Accepted Article
Article Type: Original Article
The role of endoplasmic reticulum stress in endothelial dysfunction induced by homocysteine thiolactone
Running head : ER stress induced by HTL
Shujin Wu, Xiang Gao, ShehuaYang, Min Meng, Xiaolai Yang, Bin Ge*
Department of Pharmacy, Gan Su Provincial Hospital, Lan Zhou 73000, China.
* Correspondence to: Bin Ge, PHD Department of Pharmacy Gan Su Provincial Hospital No.204 West Dong-gang Road Lan Zhou, Gan Su, 730000, China Tel: 086-931-8281754 Fax: 086-931-8281345 E-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/fcp.12101 This article is protected by copyright. All rights reserved.
Accepted Article
ABSTRACT Our and other studies have reported that homocystein thiolactone (HTL) could induce endothelial dysfunction. However, the precise mechanism was largely unknown. In this study we tested the most possible factor-endoplasmic reticulum (ER) stress, which was demonstrated to be involved in endothelial dysfunction in cardiovascular disease. Acetylcholine (Ach)-induced endothelium-dependent relaxation (EDR) and biochemical parameters were measured in rat isolated aorta. The level of reactive oxygen species (ROS) and NO was designed by specific fluorescent probe DCFH-DA and DAF-FM DA separately. The nuclear translocation of the NF-κB was studied by immune-fluorescence. The mRNA and protein expression of GRP78 -a key indicator for the induction of ER stress- were assessed by real-time PCR and western blot. Two ER stress inhibitors-4-PBA (5mM) and Tudca（500μg/mL) -significantly prevented HTL- impaired EDR and increased NO release, endothelial nitric oxide synthase
(eNOS) and SOD activity,
decreased ROS production,
NADPH activity, NOX-4 mRNA and MDA level. We also found that 4-PBA and Tudca blocked HTL-induced NF-κB activation thus inhibiting the downstream target gene production including TNF-α and ICAM-1. Simultaneously, HTL increased the mRNA and protein level of GRP78. HTL could induce ER stress leading to a downstream enhancement of oxidative stress and inflammation, which finally caused vascular endothelial dysfunction.
Key words:
homocystein thiolactone, endoplasmic reticulum stress, endothelial
dysfunction, oxidative stress，NF-κB
INTRODUCTION Hyperhomocysteinemia (HHcy) is a significant clinical problem. It is an independent risk factor for cardiovascular disease (CVD) [1,2]. It has recently been reported that homocysteine thiolactone (HTL) -formed by enzymatic conversion of Hcy in all cell types- might be the This article is protected by copyright. All rights reserved.
Accepted Article
4. Bełtowski J. Protein homocysteinylation: a new mechanism of atherogenesis? Postepy Hig. Med. Dosw.(2005) 59 392-404. 5. Landmesser U., Hornig B., Drexler H. Endothelial dysfunction in hypercholesterolemia: mechanisms, pathophysiological importance, and therapeutic interventions. Semin. Thromb. Hemost. (2000) 26 529-537. 6. Sena C.M., Pereira A.M., Seiça R. Endothelial dysfunction - a major mediator of diabetic vascular disease. Biochim. Biophys. Acta.(2013)1832 2216-2231. 7. Liu Y.H., You Y., Song T., Wu S.J., Liu L.Y. Impairment of endothelium-dependent relaxation of rat aortas by homocysteine thiolactone and attenuation by captopril. J. Cardiovasc. Pharmacol. (2007) 50 155-161. 8. Li H. , Förstermann U. Uncoupling of endothelial NO synthase in atherosclerosis and vascular disease. Curr. Opin. Pharmacol.(2013) 13 161-167. 9. Tabas I. The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ. Res. (2010) 107 839-850. 10. Zeng W., Guo Y.H., Qi W., Chen J.G., Yang L.L., Luo Z.F., Mu J., Feng B. 4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulu m stress of endothelial cells stimulated by uremic serum. Life Sci. (2014) 103 15-24. 11. Galán
M., Kassan
M., Kadowitz
P.J., Trebak
M., Belmadani
S., Matrougui
K.
Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunct-ion. Biochim. Biophys. Acta. (2014) 1843 1063-1075. 12. Minamino
T., Komuro
I., Kitakaze
M.
Endoplasmic
reticulum
stress as
a therapeutic target in cardiovascular disease. Circ. Res.( 2010) 107 1071-1082. 13.
Austin
R.C., Lentz
S.R., Werstuck
G.H.
Role
of
hyperhomocysteinemia
in endothelial dysfunction and atherothrombotic disease. Cell Death Differ.(2004) 11 56-64. 14. Dionisio N., Jardín I., Salido G.M., Rosado J.A. Homocysteine, intracellular signaling and thrombotic disorders. Curr. Med. Chem. (2010) 17 3109-3119.
This article is protected by copyright. All rights reserved.
Accepted Article
The endothelial function played such an important role in maintaining blood pressure and vascular function that this study was aimed to determine whether the ER stress was linked to HTL-induced endothelial dysfunction. The finding of this study could suggest a new strategy to prevent or treat HTL-induced vascular disease.
Reagents
Sodiumnitroprusside (SNP), Acetylcholine (Ach), homocysteine thiolactone (HTL) and phenylephrine (Phe), 4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (Tudca) were purchased from Sigma Chemical Co (Saint Louis, Mo, USA). The kits for measurement of nitric oxide (NO), endothelial nitric oxide synthase (eNOS), reactive oxygen species (ROS),
superoxide
dismutase
(SOD)
，
malondiadehyde
(MDA),
3-Amino,4-aminomethyl-2',7'-difluorescein, diacetate (DAF-FM DA) were purchased from Beyotime Institute of Biotechnology, China. All other reagents were of the highest purity available.
Animal
Male Sprague-Dawley rats, 180±20g, 6-8 weeks of age, were obtained from the Animal Department of Gan Su University of Chinese Traditional Medicine (Lan Zhou, China). All animal experimental procedures were approved by the institutional animal care and use committee at the University of Gan Su Chinese Traditional Medicine and performed
This article is protected by copyright. All rights reserved.
Accepted Article
according to the guidelines of animal ethics committee for use of experimental animals in China.
Organ chamber
Organ chamber experiments were performed as described previously [17]. Briefly, rings (3-5 mm in length) from rat aortas, free of fat and connective tissue, were mounted in organ bath in 5 mL Kreb’s solution at 37℃, gassed with 95%O2+5%CO2, under attention of 2 g, for 1 h equilibration period. The tension of the aortic ring was monitored by a force transducer and recorded on a polygraph (Model RM6240B/C; Chengdu Instruments, China). After the equilibration, rings were contracted with 60mM KCl. After washing and another 30 min equilibration, contractile response was evoked by Phe (1mM) to elicit reproducible responses. At the plateau of contraction, accumulative Ach (0.003-3 mM) or SNP (0.001-1 mM) was added into the organ bath to induce the endothelium dependent/independent relaxation. The rings with maximal relaxation ratio (Emax) to Ach of 3 mM more than 75% were considered to have intact endothelium and were used in the study.
Determination of MDA concentration and SOD activity in aortic rings
After a 6h incubation of aortic segments, the aortic segments were blotted dry and weighed, then made into 5% tissue homogenate in ice-cold 0.9% NaCl solution. A supernatant was obtained from tissue homogenate by centrifugalization (1000×g, 4 ºC, 10 min). The MDA concentration (thiobarbituric acid reactive substances, TBARS) in the supernatant was measured. TBARS results were expressed as MDA equivalents using tetraethoxypropaneas This article is protected by copyright. All rights reserved.
Accepted Article
standard. The SOD activity was assayed by the method of nitrotetrazolium blue chloride (NBT). The amount of protein that inhibited NBT reduction to 50% maximum was defined as 1 nitrite unit (NU) of SOD activity.
Assay of aorta NO concentration Aorta NO production was assayed using specific fluorescent dye DAF-FM DA as described previously [18]. Briefly, the fresh aorta sections were placed on a slide. The tissue was loaded with 5μM DAF-FM DA and maintained in a humidified 37℃ incubator gassed with 5% CO2 for 20min and washed three times with PBS buffer. The production of NO was assessed using a confocal scanning laser microscope (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Germany). The fluorescence was read at 495nm for excitation and 515 nm for emission.
Primary Cell culture
Collagenase was used to digest Human Umbilical Vein Endothelial Cell (HUVEC). The suspension was centrifuged and cells were re-suspended. After 1h incubation, the medium was refreshed to remove unattached cells. Thereafter, the cells were placed in an incubator at 37°C with 5% CO2 and transferred to a 12-well plate until 70–80% confluence. The cells were seeded in various standard-well plates for testing biochemical indexes. One day after seeding, the culture wells were pretreated with 4-PBA (5mM) and Tudca（500μg/mL) for 2 h,
This article is protected by copyright. All rights reserved.
Accepted Article
and then treated with HTL (10mmol/L) for an additional 48 h. Endothelial nitric oxide synthase (eNOS) activity assay eNOS activity was monitored by L-[3H] coralline production from L-[3H] arginine. Briefly, protein samples were incubated in reaction buffer [1 mM L-arginine/100 mM NADPH/1 mM tetrahydrobiopterin/0.2µCi of L-[3H] arginine (>66 Ci/mmol) per reaction] for 15 min at 37℃, separated by Dowex-50W ion-exchange chromatography in 20 mM HEPES (pH 5.5), 2 mM ethylene-diamine-tetra-acetic acid, and 2 mM ethyleneglycol tetra-acetic acid, and the flow-through was used for liquid scintillation counting.
Assay of ROS Production ROS were measured with the fluorescence probe DCFH-DA followed by the instruction manual. Briefly, the fresh aorta sections and the cells were placed or seeded on a slide in 6-well plates. The tissue or cells were loaded with 5μM DCFH-DA and maintained in a humidified 37℃ incubator gassed with 5% CO2 for 20min and washed three times with PBS buffer. The fluorescence was read at 488 nm for excitation and 525 nm for emission with a confocal scanning laser microscope (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Germany).
This article is protected by copyright. All rights reserved.
Accepted Article
Determination of NF-κB translocation
After the indicated treatments, the cells were fixed with stationary liquid for 5 min and washed with PBS and then treated with immune-staining confining liquid for 1h at room temperature. After removing the immune-staining confining liquid, the cells were incubated with the NF-κB p65 antibody at 4℃ over night. After removing the antibody and washing with PBS, the cells were treated with DAPI for 5 min at room temperature. Last appropriate anti-fluorescence quench mounting liquid was added and mounted on slides. Fluorescence staining was evaluated using Nikon Eclipse E800 epifluorescence microscope connected to a digital camera and interfaced with a computer.
Western Analysis
After the indicated treatments, cell extracts were prepared in phosphate-buffered saline that contained 25 μl of protease inhibitor cocktail. Aliquots of the cell extract (40–100 μg of protein) were separated by SDS-PAGE, and western blot analyses were carried out using the indicated antibodies. Antibody binding was detected by enhanced chemiluminescence. The bands were scanned and densitometrically analyzed using an automaticimage analysis system (Alpha Innotech Corp., San Leandro,Calif., USA). These quantitative analyses were normalized to β-actin.
This article is protected by copyright. All rights reserved.
Accepted Article
NADPH oxidase activity assay
NADPH oxidase activity was measured by using a lucigenin-derived chemiluminescence method. Briefly, the cells were harvested, washed with PBS (0.1 mmol/L, pH 7.0), lysed in 1.5 mL of 50 mmol/L phosphate buffer, and then homogenized 100 times on ice. The lysates were centrifuged at 16000 g for 25 min. Pellets were stored at -80 °C until use. An aliquot (50μg) of the pellets was diluted in 500μL of the same phosphate buffer. Dark-adapted lucigenin (20μmol/L) was added to the sample, and chemiluminescence was immediately measured. Chemiluminescence was measured at 30s intervals for 5 min by using a GloMax-20/20 luminometer (Turner Biosystems, Inc., USA). The results are expressed as a percentage of the control value.
Real-time RT- PCR analysis Total RNA was extracted with TRIzol. The quantitative real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems). The final reaction contained 10.7 μl of SYBR green/enzyme reaction mix, 0.4 μM of primer and 1 μl of cDNA in a total volume of 25 μl. The typical profile times used were an initial step of 95 °C for 10 min, followed by a second step at 95 °C for 15 s and 60 °C for 60 s for 40 cycles. GAPDH was used as an internal control. All results were repeated in six independent experiments and performed in triplicate each time. The following primers for realtime-PCR were used: forward primer for NOX4, 5’-GCCAACGAAGGGGTTAAACA-3’; reverse primer for NOX4, 5’-CGGGAACCAATATGTTCGTTCTTC-3’；GRP78 forward primer: 5’-CGG GCA AAG ATG TCA GGA AAG-3’, reverse primer: 5’-TTC TGG ACG GGCTTC This article is protected by copyright. All rights reserved.
Accepted Article
ATA GTA GAC-3’; GAPDH sense 5’-TCG ACA GTCAGC CGC ATC TTC TTT-3’, antisense 5’-ACC AAA TCC GTTGAC TCC GAC CTT-3’.
Statistical Analysis Relaxation response was calculated and expressed as percentage of the contraction elicited by Phe. Results are expressed as mean±SD. Data were analyzed using a one-way or two-way ANOVA followed by Newman-Student’s t-test. P< 0.05 was considered significant.
RESULTS
Effects of 4-PBA and Tudca on the impairment of endothelium dependent- relaxation (EDR) induced by HTL
As shown in Figure1A, incubation of aortic rings with 0.1, 1, and 10 mmol/L HTL for 90 min attenuated relaxation to ACh in a concentration-dependent mode compared with control group. The Emax response was reduced from 86.7± 6.0% to 69.0 ± 4.9%, 56.8 ± 3.7%, 47.0 ± 4.2 %, respectively (n=8, P < 0.05).
Treatment with ER stress inhibitors-4-PBA (5mM) and Tudca（500μg/mL)- significantly prevented inhibition of EDR induced by HTL (10mmol/L). The Emax increased from 48.0±4.1% to 73.1 ±4.0%, 69.8±3.8%, respectively (Figure1B). There was a significant difference (PT mutation. Mol. Genet. Metab.（2011）104 566-573. 25.
G.B., Kim
Park
Y.S., Lee
H.K., Song
H., Cho
D.H., Lee
W.J., Hur
D.Y.
Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-l inking of CD70 isdependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway. J. Immunol.（ 2010）185 7274-7284. 26.
Lentz S.R. Mechanisms of homocysteine-induced atherothrombosis. J. Thromb. Haemost. （2005）3 1646-1654.
FIGURE LEGENDS Figure 1.
After a 90 min incubation of aortic rings, the ACh (0.003-3 μmol/L) or SNP (0.001 to1mmol/L)-induced endothelium dependent or independent relaxations (EDR/EIDR) were measured. A Effects of HTL (0.1, 1,10μmol/L) on EDR. B Effects of 4-PBA (5mM) and Tudca（500μg/mL) on EDR impaired by HTL (10μmol/L). Data are expressed as means±S.E.M (n=8). +P
Article Type: Original Article
The role of endoplasmic reticulum stress in endothelial dysfunction induced by homocysteine thiolactone
Running head : ER stress induced by HTL
Shujin Wu, Xiang Gao, ShehuaYang, Min Meng, Xiaolai Yang, Bin Ge*
Department of Pharmacy, Gan Su Provincial Hospital, Lan Zhou 73000, China.
* Correspondence to: Bin Ge, PHD Department of Pharmacy Gan Su Provincial Hospital No.204 West Dong-gang Road Lan Zhou, Gan Su, 730000, China Tel: 086-931-8281754 Fax: 086-931-8281345 E-mail: [email protected] This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/fcp.12101 This article is protected by copyright. All rights reserved.
Accepted Article
ABSTRACT Our and other studies have reported that homocystein thiolactone (HTL) could induce endothelial dysfunction. However, the precise mechanism was largely unknown. In this study we tested the most possible factor-endoplasmic reticulum (ER) stress, which was demonstrated to be involved in endothelial dysfunction in cardiovascular disease. Acetylcholine (Ach)-induced endothelium-dependent relaxation (EDR) and biochemical parameters were measured in rat isolated aorta. The level of reactive oxygen species (ROS) and NO was designed by specific fluorescent probe DCFH-DA and DAF-FM DA separately. The nuclear translocation of the NF-κB was studied by immune-fluorescence. The mRNA and protein expression of GRP78 -a key indicator for the induction of ER stress- were assessed by real-time PCR and western blot. Two ER stress inhibitors-4-PBA (5mM) and Tudca（500μg/mL) -significantly prevented HTL- impaired EDR and increased NO release, endothelial nitric oxide synthase
(eNOS) and SOD activity,
decreased ROS production,
NADPH activity, NOX-4 mRNA and MDA level. We also found that 4-PBA and Tudca blocked HTL-induced NF-κB activation thus inhibiting the downstream target gene production including TNF-α and ICAM-1. Simultaneously, HTL increased the mRNA and protein level of GRP78. HTL could induce ER stress leading to a downstream enhancement of oxidative stress and inflammation, which finally caused vascular endothelial dysfunction.
Key words:
homocystein thiolactone, endoplasmic reticulum stress, endothelial
dysfunction, oxidative stress，NF-κB
INTRODUCTION Hyperhomocysteinemia (HHcy) is a significant clinical problem. It is an independent risk factor for cardiovascular disease (CVD) [1,2]. It has recently been reported that homocysteine thiolactone (HTL) -formed by enzymatic conversion of Hcy in all cell types- might be the This article is protected by copyright. All rights reserved.
Accepted Article
4. Bełtowski J. Protein homocysteinylation: a new mechanism of atherogenesis? Postepy Hig. Med. Dosw.(2005) 59 392-404. 5. Landmesser U., Hornig B., Drexler H. Endothelial dysfunction in hypercholesterolemia: mechanisms, pathophysiological importance, and therapeutic interventions. Semin. Thromb. Hemost. (2000) 26 529-537. 6. Sena C.M., Pereira A.M., Seiça R. Endothelial dysfunction - a major mediator of diabetic vascular disease. Biochim. Biophys. Acta.(2013)1832 2216-2231. 7. Liu Y.H., You Y., Song T., Wu S.J., Liu L.Y. Impairment of endothelium-dependent relaxation of rat aortas by homocysteine thiolactone and attenuation by captopril. J. Cardiovasc. Pharmacol. (2007) 50 155-161. 8. Li H. , Förstermann U. Uncoupling of endothelial NO synthase in atherosclerosis and vascular disease. Curr. Opin. Pharmacol.(2013) 13 161-167. 9. Tabas I. The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ. Res. (2010) 107 839-850. 10. Zeng W., Guo Y.H., Qi W., Chen J.G., Yang L.L., Luo Z.F., Mu J., Feng B. 4-Phenylbutyric acid suppresses inflammation through regulation of endoplasmic reticulu m stress of endothelial cells stimulated by uremic serum. Life Sci. (2014) 103 15-24. 11. Galán
M., Kassan
M., Kadowitz
P.J., Trebak
M., Belmadani
S., Matrougui
K.
Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunct-ion. Biochim. Biophys. Acta. (2014) 1843 1063-1075. 12. Minamino
T., Komuro
I., Kitakaze
M.
Endoplasmic
reticulum
stress as
a therapeutic target in cardiovascular disease. Circ. Res.( 2010) 107 1071-1082. 13.
Austin
R.C., Lentz
S.R., Werstuck
G.H.
Role
of
hyperhomocysteinemia
in endothelial dysfunction and atherothrombotic disease. Cell Death Differ.(2004) 11 56-64. 14. Dionisio N., Jardín I., Salido G.M., Rosado J.A. Homocysteine, intracellular signaling and thrombotic disorders. Curr. Med. Chem. (2010) 17 3109-3119.
This article is protected by copyright. All rights reserved.
Accepted Article
The endothelial function played such an important role in maintaining blood pressure and vascular function that this study was aimed to determine whether the ER stress was linked to HTL-induced endothelial dysfunction. The finding of this study could suggest a new strategy to prevent or treat HTL-induced vascular disease.
Reagents
Sodiumnitroprusside (SNP), Acetylcholine (Ach), homocysteine thiolactone (HTL) and phenylephrine (Phe), 4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (Tudca) were purchased from Sigma Chemical Co (Saint Louis, Mo, USA). The kits for measurement of nitric oxide (NO), endothelial nitric oxide synthase (eNOS), reactive oxygen species (ROS),
superoxide
dismutase
(SOD)
，
malondiadehyde
(MDA),
3-Amino,4-aminomethyl-2',7'-difluorescein, diacetate (DAF-FM DA) were purchased from Beyotime Institute of Biotechnology, China. All other reagents were of the highest purity available.
Animal
Male Sprague-Dawley rats, 180±20g, 6-8 weeks of age, were obtained from the Animal Department of Gan Su University of Chinese Traditional Medicine (Lan Zhou, China). All animal experimental procedures were approved by the institutional animal care and use committee at the University of Gan Su Chinese Traditional Medicine and performed
This article is protected by copyright. All rights reserved.
Accepted Article
according to the guidelines of animal ethics committee for use of experimental animals in China.
Organ chamber
Organ chamber experiments were performed as described previously [17]. Briefly, rings (3-5 mm in length) from rat aortas, free of fat and connective tissue, were mounted in organ bath in 5 mL Kreb’s solution at 37℃, gassed with 95%O2+5%CO2, under attention of 2 g, for 1 h equilibration period. The tension of the aortic ring was monitored by a force transducer and recorded on a polygraph (Model RM6240B/C; Chengdu Instruments, China). After the equilibration, rings were contracted with 60mM KCl. After washing and another 30 min equilibration, contractile response was evoked by Phe (1mM) to elicit reproducible responses. At the plateau of contraction, accumulative Ach (0.003-3 mM) or SNP (0.001-1 mM) was added into the organ bath to induce the endothelium dependent/independent relaxation. The rings with maximal relaxation ratio (Emax) to Ach of 3 mM more than 75% were considered to have intact endothelium and were used in the study.
Determination of MDA concentration and SOD activity in aortic rings
After a 6h incubation of aortic segments, the aortic segments were blotted dry and weighed, then made into 5% tissue homogenate in ice-cold 0.9% NaCl solution. A supernatant was obtained from tissue homogenate by centrifugalization (1000×g, 4 ºC, 10 min). The MDA concentration (thiobarbituric acid reactive substances, TBARS) in the supernatant was measured. TBARS results were expressed as MDA equivalents using tetraethoxypropaneas This article is protected by copyright. All rights reserved.
Accepted Article
standard. The SOD activity was assayed by the method of nitrotetrazolium blue chloride (NBT). The amount of protein that inhibited NBT reduction to 50% maximum was defined as 1 nitrite unit (NU) of SOD activity.
Assay of aorta NO concentration Aorta NO production was assayed using specific fluorescent dye DAF-FM DA as described previously [18]. Briefly, the fresh aorta sections were placed on a slide. The tissue was loaded with 5μM DAF-FM DA and maintained in a humidified 37℃ incubator gassed with 5% CO2 for 20min and washed three times with PBS buffer. The production of NO was assessed using a confocal scanning laser microscope (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Germany). The fluorescence was read at 495nm for excitation and 515 nm for emission.
Primary Cell culture
Collagenase was used to digest Human Umbilical Vein Endothelial Cell (HUVEC). The suspension was centrifuged and cells were re-suspended. After 1h incubation, the medium was refreshed to remove unattached cells. Thereafter, the cells were placed in an incubator at 37°C with 5% CO2 and transferred to a 12-well plate until 70–80% confluence. The cells were seeded in various standard-well plates for testing biochemical indexes. One day after seeding, the culture wells were pretreated with 4-PBA (5mM) and Tudca（500μg/mL) for 2 h,
This article is protected by copyright. All rights reserved.
Accepted Article
and then treated with HTL (10mmol/L) for an additional 48 h. Endothelial nitric oxide synthase (eNOS) activity assay eNOS activity was monitored by L-[3H] coralline production from L-[3H] arginine. Briefly, protein samples were incubated in reaction buffer [1 mM L-arginine/100 mM NADPH/1 mM tetrahydrobiopterin/0.2µCi of L-[3H] arginine (>66 Ci/mmol) per reaction] for 15 min at 37℃, separated by Dowex-50W ion-exchange chromatography in 20 mM HEPES (pH 5.5), 2 mM ethylene-diamine-tetra-acetic acid, and 2 mM ethyleneglycol tetra-acetic acid, and the flow-through was used for liquid scintillation counting.
Assay of ROS Production ROS were measured with the fluorescence probe DCFH-DA followed by the instruction manual. Briefly, the fresh aorta sections and the cells were placed or seeded on a slide in 6-well plates. The tissue or cells were loaded with 5μM DCFH-DA and maintained in a humidified 37℃ incubator gassed with 5% CO2 for 20min and washed three times with PBS buffer. The fluorescence was read at 488 nm for excitation and 525 nm for emission with a confocal scanning laser microscope (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Germany).
This article is protected by copyright. All rights reserved.
Accepted Article
Determination of NF-κB translocation
After the indicated treatments, the cells were fixed with stationary liquid for 5 min and washed with PBS and then treated with immune-staining confining liquid for 1h at room temperature. After removing the immune-staining confining liquid, the cells were incubated with the NF-κB p65 antibody at 4℃ over night. After removing the antibody and washing with PBS, the cells were treated with DAPI for 5 min at room temperature. Last appropriate anti-fluorescence quench mounting liquid was added and mounted on slides. Fluorescence staining was evaluated using Nikon Eclipse E800 epifluorescence microscope connected to a digital camera and interfaced with a computer.
Western Analysis
After the indicated treatments, cell extracts were prepared in phosphate-buffered saline that contained 25 μl of protease inhibitor cocktail. Aliquots of the cell extract (40–100 μg of protein) were separated by SDS-PAGE, and western blot analyses were carried out using the indicated antibodies. Antibody binding was detected by enhanced chemiluminescence. The bands were scanned and densitometrically analyzed using an automaticimage analysis system (Alpha Innotech Corp., San Leandro,Calif., USA). These quantitative analyses were normalized to β-actin.
This article is protected by copyright. All rights reserved.
Accepted Article
NADPH oxidase activity assay
NADPH oxidase activity was measured by using a lucigenin-derived chemiluminescence method. Briefly, the cells were harvested, washed with PBS (0.1 mmol/L, pH 7.0), lysed in 1.5 mL of 50 mmol/L phosphate buffer, and then homogenized 100 times on ice. The lysates were centrifuged at 16000 g for 25 min. Pellets were stored at -80 °C until use. An aliquot (50μg) of the pellets was diluted in 500μL of the same phosphate buffer. Dark-adapted lucigenin (20μmol/L) was added to the sample, and chemiluminescence was immediately measured. Chemiluminescence was measured at 30s intervals for 5 min by using a GloMax-20/20 luminometer (Turner Biosystems, Inc., USA). The results are expressed as a percentage of the control value.
Real-time RT- PCR analysis Total RNA was extracted with TRIzol. The quantitative real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems). The final reaction contained 10.7 μl of SYBR green/enzyme reaction mix, 0.4 μM of primer and 1 μl of cDNA in a total volume of 25 μl. The typical profile times used were an initial step of 95 °C for 10 min, followed by a second step at 95 °C for 15 s and 60 °C for 60 s for 40 cycles. GAPDH was used as an internal control. All results were repeated in six independent experiments and performed in triplicate each time. The following primers for realtime-PCR were used: forward primer for NOX4, 5’-GCCAACGAAGGGGTTAAACA-3’; reverse primer for NOX4, 5’-CGGGAACCAATATGTTCGTTCTTC-3’；GRP78 forward primer: 5’-CGG GCA AAG ATG TCA GGA AAG-3’, reverse primer: 5’-TTC TGG ACG GGCTTC This article is protected by copyright. All rights reserved.
Accepted Article
ATA GTA GAC-3’; GAPDH sense 5’-TCG ACA GTCAGC CGC ATC TTC TTT-3’, antisense 5’-ACC AAA TCC GTTGAC TCC GAC CTT-3’.
Statistical Analysis Relaxation response was calculated and expressed as percentage of the contraction elicited by Phe. Results are expressed as mean±SD. Data were analyzed using a one-way or two-way ANOVA followed by Newman-Student’s t-test. P< 0.05 was considered significant.
RESULTS
Effects of 4-PBA and Tudca on the impairment of endothelium dependent- relaxation (EDR) induced by HTL
As shown in Figure1A, incubation of aortic rings with 0.1, 1, and 10 mmol/L HTL for 90 min attenuated relaxation to ACh in a concentration-dependent mode compared with control group. The Emax response was reduced from 86.7± 6.0% to 69.0 ± 4.9%, 56.8 ± 3.7%, 47.0 ± 4.2 %, respectively (n=8, P < 0.05).
Treatment with ER stress inhibitors-4-PBA (5mM) and Tudca（500μg/mL)- significantly prevented inhibition of EDR induced by HTL (10mmol/L). The Emax increased from 48.0±4.1% to 73.1 ±4.0%, 69.8±3.8%, respectively (Figure1B). There was a significant difference (PT mutation. Mol. Genet. Metab.（2011）104 566-573. 25.
G.B., Kim
Park
Y.S., Lee
H.K., Song
H., Cho
D.H., Lee
W.J., Hur
D.Y.
Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-l inking of CD70 isdependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway. J. Immunol.（ 2010）185 7274-7284. 26.
Lentz S.R. Mechanisms of homocysteine-induced atherothrombosis. J. Thromb. Haemost. （2005）3 1646-1654.
FIGURE LEGENDS Figure 1.
After a 90 min incubation of aortic rings, the ACh (0.003-3 μmol/L) or SNP (0.001 to1mmol/L)-induced endothelium dependent or independent relaxations (EDR/EIDR) were measured. A Effects of HTL (0.1, 1,10μmol/L) on EDR. B Effects of 4-PBA (5mM) and Tudca（500μg/mL) on EDR impaired by HTL (10μmol/L). Data are expressed as means±S.E.M (n=8). +P
