Reactive oxygen species mediate oxidized low-density lipoprotein-induced endothelin-1 gene expression via extracellular signal-regulated kinase in vascular endothelial cells.

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) promotes expression and secretion of endothelin-1 (ET-1), however, the precise mechanism involved is unclear. This study was designed to identify the regulatory mechanism of oxLDL-induced ET-1 expression in endothelial cells. METHODS: ET-1 mRNA expression, secretion and promoter activity were evaluated by reverse transcriptase-PCR (RT-PCR), enzyme immunometric and luciferase assays, respectively. RESULTS: oxLDL (35 microg/ml) significantly enhanced reactive oxygen species (ROS), mRNA expression, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVECs), all of which were nullified by the antioxidant N-acetyl cysteine (NAC). oxLDL stimulated the extracellular signal-regulated kinase (ERK) phosphorylation in HUVECs, which was blocked by NAC and the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD98059. NAC and PD98059 stopped oxLDL-elicited increase in mRNA expression, secretion and promoter activity of ET-1. Fusion plasmids with decreasing length of 5'-flanking sequence of ET-1 from -566 bpLuc to -250 bpLuc displayed increased luciferase activity after 24 h of oxLDL treatment. Interestingly, fusion plasmid from -233 and -185 bpLuc significantly reduced the luciferase activity in control and oxLDL-treated HUVECs. In addition, transfection of the reporter construct -250Luc, which contains a 2 bp mutation at activator protein-1 site, abolished both basal and oxLDL-stimulated ET-1 promoter activities. CONCLUSION: Collectively, our data favor the notion that oxLDL stimulates ERK phosphorylation via ROS accumulation, which in turn stimulates vascular endothelial transcriptional factor activator protein-1 and ET-1 expression as well as secretion.

alpha-Zearalanol attenuates oxLDL-induced ET-1 gene expression, ET-1 secretion and redox-sensitive intracellular signaling activation in human umbilical vein endothelial cells.

alpha-Zearalanol (alpha-ZAL), a phytochemical with both antioxidant and estrogen-like properties, has been shown to retard progression of atherosclerosis and regulate cardiovascular function in part through suppression of endothelin-1 (ET-1) secretion. However, the precise nature behind alpha-ZAL-elicited inhibition on ET-1 cascade is not largely known. Oxidized low density lipoprotein (oxLDL) plays a critical role in the expression and secretion of ET-1 as well as the onset and progression of atherosclerosis through accumulation of reactive oxygen species (ROS) and activation of mitogen-activated protein kinase stress signaling cascade. Therefore, this study was designed to examine the effect of alpha-ZAL on oxLDL-induced extracellular signal-regulated kinase (ERK) phosphorylation, ROS generation, activation of the transcriptional factor activator protein-1 (AP-1), expression, secretion and promoter activity of ET-1 in human umbilical vein endothelial cells (HUVEC). ROS generation was monitored using 2,7-dichlorofluorescin fluorescence. ET-1 expression and promoter activity were evaluated by RT-PCR and luciferase assays, respectively. oxLDL (35 microg/ml) significantly enhanced ERK phosphorylation, ROS generation, AP-1 activity, mRNA expression, secretion and promoter activity of ET-1 in HUVECs, all of which were abrogated by alpha-ZAL and the antioxidant N-acetyl-l-cysteine. Collectively, these data favor the notion that alpha-ZAL antagonizes oxLDL-induced upregulation of ET-1 gene expression and secretion via suppression of oxLDL-induced ROS accumulation, ERK phosphorylation, and activation of the endothelial transcriptional factor AP-1.

Phytoestrogen alpha-zearalanol antagonizes homocysteine-induced imbalance of nitric oxide/endothelin-1 and apoptosis in human umbilical vein endothelial cells.

Although the issue of estrogen replacement therapy on cardiovascular health is debatable, it has presumable benefits for endothelial function in postmenopausal women. However, the fear of breast cancer has intimidated women contemplating estrogen treatment and limited its long-term application. An effective alternative remedy not associated with breast carcinoma is in serious demand. This study was designed to examine the effect of phytoestrogen alpha-zearalanol (alpha-ZAL) and 17beta-estradiol (E2) on nitric oxide (NO) and endothelin (ET)-1 levels, apoptosis, and apoptotic enzymes in human umbilical vein endothelial cells (HUVEC). HUVEC cells were challenged for 24 h with homocysteine (10-3 M), an independent risk factor for a variety of vascular diseases, in the presence of alpha-ZAL or E2 (10-9 to 10-6 M). Release of NO and ET-1 were measured with enzyme immunoassay. Apoptosis was evaluated by fluorescence-activated cell sorter analysis. Expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were determined using Western blot. NOS activity was evaluated with 3H-arginine to 3H-citrulline conversion. Our results indicated that Hcy significantly reduced NO production, NOS activity, enhanced ET-1/NO ratio and apoptosis, upregulated iNOS, Bax, and downregulated eNOS, Bcl-2 expression. These effects were significantly attenuated by alpha-ZAL and E2. ZAL displayed a similar potency compared with E2 in antagonizing Hcy-induced effects. In summary, these results suggested that alpha-ZAL may effectively preserve Hcy-induced decrease in NO, increase in ET-1/NO ratio and apoptosis, which contributes to protective effects of phytoestrogens on endothelial function.

[Signaling pathways in expression of inducible nitric oxide synthase induced by high mobility group box 1 in rat alveolar macrophages].

OBJECTIVE: To explore roles of extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen activated protein kinase (p38 MAPK) and nuclear factor (NF) -KB in expression of inducible nitric oxide synthase (iNOS) in rat alveolar macrophages induced by high mobility group box 1 (HMGB1 ). METHODS: Primary rat alveolar macrophages (PRAMs) cultured in vitro were incubated with PD98059 ( inhibitor against ERK), SB203580 (inhibitor against p38 MAPK) , PDTC (inhibitor against NF-kappaB), or PD98059 plus SB203580 for 2 hours, respectively. HMGB1 was added into the cultures and incubated with cells for 6 hours. Total RNA of PRAMs was extracted and iNOS mRNA expression was semi-quantified with reverse transcription-polymerase chain reaction ( RT-PCR). Greiss reaction was applied to determine nitrite/nitrate (NO2-/NO3- ) concentration in PRAMs culture supernatants. RESULTS: Expression of iNOS mRNA and NO production in PRAMs culture supernatants were down-regulated by inhibition of ERK or p38 MAPK by PD98059 or SB203580, respectively (P <0. 05). Moreover, inhibition of iNOS expression and NO production was observed after simultaneous pretreatment with PD98059 and SB203580 (P < 0. 05). Expression of iNOS mRNA in PRAMs and NO production in PRAMs culture supernatants were down-regulated by inhibition of NF-kappaB by PDTC (P <0. 05). CONCLUSION: Cellular signal molecules of ERK, p38 MAPK, and NF-kappaB all participate in the expression of iNOS and NO production in PRAMs induced by HMGB1.

Inhibitory effects of alpha-zearalenol on angiotensin II-induced integrin beta3 mRNA via suppression of nuclear factor-kappaB.

OBJECTIVE: To investigate the effect of alpha-zearalenol on angiotensin II-induced beta3 integrin mRNA expression in human umbilical vein endothelial cells (HUVECs). METHODS: The mRNA level in integrin beta3 was determined by reverse transcription-polymerase chain reaction. Endothelial NF-kappaB activity was determined by the luciferase activity assay of plasmid NF-kappaB-LUC. RESULTS: The angiotensin II-induced beta3 integrin mRNA expression was inhibited by alpha-zearalenol and 17beta-estradiol (10 nmol/L -1 micromol/L), but not influenced by ICI 182, 780, a pure competitive antagonist for estrogen receptor or a nitric oxide inhibitor Nomega-Nitro-L-arginine methyl ester hydrochloride. Alpha-zearalenol and 17beta-estradiol suppressed the angiotensin II-induced activation of NF-kappaB in endothelial cells. CONCLUSION: Alpha-zearalenol inhibits angiotensin II-induced integrin beta3 mRNA expression by suppressing NF-kappaB activation in endothelial cells.

[Cardiovascular effects of phytoestrogens].

Phytoestrogens are bioactive substances existing in natural plants. They have similar molecular structure to those of estrogens. In this article we introduced their classification and sources, and elucidated their effects on heart from aspects involving cardiac function and myocardial electrophysiology. By regulating serum lipid metabolism, arterial vessels, cytokine levels, and coagulation/fibrinolysis system, phytoestrogens possess the effects of anti-atherosclerosis and may be used to prevent and treat cardiovascular diseases.

[Effect of xianzhen tablet on content of advanced glycosylation end products (AGEs) and mRNA expression of AGE-specific cellular receptor in renal cortex of diabetic rats].

OBJECTIVE: To investigate the effect of Xianzhen tablet (XZT, a Chinese patent compound recipe) on advanced glycosylation end products (AGEs) and mRNA expression of AGE-specific cellular receptor (RAGE) in renal cortex of diabetic rats in order to explore the mechanism of XZT in protecting kidney. METHODS: The diabetic rat model with persistent hyperglycemic renal damage was reproduced by streptozotocin. Fluorescent assay and RT-PCR were used to determine the content of AGEs and expression of RAGE mRNA in renal cortex in model rats, which were treated with XZT and controlled by aminoguanidine (AG) administration. RESULTS: The relative content of AGEs and RAGE mRNA expression in renal cortex of model rats 12 weeks after modeling were significantly higher than those in the normal group (P < 0.05), while those in model rats treated by XZT or AG were markedly lower than those in non-treated model rats (P < 0.05), the effect of the both groups showed insignificant difference (P > 0.05). CONCLUSION: XZT could reduce the accumulation of AGEs in renal cortex of diabetic rats, down-regulate the over-expression of RAGE mRNA, with the effects similar to that of AG, the inhibition of XZT on protein non-enzymatic glycosylation might be one of the mechanisms of its effect in protecting kidney.

[Acute lung injury and changes of myocardial ATP enzymes induced by lipopolysaccharide in aging rats].

OBJECTIVE: To investigate whether acute lung injury (ALI) and changes of myocardial ATP enzymes were induced by intravenous or intraventricle of left heart injection of lipopolysaccharide (LPS) in aging rats. METHODS: 40 male Wistar rats were used for reproducing aging animal model. Aging rats were randomly divided into aging control group (n = 8), ALI group (LPS, 5 mg/kg body weight intravenous injection, n = 16), and LPS group (same dosage LPS, intraventricle of left heart injection, n = 16). The samples (blood, lung and heart) were collected at 2, 6 hours after LPS or saline administration. RESULTS: Compared with aging control, protein content in bronchial alveolar lavage fluid (BALF), ratio of lung wet/dry weight and the LA, NO2-/NO3- and MDA contents in blood were increased markedly (P < 0.01) at 2, 6 hours in ALI group. The GSH-Px, Na(+)-K(+)-ATPase activities in lung tissue were decreased significantly (P < 0.01), but NO2-/NO3- content in lung tissue was increased obviously (P < 0.01) at 2 hours in ALI group. These changes were maintained until at 6 hours after LPS administration. The above parameters were no obviously changes in myocardium at 2 hours after LPS administration in ALI group. But at 6 hours, MDA content was increased obviously (P < 0.01); Na(+)-K(+)-ATPase, Ca(2+)-Mg(2+)-ATPase and GSH-Px activities in myocardium were decreased markedly (P < 0.01). While in LPS group, only NO2-/NO3- contents were increased (P < 0.05) in blood and lung tissue as well as Na(+)-K(+)-ATPase activity in lung tissue were decreased (P < 0.05), another parameters had no obvious changes. CONCLUSIONS: ALI was obviously formed by intravenous injection LPS after 2, 6 hours in aging rats. Myocardial enzyme etc decreased only at 6 hours in ALI group. But above parameters were no obviously changes in LPS group. It was suggested that there was probable myocardial damage in rats of ALI group, and it was mainly induced by ALI.

[Hepatic injury induced by acute lung injury in aging rats].

OBJECTIVE: To investigate the induction of hepatic function damage by acute lung injury (ALI) in aging rats and the effect of Ginkgo Biloba extract (GBE) on this process. METHODS: Thirty male Wistar rats were used to produce the aging animal model. Aging rats were randomly divided into three groups: the control group, the lipopolysaccharide (LPS, intravenous injection) group, and the GBE + LPS group (GBE given 7 days before experiment, once a day, via the esophagus). Samples from the blood, the lung and the liver were collected 2 and 6 h after LPS or saline administration. RESULTS: ALI was induced by intravenous injection of LPS in aging rats. Compared with the aging control, the total bilirubin content and the glutamic pyruvic transaminase (GPT) activity in serum did not change at 2 h after LPS administration. But at 6 h, they were increased, respectively from (10.9 +/- 0.6) mg/L and (26 +/- 3) U in the control group to (30.1 +/- 2.1) mg/L and (88 +/- 12) U in the LPS group (P < 0.001). MDA content increased in the blood and the lung tissue at 2 has compared to the control group, from (15.9 +/- 1.8) micro mol/L and (18.8 +/- 2.1) nmol/mg protein to (22.1 +/- 1.9) micro mol/L and (28.8 +/- 3.1) nmol/mg protein (all P < 0.001), respectively. SOD activity in the lung tissue was decreased significantly, from (25.5 +/- 2.6) mU/L and (36.1 +/- 2.4) U/mg protein to (20.6 +/- 1.9) mU/L and (32.0 +/- 2.7) U/mg protein, respectively (P < 0.05, P < 0.001). The GSH-P(X) activity and the Na(+)-K(+)-ATPase activity in the lung tissue at 2 hours after LPS administration were decreased markedly, from (28.2 +/- 2.8) U/mg protein and (4.9 +/- 0.5) micromol Pi x mg(-1) protein x h(-1). to (21.1 +/- 2.7) U/mg protein and (3.1 +/- 0.3) micromol Pi x mg(-1) protein x h(-1). These changes lasted 6 h after LPS administration. These parameters did not change significantly in the hepatic tissue at 2 h after LPS administration. But after 6 h, MDA content was increased from (7.9 +/- 0.9) nmol/mg protein to (10.9 +/- 0.7) nmol/mg protein; while the GSH-P(X) and the Na(+)-K(+)-ATPase activities were decreased markedly, from (59.0 +/- 3.9) U/mg protein and (0.87 +/- 0.04) micromol Pi x mg(-1) protein x h(-1) to (49.2 +/- 3.0) U/mg protein and (0.77 +/- 0.04) micromol Pi x mg(-1) protein x h(-1) (P < 0.001, P < 0.01). There was no obvious change in the SOD activity. All the changes were significantly attenuated in the GBE + LPS group (P < 0.05, P < 0.01). CONCLUSION: Hepatic function damage could be induced by ALI in aging rats. GBE showed a protective effect on ALI and hepatic function damage in this animal model.

[Protective effect of Ginkgo biloba extract on acute lung injury induced by lipopolysaccharide in D-galactose aging rats].

OBJECTIVE: To investigate the effect of Ginkgo biloba extract (GBE) on acute lung injury(ALI) induced by lipopolysaccharide (LPS) in aging rats. METHODS: The rats were randomly divided into two parts: six rats served as normal controls; 18 rats were used for producing the aging animal model ( D-gal 50 mg/kg body weight was injected intraperitoneally, once a day for 6 weeks). The aging rats were then randomly divided into 3 groups: 6 rats as the aging control group; another 6 as the LPS treated aging group (LPS,5 mg/kg body weight intravenous injection); and the third as the GBE+LPS group (6 rats, GBE was started 7 days before the experiment,given once a day via the esophagus, the amount of flavone glycosides administered being 8 mg/kg body weight, and on the day of experiment, one dose of GBE was given 2 hours before LPS administration ). The samples were collected 2 hours after LPS or saline administration. RESULTS: Compared with normal controls, the SOD activity in red cells and the Na(+) -K(+) -ATPase activity in the lung tissue decreased markedly (all P < 0.05), but the LDH activity increased (P < 0.05) in the aging rats. ALI was observed in the aging rats 2 hours after LPS administration. Compared with the aging control, in the LPS treated rats, there were more inflammatory cells in the lung tissue; protein content in bronchial alveolar lavage fluid (BALF) and the pulmonary permeability index (PPI) increased significantly (all P < 0.001); LD, MDA, NO(2) (-)/NO(3)(-), ET-1 and TNF-alpha content and LDH activity in blood, and MPO activity in lung tissue all increased significantly. On the contrary, SOD activity in red cells and Na(+) -K(+) -ATPase activity in lung tissue decreased markedly (P < 0.05, P < 0.01). These changes, except SOD, were markedly attenuated in the GBE+LPS rats. CONCLUSIONS: The anti-oxidant activity was decreased in D-gal-induced aging rats. Intravenous administration of LPS caused acute lung injury in aging rats. GBE had protective effect on ALI induced by LPS.

RETRACTED: Phytoestrogen alpha-zearalanol inhibits homocysteine-induced endothelin-1 expression and oxidative stress in human umbilical vein endothelial cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. After an institutional investigation into the work of Dr. Jun Ren, University of Wyoming subsequently conducted an examination of other selected publications of Dr. Ren's under the direction of the HHS Office of Research Integrity. Based on the findings of this examination, the University of Wyoming recommended this article be retracted due to data irregularities in Figures 3 and 5 that significantly affect the results and conclusions reported in the manuscript.

Angiotensin II stimulates endothelial integrin beta3 expression via nuclear factor-kappaB activation.

The present study examined the effect and part mechanism of angiotensin II-stimulated integrin beta3 gene expression in human umbilical vein endothelial cells. Protein level and mRNA level of integrin beta3 expression were determined using enzyme-linked immunoadsorbent assay and reverse transcription-polymerase chain reaction. Four plasmids of 5'-different deletion of integrin beta3 gene promoter were constructed to transiently transfected into cells to uncover the region in response to angiotensin II. Blockade of nuclear factor-kappaB (NF-kappaB) signaling pathway effect on integrin beta3 expression was analyzed by cotransfection with mutant plasmids for NF-kappaB-inducing kinase, inhibitory proteins alpha and beta of NF-kappaB kinase, respectively, together with the integrin beta3 plasmid including the sequence -1486 approximately - 900. The study found that 10(-8) mol/L,10(-7) mol/L, 10(-6) mol/L, and 10(-5) mol/L angiotensin II increased integrin beta(3) protein level by 45%, 52%, 62%, and 73% respectively. Angiotensin II at 10(-6) mol/L increased integrin beta3 mRNA level by 67%. The luciferase activity of the integrin beta3 plasmid PGL3 - 1486 approximately - 900 increased by 84.72% in response to angiotensin II. N-acetylcysteine blocked angiotensin II-induced NF-kappaB activity and integrin beta3 expression. Blockade of NF-kappaB signaling pathway abolished the stimulation of angiotensin II. These results suggest that angiotensin II stimulates integrin beta3 expression partly by NF-kappaB activation.

TSC-36/FRP inhibits vascular smooth muscle cell proliferation and migration.

OBJECTIVE: In-stent restenosis is a vascular proliferation/migration disorder characterized by hyperplasia of vascular smooth muscle cells (VSMCs). Because mounting evidence suggests that the therapeutic potential of anti-proliferation and anti-migration therapy, we investigated possible inhibitory effects of the matricellular protein TGF-beta-stimulated clone 36 (TSC-36) on vascular smooth muscle cell proliferation and migration in vitro and in vivo. METHODS: Human umbilical artery smooth muscle cells (SMCs) were treated with inducting agents daidzein or estradiol. TSC-36 expression was detected by nested competitive PCR and in situ hybridization. TSC-36 was expressed in Origami (DE3) cells. The recombinant protein was used to immunize rabbits to produce polyclonal antibodies. VSMCs were treated with various concentrations of recombinant TSC-36 (rTSC-36) protein and daidzein. The MTT assay was used to analyze for cell proliferation. A transwell system was used to detect cell migration. Flow cytometry was used to detect cell phase. A rat carotid artery balloon injury model was duplicated. The rats were treated with daidzein or solvent control. Animals were sacrificed 5 weeks later, and injured arteries were taken for pathology and histology. RESULTS: TSC-36 mRNA and protein expression was induced in SMCs. Cell proliferation and migration were inhibited by rTSC-36. rTSC-36 caused accumulation of SMCs in G2 phase. The inducting agent daidzein decreased neo-intima proliferation. TSC-36 mRNA and protein expression was induced and expressed in the neo-intima. CONCLUSION: TSC-36 can be induced in VSMCs and inhibits VSMCs proliferation in vitro and in vivo.

Roles of NF-kappaB and SP-1 in oxidative stress-mediated induction of platelet-derived growth factor-B by TNFalpha in human endothelial cells.

Platelet-derived growth factor-B (PDGF-B) is upregulated by proinflamatory stimuli in the early stages of atherosclerosis. However, its mechanisms are not fully elucidated. In the present study, by using the antioxidant N-acetylcysteine (NAC), we investigated in human umbilical vein endothelial cells (HUVECs) the roles of oxidative stress in PDGF-B expression induced by tumor necrosis factor alpha (TNFalpha) and its underlying mechanisms. Exposure of HUVECs to TNFalpha (200 U/ml) for 24 hours caused significant increases of both the PDGF-B expression and its promoter/enhancer activity, which were abolished by NAC (20 mmol/L). Accordingly, a prolonged oxidative stress was induced by TNFalpha and that was prevented by pretreatment with NAC. Electrophoresis mobility shift assay (EMSA) and Western blot analysis showed that both the nuclear factor-kappaB (NF-kappaB) and the specificity protein-1 (SP-1) were activated by TNFalpha. However, NAC only partially inhibited the TNFalpha-induced activation of NF-kappaB, but abolished the activation of SP-1. Mutation of the NF-kappaB binding site resulted in a moderate reduction in the TNFalpha-induced activity of PDGF-B promoter/enhancer, whereas mutation of SP-1 binding site resulted in an absence of induction by TNFalpha. These results suggest that oxidative stress mediates the TNFalpha-induced expression of PDGF-B in HUVECs through redox-sensitive transcription factors, predominantly the SP-1 and possibly, to some extent of NF-kappaB.

Phytoestrogen alpha-zearalanol antagonizes oxidized LDL-induced inhibition of nitric oxide production and stimulation of endothelin-1 release in human umbilical vein endothelial cells.

Oxidative modification of low-density lipoprotein (LDL) leads to formation of the atherogenic molecule oxidized LDL (oxLDL), which is considered to be an important mediator for vascular endothelial dysfunction and atherosclerosis. It is speculated that reduced nitric oxide (NO) release/bioavailability and enhanced release of endothelin-1 (ET-1) may contribute to oxLDL-induced endothelial dysfunction. Estrogen may improve lipid profile and inhibit oxLDL-induced endothelial damage. However, estrogen replacement therapy has been suspended due to uncertainty in benefits versus risk (such as cancer progression) in postmenopausal women. This study was designed to evaluate the effect of a novel phytoestrogen, alpha-zearalanol (alpha-ZAL), on oxLDL-induced effect on NO and ET-1 production in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with oxLDL (50 microg/mL) for 24 h in the absence or presence of alpha-ZAL (0-1000 nM), 17beta-estradiol (E2, 10 nM), or the E2 receptor antagonist ICI182780 (1 microM). Levels of NO and ET-1 were measured by spectrophotometry and enzymatic immunoassay, respectively. NOS activity was evaluated by conversion of 3H-arginine to 3H-citrulline. Protein and mRNA expression of NOS and ET-1 were measured by Western blot and RT-PCR. Our results indicated that oxLDL significantly reduced NO release and NOS activity, and enhanced ET-1 pro-duction associated with reduced NOS3 (but not NOS2) expression and enhanced ET-1 mRNA expression. All these oxLDL-induced alterations were significantly attenuated or abolished by co-incubation with alpha-ZAL or E2, both through an E2 receptor-dependent mechanism. alpha-ZAL, E2, and ICI182780 had no effect on NO/ET-1 release, NOS activity, or expression of NOS and ET-1. These data suggested that the phytoestrogen alpha-ZAL, like E2, may effectively antagonize oxLDL-induced decrease in NO and increase in ET-1, which may be protective for endothelial function.