Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IK <subscript>Ca</subscript> channel-dependent Ca <superscript>2+</superscript> signaling. - OpacWWW

Szczegóły
Abstrakt

Tytuł:: Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IK Ca channel-dependent Ca signaling.
Autorzy:: Jia X; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.; School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Yang Q; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Gao C; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Chen X; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Li Y; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Su H; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Zheng Y; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Zhang S; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Wang Z; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Wang H; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Jiang LH; School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, UK.; Sino-UK Joint Laboratory of Brain Function and Injury of Henan Province and Department of Physiology and Pathophysiology, Xinxiang Medical University, Xinxiang, China.
Sun Y; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Fan Y; Beijing Advanced Innovation Centre for Biomedical Engineering, Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical Engineering, Beihang University, Beijing, China.; School of Engineering Medicine, Beihang University, No.37, Xueyuan Road, haidian district, Beijing, China.
Źródło:: Journal of cellular physiology [J Cell Physiol] 2021 Oct; Vol. 236 (10), pp. 6897-6906. Date of Electronic Publication: 2021 Mar 01.
Typ publikacji:: Journal Article; Research Support, Non-U.S. Gov't
Język:: English
Imprint Name(s):: Publication: New York, NY : Wiley-Liss
Original Publication: Philadelphia, Wistar Institute of Anatomy and Biology.
MeSH Terms:: Calcium Signaling*
Cell Proliferation*
Mechanotransduction, Cellular*
Dimethylpolysiloxanes/*chemistry
Intermediate-Conductance Calcium-Activated Potassium Channels/*metabolism
Muscle, Smooth, Vascular/*metabolism
Myocytes, Smooth Muscle/*metabolism
Animals ; Cell Culture Techniques ; Cell Line ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Intermediate-Conductance Calcium-Activated Potassium Channels/genetics ; Rats
References:: Bi, D. , Toyama, K. , Lemaître, V. , Takai, J. , Fan, F. , Jenkins, D. P. , Wulff, H. , Gutterman, D. D. , Park, F. , & Miura, H. (2013). The intermediate conductance calcium-activated potassium channel KCa3.1 regulates vascular smooth muscle cell proliferation via controlling calcium-dependent signaling. Journal of Biological Chemistry, 288(22), 15843-15853. https://doi.org/10.1074/jbc.M112.427187.
Brakemeier, S. (2003). Shear stress-induced up-regulation of the intermediate-conductance Ca²⁺-activated K+ channel in human endothelium. Cardiovascular Research, 60(3), 488-496. https://doi.org/10.1016/j.cardiores.2003.09.010.
Brown, X. Q. , Bartolak-Suki, E. , Williams, C. , Walker, M. L. , Weaver, V. M. , & Wong, J. Y. (2010). Effect of substrate stiffness and PDGF on the behavior of vascular smooth muscle cells: Implications for atherosclerosis. Journal of Cellular Physiology, 225(1), 115-122. https://doi.org/10.1002/jcp.22202.
Cavalcante, J. L. , Lima, J. A. , Redheuil, A. , & Al-Mallah, M. H. (2011). Aortic stiffness: Current understanding and future directions. Journal of the American College of Cardiology, 57(14), 1511-1522. https://doi.org/10.1016/j.jacc.2010.12.017.
Cecelja, M. , & Chowienczyk, P. (2012). Role of arterial stiffness in cardiovascular disease. JRSM Cardiovascular Disease, 1(4), 1-10. https://doi.org/10.1258/cvd.2012.012016.
D. L. Tharp, B. R. W. , Turk, J. R. , & Bowles, D. K. (2006). Upregulation of intermediate-conductance Ca2+ activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle. American Journal of Physiology: Heart and Circulatory Physiology, 291(H2493), H2503-H2503. https://doi.org/10.1152/ajpheart.01254.2005.-A.
Ebrahimi, A. P. (2009). Mechanical properties of normal and diseased cerebrovascular system. Journal of Vascular and Interventional Neurology, 2(2), 155-162.
Faouzi, M. , Hague, F. , Geerts, D. , Ay, A. S. , Potier-Cartereau, M. , Ahidouch, A. , & Ouadid-Ahidouch, H. (2016). Functional cooperation between KCa3.1 and TRPC1 channels in human breast cancer: Role in cell proliferation and patient prognosis. Oncotarget, 7(24), 36419-36435. https://doi.org/10.18632/oncotarget.9261.
Fioretti, B. , Catacuzzeno, L. , Sforna, L. , Aiello, F. , Pagani, F. , Ragozzino, D. , Castigli, E. , & Franciolini, F. (2009). Histamine hyperpolarizes human glioblastoma cells by activating the intermediate-conductance Ca2+-activated K+ channel. American Journal of Physiology: Cell Physiology, 297(1), C102-C110. https://doi.org/10.1152/ajpcell.00354.2008.
Funabashi, K. , Ohya, S. , Yamamura, H. , Hatano, N. , Muraki, K. , Giles, W. , & Imaizumi, Y. (2010). Accelerated Ca2+ entry by membrane hyperpolarization due to Ca2+-activated K+ channel activation in response to histamine in chondrocytes. American Journal of Physiology: Cell Physiology, 298(4), C786-C797. https://doi.org/10.1152/ajpcell.00469.2009.
Ghosh, D. , Syed, A. U. , Prada, M. P. , Nystoriak, M. A. , Santana, L. F. , Nieves-Cintron, M. , & Navedo, M. F. (2017). Calcium channels in vascular smooth muscle. Advances in Pharmacology, 78, 49-87. https://doi.org/10.1016/bs.apha.2016.08.002.
Gonzalez-Cobos, J. C. , & Trebak, M. (2010). TRPC channels in smooth muscle cells. Frontiers in Bioscience, 15, 1023-1039. https://doi.org/10.2741/3660.
Gudipaty, S. A. , Lindblom, J. , Loftus, P. D. , Redd, M. J. , Edes, K. , Davey, C. F. , Krishnegowda, V. , & Rosenblatt, J. (2017). Mechanical stretch triggers rapid epithelial cell division through Piezo1. Nature, 543(7643), 118-121. https://doi.org/10.1038/nature21407.
Guo, S. , Shen, Y. , He, G. , Wang, T. , Xu, D. , & Wen, F. (2017). Involvement of Ca2+-activated K* channel 3.1 in hypoxia-induced pulmonary arterial hypertension and therapeutic effects of TRAM-34 in rats. Bioscience Reports, 37(4). https://doi.org/10.1042/BSR20170763.
Haga, J. H. , Li, Y. S. , & Chien, S. (2007). Molecular basis of the effects of mechanical stretch on vascular smooth muscle cells. Journal of Biomechanics, 40(5), 947-960. https://doi.org/10.1016/j.jbiomech.2006.04.011.
Hara, M. , Tabata, K. , Suzuki, T. , Do, M. K. Q. , Mizunoya, W. , Nakamura, M. , Nishimura, S. , Tabata, S. , Ikeuchi, Y. , Sunagawa, K. , Anderson, J. E. , Allen, R. E. , & Tatsumi, R. (2012). Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch. American Journal of Physiology: Cell Physiology, 302(12), C1741-C1750. https://doi.org/10.1152/ajpcell.00068.2012.
Hayabuchi, Y. , Nakaya, Y. , Mawatari, K. , Inoue, M. , Sakata, M. , & Kagami, S. (2011). Cell membrane stretch activates intermediate-conductance Ca2+-activated K* channels in arterial smooth muscle cells. Heart and Vessels, 26(1), 91-100. https://doi.org/10.1007/s00380-010-0025-0.
Hoffman, A. H. , Teng, Z. , Zheng, J. , Wu, Z. , Woodard, P. K. , Billiar, K. L. , Wang, L. , & Tang, D. (2017). Stiffness properties of adventitia, media, and full thickness human atherosclerotic carotid arteries in the axial and circumferential directions. Journal of Biomechanical Engineering, 139(12). https://doi.org/10.1115/1.4037794.
House, S. J. , Potier, M. , Bisaillon, J. , Singer, H. A. , & Trebak, M. (2008). The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease. Pflugers Archiv. European Journal of Physiology, 456(5), 769-785. https://doi.org/10.1007/s00424-008-0491-8.
Jia, X. , Su, H. , Chen, X. , Huang, Y. , Zheng, Y. , Ji, P. , Gao, C. , Gong, X. , Huang, Y. , Jiang, L. H. , & Fan, Y. (2020). A critical role of the KCa3.1 channel in mechanical stretch-induced proliferation of rat bone marrow-derived mesenchymal stem cells. Journal of Cellular and Molecular Medicine, 24, 00 1-6. https://doi.org/10.1111/jcmm.15014.
Jia, X. , Yang, J. , Song, W. , Li, P. , Wang, X. , Guan, C. , Yang, L. , Huang, Y. , Gong, X. , Liu, M. , Zheng, L. , & Fan, Y. (2013). Involvement of large conductance Ca2+-activated K* channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation. Pflugers Archiv: European Journal of Physiology, 465(2), 221-232. https://doi.org/10.1007/s00424-012-1182-z.
Karimi, A. , Navidbakhsh, M. , Shojaei, A. , & Faghihi, S. (2013). Measurement of the uniaxial mechanical properties of healthy and atherosclerotic human coronary arteries. Materials Science and Engineering C: Materials for Biological Applications, 33(5), 2550-2554. https://doi.org/10.1016/j.msec.2013.02.016.
Kohn, J. C. , Lampi, M. C. , & Reinhart-King, C. A. (2015). Age-related vascular stiffening: Causes and consequences. Frontiers in Genetics, 6, 112. https://doi.org/10.3389/fgene.2015.00112.
Kumar, B. , Dreja, K. , Shah, S. S. , Cheong, A. , Xu, S. Z. , Sukumar, P. , Naylor, J. , Forte, A. , Cipollaro, M. , McHugh, D. , Kingston, P. A. , Heagerty, A. M. , Munsch, C. M. , Bergdahl, A. , Hultgårdh-Nilsson, A. , Gomez, M. F. , Porter, K. E. , Hellstrand, P. , & Beech, D. J. (2006). Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia. Circulation Research, 98(4), 557-563. https://doi.org/10.1161/01.RES.0000204724.29685.db.
Lallet-Daher, H. , Roudbaraki, M. , Bavencoffe, A. , Mariot, P. , Gackière, F. , Bidaux, G. , Urbain, R. , Gosset, P. , Delcourt, P. , Fleurisse, L. , Slomianny, C. , Dewailly, E. , Mauroy, B. , Bonnal, J. L. , Skryma, R. , & Prevarskaya, N. (2009). Intermediate-conductance Ca2+-activated K* channels (IKCa1) regulate human prostate cancer cell proliferation through a close control of calcium entry. Oncogene, 28(15), 1792-1806. https://doi.org/10.1038/onc.2009.25.
Laurent, S. , Kingwell, B. , Bank, A. , Weber, M. , & Struijker-Boudier, H. (2002). Clinical applications of arterial stiffness: Therapeutics and pharmacology. American Journal of Hypertension, 15(5), 453-458. https://doi.org/10.1016/s0895-7061(01)02329-9.
Llaurado, G. , Ceperuelo-Mallafre, V. , Vilardell, C. , Simo, R. , Freixenet, N. , Vendrell, J. , & Gonzalez-Clemente, J. M. (2012). Arterial stiffness is increased in patients with type 1 diabetes without cardiovascular disease: A potential role of low-grade inflammation. Diabetes Care, 35(5), 1083-1089. https://doi.org/10.2337/dc11-1475.
Matsumoto, T. , & Nagayama, K. (2012). Tensile properties of vascular smooth muscle cells: Bridging vascular and cellular biomechanics. Journal of Biomechanics, 45(5), 745-755. https://doi.org/10.1016/j.jbiomech.2011.11.014.
O'Rourke, M. F. , Staessen, J. A. , Vlachopoulos, C. , Duprez, D. , & Plante, G. E. (2002). Clinical applications of arterial stiffness; definitions and reference values. American Journal of Hypertension, 15(5), 426-444. https://doi.org/10.1016/s0895-7061(01)02319-6.
Palchesko, R. N. , Zhang, L. , Sun, Y. , & Feinberg, A. W. (2012). Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve. PLOS One, 7(12), e51499. https://doi.org/10.1371/journal.pone.0051499.
Peyton, S. R. , Raub, C. B. , Keschrumrus, V. P. , & Putnam, A. J. (2006). The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells. Biomaterials, 27(28), 4881-4893. https://doi.org/10.1016/j.biomaterials.2006.05.012.
Sazonova, O. V. , Isenberg, B. C. , Herrmann, J. , Lee, K. L. , Purwada, A. , Valentine, A. D. , Buczek-Thomas, J. A. , Wong, J. Y. , & Nugent, M. A. (2015). Extracellular matrix presentation modulates vascular smooth muscle cell mechanotransduction. Matrix Biology, 41, 36-43. https://doi.org/10.1016/j.matbio.2014.11.001.
Shukla, N. , Rowe, D. , Hinton, J. , Angelini, G. D. , & Jeremy, J. Y. (2005). Calcium and the replication of human vascular smooth muscle cells: Studies on the activation and translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression. European Journal of Pharmacology, 509(1), 21-30. https://doi.org/10.1016/j.ejphar.2004.12.036.
Si, H. , Grgic, I. , Heyken, W. T. , Maier, T. , Hoyer, J. , Reusch, H. P. , & Kohler, R. (2006). Mitogenic modulation of Ca2+-activated K* channels in proliferating A7r5 vascular smooth muscle cells. British Journal of Pharmacology, 148(7), 909-917. https://doi.org/10.1038/sj.bjp.0706793.
Takai, J. , Santu, A. , Zheng, H. , Koh, S. D. , Ohta, M. , Filimban, L. M. , Lemaître, V. , Teraoka, R. , Jo, H. , & Miura, H. (2013). Laminar shear stress upregulates endothelial Ca2+-activated K* channels KCa2.3 and KCa3.1 via a Ca2+/calmodulin-dependent protein kinase kinase/Akt/p300 cascade. American Journal of Physiology: Heart and Circulatory Physiology, 305(4), H484-H493. https://doi.org/10.1152/ajpheart.00642.2012.
Tarasov, M. V. , Bystrova, M. F. , Kotova, P. D. , Rogachevskaja, O. A. , Sysoeva, V. Y. , & Kolesnikov, S. S. (2017). Calcium-gated K+ channels of the KCa1.1- and KCa3.1-type couple intracellular Ca2+ signals to membrane hyperpolarization in mesenchymal stromal cells from the human adipose tissue. Pflügers Archiv: European Journal of Physiology, 469(2), 349-362. https://doi.org/10.1007/s00424-016-1932-4.
Tharp, D. L. , Wamhoff, B. R. , Turk, J. R. , & Bowles, D. K. (2006). Upregulation of intermediate-conductance Ca2+-activated K* channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle. American Journal of Physiology: Heart and Circulatory Physiology, 291(5), H2493-H2503. https://doi.org/10.1152/ajpheart.01254.2005.
Tharp, D. L. , Wamhoff, B. R. , Wulff, H. , Raman, G. , Cheong, A. , & Bowles, D. K. (2008). Local delivery of the KCa3.1 blocker, TRAM-34, prevents acute angioplasty-induced coronary smooth muscle phenotypic modulation and limits stenosis. Arteriosclerosis, Thrombosis, and Vascular Biology, 28(6), 1084-1089. https://doi.org/10.1161/ATVBAHA.107.155796.
Toyama, K. , Wulff, H. , Chandy, K. G. , Azam, P. , Raman, G. , Saito, T. , Fujiwara, Y. , Mattson, D. L. , Das, S. , Melvin, J. E. , Pratt, P. F. , Hatoum, O. A. , Gutterman, D. D. , Harder, D. R. , & Miura, H. (2008). The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans. Journal of Clinical Investigation, 118(9), 3025-3037. https://doi.org/10.1172/JCI30836.
Wagenseil, J. E. , & Mecham, R. P. (2009). Vascular extracellular matrix and arterial mechanics. Physiological Reviews, 89(3), 957-989. https://doi.org/10.1152/physrev.00041.2008.
Wykretowicz, A. , Gerstenberger, P. , Guzik, P. , Milewska, A. , Krauze, T. , Adamska, K. , Rutkowska, A. , & Wysocki, H. (2009). Arterial stiffness in relation to subclinical atherosclerosis. European Journal of Clinical Investigation, 39(1), 11-16. https://doi.org/10.1111/j.1365-2362.2008.02057.x.
Xie, S. A. , Zhang, T. , Wang, J. , Zhao, F. , Zhang, Y. P. , Yao, W. J. , Hur, S. S. , Yeh, Y. T. , Pang, W. , Zheng, L. S. , Fan, Y. B. , Kong, W. , Wang, X. , Chiu, J. J. , & Zhou, J. (2018). Matrix stiffness determines the phenotype of vascular smooth muscle cell in vitro and in vivo: Role of DNA methyltransferase 1. Biomaterials, 155, 203-216. https://doi.org/10.1016/j.biomaterials.2017.11.033.
Xinghua Lu, A. F. , Feinstein, M. B. , & O'Rourke, F. A. (1999). Antisense knock out of the inositol 1,3,4,5-tetrakisphosphate receptor GAP1 IP4BP in the human erythroleukemia cell line leads to the appearance of intermediate conductance KCa channels that hyperpolarize the membrane and enhance calcium influx. Journal of General Physiology, 113, 81-95.
Zhang, Q. Y. , Zhang, Y. Y. , Xie, J. , Li, C. X. , Chen, W. Y. , Liu, B. L. , Wu, X. , Li, S. N. , Huo, B. , Jiang, L. H. , & Zhao, H. C. (2014). Stiff substrates enhance cultured neuronal network activity. Scientific Reports, 4, 6215. https://doi.org/10.1038/srep06215.
Contributed Indexing:: Keywords: Ca2+; extracellular signal-regulated kinases; intermediate-conductance Ca2+-activated K+ channel; matrix stiffness; vascular smooth muscle cell proliferation
Substance Nomenclature:: 0 (Dimethylpolysiloxanes)
0 (Intermediate-Conductance Calcium-Activated Potassium Channels)
63148-62-9 (baysilon)
EC 2.7.11.24 (Extracellular Signal-Regulated MAP Kinases)
Entry Date(s):: Date Created: 20210302 Date Completed: 20211129 Latest Revision: 20211129
Update Code:: 20240105
DOI:: 10.1002/jcp.30349
PMID:: 33650160
: Czasopismo naukowe

Full Text Finder

Vascular stiffening, an early and common characteristic of cardiovascular diseases (CVDs), stimulates vascular smooth muscle cell (VSMC) proliferation which reciprocally accelerates the progression of CVDs. However, the mechanisms by which extracellular matrix stiffness accompanying vascular stiffening regulates VSMC proliferation remain largely unknown. In the present study, we examined the role of the intermediate-conductance Ca 2+ -activated K + (IK Ca ) channel in the matrix stiffness regulation of VSMC proliferation by growing A7r5 cells on soft and stiff polydimethylsiloxane substrates with stiffness close to these of arteries under physiological and pathological conditions, respectively. Stiff substrates stimulated cell proliferation and upregulated the expression of the IK Ca channel. Stiff substrate-induced cell proliferation was suppressed by pharmacological inhibition using TRAM34, an IK Ca channel blocker, or genetic depletion of the IK Ca channel. In addition, stiff substrate-induced cell proliferation was also suppressed by reducing extracellular Ca 2+ concentration using EGTA or intracellular Ca 2+ concentration using BAPTA-AM. Moreover, stiff substrate induced activation of extracellular signal-regulated kinases (ERKs), which was inhibited by treatment with TRAM34 or BAPTA-AM. Stiff substrate-induced cell proliferation was suppressed by treatment with PD98059, an ERK inhibitor. Taken together, these results show that substrates with pathologically relevant stiffness upregulate the IK Ca channel expression to enhance intracellular Ca 2+ signaling and subsequent activation of the ERK signal pathway to drive cell proliferation. These findings provide a novel mechanism by which vascular stiffening regulates VSMC function.
(© 2021 Wiley Periodicals LLC.)

Informacja

Stimulation of vascular smooth muscle cell proliferation by stiff matrix via the IK Ca channel-dependent Ca 2+ signaling.