Author’s Accepted Manuscript Inhibition of angiotension II type 1 receptor reduced human endothelial inflammation induced by low shear stress Yuelin Chao, Linlin Zhu, Xinliang Qu, Junxia Zhang, Wen Wu, Peng Ye, Jie Luo, Hongfeng Yang, Shaoliang Chen www.elsevier.com/locate/yexcr
PII: DOI: Reference:
S0014-4827(17)30451-2 http://dx.doi.org/10.1016/j.yexcr.2017.08.030 YEXCR10716
To appear in: Experimental Cell Research Received date: 4 June 2017 Revised date: 19 August 2017 Accepted date: 21 August 2017 Cite this article as: Yuelin Chao, Linlin Zhu, Xinliang Qu, Junxia Zhang, Wen Wu, Peng Ye, Jie Luo, Hongfeng Yang and Shaoliang Chen, Inhibition of angiotension II type 1 receptor reduced human endothelial inflammation induced by low shear stress, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2017.08.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of angiotension II type 1 receptor reduced human endothelial inflammation induced by low shear stress Yuelin Chao*, Linlin Zhu*, Xinliang Qu*, Junxia Zhang*, Wen Wu*, Peng Ye*, JieLuo*, *Department
Hongfeng Yang*, Shaoliang Chen*,1
of Cardiology, Nanjing First Hospital, Nanjing Medical
University, Nanjing, China 1
Correspondence: Department of Cardiology, Nanjing First Hospital,
Nanjing Medical University, #68 Changle Road, Nanjing, China E-mail: [email protected]
Abstract Low shear stress (LSS)-induced endothelial inflammation is the basis for the development of atherosclerosis. However, the mechanism underlying LSS-induced inflammation is not well understood. The angiotensin II type 1 receptor (AT1R), a component of the renin-angiotensin system, participates in atherosclerotic plaque progression. The aim of this study was to investigate the role of AT1R in LSS-induced endothelial activation. Using immunohistochemistry, we noted significant increases in AT1R, vascular endothelial adhesion cell-1 (VCAM1), and intercellular adhesion molecule-1 (ICAM1) expression in the inner curvature of the aortic arch in C57BL/6 mice compared to the descending aorta in these mice. Moreover, western blotting revealed that these LSS-induced increases in AT1R, ICAM1 and VCAM1 expression were time dependent. However, the expression of these proteins was significantly abolished by treatment with the AT1R antagonist Losartan (1 μM) or AT1R small interfering RNA (siRNA). AT1R inhibition significantly suppressed extracellular signal-regulated kinase 1/2 (ERK) upregulation, which also resulted in decreases in ICAM1 and VCAM1 protein expression. These findings demonstrate that LSS induces endothelial inflammation via AT1R/ERK signaling and that Losartan has beneficial effects on endothelial inflammation.
Key words:
Low shear stress•Angiotensin II type 1 receptor•Inflammation•
Mechanotransduction
Introduction Atherosclerosis is chronic vascular inflammation, to which specific sites within the vascular tree, such as arterial bifurcations, branch points, and curvatures, are predisposed. These sites are characterized by low shear stress [1-3] . Low shear stress (LSS), a force to which endothelial cells are subjected, plays important roles in the initial formation and progression of atherosclerotic plaques and the development of vascular inflammation [4]. Ishibazawa et al determined that LSS induced the expression of proinflammatory genes and proteins, such as vascular endothelial adhesion cell-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-selectin [5]. Similarly, evidence by Mohan S showed LSS increased markedly VCAM1 mRNA expression in HAEC exposed to 6 hours of LSS [6]. However, the specific molecular mechanism underlying this phenomenon is unknown. The angiotensin II type 1 receptor (AT1R), one of members of the G protein-coupled receptors (GPCRs) family that sense mechanical stress, interacts with angiotensin II (Ang II) and exerts intracellular effects [2]. Recent studies have shown that Ang II induces inflammation through AT1R and that Losartan induces plaque regression by decreasing the expression of pro-inflammatory proteins, such as VCAM1 [7]. Therefore, we propose that LSS induces inflammation through AT1R. In the present study, we investigated the role of AT1R in LSS-induced inflammation and identified the downstream molecules that are potentially involved in this process.
Materials and methods Animals The protocols for all of the animal experiments were approved by the Institutional Animal Care
and
Use
Committee
of
Nanjing
Medical
University
(permit
number:
SYXK2016-0006 ). Eight-week-old C57BL/6 mice from the Animal Center of Nanjing Medical University were euthanized by a lethal dose of sodium pentobarbital. Their aortas were rinsed with cold PBS thrice using a cannula inserted into the left ventricle and then fixed with 4% paraformaldehyde for 30 min [3]. The inner curvature of the aortic arch and the descending aorta were separated and embedded in OCT compound. Successive lateral tissue sections were cut using a microtome and transferred onto slides, which were blocked with 5% BSA after three treatments with PBS. Then, an anti-AT1R antibody (CST), an anti-CD31 antibody and an anti-VCAM-1 or anti-ICAM1 antibody (Abcam) were successively applied to the tissues, after which the appropriate secondary antibodies (CST) were applied to the tissues. The segments were then cultured with medium containing DAPI (Beyotime).
Cell culture The human vein endothelial cell (HUVEC) line EA.hy926 (Cellbank of Chinese Academy of Sciences, Shanghai) was grown to confluence in a humidified incubator at 37°C with 5% CO2 incubator. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBICO). Upon reaching 100% confluence, the cells were placed on separate slides for subsequent experiments. In some of the experiments, the cells were pretreated with Losartan and PD98059 (TOCRIS) for 30 minutes and subsequently applied to shear stress.
Application of shear stress A parallel flow chamber (Shanghai Medical Instrument School, Shanghai, China), which consists of two stainless-steel plates and a silicon gasket, as described elsewhere [8], was used in this study. A glass slide with confluent cells was placed on the lower plate of the chamber and then subjected to LSS induced by continuous fluid flow. Shear stress values (3 dyn/cm2) were modulated by the flow though the chamber.
Materials and reagents Losartan and PD98059 were purchased from TOCRICS, while Trizol reagent was purchased from Invitrogen. The following primary antibodies were obtained commercially: anti-AT1R (sc-1173, 1:100, Cruz for western blotting; 9391, 1:50, Abcam for immunofluorescence), anti-VCAM1 (ab98954, 1:1000, Abcam), anti-ICAM1 (sc-1511, 1:100 for western blotting, Santa Cruz, 2213, 1:40, Abcam for immunoflurescence), anti-Phospho-NF-kB p65 (3031, 1:1000, CST), anti-NF-κB p65 (6956, 1:1000, CST), anti-Phospho-ERK (4370, 1:1000, CST), anti-ERK (9102, 1:1000, CST), anti-P-JNK (9251, 1:1000, CST), anti-JNK (9252, 1:1000, CST), anti-Phospho-P38 (9211, 1:1000, CST), anti-P38 (8690, 1:1000, CST) and GAPDH (2118, 1:1000, CST).
Immunofluorescence HUVECs were fixed in 4% paraformaldehyde at room temperature for 30 min and then permeabilized in 0.1%Triton X-100/PBS for 10 min. After being blocked with BSA for 30 min, the cells were incubated with the following primary antibodies overnight at 4°C: mouse monoclonal anti-AT1R (9391, 1:50, Abcam), anti-ICAM1 (2213, 1:40, Abcam), and anti-VCAM1 (98954, 1:40; Abcam). After being washed with TBS-T, the cells were incubated with the appropriate secondary antibodies (1:200; CST). The slides were then incubated with DAPI (1:50; Beyotime), according to the manufacturer’s instructions. Images were acquired by laser scanning confocal microscopy (LSM 710; Carl Zeiss, Germany).
Western blotting Confluent cell monolayers supplemented with 5% FBS and 1% penicillin-streptomycin liquid on glass slides were subjected to LSS for different times. The cells were washed with ice-cold PBS twice and lysed in a mixture of RIPA, phosphatase inhibitor and proteinase inhibitor (100:10:1). The lysates were centrifuged at 12 000×g for 20 min at 4°C after incubating for 20 min on ice. To get Nuclear proteins, cells were treated with nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, China) according to the manufacture's instructions. Protein concentrations were quantified using bicinchoninic acid protein assay. The cell lysate (50 μg) supernatants were treated with SDS–PAGE sample buffer and then separated by 10% SDS-PAGE before being transferred to PVDF membranes, which were incubated with the appropriate primary antibodies overnight after being blocked with 5% milk for two hours. The signals were detected by a chemiluminescent HRP Substrate Kit (ECL, Millipore), and the bands were revealed using the GeneGnome (SYNGENE). The intensities of the protein bands were also measured using gene software.
Quantitative real-time PCR and small interfering RNA (siRNA) Total RNA was isolated with Trizol reagent, according to the manufacturers’ specifications, and then reverse transcribed to cDNA using a TaKaRa kit (RR037A). cDNA was amplified using a TaKaRa kit (RR420A), according to the manufacturer’s instructions, as well as the primers listed below and a Fast 7500. Amplification of GAPDH, which served as a control, was performed for normalization. The following PCR primers used were used in the study: ICAM1, F: 5-GTCCCCTCAAAAGTCATCC-3 and R: 5-AACCCCATTCAGCGTCACC-3; VCAM1, F:5-GGTGGGACACAAATAAGGGTTTTGG-3 and R: 5-CTTGCAATTCTTTTACA GCCTGCC-3. AT1R, F: 5-GCACCAGGTGTATTTGATAG, and AT1R, R: 5-CTGTTTCCAA ATATTCCCACC. GAPDH, F: 5-CCACCCATGGCAAATTCCATGGCA, and GAPDH, R: 5TCTACACGGCAGGTCAGGTCCACC. After the cells reached 50% confluency, we diluted 30 µl of Lipofectamine-RNAIMAX reagent (Invitrogen) and 20 µl of siRNA solution (20 µM) in two separate tubes containing 200 µl of opti-MEMI medium without FBS and incubated the mixtures for 5 min. Then, the mixtures were added together in another tube and incubated for an additional 20 minutes at room temperature. The cells were placed on several slides after culturing for 24 hours. Every experiment was performed independently and repeated three times. The sequences of the siRNAs used for the experiments are as follows:
Negative control siRNA sequence, 5-UUCUCCGAAGGUGUCACGUTT-3; AT1R siRNA sequence,
5-GUAUGCCUUCCUGUUUAAATT-3;
and
ERK2
siRNA
sequence,
5-CACCAACCAUCGAGCAAAU-3.
Flow cytometry The HUVECs were divided into the following four groups: i) a control group, ii) a 1 μM Losartan without shear stress group, iii) a 30-minute shear stress group, and iv) a 1 μM Losartan with 30 minutes of shear stress group. These groups received various special treatments before undergoing flow cytometric evaluation. A total of 1x106 cells were collected after these treatments, washed three times, and then prepared for flow cytometry via the following procedure: first, the cells were resuspended in 100 µl of 1x binding buffer, to which 5 µl of Annexin V-FITC and 5 µl of PI were added, according the manufacturer’s instructions. Second, the mixtures were incubated for 15 min at room temperature in the absence of bright light. Finally, the mixtures were added 400 µl of 1x binding buffer and shaken slightly. The samples were uploaded for flow cytometry within 1 h of the above procedure, and all analyses were performed in triplicate.
TUNEL staining Cells on slides were washed in PBS twice after incubating with 4% paraformaldehyde for 25 minutes. Then, we exposed the cells to 0.2% Triton X-100 for 10 minutes before treating them with a TDT mixture (Vazyme Biotech) for 1 hour at 37°C, according to the manufacturer’s instructions. The slides were cultured in DAPI (Beyotime Biotechnology) for another 5 minutes after being treated with the TDT mixture and then analyzed via fluorescence microscopy and photographed.
Statistical analysis Data are shown as the mean±SD of at least three repeated experiments. Student’s t-test or one-way ANOVA was performed where appropriate. P
PII: DOI: Reference:
S0014-4827(17)30451-2 http://dx.doi.org/10.1016/j.yexcr.2017.08.030 YEXCR10716
To appear in: Experimental Cell Research Received date: 4 June 2017 Revised date: 19 August 2017 Accepted date: 21 August 2017 Cite this article as: Yuelin Chao, Linlin Zhu, Xinliang Qu, Junxia Zhang, Wen Wu, Peng Ye, Jie Luo, Hongfeng Yang and Shaoliang Chen, Inhibition of angiotension II type 1 receptor reduced human endothelial inflammation induced by low shear stress, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2017.08.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of angiotension II type 1 receptor reduced human endothelial inflammation induced by low shear stress Yuelin Chao*, Linlin Zhu*, Xinliang Qu*, Junxia Zhang*, Wen Wu*, Peng Ye*, JieLuo*, *Department
Hongfeng Yang*, Shaoliang Chen*,1
of Cardiology, Nanjing First Hospital, Nanjing Medical
University, Nanjing, China 1
Correspondence: Department of Cardiology, Nanjing First Hospital,
Nanjing Medical University, #68 Changle Road, Nanjing, China E-mail: [email protected]
Abstract Low shear stress (LSS)-induced endothelial inflammation is the basis for the development of atherosclerosis. However, the mechanism underlying LSS-induced inflammation is not well understood. The angiotensin II type 1 receptor (AT1R), a component of the renin-angiotensin system, participates in atherosclerotic plaque progression. The aim of this study was to investigate the role of AT1R in LSS-induced endothelial activation. Using immunohistochemistry, we noted significant increases in AT1R, vascular endothelial adhesion cell-1 (VCAM1), and intercellular adhesion molecule-1 (ICAM1) expression in the inner curvature of the aortic arch in C57BL/6 mice compared to the descending aorta in these mice. Moreover, western blotting revealed that these LSS-induced increases in AT1R, ICAM1 and VCAM1 expression were time dependent. However, the expression of these proteins was significantly abolished by treatment with the AT1R antagonist Losartan (1 μM) or AT1R small interfering RNA (siRNA). AT1R inhibition significantly suppressed extracellular signal-regulated kinase 1/2 (ERK) upregulation, which also resulted in decreases in ICAM1 and VCAM1 protein expression. These findings demonstrate that LSS induces endothelial inflammation via AT1R/ERK signaling and that Losartan has beneficial effects on endothelial inflammation.
Key words:
Low shear stress•Angiotensin II type 1 receptor•Inflammation•
Mechanotransduction
Introduction Atherosclerosis is chronic vascular inflammation, to which specific sites within the vascular tree, such as arterial bifurcations, branch points, and curvatures, are predisposed. These sites are characterized by low shear stress [1-3] . Low shear stress (LSS), a force to which endothelial cells are subjected, plays important roles in the initial formation and progression of atherosclerotic plaques and the development of vascular inflammation [4]. Ishibazawa et al determined that LSS induced the expression of proinflammatory genes and proteins, such as vascular endothelial adhesion cell-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-selectin [5]. Similarly, evidence by Mohan S showed LSS increased markedly VCAM1 mRNA expression in HAEC exposed to 6 hours of LSS [6]. However, the specific molecular mechanism underlying this phenomenon is unknown. The angiotensin II type 1 receptor (AT1R), one of members of the G protein-coupled receptors (GPCRs) family that sense mechanical stress, interacts with angiotensin II (Ang II) and exerts intracellular effects [2]. Recent studies have shown that Ang II induces inflammation through AT1R and that Losartan induces plaque regression by decreasing the expression of pro-inflammatory proteins, such as VCAM1 [7]. Therefore, we propose that LSS induces inflammation through AT1R. In the present study, we investigated the role of AT1R in LSS-induced inflammation and identified the downstream molecules that are potentially involved in this process.
Materials and methods Animals The protocols for all of the animal experiments were approved by the Institutional Animal Care
and
Use
Committee
of
Nanjing
Medical
University
(permit
number:
SYXK2016-0006 ). Eight-week-old C57BL/6 mice from the Animal Center of Nanjing Medical University were euthanized by a lethal dose of sodium pentobarbital. Their aortas were rinsed with cold PBS thrice using a cannula inserted into the left ventricle and then fixed with 4% paraformaldehyde for 30 min [3]. The inner curvature of the aortic arch and the descending aorta were separated and embedded in OCT compound. Successive lateral tissue sections were cut using a microtome and transferred onto slides, which were blocked with 5% BSA after three treatments with PBS. Then, an anti-AT1R antibody (CST), an anti-CD31 antibody and an anti-VCAM-1 or anti-ICAM1 antibody (Abcam) were successively applied to the tissues, after which the appropriate secondary antibodies (CST) were applied to the tissues. The segments were then cultured with medium containing DAPI (Beyotime).
Cell culture The human vein endothelial cell (HUVEC) line EA.hy926 (Cellbank of Chinese Academy of Sciences, Shanghai) was grown to confluence in a humidified incubator at 37°C with 5% CO2 incubator. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBICO). Upon reaching 100% confluence, the cells were placed on separate slides for subsequent experiments. In some of the experiments, the cells were pretreated with Losartan and PD98059 (TOCRIS) for 30 minutes and subsequently applied to shear stress.
Application of shear stress A parallel flow chamber (Shanghai Medical Instrument School, Shanghai, China), which consists of two stainless-steel plates and a silicon gasket, as described elsewhere [8], was used in this study. A glass slide with confluent cells was placed on the lower plate of the chamber and then subjected to LSS induced by continuous fluid flow. Shear stress values (3 dyn/cm2) were modulated by the flow though the chamber.
Materials and reagents Losartan and PD98059 were purchased from TOCRICS, while Trizol reagent was purchased from Invitrogen. The following primary antibodies were obtained commercially: anti-AT1R (sc-1173, 1:100, Cruz for western blotting; 9391, 1:50, Abcam for immunofluorescence), anti-VCAM1 (ab98954, 1:1000, Abcam), anti-ICAM1 (sc-1511, 1:100 for western blotting, Santa Cruz, 2213, 1:40, Abcam for immunoflurescence), anti-Phospho-NF-kB p65 (3031, 1:1000, CST), anti-NF-κB p65 (6956, 1:1000, CST), anti-Phospho-ERK (4370, 1:1000, CST), anti-ERK (9102, 1:1000, CST), anti-P-JNK (9251, 1:1000, CST), anti-JNK (9252, 1:1000, CST), anti-Phospho-P38 (9211, 1:1000, CST), anti-P38 (8690, 1:1000, CST) and GAPDH (2118, 1:1000, CST).
Immunofluorescence HUVECs were fixed in 4% paraformaldehyde at room temperature for 30 min and then permeabilized in 0.1%Triton X-100/PBS for 10 min. After being blocked with BSA for 30 min, the cells were incubated with the following primary antibodies overnight at 4°C: mouse monoclonal anti-AT1R (9391, 1:50, Abcam), anti-ICAM1 (2213, 1:40, Abcam), and anti-VCAM1 (98954, 1:40; Abcam). After being washed with TBS-T, the cells were incubated with the appropriate secondary antibodies (1:200; CST). The slides were then incubated with DAPI (1:50; Beyotime), according to the manufacturer’s instructions. Images were acquired by laser scanning confocal microscopy (LSM 710; Carl Zeiss, Germany).
Western blotting Confluent cell monolayers supplemented with 5% FBS and 1% penicillin-streptomycin liquid on glass slides were subjected to LSS for different times. The cells were washed with ice-cold PBS twice and lysed in a mixture of RIPA, phosphatase inhibitor and proteinase inhibitor (100:10:1). The lysates were centrifuged at 12 000×g for 20 min at 4°C after incubating for 20 min on ice. To get Nuclear proteins, cells were treated with nuclear and cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology, China) according to the manufacture's instructions. Protein concentrations were quantified using bicinchoninic acid protein assay. The cell lysate (50 μg) supernatants were treated with SDS–PAGE sample buffer and then separated by 10% SDS-PAGE before being transferred to PVDF membranes, which were incubated with the appropriate primary antibodies overnight after being blocked with 5% milk for two hours. The signals were detected by a chemiluminescent HRP Substrate Kit (ECL, Millipore), and the bands were revealed using the GeneGnome (SYNGENE). The intensities of the protein bands were also measured using gene software.
Quantitative real-time PCR and small interfering RNA (siRNA) Total RNA was isolated with Trizol reagent, according to the manufacturers’ specifications, and then reverse transcribed to cDNA using a TaKaRa kit (RR037A). cDNA was amplified using a TaKaRa kit (RR420A), according to the manufacturer’s instructions, as well as the primers listed below and a Fast 7500. Amplification of GAPDH, which served as a control, was performed for normalization. The following PCR primers used were used in the study: ICAM1, F: 5-GTCCCCTCAAAAGTCATCC-3 and R: 5-AACCCCATTCAGCGTCACC-3; VCAM1, F:5-GGTGGGACACAAATAAGGGTTTTGG-3 and R: 5-CTTGCAATTCTTTTACA GCCTGCC-3. AT1R, F: 5-GCACCAGGTGTATTTGATAG, and AT1R, R: 5-CTGTTTCCAA ATATTCCCACC. GAPDH, F: 5-CCACCCATGGCAAATTCCATGGCA, and GAPDH, R: 5TCTACACGGCAGGTCAGGTCCACC. After the cells reached 50% confluency, we diluted 30 µl of Lipofectamine-RNAIMAX reagent (Invitrogen) and 20 µl of siRNA solution (20 µM) in two separate tubes containing 200 µl of opti-MEMI medium without FBS and incubated the mixtures for 5 min. Then, the mixtures were added together in another tube and incubated for an additional 20 minutes at room temperature. The cells were placed on several slides after culturing for 24 hours. Every experiment was performed independently and repeated three times. The sequences of the siRNAs used for the experiments are as follows:
Negative control siRNA sequence, 5-UUCUCCGAAGGUGUCACGUTT-3; AT1R siRNA sequence,
5-GUAUGCCUUCCUGUUUAAATT-3;
and
ERK2
siRNA
sequence,
5-CACCAACCAUCGAGCAAAU-3.
Flow cytometry The HUVECs were divided into the following four groups: i) a control group, ii) a 1 μM Losartan without shear stress group, iii) a 30-minute shear stress group, and iv) a 1 μM Losartan with 30 minutes of shear stress group. These groups received various special treatments before undergoing flow cytometric evaluation. A total of 1x106 cells were collected after these treatments, washed three times, and then prepared for flow cytometry via the following procedure: first, the cells were resuspended in 100 µl of 1x binding buffer, to which 5 µl of Annexin V-FITC and 5 µl of PI were added, according the manufacturer’s instructions. Second, the mixtures were incubated for 15 min at room temperature in the absence of bright light. Finally, the mixtures were added 400 µl of 1x binding buffer and shaken slightly. The samples were uploaded for flow cytometry within 1 h of the above procedure, and all analyses were performed in triplicate.
TUNEL staining Cells on slides were washed in PBS twice after incubating with 4% paraformaldehyde for 25 minutes. Then, we exposed the cells to 0.2% Triton X-100 for 10 minutes before treating them with a TDT mixture (Vazyme Biotech) for 1 hour at 37°C, according to the manufacturer’s instructions. The slides were cultured in DAPI (Beyotime Biotechnology) for another 5 minutes after being treated with the TDT mixture and then analyzed via fluorescence microscopy and photographed.
Statistical analysis Data are shown as the mean±SD of at least three repeated experiments. Student’s t-test or one-way ANOVA was performed where appropriate. P
