Citation: Ning Li, Yating Huo, Haojun Xie, Yuanxiong Cheng. Angiotensin II induces the secretion of ICAM-1 and MCP-1 in human airway smooth muscle cells in vitro[J]. AIMS Medical Science, 2018, 5(3): 259-267. doi: 10.3934/medsci.2018.3.259
| [1] | Hong-Yi Chang, Hung-Wen Tsai, Chiao-Fang Teng, Lily Hui-Ching Wang, Wenya Huang, Ih-Jen Su . Ground glass hepatocytes provide targets for therapy or prevention of hepatitis B virus-related hepatocellular carcinoma. AIMS Medical Science, 2018, 5(2): 90-101. doi: 10.3934/medsci.2018.2.90 |
| [2] | Teruo Sekimoto, Takamasa Tanaka, Tatsuya Shiraki, Renu Virmani, Aloke V. Finn . How does atherosclerotic plaque become calcified, and why?. AIMS Medical Science, 2024, 11(4): 421-438. doi: 10.3934/medsci.2024029 |
| [3] | Margarida Pujol-López, Luis Ortega-Paz, Manel Garabito, Salvatore Brugaletta, Manel Sabaté, Ana Paula Dantas . miRNA Update: A Review Focus on Clinical Implications of miRNA in Vascular Remodeling. AIMS Medical Science, 2017, 4(1): 99-112. doi: 10.3934/medsci.2017.1.99 |
| [4] | Nádia C. M. Okuyama, Fernando Cezar dos Santos, Kleber Paiva Trugilo, Karen Brajão de Oliveira . Involvement of CXCL12 Pathway in HPV-related Diseases. AIMS Medical Science, 2016, 3(4): 417-440. doi: 10.3934/medsci.2016.4.417 |
| [5] | Luis Miguel Juárez-Salcedo, Luis Manuel González, Samir Dalia . Immunotherapy for diffuse large B-cell lymphoma: current use of immune checkpoint inhibitors therapy. AIMS Medical Science, 2023, 10(3): 259-272. doi: 10.3934/medsci.2023020 |
| [6] | Wenping Lin, Kai Dai, Luokun Xie . Recent Advances in αβ T Cell Biology: Wnt Signaling, Notch Signaling, Hedgehog Signaling and Their Translational Perspective. AIMS Medical Science, 2016, 3(4): 312-328. doi: 10.3934/medsci.2016.4.312 |
| [7] | Somayeh Boshtam, Mohammad Shokrzadeh, Nasrin Ghassemi-Barghi . Fluoxetine induces oxidative stress-dependent DNA damage in human hepatoma cells. AIMS Medical Science, 2023, 10(1): 69-79. doi: 10.3934/medsci.2023007 |
| [8] | Sabrina K Uppal, Toni L Uhlendorf, Ruslan L Nuryyev, Jacqueline Saenz, Menaga Shanmugam, Jessica Ochoa, William Van Trigt, Cindy S Malone, Andrew P St. Julian, Oleg Kopyov, Alex Kopyov, Randy W Cohen . Human neural progenitor cells ameliorate NMDA-induced hippocampal degeneration and related functional deficits. AIMS Medical Science, 2021, 8(3): 252-268. doi: 10.3934/medsci.2021021 |
| [9] | Michiro Muraki . Sensitization to cell death induced by soluble Fas ligand and agonistic antibodies with exogenous agents: A review. AIMS Medical Science, 2020, 7(3): 122-203. doi: 10.3934/medsci.2020011 |
| [10] | Michiro Muraki . Human Fas ligand extracellular domain: current status of biochemical characterization, engineered-derivatives production, and medical applications. AIMS Medical Science, 2022, 9(4): 467-485. doi: 10.3934/medsci.2022025 |
Approximately 300 million people worldwide suffer from asthma [2], and the number of asthma patients is estimated to increase to 400 million by 2025, which will remarkably increase the human and social financial burden [3]. Although airway narrowing in lung is the final common pathway that leads to physiological changes and associated symptoms in asthma, the chronic inflammation of the conducting zone in airway is considered to be responsible for the increased contractibility of the airway smooth muscle [4].
In asthma, the inflammation in lung airway requires integrated responses from various cell types when exposing to pathogens, viruses, and bacteria [5]. Cell types such as bronchial epithelial cell, eosinophil, and mast cell have been reported to be involved in the airway inflammation of asthma [6]. The airway smooth muscle cell (ASMC), which once was considered as a solo target of the inflammatory process, however, being demonstrated by growing evidences that it might be a contributor of secretion of pro-inflammatory mediators, and a crucial source of chemokines [7,8,9], thereby indicating its potential functions both in inflammatory and immunomodulatory processes [10,11].
The renal renin angiotensin system (RAS), which is characterized by elevated expressions of angiotensin converting enzyme and angiotensin [1] II, has been found to involve in inflammation process in various human organs such as kidney [12], Heart and Vascular [13]. Ang II is the main peptide of RAS, previous studies have reported that Ang II acts as a true cytokine to regulate cell growth, inflammation and fibrosis [14]. Emerging evidences also demonstrate that Ang II plays crucial roles in regulating cytokines secretion by vascular SMC [15,16,17]. However, the effect of Ang II on regulating cytokines secretion by ASMC has not yet been explored. In this study, we aimed to investigate the effect of Ang II on the expression level of cytokines, intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) in human ASMC (HASMC), for better understanding the contribution of Ang II in ASMC inflammation.
This study was approved by the Ethics Committee in Nanfang Hospital, Southern Medical University (NFEC-201109-K1). Written consent was obtained from each patient who participated in the study.
HASMC were obtained from the lobar or main bronchus of lungs in patients who received lung resection. Isolated HASMCs were cultivated as primary culture using Dulbecco's modied Eagle's medium (DMEM; Hyclone), 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin and 100 U/ml streptomycin according to the previous studies [18,19]. The phenotype of cells was identified performed with immunofluorescence. Cells were stimulated with Ang II (10−7 mol/L, 24 h), co-treatment with Ang II and Ang-(1-7) (both were 10−7 mol/L, 24 h), or co-treatment with irbesartan (IRB) and Ang II (first 10−5 mol/L of IRB for 30 min, afterwards 10−7 mol/L of Ang II for 24 h), respectively. Cells in control group were incubated only with medium. Stimulated cells and culture supernatants were collected for subsequent molecular experiments.
To observe the nuclear translocation of the NF-κB p65 subunit, HASMCs were cultured and stimulated with Ang II, Ang-(1-7), or IRB using the aforementioned procedure. Cells monolayers grown on six-well chamber slides were fix with 4% paraformaldehyde for 10 min, and then washed with PBS for three times, followed by permeablilized in 0.1% Trixton X-100 for 5 min, then washed with PBS for three times, blocked with 10% BSA for 1 h, and further incubated with the rabbit polyclonal antibody against NF-κB p65 (1:100 dilution) in blocking buffer overnight at 4 °C. Cells were rewarmed to 37 °C and washed with PBS, then incubated with Cy3-labeled Goat Anti-Rabbit IgG in blocking buffer for 30 min at 37 °C. After being washed with PBS for three times, cells were incubated with DAPI (Weijia technology co. Ltd, China) and diluted in PBS (10 ng/ml) for 20 min, and then washed with PBS for three times. Immunofluorescence images were detected performed with an inverted fluorescence microscope (Olympus, Japan).
Total RNA was isolated from the stimulated HASMCs using RNAiso Plus kit according to the manufacturer's instruction. Primers for MCP-1 (NM_002982.3) and ICAM-1 (NM_000201.2) were designed by Primer 5.0 software (Table 1). The cDNA was generated by the PrimeScript® RT reagent kit (Takara Biotechnology Co., Ltd, Japan) and synthesized for the subsequent PCR analysis. Real-time PCR analysis was performed by using SYBR-Green-based assays with the ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The concentration of primer used in each reaction was 0.4 µM. Cycling conditions were described as follows: Step 1, 95 °C for 30 sec; step 2, 95 °C for 5 sec and 60 °C for 34 sec; step 3, 95 °C for 15 sec, 60 °C for 1 min and 95 °C for 15 sec, with repetition of step 2 up to 35 times. Data were collected and analyzed by the 7500 software v2.0.4 by using a standard curve. GAPDH was used to normalize he relative quantitation of gene expression.
| Gene name | Accession no | Primer | Size (bp) |
| ICAM-1 | NM_000201.2 | 5′-GGACACTTGCTGCTGGTGATTC-3′ | 136 |
| 5′-CCAAGAGGAAGGAGCAAG-3′ | |||
| MCP-1 | NM_002982.3 | 5′-GCTCATAGCAGCCACCTTCATTC-3′ | 147 |
| 5′-GGACACTTGCTGCTGGTGATTC-3′ | |||
| GAPDH | NM_002046.5 | 5′-GCACCGTCAAGGCTGAGAAC-3′ | 138 |
| 5′-TGGTGAAGACGCCAGTGGA-3′ |
DownLoad: CSVTo explore the effects of Ang II, Ang-(1-7) and IRB on airway inflammation in vitro, expression levels of MCP-1 and ICAM-1 in culture supernatants were measured, respectively. HASMCs were cultured and stimulated with Ang II, Ang-(1-7), or IRB using the aforementioned procedure. After 24 h of simulation, culture supernatants were collected, and ELISAs of collected culture supernatants were performed according to the manufacturer's instruction (Boster Biological Technology co. ltd, USA). Absorbance of each sample was measured at 490 nm performed with ELISA reader (Model 5501, BIO-RAD, USA).
All values were presented as the means ± standard deviations (SD). One-way analysis of variance (ANOVA) was used to analyze the difference between four groups, and post-hoc multiple comparisons were used to compare the difference between any two groups. All data were analyzed performed with the Statistical Package for Social Sciences (SPSS) 13.0 (Statistical Software for Social Sciences, Chicago, IL). p-value < 0.05 was considered as statistical significantly different.
Primary cultured HASMCs confluenced and displayed the typical appearance of hill and valley under an inverted light microscope. HASMCs were characterized by positive immunostaining for α-smooth muscle actin, thereby were identified to be a degree of purity ≥ 98% according to their typical morphology and phenotype (Figure 1).
Figure 1. Primary cultured HASMCs were identified in morphology and phenotype. A: The confluent HASMCs displayed the typical “hill and valley” appearance from normal light. B: HASMCs immunostained with anti-α-actin antibody revealed the positive expression of α-actin (scale bar, 200 µm). HASMCs, human airway smooth muscle cells.The NF-κB pathway is a crucial mediator in the inflammatory process. We hereby estimated the effect of Ang II on inducing NF-κB pathway. As showed in Figure 2, Ang II sharply increased translocation of NF-κB p65 from the cytosol to nucleus, which could be inhibited by Ang-(1-7) and IRB.
Figure 2. Different appearances of nuclear translocation of p65 triggered by different stimuli. All scale bars: 200 µm. A: Control; B: Ang II (10−7 mol/L, 24 h); C: Ang II + Ang-(1-7) (both were 10−7 mol/L, 24 h); and D: Ang II + IRB (first 10−5 mol/L of IRB for 30 min, afterwards 10−7 mol/L of Ang II for 24 h).To estimate the effect of Ang II on inducing expression of cytokines, expression of ICAM-1 and MCP-1 in HASMCs after being treated with Ang II, Ang-(1-7) or IRB were evaluated by real-time PCR, respectively. Significantly increased expression levels of both ICAM-1 and MCP-1 induced by Ang II were found when comparing with other groups, respectively (both were p < 0.01) (Figure 3). Comparing with control group, the two co-treatment groups showed no significant different expression levels of ICAM-1 or MCP-1 (p > 0.05).
To estimate the effect of Ang II on inducing expression of ICAM-1 and MCP-1 proteins, culture supernatants of HASMCs were measured. Significantly increased expression levels of both protein ICAM-1 and MCP-1 induced by Ang II were found when comparing with other groups, respectively (both were p < 0.01) (Figure 4). Comparing with control group, the two co-treatment groups showed no significant different expression levels of protein ICAM-1 or MCP-1 (p > 0.05).
Figure 3. Expressions of ICAM-1 and MCP-1 in HASMCs induced by different stimuli. HASMCs were treated by Ang II (10−7 mol/L, 24 h) with Ang-(1-7) (10−7 mol/L, 24 h) or IRB (10−5 mol/L of IRB for 30 min, afterwards Ang II for 24 h). Relative gene expressions (RQ) of ICAM-1 (Figure 3A) and MCP-1 (Figure 3B) were evaluated (*p < 0.01 vs. the other groups). ICAM-1, intercellular adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1.
Figure 4. Expressions of proteins ICAM-1 and MCP-1 in HASMCs induced by different stimuli. HASMCs were treated by Ang II (10−7 mol/L, 24 h) with Ang-(1-7) (10−7 mol/L, 24 h) or IRB (10−5 mol/L of IRB for 30 min, afterwards Ang II for 24 h). Relative expression of protein ICAM-1 (Figure 4A) and MCP-1 (Figure 4B) were evaluated (*p < 0.01 vs. the other groups). ICAM-1, intercellular adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1.The results from our present study demonstrated that Ang II caused significant elevated expression of ICAM-1 and MCP-1 secreted by HASMCs in vitro, and this effect could be inhibited by either Ang-(1-7) or IRB.
Growing evidences have demonstrated that the HASMC is an important source of chemokines, biological active cytokines, and growth factors [20]. The synthetic function of HASMC is thereby considered to be related to the intensity and perpetuation of airway inflammation, which might be via chemotactic, autocrine, or paracrine signaling pathway [9].
MCP-1 belongs to the CC chemokine family, it exhibits a chemotactic activity that recruits monocytes, T cells, and dendritic cells to the sites of inflammation to enhance the inflammatory process [21]. Stronger MCP-1 staining in the asthmatic bronchial smooth muscle from biopsy sample was observed comparing with nonasthmatic sample [22]. Watson et al. [23] first reported that MCP-1 can be secret by HASMCs, and the secretion function of HASMCs is regulated by various stimuli including TNFα, endothelin-1, and IL-1β [19,23,24]. Later study demonstrated that MPC-1 can also be secreted by vascular SMCs induced by Ang II via the NFκB inflammation pathway [17].
ICAM-1 is a member of the immunoglobulin superfamily, it is mainly expressed in endothelin cells, monocytes, and lymph cells. ICAM-1 can mediate adhesion between the above cells, and can activate the adhesion and aggregation of leukocytes which cause release of pro-inflammatory cytokines and enhance the inflammatory process [25]. Previous study has suggested that the expression of ICAM-1 could be upregulated by Ang II in vascular SMCs [26].
In our previous study, Ang II was found to cause significant contraction of HASMCs in embedded in collagen gels via RhoA/ROCK2 signaling pathway, and its effect could be inhibited by Ang-(1-7) and IRB [17]. In the present study, the effect of Ang II on HASMCs was further explored. We found that, Ang II can sharply increase translocation of NF-κB p65 from the cytosol to nucleus in HASMCs, while in each combination treatment group (Ang II + Ang-(1-7) and Ang II + IRB, respectively), only slightly increased nuclear translocation was observed. This may possibly indicate an inflammatory effect of Ang II on HASMCs via activating AT-1 receptor.
As mentioned above, Ang II was reported to be able to cause vascular SMCs to secret MCP-1 and ICAM-1, interestingly, similar effect of Ang II was observed in HASMCs. Significant evaluated expressions of both MCP-1 and ICAM-1 were detected in HASMCs after being treated with Ang II, and the effect of Ang II could be inhibited by Ang-(1-7) and IRB, respectively. Expression levels of MCP-1 and ICAM-1 in Ang II + IRB group were lower, but not significantly, than Ang II + IRB group, we hypothesized that this may due to the incomplete inhibiting effect of Ang-(1-7) on Ang II.
To conclude, our present study first reveals the function of Ang II in regulating HASMCs to secret MCP-1 and ICAM-1, and this effect can be inhibited by Ang-(1-7) and IRB. This may provide a new sight for the relation between Ang II and HASMCs. Further studies to better understand more effects and the underlying mechanism of Ang II on regulating HASMCs secretion function are warranted.
This project was supported by the Natural Science Foundation of Guangdong Province [No.2012010009036]. We would like to thank the Department of Thoracic Surgery in Nanfang Hospital for its valuable contribution of the lung specimens.
All authors declared that they have no conflicts of interest to this work.
| [1] | Kaiya H, Kangawa K, Miyazato M (2013) What is the general action of ghrelin for vertebrates? -comparisons of ghrelin's effects across vertebrates. Gen Comp Endocrinol 181: 187-191. |
| [2] |
Braman SS (2006) The global burden of asthma. Chest 130: 4s-12s. doi: 10.1378/chest.130.1_suppl.4S
|
| [3] |
Croisant S (2014) Epidemiology of asthma: prevalence and burden of disease. Adv Exp Med Biol 795: 17-29. doi: 10.1007/978-1-4614-8603-9_2
|
| [4] |
Holgate ST (2011) Asthma: a simple concept but in reality a complex disease. Eur J Clin Invest 41: 1339-1352. doi: 10.1111/j.1365-2362.2011.02534.x
|
| [5] |
Webley WC, Hahn DL (2017) Infection-mediated asthma: etiology, mechanisms and treatment options, with focus on Chlamydia pneumoniae and macrolides. Respir Res 18: 98. doi: 10.1186/s12931-017-0584-z
|
| [6] |
Schuijs MJ, Willart MA, Hammad H, et al. (2013) Cytokine targets in airway inflammation. Curr Opin Pharmacol 13: 351-361. doi: 10.1016/j.coph.2013.03.013
|
| [7] |
Tliba O, Amrani Y, Panettieri RA, et al. (2008) Is airway smooth muscle the "missing link" modulating airway inflammation in asthma? Chest 133: 236-242. doi: 10.1378/chest.07-0262
|
| [8] |
Singh SR, Sutcliffe A, Kaur D, et al. (2014) CCL2 release by airway smooth muscle is increased in asthma and promotes fibrocyte migration. Allergy 69: 1189-1197. doi: 10.1111/all.12444
|
| [9] | McKay S, Sharma HS (2002) Autocrine regulation of asthmatic airway inflammation: role of airway smooth muscle. Respir Res 3: 11. |
| [10] |
Koziol-White CJ, Panettieri RA, et al. (2011) Airway smooth muscle and immunomodulation in acute exacerbations of airway disease. Immunol Rev 242: 178-185. doi: 10.1111/j.1600-065X.2011.01022.x
|
| [11] | Ozier A, Allard B, Bara I, et al. (2011) The pivotal role of airway smooth muscle in asthma pathophysiology. J Allergy (Cairo) 2011: 742710. |
| [12] |
Zheng Y, Tang L, Huang W, et al. (2015) Anti-Inflammatory Effects of Ang-(1–7) in Ameliorating HFD-Induced Renal Injury through LDLr-SREBP2-SCAP Pathway. PLoS One 10: e0136187. doi: 10.1371/journal.pone.0136187
|
| [13] | Padda RS, Shi Y, Lo CS, et al. (2015) Angiotensin-(1–7): A Novel Peptide to Treat Hypertension and Nephropathy in Diabetes? J Diabetes Metab 6. |
| [14] |
Ruiz-Ortega M, Esteban V, Ruperez M, et al. (2006) Renal and vascular hypertension-induced inflammation: role of angiotensin II. Curr Opin Nephrol Hypertens 15: 159-166. doi: 10.1097/01.mnh.0000203190.34643.d4
|
| [15] | Li C, He J, Zhong X, et al. (2018) CX3CL1/CX3CR1 Axis Contributes to Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation and Inflammatory Cytokine Production. Inflammation. |
| [16] |
Li HY, Yang M, Li Z, et al. (2017) Curcumin inhibits angiotensin II-induced inflammation and proliferation of rat vascular smooth muscle cells by elevating PPAR-gamma activity and reducing oxidative stress. Int J Mol Med 39: 1307-1316. doi: 10.3892/ijmm.2017.2924
|
| [17] |
Bihl JC, Zhang C, Zhao Y, et al. (2015) Angiotensin-(1–7) counteracts the effects of Ang II on vascular smooth muscle cells, vascular remodeling and hemorrhagic stroke: Role of the NFsmall ka, CyrillicB inflammatory pathway. Vascul Pharmacol 73: 115-123. doi: 10.1016/j.vph.2015.08.007
|
| [18] |
Pang L, Nie M, Corbett L, et al. (2006) Mast cell beta-tryptase selectively cleaves eotaxin and RANTES and abrogates their eosinophil chemotactic activities. J Immunol 176: 3788-3795. doi: 10.4049/jimmunol.176.6.3788
|
| [19] |
Sutcliffe AM, Clarke DL, Bradbury DA, et al. (2009) Transcriptional regulation of monocyte chemotactic protein-1 release by endothelin-1 in human airway smooth muscle cells involves NF-kappaB and AP-1. Br J Pharmacol 157: 436-450. doi: 10.1111/j.1476-5381.2009.00143.x
|
| [20] |
Deacon K, Knox AJ (2015) Human airway smooth muscle cells secrete amphiregulin via bradykinin/COX-2/PGE2, inducing COX-2, CXCL8, and VEGF expression in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 309: L237-249. doi: 10.1152/ajplung.00390.2014
|
| [21] |
Gangur V, Oppenheim JJ (2000) Are chemokines essential or secondary participants in allergic responses? Ann Allergy Asthma Immunol 84: 569-579. doi: 10.1016/S1081-1206(10)62403-9
|
| [22] |
Sousa AR, Lane SJ, Nakhosteen JA, et al. (1994) Increased expression of the monocyte chemoattractant protein-1 in bronchial tissue from asthmatic subjects. Am J Respir Cell Mol Biol 10: 142-147. doi: 10.1165/ajrcmb.10.2.8110469
|
| [23] |
Watson ML, Grix SP, Jordan NJ, et al. (1998) Interleukin 8 and monocyte chemoattractant protein 1 production by cultured human airway smooth muscle cells. Cytokine 10: 346-352. doi: 10.1006/cyto.1997.0350
|
| [24] |
Nie M, Corbett L, Knox AJ, et al. (2005) Differential regulation of chemokine expression by peroxisome proliferator-activated receptor gamma agonists: interactions with glucocorticoids and beta2-agonists. J Biol Chem 280: 2550-2561. doi: 10.1074/jbc.M410616200
|
| [25] | Wang YX, Ji ML, Jiang CY, et al. (2016) Upregulation of ICAM-1 and IL-1beta protein expression promotes lung injury in chronic obstructive pulmonary disease. Genet Mol Res 15. |
| [26] |
Liang B, Wang X, Zhang N, et al. (2015) Angiotensin-(1–7) Attenuates Angiotensin II-Induced ICAM-1, VCAM-1, and MCP-1 Expression via the MAS Receptor Through Suppression of P38 and NF-kappaB Pathways in HUVECs. Cell Physiol Biochem 35: 2472-2482. doi: 10.1159/000374047
|
Figures(4) / Tables(1)
Ning Li, Yating Huo, Haojun Xie, Yuanxiong Cheng. Angiotensin II induces the secretion of ICAM-1 and MCP-1 in human airway smooth muscle cells in vitro[J]. AIMS Medical Science, 2018, 5(3): 259-267. doi: 10.3934/medsci.2018.3.259
| Gene name | Accession no | Primer | Size (bp) |
| ICAM-1 | NM_000201.2 | 5′-GGACACTTGCTGCTGGTGATTC-3′ | 136 |
| 5′-CCAAGAGGAAGGAGCAAG-3′ | |||
| MCP-1 | NM_002982.3 | 5′-GCTCATAGCAGCCACCTTCATTC-3′ | 147 |
| 5′-GGACACTTGCTGCTGGTGATTC-3′ | |||
| GAPDH | NM_002046.5 | 5′-GCACCGTCAAGGCTGAGAAC-3′ | 138 |
| 5′-TGGTGAAGACGCCAGTGGA-3′ |
DownLoad: CSV
