LOX-1 mRNA Expression Research Articles

Lectin-like oxidized low density lipoprotein receptor-1 mediates effects of bezatibrate on the apoptosis of endothelial cells

Objective To observe the influence of bezafibrate on the apoptosis and expression of lectin-like oxidized low density lipoprotein receptor-1(LOX-1) mediated by oxidized low density lipoprotein(ox-LDL) in cultured human umbilical vein endothelial cells(HUVECs).Methodes The apoptosis of HUVECs mediated by ox-LDL were evaluated by flow cytometry and the expression of LOX-1 mRNA were detected by RT-PCR.Results Compared with the control group,ox-LDL could increase apoptosis and the expression of LOX-1(P<0.05),and Bezafibrate could decrease the apoptosis and the expression of LOX-1 in a concentration -dependent manner(P<0.05).Preincubation of HUVECs with polyinosinic acid for 2 hours,the apoptosis and the expression of LOX-1 decreased(P<0.05).Conclusions Bezafibrate inhibits the apoptosis of HUVECs mediated by ox-LDL by reducing the expression of LOX-1,which may be part of the reasons for bezafibrate to prevent and treat atherosclerosis. Key words: Bezafibrate/PD; Receptors,LDL; Apoptosis

Differential effect of surfactant and its saturated phosphatidylcholines on human blood macrophages

Blood monocyte-derived macrophages invading the alveolus encounter pulmonary surfactant, a phospholipoprotein complex that changes composition during lung development. We tested the hypothesis that characteristic phosphatidylcholine (PC) components differentially influence macrophage phenotype and function, as determined by phagocytosis of green fluorescent protein-labeled Escherichia coli and alphaCD3-induced T cell proliferation. Human macrophages were exposed to surfactant (Curosurf(R)), to two of its characteristic phosphadidylcholine (PC) components (dipalmitoyl-PC and palmitoylmyristoyl-PC), and to a ubiquituous PC (palmitoyloleoyl-PC) as control. Interaction of Curosurf and PC species with macrophages was assessed using Lissaminetrade mark-dihexadecanoyl-phosphoethanolamine-labeled liposomes. Curosurf and both saturated surfactant PC species downregulated CD14 expression and upregulated CD206. HLA-DR and CD80 were upregulated by Curosurf and palmitoylmyristoyl-PC, whereas dipalmitoyl-PC showed no effect. The latter upregulated TLR2 and TLR4 expression, whereas Curosurf and palmitoylmyristoyl-PC had no effect. PC species tested were incorporated in comparable amounts by macrophages. Curosurf and PC species inhibited phagocytosis of E. coli. Scavenger receptor CD36, CD68, SR-A, and LOX-1 mRNA expression was upregulated by Curosurf, whereas PC species only upregulated SR-A. Curosurf and palmitoylmyristoyl-PC inhibited alphaCD3-induced T cell proliferation by 50%, whereas dipalmitoyl-PC and palmitoyloleoyl-PC showed no effect. These data identify individual surfactant PC species as modifiers of macrophage differentiation and suggest differential effects on innate and adaptive immune functions.

Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase

Oxidized low density lipoprotein (OxLDL) is one of the most important risk factors of cardiovascular disease. Here, we study the impact of OxLDL on endothelial progenitor cells (EPCs) and determine whether OxLDL affects EPCs by an inhibitory effect on endothelial nitric oxide synthase (eNOS). It was found that OxLDL decreased EPC survival and impaired its adhesive, migratory, and tube-formation capacities in a dose-dependent manner. However, all of the detrimental effects of OxLDL were attenuated by pretreatment of EPCs with lectin-like oxidized low density lipoprotein receptor (LOX-1) monoclonal antibody or l-arginine. Western blot analysis revealed that OxLDL dose-dependently decreased Akt phosphorylation and eNOS protein expression and increased LOX-1 protein expression. Furthermore, OxLDL caused a decrease in eNOS mRNA expression and an increase in LOX-1 mRNA expression. These data indicate that OxLDL inhibits EPC survival and impairs its function, and this action is attributable to an inhibitory effect on eNOS.

Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1-Mediated Autophagy in Human Granulosa Cells as an Alternative of Programmed Cell Death

The LOX-1 receptor, identified on endothelial cells, mediates the uptake of oxidized low-density lipoprotein (oxLDL). The oxLDL-dependent LOX-1 activation causes endothelial cell apoptosis. We here investigated the presence of LOX-1 in granulosa cells from patients under in vitro fertilization therapy. We were interested in the oxLDL-dependent LOX-1 receptor biology, in particular in the induction of apoptosis. In the human ovary, LOX-1 was localized in regressing antral follicles. In granulosa cell cultures, oxLDL-induced mRNA expression of LOX-1 in a time- and dose-dependent manner. The LOX-1 inhibitors (anti-LOX-1 antibody and kappa-carrageenan) abrogated the up-regulation of LOX-1. The oxLDL (100 microg/ml) treatment caused the autophagy form of programmed cell death: 1) reorganization of the actin cytoskeleton at the 6-h time point; 2) uptake of YO-PRO, a marker for the early step of programmed cell death, before propidium iodide staining to signify necrosis; 3) absence of apoptotic bodies and cleaved caspase-3; 4) abundant vacuole formation at the ultrastructural level; and 5) decrease of the autophagosome marker protein MAP LC3-I at the 6-h time point indicative of autophagosome formation. We conclude that follicular atresia is not under the exclusive control of apoptosis. The LOX-1-dependent autophagy represents an alternate form of programmed cell death. Obese women with high blood levels of oxLDL may display an increased rate of autophagic granulosa cell death.

Pravastatin Inhibits Expression of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 (LOX-1) in Watanabe Heritable Hyperlipidemic Rabbits

LOX-1, a receptor for oxidized low-density lipoprotein (OxLDL), seems to play a critical role in foam cell formation of macrophages (Mphis) and smooth muscle cells (SMC). Inhibition of LOX-1 expression reduces foam cell formation and might influence lipid core formation in atherosclerotic lesions. Because statins are able to downregulate LOX-1 expression in vitro, we examined if pravastatin can be used to reduce LOX-1 expression and lipid core formation in lesions of Watanabe heritable hyperlipidemic (WHHL) rabbits. Pravastatin downregulated LOX-1 expression in cultured human Mphis and in cultured human aortic SMCs. Homozygous WHHL rabbits were treated with 50 mg kg(-1) d(-1) pravastatin for 32 weeks. Immunohistochemical studies revealed that LOX-1 was expressed in intimal Mphis and SMCs of atherosclerotic lesions. The pravastatin-treated rabbits showed, compared with untreated rabbits, a significantly reduced LOX-1 protein and mRNA expression in the aortic arch. Lipid labeling of this aorta region also demonstrated a strong reduction of the ratio of lipid core area/total lesion area in pravastatin-treated rabbits. The in vivo inhibition of LOX-1 expression by pravastatin demonstrated here represents a new pleiotropic effect of pravastatin. This in vivo inhibition of LOX-1 might be one mechanism for the lipid core reducing effect of pravastatin in atherogenesis.

Doxorubicin-induced expression of LOX-1 in H9c2 cardiac muscle cells and its role in apoptosis

Up-regulation of LOX-1 is implicated in apoptosis in both vascular smooth muscle cells and in endothelial cells. We examined the effects of doxorubicin on LOX-1 expression in H9c2 cardiomyocytes and the role played by LOX-1 up-regulation in doxorubicin-induced apoptosis. Reactive oxygen species (ROS) formation was assessed by DCF flow cytometry. LOX-1 mRNA and protein expression was assessed by RT-PCR and Western blotting. Apoptosis was evaluated by flow cytometry with annexin/PI double staining. Doxorubicin-induced LOX-1 expression in a concentration- and time-dependent fashion. The doxorubicin-induced ROS formation and the LOX-1 expression were significantly attenuated by pre-treatment with antioxidants. By exposing cells that had been pre-treated with doxorubicin to oxidized-LDL, a LOX-1 agonist, in the presence or in the absence of k-carrageenan, a LOX-1 receptor antagonist, we documented that doxorubicin-induced LOX-1 expression plays a role in inducing apoptosis. These findings suggest that LOX-1 up-regulation is redox-sensitive and may contribute to doxorubicin-induced cardiotoxicity.

Herpes Simplex Virus 1 Induced LOX-1 Expression in an Endothelial Cell Line, ECV 304

Infections, such as by Chlamydophilia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori, have been shown to be involved in atherogenesis. Herpes simplex virus I (HSV-1) could infect vascular endothelial cells, and it has been shown that, when endothelial cells were activated with oxidized LDL (oxLDL), a number of cellular events are occurred, leading to endothelial cell dysfunction. Since LOX-1 is a major receptor for oxLDL on endothelial cells and its expression was increased in atherosclerosis, we investigated whether HSV1 infection can lead to the increase expression of LOX-1 in endothelial cells. LOX-1 mRNA expression determined by RT-PCR and LOX-1 promoter activity measured by luciferase assay were increased in endothelial cells following HSV-1 infection. This suggests that one of the mechanisms by which HSV-1 is involved in atherogenesis maybe the enhanced uptake of oxLDL via the increased expression of LOX-1 in endothelial cells.

Inhibition of oxidative stress and improvement of endothelial function by amlodipine in angiotensin II-infused rats

Background Calcium channel blockers such as amlodipine are effective antihypertensive agents. In this study we investigated the effects of amlodipine on vascular oxidative stress, expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1), and endothelial function in angiotensin (Ang) II-infused rats. Methods Sprague-Dawley rats were treated with Ang II (0.7 mg/kg/day subcutaneously injected by mini-pump), with or without amlodipine (10 mg/kg/day by gavage), for 5 days and compared with control rats. Levels of aortic ring superoxide (O 2 −) and peroxynitrite (ONOO −) were determined, and systolic blood pressure (SBP) and endothelium-dependent relaxation were evaluated. Results Compared with control rats, Ang II-infused rats developed hypertension (175 ± 3 v 135 ± 2 mm Hg, P < .05), aortic hypertrophy (16.9 ± 1.3 v 13.2 ± 0.3 mg/cm, P < .05), left ventricular hypertrophy (0.236 ± 0.003 v 0.204 ± 0.004 g/100 g body weight, P < .05), and impaired endothelium-dependent relaxation (ED 50: 6.6 ± 0.2 v 8.0 ± 0.2 −log mol/L acetylcholine concentration, P < .05). Compared with control rats, Ang II-infused rats also had higher aortic levels of LOX-1 mRNA expression, O 2 −production (1005 ± 140 v 608 ± 159 counts/min/mg, P < .05), ONOO − production (1875 ± 295 v 782 ± 115 counts/min/mg, P < .05), and plasma free 8-F 2α-isoprostanes (67.4 ± 19.1 v 27.2 ± 6.1 pg/mL, P < .05). In Ang II-infused rats SBP, aortic hypertrophy, endothelial dysfunction, LOX-1 expression, aortic O 2 − and ONOO − production, and plasma free 8-F 2α-isoprostane levels were significantly reduced by amlodipine treatment. Conclusions Amlodipine has antihypertensive and antioxidant activity in vivo, which effectively inhibits many of the oxidative stress-dependent mechanisms involved in Ang II-mediated cardiovascular injury.

Induction of LOX‐1 and iNOS expressions by ischemia‐reperfusion of rat kidney and the opposing effect of L‐arginine

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a newly identified endothelial cell surface major receptor for oxidatively modified low-density lipoprotein. Progression of arthrosclerosis in the donor organ after organ transplantation is a major problem. We hypothesized that ischemia-reperfusion induces LOX-1. After 1 h ischemia of bilateral kidneys plus 3, 6, or 12 h reperfusion, we first revealed that LOX-1 mRNA expression was increased in renal cortex and medulla at 6 h after reperfusion, which was decreased by L-arginine supplement. Plasma nitric oxide (NO) end-product nitrite plus nitrate and inducible nitric oxide synthase (NOS) expression were increased after reperfusion of 6 h. However, NOS substrate L-arginine did not augment but markedly decreased plasma NO end product, because L-arginine supplement suppressed inducible NOS expression in kidney. We hypothesized that available L-arginine is depleted by ischemia-reperfusion, leading to inducible NOS induction. Ischemia decreased L-arginine levels in kidney and L-arginine supplement increased NO end products in renal cortex in the earliest phase of reperfusion. These results disclosed for the first time that a deficiency in L-arginine by ischemia reperfusion causes uncoupling of constitutive NOS, which induces inducible NOS and LOX-1, implying why L-arginine is effective for stroke or transplantation in preventing atherosclerotic progress.

Angiotensin II increases the expression of lectin-like oxidize low-density lipoprotein receptor- 1 in cultured human umbilical vein endothlial cells

OBJECTIVE: To observe whether angiotensin II (AngII) influences expression of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) in cultured human umbilical vein endothlial cells(HUVECs). METHODS: Cultured HUVECs were incubated with 10(-5)mol/L approximate, equals 10(-10) mol/L AngII for 24 h or with 10(-6) mol/L AngII for various time up to 48 h. Then HUVECs LOX-1 protein expression was measured by endothlial cell enzyme linked immunosorbent assay, and mRNA level of LOX-1 was detected by quantitative competitive reverse transcription-polymerase chain reaction. RESULTS: Incubation of HUVECs with AngII for 24 h significantly increased LOX-1 protein expression (P<0.001). The increase was dependent on AngII concentration (10(-5)mol/L approximate, equals 10(-9)mol/L). LOX-1mRNA expression was also induced by AngII, and after 24 h incubation of AngII(10(-5)mol/L approximate, equals 10(-8)mol/L), LOX-1mRNA expression increased 4.43, 4.25, 2.71, 1.84 times, respectively. After treatment with 10(-6) mol/L AngII for 3 h, LOX-1 expression (protein and mRNA) was elevated (P<0.001) in HUVECs, reaching its maxium at 24 approximate, equals 36 h. CONCLUSION: AngII upregulates LOX-1 expression concentration-dependently and time-dependently in HUVECs.

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

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). 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. 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.

Induction of the OxLDL Receptor LOX-1 by Endothelin-1 in Human Endothelial Cells

In this study, we analyzed the effect of endothelin-1 (ET-1) on expression of the lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 LOX-1 and on oxLDL uptake in primary cultures of human umbilical vein endothelial cells (HUVEC). LOX-1 mRNA was quantified by standard-calibrated competitive RT-PCR, LOX-1 protein expression by Western analysis and endothelial oxLDL uptake using DiI-labeled oxLDL. ET-1 induces LOX-1 mRNA expression, reaching its maximum after 1 h (160 ± 14% of control, 100 nM ET-1, P < 0.05). This increased ET-1-mediated LOX-1 mRNA expression could be inhibited by endothelin receptor B antagonist BQ-788. In addition, ET-1 stimulates LOX-1 protein expression and oxLDL uptake in HUVEC. The augmented oxLDL uptake by ET-1 is mediated by endothelin receptor B, but not by protein kinases. These data support a new pathophysiological mechanism how locally and systemically increased ET-1 levels could promote LOX-1-mediated oxLDL uptake in human endothelial cells and the development and progression of endothelial dysfunction and atherosclerosis.

Redox-Sensitive Regulation of LOX-1 Gene Expression in Vascular Endothelium

Oxidative stress has been implicated in atherosclerosis and its underlying conditions. LOX-1 is a novel endothelial receptor for oxidized low-density lipoprotein which might mediate endothelial dysfunction and subsequent atherogenesis. In the present study, we examined LOX-1 gene regulation by oxidative stress. First, superoxide anions generated by hypoxanthine and xanthine oxidase as well as hydrogen peroxide increased LOX-1 mRNA expression in cultured aortic endothelial cells. Homocysteine, an atherogenic substance believed to exert its effects through oxidative stress, enhanced endothelial LOX-1 gene expression, which was suppressed by N-acetylcysteine. Second, rats receiving angiotensin II for 10 days manifested hypertension and LOX-1 upregulation in aortic endothelium via AT1 receptor. Tempo, a superoxide dismutase mimetic, alleviated LOX-1 augmentation induced by angiotensin II. These results indicated redox-sensitive upregulation of LOX-1 mRNA in both in vitro and in vivo systems, suggesting its potential role in atherosclerosis.

Upregulation of endothelial receptor for oxidized low-density lipoprotein (LOX-1) in cultured human coronary artery endothelial cells by angiotensin II type 1 receptor activation.

Cross talk between oxidized LDL (ox-LDL) and angiotensin II (Ang II) may be relevant in atherosclerosis. In this study, we examined the presence of a specific endothelial receptor for ox-LDL (LOX-1) and Ang II receptors in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of Ang II on LOX-1 gene and protein expression. LOX-1 was consistently identified in HCAECs by reverse transcriptase-polymerase chain reaction (RT-PCR), cDNA sequence, Western blot, and 125I-labeled ox-LDL binding assay (Bmax, 29.7 ng/mg protein). The HCAECs also exhibited Ang II receptors (AT1>AT2), as determined by RT-PCR and 125I-labeled Ang II binding assay (Bmax, 2.21 and 1.19 fmol/mg protein, respectively). Incubation of HCAECs with Ang II markedly increased LOX-1 mRNA (RT-PCR) and protein (Western blot) expression. The increase in LOX-1 expression was dependent on Ang II concentration (10(-12) to 10(-6) mol/L). Ang II caused a concentration-dependent increase in 125I-labeled ox-LDL uptake by HCAECs and enhanced ox-LDL-mediated cell injury, as evident from an increase in LDH release and a decrease in cell viability. These effects of Ang II were completely blocked by pretreatment of HCAECs with losartan, a specific AT1 blocker, but not by PD123319, a specific AT2 blocker. These observations indicate the following: (1) HCAECs possess abundant LOX-1 as well as Ang II (AT1>AT2) receptors, (2) Ang II upregulates LOX-1 receptor and ox-LDL uptake, (3) the effects of Ang II are mediated by AT1 activation, and (4) Ang II enhances ox-LDL-mediated injury to HCAECs.

The expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) on human vascular smooth muscle cells and monocytes and its down-regulation by lovastatin

Accumulation of oxidatively modified low-density lipoprotein (oxLDL) in the vascular wall is a characteristic feature of atherosclerosis. oxLDL can be taken up into monocytes, smooth muscle cells, and endothelial cells by several known scavenger receptors such as scavenger receptor class A I and II, CD36, and CD68. A new lectin-like oxLDL receptor (LOX-1) was recently found in bovine and human endothelial cells. We studied whether LOX-1 is also expressed in other cells present in the atherosclerotic lesion and whether its expression can be modified. We found LOX-1 expression in human blood monocytes, umbilical smooth muscle and endothelial cells, and 3T3 fibroblasts. LOX-1 mRNA expression in monocytes could be significantly suppressed by lovastatin. Thus, LOX-1 expression is not restricted to endothelial cells and its down-regulation by HMG-CoA reductase inhibitors could contribute to the clinical benefits of these drugs.