Scilight Press

Author Information

Received: 06 Apr 2024 | Revised: 28 May 2024 | Accepted: 29 May 2024 | Published: 21 Jun 2024

Abstract

Keywords

Integrins | cardiovascular disease | integrin therapeutics

References 

• 1.
  Takada, Y.; Ye, X.; Simon, S. The integrins. Genome Biol. 2007, 8, 215.
• 2.
  Hynes, R.O. The emergence of integrins: A personal and historical perspective. Matrix Biol. 2004, 23, 333–340, https://doi.org/10.1016/j.matbio.2004.08.001.
• 3.
  Morse, E.M.; Brahme, N.N.; Calderwood, D.A. Integrin Cytoplasmic Tail Interactions. Biochemistry 2014, 53, 810–820, https://doi.org/10.1021/bi401596q.
• 4.
  Laeremans, T.; Sands, Z.A.; Claes, P.; et al. Accelerating GPCR drug discovery with conformation-stabilizing VHHs. Front. Mol. Biosci. 2022, 9, 863099, https://doi.org/10.3389/fmolb.2022.863099.
• 5.
  Zhang, H.; Zhu, D.S.; Zhu, J. Family-wide analysis of integrin structures predicted by αFold2. Comput. Struct. Biotechnol. J. 2023, 21, 4497–4507, https://doi.org/10.1016/j.csbj.2023.09.022.
• 6.
  Trimm, E.; Red-Horse, K. Vascular endothelial cell development and diversity. Nat. Rev. Cardiol. 2022, 20, 197–210, https://doi.org/10.1038/s41569-022-00770-1.
• 7.
  Baeyens, N.; Bandyopadhyay, C.; Coon, B.G.; et al. Endothelial fluid shear stress sensing in vascular health and disease. J. Clin. Investig. 2016, 126, 821–828, doi:10.1172/jci83083.
• 8.
  Li, F.; Yan, K.; Wu, L.; et al. Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow. Cell. Death Discov. 2021, 7, 1–14, https://doi.org/10.1038/s41420-021-00567-0.
• 9.
  Tzima, E.; del Pozo, M.A.; Shattil, S.J.; et al. Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment. EMBO J. 2001, 20, 4639–4647, https://doi.org/10.1093/emboj/20.17.4639.
• 10.
  Finney, A.C.; Stokes, K.Y.; Pattillo, C.B.; et al. Integrin signaling in atherosclerosis. Cell. Mol. Life Sci. 2017, 74, 2263–2282, https://doi.org/10.1007/s00018-017-2490-4.
• 11.
  Chen, P.Y.; Qin, L.; Baeyens, N.; et al. Endothelial-to-mesenchymal transition drives atherosclerosis progression. J. Clin. Invest. 2015, 125, 4514–4528.
• 12.
  Yun, S.; Budatha, M.; Dahlman, J.E.; et al. Interaction between integrin α5 and PDE4D regulates endothelial inflammatory signalling. Nature 2016, 18, 1043–1053, https://doi.org/10.1038/ncb3405.
• 13.
  Yun, S.; Hu, R.; Schwaemmle, M.E.; et al. Integrin α5β1 regulates PP2A complex assembly through PDE4D in atherosclerosis. J. Clin. Invest. 2019, 129, 4863–4874.
• 14.
  Piera-Velazquez, S.; Jimenez, S.A. Endothelial to mesenchymal transition: Role in physiology and in the pathogenesis of human diseases. Physiol. Rev. 2019, 99, 1281–1324, https://doi.org/10.1152/physrev.00021.2018.
• 15.
  Allahverdian, S.; Chaabane, C.; Boukais, K.; et al. Smooth muscle cell fate and plasticity in atherosclerosis. Cardiovasc. Res. 2018, 114, 540–550, doi:10.1093/cvr/cvy022.
• 16.
  Misra, A.; Feng, Z.; Chandran, R.R.; et al. Integrin beta3 regulates clonality and fate of smooth muscle-derived atherosclerotic plaque cells. Nat. Commun. 2018, 9, 2073, https://doi.org/10.1038/s41467-018-04447-7.
• 17.
  Zhang, Y.; Wang, H. Integrin signalling and function in immune cells. Immunology 2012, 135, 268–275, https://doi.org/10.1111/j.1365-2567.2011.03549.x.
• 18.
  Renshall, L.; Arnold, N.; West, L.; et al. Selective improvement of pulmonary arterial hypertension with a dual ET(A)/ET(B) receptors antagonist in the apolipoprotein E-/- model of PAH and atherosclerosis. Pulm. Circ. 2018, 8, 2045893217752328.
• 19.
  Reed, D.; Reed, C.; Stemmermann, G.; et al. Are aortic aneurysms caused by atherosclerosis? Circulation 1992, 85, 205–211.
• 20.
  Nickel, N.P.; Yuan, K.; Dorfmuller, P.; et al., Beyond the lungs: Systemic manifestations of pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 2020, 201, 148–157.
• 21.
  Maleki, S.; Poujade, F.-A.; Bergman, O.; et al. Endothelial/epithelial mesenchymal transition in ascending aortas of patients with bicuspid aortic valve. Front. Cardiovasc. Med. 2019, 6, 182, https://doi.org/10.3389/fcvm.2019.00182.
• 22.
  Jia, D.; Zhu, Q.; Liu, H.; et al. Osteoprotegerin disruption attenuates hysu-induced pulmonary hypertension through integrin αvβ3/FAK/AKT pathway suppression. Circ. Cardiovasc Genet. 2017, 10, e001591.
• 23.
  Blanchard, N.; Link, P.A.; Farkas, D.; et al., Dichotomous role of integrin-β5 in lung endothelial cells. Pulm. Circ. 2022, 12, e12156.
• 24.
  Andre, P.; Joshi, S.R.; Briscoe, S.D.; et al. Therapeutic approaches for treating pulmonary arterial hypertension by correcting imbalanced TGF-β superfamily signaling. Front. Med. 2021, 8, 814222.
• 25.
  Shochet, G.E.; Brook, E.; Bardenstein-Wald, B.; et al. Integrin α-5 silencing leads to myofibroblastic differentiation in IPF-derived human lung fibroblasts. Ther. Adv. Chronic Dis. 2020, 11, 2040622320936023, https://doi.org/10.1177/2040622320936023.
• 26.
  Chen, H.; Qu, J.; Huang, X.; et al. Mechanosensing by the α6-integrin confers an invasive fibroblast phenotype and mediates lung fibrosis. Nat. Commun. 2016, 7, 12564, https://doi.org/10.1038/ncomms12564.
• 27.
  Manso, A.M.; Kang, S.M.; Ross, R.S. Integrins, focal adhesions, and cardiac fibroblasts. J. Investig. Med. 2009, 57, 856–60.
• 28.
  Guo, D.C.; Papke, C.L.; He, R.; et al. Pathogenesis of thoracic and abdominal aortic aneurysms. Ann. N.Y. Acad. Sci. 2006, 1085, 339–352.
• 29.
  Chen, M.; Cavinato, C.; Hansen, J.; et al. FN (Fibronectin)-Integrin α5 signaling promotes thoracic aortic aneurysm in a mouse model of marfan syndrome. Arter. Thromb. Vasc. Biol. 2023, 43, E132–E150, https://doi.org/10.1161/atvbaha.123.319120.
• 30.
  Nakamura, K.; Dalal, A.R.; Yokoyama, N.; et al. Lineage-Specific induced pluripotent stem cell–derived smooth muscle cell modeling predicts integrin alpha-v antagonism reduces aortic root aneurysm formation in marfan syndrome mice. Arter. Thromb. Vasc. Biol. 2023, 43, 1134–1153, https://doi.org/10.1161/atvbaha.122.318448.
• 31.
  Pan, L.; Bai, P.; Weng, X.; et al., Legumain is an endogenous modulator of integrin αvβ3 triggering vascular degeneration, dissection, and rupture. Circulation 2022, 145, 659–674.
• 32.
  Wu, P.H.; Opadele, A.E.; Onodera, Y.; et al., Targeting integrins in cancer nanomedicine: Applications in cancer diagnosis and therapy. Cancers 2019, 11, 1783.
• 33.
  Hamidi, H.; Ivaska, J. Every step of the way: Integrins in cancer progression and metastasis. Nat. Rev. Cancer 2018, 18, 533–548, https://doi.org/10.1038/s41568-018-0038-z.
• 34.
  Sloan, E.K.; Pouliot, N.; Stanley, K.L.; et al., Tumor-specific expression of αvβ3 integrin promotes spontaneous metastasis of breast cancer to bone. Breast Cancer Res. 2006, 8, 1–14.
• 35.
  Bergonzini, C.; Kroese, K.; Zweemer, A.J.M.; et al. Targeting integrins for cancer therapy - disappointments and opportunities. Front. Cell. Dev. Biol. 2022, 10, 863850, https://doi.org/10.3389/fcell.2022.863850.
• 36.
  Janus-Bell, E.; Mangin, P.H. The relative importance of platelet integrins in hemostasis, thrombosis and beyond. Haematologica 2023, 108, 1734–1747, https://doi.org/10.3324/haematol.2022.282136.
• 37.
  Stark, K.; Massberg, S. Interplay between inflammation and thrombosis in cardiovascular pathology. Nat. Rev. Cardiol. 2021, 18, 666–682, https://doi.org/10.1038/s41569-021-00552-1.
• 38.
  Estevez, B.; Shen, B.; Du, X.; et al. Targeting integrin and integrin signaling in treating thrombosis. Arter. Thromb. Vasc. Biol. 2015, 35, 24–29, https://doi.org/10.1161/atvbaha.114.303411.
• 39.
  Goligorsky, M.S. Microvascular rarefaction: The decline and fall of blood vessels. Organogenesis 2010, 6, 1–10.
• 40.
  Honce, J.M.; Nagae, L.; Nyberg, E. Neuroimaging of natalizumab complications in multiple sclerosis: PML and other associated entities. Mult. Scler. Int. 2015, 2015, 1–14, https://doi.org/10.1155/2015/809252.
• 41.
  Kawamoto, E.; Nakahashi, S.; Okamoto, T.; et al. Anti-integrin therapy for multiple sclerosis. Autoimmune Dis. 2012, 2012, 1–6, https://doi.org/10.1155/2012/357101.
• 42.
  Cohen, S.A.; Trikha, M.; Mascelli, M.A. Potential future clinical applications for the GPIIb/IIIa antagonist, abciximab in thrombosis, vascular and oncological indications. Pathol. Oncol. Res. 2000, 6, 163–174, https://doi.org/10.1007/bf03032368.
• 43.
  Nishimura, S.L. Integrin-mediated transforming growth factor-beta activation, a potential therapeutic target in fibrogenic disorders. Am. J. Pathol. 2009, 175, 1362–1370.
• 44.
  Miller, P.B.; Parnell, B.A.; Bushnell, G.; et al. Endometrial receptivity defects during IVF cycles with and without letrozole. Hum. Reprod. 2012, 27, 881–888.
• 45.
  Chen, Y.F.; Yang, Y.N.; Chu, H.R.; et al. Role of Integrin alphavbeta3 in Doxycycline-Induced Anti-Proliferation in Breast Cancer Cells. Front. Cell Dev. Biol. 2022, 10, 829788.
• 46.
  Kondo, K.; Umemura, K. Clinical pharmacokinetics of tirofiban, a nonpeptide glycoprotein IIb/IIIa receptor antagonist: Comparison with the monoclonal antibody abciximab. Clin. Pharmacokinet. 2002, 41, 187–195.
• 47.
  Tonin, G.; Klen, J. Eptifibatide, an older therapeutic peptide with new indications: From clinical pharmacology to everyday clinical practice. Int. J. Mol. Sci. 2023, 24, 5446, https://doi.org/10.3390/ijms24065446.
• 48.
  Stuve, O.; Bennett, J.L. Pharmacological properties, toxicology and scientific rationale for the use of natalizumab (Tysabri) in inflammatory diseases. CNS Drug Rev. 2007, 13, 79–95.
• 49.
  Zaheer, F.; Berger, J.R. Treatment-related progressive multifocal leukoencephalopathy: Current understanding and future steps. Ther. Adv. Drug Saf. 2012, 3, 227–239, https://doi.org/10.1177/2042098612453849.
• 50.
  Monschein, T.; Dekany, S.; Zrzavy, T.; et al. Real-world use of natalizumab in Austria: Data from the Austrian Multiple Sclerosis Treatment Registry (AMSTR). J. Neurol. 2023, 270, 3779–3786.
• 51.
  Moser, T.; Zimmermann, G.; Baumgartner, A.; et al., Long-term outcome of natalizumab-associated progressive multifocal leukoencephalopathy in Austria: A nationwide retrospective study. J. Neurol. 2024, 271, 374–385.
• 52.
  Kim, J.; Kim, C.; Lee, J.A.; et al. Long-term prognosis and overall mortality in patients with progressive multifocal leukoencephalopathy. Sci. Rep. 2023, 13, 14291.
• 53.
  Anton, R.; Haas, M.; Arlett, P.; et al. Drug-induced progressive multifocal leukoencephalopathy in multiple sclerosis: European regulators’ perspective. Clin. Pharmacol. Ther. 2017, 102, 283–289.
• 54.
  McClellan, K.J.; Goa, K.L. Tirofiban. A review of its use in acute coronary syndromes. Drugs 1998, 56, 1067–1080.
• 55.
  Zhou, X.; Wu, X.; Sun, H.; et al. Efficacy and safety of eptifibatide versus tirofiban in acute coronary syndrome patients: A systematic review and meta-analysis. J. Evid. Based Med. 2017, 10, 136–144.
• 56.
  Puri, A.; Bansal, A.; Narain, V.; et al. Comparative assessment of platelet GpIIb/IIIa receptor occupancy ratio with Eptifibatide/Tirofiban in patients presenting with ACS and undergoing PCI. Indian Hear. J. 2012, 65, 152–157, https://doi.org/10.1016/j.ihj.2012.08.007.
• 57.
  Movva, H.; Rabah, R.; Tekle, W.; et al. There is no difference in safety and efficacy with Tirofiban or Eptifibatide for patients undergoing treatment of large vessel occlusion and underlying intracranial atherosclerosis. Interdiscip. Neurosurg. 2020, 23, 100927, https://doi.org/10.1016/j.inat.2020.100927.
• 58.
  Lin, F.Y.; Li, J.; Xie, Y.; et al., A general chemical principle for creating closure-stabilizing integrin inhibitors. Cell. 2022, 185, 3533–3550.
• 59.
  Zhu, J.; Zhu, J.; Springer, T.A. Complete integrin headpiece opening in eight steps. J. Cell. Biol. 2013. 201, 1053–1068.
• 60.
  Abrams, C.S.; Cines, D.B. Thrombocytopenia after treatment with platelet glycoprotein IIb/IIIa inhibitors. Curr. Hemat. Rep. 2004, 3, 143–147.
• 61.
  Blue, R.; Murcia, M.; Karan, C.; et al. Application of high-throughput screening to identify a novel αIIb-specific small- molecule inhibitor of αIIbβ3-mediated platelet interaction with fibrinogen. Blood 2008, 111, 1248–1256.
• 62.
  Zhu, J.; Choi, W.S.; McCoy, J.G.; et al. Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg2+ binding to the MIDAS. Sci. Transl. Med. 2012, 4, 125ra32.
• 63.
  Hülskamp, M.D.; Kronenberg, D.; Stange, R. The small-molecule protein ligand interface stabiliser E7820 induces differential cell line specific responses of integrin α2 expression. BMC Cancer 2021, 21, 1–12, https://doi.org/10.1186/s12885-021-08301-w.
• 64.
  Funahashi, Y.; Sugi, N.H.; Semba, T.; et al. Sulfonamide derivative, E7820, is a unique angiogenesis inhibitor suppressing an expression of integrin α2 subunit on endothelium. Cancer Res. 2002, 62, 6116–6123.
• 65.
  Mita, M.; Kelly, K.R.; Mita, A.; et al. Phase I study of E7820, an oral inhibitor of integrin α-2 expression with antiangiogenic properties, in patients with advanced malignancies. Clin. Cancer Res. 2011, 17, 193–200.
• 66.
  McCarty, J.H. αvβ8 integrin adhesion and signaling pathways in development, physiology and disease. J. Cell. Sci. 2020, 133, jcs239434.
• 67.
  Ansari, M.J.; Bokov, D.; Markov, A.; et al. Cancer combination therapies by angiogenesis inhibitors; a comprehensive review. Cell. Commun. Signal. 2022, 20, 1–23, https://doi.org/10.1186/s12964-022-00838-y.
• 68.
  Lokugamage, N.; Chowdhury, I.H.; Biediger, R.J.; et al. Use of a small molecule integrin activator as a systemically administered vaccine adjuvant in controlling Chagas disease. npj Vaccines 2021, 6, 1–14, https://doi.org/10.1038/s41541-021-00378-5.
• 69.
  Bondarev, A.D.; Attwood, M.M.; Jonsson, J.; et al. Recent developments of phosphodiesterase inhibitors: Clinical trials, emerging indications and novel molecules. Front. Pharmacol. 2022, 13, 1057083.
• 70.
  Shen, B.; Zhao, X.; O’brien, K.A.; et al. A directional switch of integrin signalling and a new anti-thrombotic strategy. Nature 2013, 503, 131–135, https://doi.org/10.1038/nature12613.
• 71.
  Kapp, T.G.; Rechenmacher, F.; Neubauer, S.; et al. A comprehensive evaluation of the activity and selectivity profile of ligands for rgd-binding integrins. Sci. Rep. 2017, 7, 39805.
• 72.
  Slack, R.J.; Macdonald, S.J.F.; Roper, J.A.; et al. Emerging therapeutic opportunities for integrin inhibitors. Nat. Rev. Drug Discov. 2021, 21, 60–78, https://doi.org/10.1038/s41573-021-00284-4.
• 73.
  Hersey, P.; Sosman, J.; O’Day, S.; et al. A randomized phase 2 study of etaracizumab, a monoclonal antibody against integrin αvβ3, + or - dacarbazine in patients with stage IV metastatic melanoma. Cancer 2010, 116, 1526–1534.
• 74.
  Park, C.H.; Yoo, T.H. TGF-beta inhibitors for therapeutic management of kidney fibrosis. Pharmaceuticals 2022, 15, 1485.
• 75.
  Sant, S.; Johnston, P.A. The production of 3D tumor spheroids for cancer drug discovery. Drug Discov. Today: Technol. 2017, 23, 27–36, https://doi.org/10.1016/j.ddtec.2017.03.002.
• 76.
  Ramadan, Q.; Sharma, P. Editorial: Tissue engineering for drug discovery and personalized medicine. Front. Med. Technol. 2021, 3, 663057, https://doi.org/10.3389/fmedt.2021.663057.
• 77.
  Jain, P.; Kathuria, H.; Dubey, N. Advances in 3D bioprinting of tissues/organs for regenerative medicine and in-vitro models. Biomaterials 2022, 287, 121639, https://doi.org/10.1016/j.biomaterials.2022.121639.
• 78.
  Usman Khan, M.; Cai, X.; Shen, Z.; et al. Challenges in the development and application of organ-on-chips for intranasal drug delivery studies. Pharmaceutics 2023, 15, 1557.
• 79.
  Phan, T.H.; Shi, H.; Denes, C.E.; et al. Advanced pathophysiology mimicking lung models for accelerated drug discovery. Biomater. Res. 2023, 27, 35.
• 80.
  Zou, W.; Teitelbaum, S.L. Absence of Dap12 and the αvβ3 integrin causes severe osteopetrosis. J. Cell. Biol. 2015, 208, 1251–36.
• 81.
  West, C.; Tobo, C.; Au, J.; et al. Combined application of biosponges and an antifibrotic agent for the treatment of volumetric muscle loss. Am. J. Physiol. Physiol. 2023, 324, C1341–C1352, https://doi.org/10.1152/ajpcell.00092.2023.
• 82.
  Dahlman, J.E.; Barnes, C.; Khan, O.F.; et al. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight. Nat. Nanotechnol. 2014, 9, 648–655, https://doi.org/10.1038/nnano.2014.84.
• 83.
  Cappelluti, M.A.; Poeta, V.M.; Valsoni, S.; et al. Durable and efficient gene silencing in vivo by hit-and-run epigenome editing. Nature 2024, 627, 416–423, https://doi.org/10.1038/s41586-024-07087-8.
• 84.
  Lenz, T.; Koch, T.; Joner, M.; et al. Ten‐Year clinical outcomes of biodegradable versus durable polymer new‐generation drug‐eluting stent in patients with coronary artery disease with and without diabetes mellitus. J. Am. Hear. Assoc. 2021, 10, e020165, https://doi.org/10.1161/jaha.120.020165.
• 85.
  Pandey, N. AXT107 an inhibitor of neovascularization, vascular leakage, and inflammation, is well-tolerated and could potentially be dosed once a year to treat retinal vascular diseases. Invest. Ophthalmology Visual Sci. 2021, 62, 3304–3304.
• 86.
  Joshi, A. Microparticulates for ophthalmic drug delivery. J. Ocul. Pharmacol. Ther. 1994, 10, 29–45, https://doi.org/10.1089/jop.1994.10.29.
• 87.
  Kim, H.S.; Kang, J.; Hwang, D.; et al. Durable polymer versus biodegradable polymer drug-eluting stents after percutaneous coronary intervention in patients with acute coronary syndrome: The HOST-REDUCE-POLYTECH-ACS trial. Circulation 2021, 143, 1081–1091.
• 88.
  Yamaji, K.; Kimura, T.; Morimoto, T.; et al. Very long-term (15 to 20 years) clinical and angiographic outcome after coronary bare metal stent implantation. Circ. Cardiovasc. Interv. 2010, 3, 468–475.
• 89.
  Blindt, R.; Vogt, F.; Astafieva, I.; et al. A novel drug-eluting stent coated with an integrin-binding cyclic arg-gly-asp peptide inhibits neointimal hyperplasia by recruiting endothelial progenitor cells. J. Am. Coll. Cardiol. 2006, 47, 1786–1795, https://doi.org/10.1016/j.jacc.2005.11.081.
• 90.
  Wang, W.; Wang, Z.; Tian, D.; et al. Integrin β3 mediates the endothelial-to-mesenchymal transition via the notch pathway. Cell. Physiol. Biochem. 2018, 49, 985.
• 91.
  Evrard, S.M.; Lecce, L.; Michelis, K.C.; et al. Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability. Nat. Commun. 2016, 7, 11853, https://doi.org/10.1038/ncomms11853.
• 92.
  Kabir, I.; Zhang, X.; Dave, J.M.; et al. The age of bone marrow dictates the clonality of smooth muscle-derived cells in atherosclerotic plaques. Nat. Aging 2023, 3, 64–81, https://doi.org/10.1038/s43587-022-00342-5.
• 93.
  Bricelj, A.; Steinebach, C.; Kuchta, R.; et al. E3 ligase ligands in successful PROTACs: An overview of syntheses and linker attachment points. Front. Chem. 2021, 9, 707317, https://doi.org/10.3389/fchem.2021.707317.
• 94.
  Layman, R.M.; Jerzak, K.J.; Hilton, J.F.; et al. TACTIVE-U: Phase 1b/2 umbrella study of ARV-471, a proteolysis targeting chimera (PROTAC) estrogen receptor (ER) degrader, combined with other anticancer treatments in ER+ advanced or metastatic breast cancer. J. Clin. Oncol. 2023, 41, TPS1121–TPS1121, https://doi.org/10.1200/jco.2023.41.16_suppl.tps1121.
• 95.
  Hynes, R.O. A reevaluation of integrins as regulators of angiogenesis. Nat. Med. 2002, 8, 918–921, https://doi.org/10.1038/nm0902-918.
• 96.
  Demircioglu, F.; Hodivala-Dilke, K. αvβ3 Integrin and tumour blood vessels-learning from the past to shape the future. Curr. Opin. Cell. Biol. 2016, 42, 121–127.
• 97.
  Mahabeleshwar, G.H.; Feng, W.; Phillips, D.R.; et al. Integrin signaling is critical for pathological angiogenesis. J. Exp. Med. 2006, 203, 2495–2507, https://doi.org/10.1084/jem.20060807.
• 98.
  Liao, Z.; Kato, H.; Pandey, M.; et al. Interaction of kindlin-2 with integrin β3 promotes outside-in signaling responses by the αVβ3 vitronectin receptor. Blood 2015, 125, 1995–2004.