX-1 expression and [125I]-ox-LDL uptake. A high concentration of statins (10 microM) gave a more potent effect than the low concentration (1 microM). The effects of the two different statins were similar. CONCLUSIONS These observations show that statins decrease LOX-1 expression, a novel oxidized-LDL endothelial receptor, and uptake of oxidized-LDL in HCAECs. The effect of statins on LOX-1 expression is associated with an increase in PKB activity in HCAECs.

Expression of lectin-like oxidized low-density lipoprotein receptors during ischemia-reperfusion and its role in determination of apoptosis and left ventricular dysfunction

OBJECTIVES The goal of this study was to determine the role of lectin-like oxidized low-density lipoprotein receptors (LOX-1), a recently identified oxidized low-density lipoprotein (ox-LDL) receptor, in ischemia-reperfusion injury to the heart. BACKGROUND Reactive oxygen species (ROS) released during ischemia-reperfusion oxidize low-density lipoproteins; LOX-1 is upregulated by ox-LDL and ROS, and is involved in cell injury. METHODS Anesthetized rats were subjected to left coronary artery ligation for 60 min (n = 10, ischemia group), or ischemia followed by 60 min of reperfusion (n = 30, ischemia-reperfusion group). Rats in the latter group were treated with saline, the LOX-1 blocking antibody JXT21 (10 mg/kg), or nonspecific anti-goat immunoglobulin G (IgG) (10 mg/kg). Ten other rats underwent thoracotomy without coronary ligation (sham control). RESULTS Ischemia-reperfusion was associated with an increase in LOX-1 expression, lipid peroxidation and apoptosis, a large infarct area, and a decrease in left ventricular function (all, p < 0.01 vs. sham control and ischemia alone groups). Treatment of rats with LOX-1 antibody prevented ischemia-reperfusion-induced upregulation of LOX-1. Importantly, the LOX-1 antibody reduced apoptosis by 48%, lipid peroxidation by 39%, and myocardial infarct size by 45%, and improved left ventricular function (first derivative of pressure measured over time: -47% to -18%, p < 0.01). Nonspecific IgG had no effect. CONCLUSIONS Lectin-like oxidized low-density lipoprotein receptors are upregulated during myocardial ischemia-reperfusion, and appear to be associated with apoptosis, necrosis, and left ventricular functional deterioration.

EPA and DHA attenuate ox-LDL-induced expression of adhesion molecules in human coronary artery endothelial cells via protein kinase B pathway

Uptake of oxidized low-density lipoprotein (ox-LDL) by endothelial cells is a critical step for the initiation and development of atherosclerosis. Adhesion molecules are inflammatory makers, which are upregulated by ox-LDL and play a pivotal role in atherogenesis. A number of studies suggest that fish and its constituents can reduce inflammation and decrease atherosclerosis. We hypothesized that fish oil constituents namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may reduce expression of adhesion molecules induced by ox-LDL. Cultured human coronary artery endothelial cells (HCAECs) were incubated with ox-LDL for 24 h. Parallel groups of cells were pretreated with DHA or EPA (10 or 50 microM) overnight before incubation with ox-LDL. Ox-LDL markedly increased the expression of P-selectin and intracellular adhesion molecule-1 (ICAM-1) (both protein and mRNA) in HCAECs, and enhanced the adhesion of monocytes to the cultured HCAECs. Both EPA and DHA decreased ox-LDL-induced upregulation of expression of P-selectin and ICAM-1, and the enhanced adhesion of monocytes to HCAECs. To determine the role of protein kinase B (PKB) as an intracellular-signaling pathway, HCAECs were treated with the PKB upstream inhibitor wortmannin (100 nM) or transfected with plasmids encoding dominant-negative mutants of PKB (PKB-DN) before treatment with DHA. Ox-LDL alone downregulated the activity of PKB; DHA attenuated this effect of ox-LDL, and both wortmannin and PKB-DN blocked the effect of DHA. The present study in human coronary endothelial cells suggests that both EPA and DHA attenuate ox-LDL-induced expression of adhesion molecules, and the adhesion of monocytes to HCAECs by modulation of PKB activation. These effects may be important mechanisms of anti-atherosclerotic effects of fish and fish oils.

Oxidized-LDL through LOX-1 increases the expression of angiotensin converting enzyme in human coronary artery endothelial cells

Background and objectives: Our previous studies have shown that oxidized low-density lipoprotein (ox-LDL) and angiotensin II (Ang II) influence each others action in endothelial cells. This study was designed to examine the regulation by ox-LDL of the expression of angiotensin converting enzyme (ACE) gene in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of the HMG CoA reductase inhibitor simvastatin on this interaction. Methods and results: Cultured HCAECs were incubated with ox-LDL (10–80 μg/ml) for 1–24 h. Ox-LDL increased the expression of ACE in a concentration- and time-dependent fashion. The upregulation of ACE expression in response to ox-LDL was mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 prevented the expression of ACE ( P <0.01). Native-LDL had no significant effect on ACE expression. In this process, ox-LDL-induced activation of mitogen-activated protein kinase (MAPK p42/44) played an important role, since pretreatment of HCAECs with the MAPK p42/44 inhibitor (PD98059, 10 μM) inhibited MAPK activation and subsequently attenuated the expression of ACE ( P <0.01 vs. ox-LDL alone). In other experiments, we pretreated HCAECs with simvastatin (10 μM) and then exposed the cells to ox-LDL. Simvastatin markedly attenuated ox-LDL-induced MAPK activation, and concurrently reduced ACE expression ( P <0.01 vs. ox-LDL alone). Conclusions: Our observations provide direct evidence that ox-LDL via LOX-1 activation induces ACE gene expression in HCAECs, and MAPK activation plays a signal transduction role in this process. Simvastatin, which inhibits MAPK activation, also blocks ox-LDL-mediated upregulation of ACE.

Eicosapentanoic acid inhibits hypoxia-reoxygenation-induced injury by attenuating upregulation of MMP-1 in adult rat myocytes.

BACKGROUND Myocardial hypoxia-reoxygenation (H-R) is associated with upregulation of metalloproteinases (MMPs). Upregulation of MMPs is associated with cell injury. Previous studies have shown that fish oil can protect myocardium from injury induced by H-R. This study was designed to examine the effect of eicosapentanoic acid (EPA), one of the major components in fish oil, on the modulation of MMP-1 expression in response to H-R in cultured adult rat myocytes. METHODS AND RESULTS Myocytes isolated from adult Sprague-Dawley rat hearts were cultured with or without EPA or arachidonic acid (AA) (10 and 50 microM) and exposed to 24 h of hypoxia followed by 3 h of reoxygenation (H-R). H-R resulted in myocyte injury (measured on LDH release), increase in p38MAPK phosphorylation (Western analysis), augmentation of lipid peroxidation, and upregulation of MMP-1 activity (zymography) and expression (RT-PCR and Western analysis) (all P<0.01 vs. control, n=5). Pretreatment of myocytes with EPA, but not AA, resulted in a reduction in LDH release, and attenuation of p38MAPK phosphorylation and MMP-1 activity and expression in response to H-R (all P<0.05 vs. H-R alone). Pretreatment of myocytes with EPA also reduced lipid peroxidation in myocytes exposed to H-R (P<0.05 vs. H-R alone). A high concentration of EPA (50 microM) was more potent than the lower concentration of EPA (10 microM). CONCLUSIONS These observations suggest that EPA attenuates an increase in MMP-1 following H-R, which may be a basis of protection of myocytes from the adverse effects of H-R. p38MAPK phosphorylation may be an important signaling event in this process.

Modulation of matrix metalloproteinase-1, its tissue inhibitor, and nuclear factor-κB by losartan in hypercholesterolemic rabbits

Upregulation of angiotensin II receptor, may be involved in the initiation and progression of atherosclerosis. To examine the contribution of AT1 receptor in the expression of matrix metalloproteinase-1 (MMP-1) and its tissue inhibitor (TIMP-2) in lipid-deposited arterial tissues, New Zealand white rabbits were given high-cholesterol chow (with losartan 25 mg/d or vehicle) for 10 weeks. Losartan reduced the areas of sudanophilia in the aorta of rabbits fed high-cholesterol diet (p < 0.01 vs. control). Losartan also significantly decreased the enhanced mRNA expression of MMP-1 and TIMP-2 in aortas of rabbits with high-cholesterol diet. Losartan-treated rabbits revealed a reduction in immunohistochemical expression of MMP-1, whereas TIMP-2 expression became localized to the intima. In addition, losartan treatment reduced the activation of NF-&kgr;B by inhibiting the degradation of its inhibitor I&kgr;-B&agr;. These observations demonstrate that AT1 receptor blockade with losartan reduces lipid deposition and exerts potent inhibitory effects on NF-&kgr;B activation and modulates the expression of MMP-1 and TIMP-2 in hypercholesterolemic rabbits.

Protection of Myocytes From Hypoxia-Reoxygenation Injury by Nitric Oxide Is Mediated by Modulation of Transforming Growth Factor-β1

Background—Reoxygenation injury is a result of several complex events, including release of reactive oxygen species, protein kinase C (PKC) activation, and altered expression of transforming growth factor-&bgr;1 (TGF-&bgr;1). Nitric oxide (NO) generally protects tissues from reperfusion injury. Methods and Results—We examined the modulation of TGF-&bgr;1 expression and activity and PKC activation in cultured rat heart myocytes exposed to a brief period of hypoxia-reoxygenation (H-R) by NO donor 3-morpholino-sydnonimine (SIN-1). H-R resulted in an increased expression of total TGF-&bgr;1 (mRNA and protein) but a decrease in the release of active TGF-&bgr;1. Myocyte PKC-&agr; protein level was not altered by H-R, but its phosphorylation was augmented. Pretreatment of myocytes with SIN-1 diminished myocyte injury quantified as lactate dehydrogenase release. Simultaneously, release of active TGF-&bgr; 1 increased and total TGF-&bgr;1 expression decreased (all P <0.05 versus H-R alone). PKC-&agr; phosphorylation increased further in cells treated with SIN-1. The effects of SIN-1 were blocked by the NO scavenger phenyl-tetramethyl-imidazoline-oxyl-oxide as well as by the PKC inhibitor staurosporine. To examine if another NO donor would have a similar effect, cardiomyocytes were treated with nitroglycerin before H-R. With nitroglycerin treatment, similar to SIN-1 treatment, myocyte injury was diminished, TGF-&bgr; 1 release increased, and total TGF-&bgr; 1 expression decreased. Conclusions—These observations suggest modulation of TGF-&bgr; 1 expression as a novel mechanism of salutary effect of NO donors. PKC-&agr; activation may play an important role in the protective effect of NO against H-R injury.

Identification, Regulation and Function of Lox-1, a Novel Receptor for Ox-Ldl

Oxidatively modified low-density lipoprotein (ox-LDL) causes endothelial activa-tion, dysfunction and injury, which are considered primary steps in atherogenesis. Recently, a novel lectin-like receptor for ox-LDL (LOX-1) has been identified, primarily in endothe-lial cells. This receptor mediates uptake of ox-LDL into the endothelial cells and activates a variety of signal transduction mechanisms that lead to endothelial cell activation and expres-sion of adhesion molecules. LOX-1 receptor is transcriptionally upregulated by various stimuli accompanying pathogenic states, including TNF-OC, angiotensin II, shear stress and ox-LDL itself. LOX-1 receptor expression has been demonstrated in animal models and humans with hypertension, diabetes mellitus and atherosclerosis. Expression of this receptor may also be pathogenically involved in arterial thrombosis and myocardial ischemia-reperfusion injury. Understanding the regulation and signal transduction pathways of this receptor may lead to new therapies in prevention and treatment of atherosclerosis, and its complications.