Вопросы вирусологии. 2021; 66: 198-210
Экспрессия эндотелиальных факторов в клетках эндотелия человека при инфекции, вызванной вирусом гриппа А(H1N1)pdm09 (Orthomyxoviridae; Alphainfluenzavirus)
https://doi.org/10.36233/0507-4088-48Аннотация
Введение. Вирус гриппа (ВГ) А (Orthomyxoviridae; Alphainfluenzavirus) способен вызывать дисфункцию эндотелия (ДЭ), апоптоз эндотелиоцитов, а также влиять на экспрессию эндотелиальных факторов, поддерживающих сосудистый гемостаз. В то же время воздействие этого патогена на характер экспрессии ключевых факторов эндотелия до настоящего времени неизвестно.
Цель исследования – выявить изменения экспрессии эндотелиальной синтазы оксида азота (NO) (eNOS) и ингибитора активатора плазминогена 1 (PAI-1, или serpin E1) в инфицированных ВГ А эндотелиоцитах. Задачи работы: изучение экспрессии указанных факторов в клетках эндотелия, инфицированных вирусом А(H1N1)pdm09; установление наличия гомологичных фрагментов в белках исследуемого патогена и эндотелиальных факторах.
Материал и методы. В экспериментах использовали клеточную линию эндотелия человека EA.hy926, которую инфицировали ВГ А/Санкт-Петербург/48/16 (H1N1)pdm09. Детекцию уровня экспрессии эндотелиальных факторов в динамике (6, 12, 18, 24, 48 и 72 ч) выполняли иммуноцитохимическим методом (ИЦХ) с помощью антител (АТ) к eNOS и PAI-1. Для количественной оценки полученного сигнала использовали программу Nis-Elements F3.2 («Nikon», Япония). Поиск гомологичных последовательностей в структуре вирусных белков и молекул eNOS и PAI-1 осуществляли путём компьютерного сравнения в них фрагментов длиной 12 а.о.
Результаты и обсуждение. Экспрессия eNOS в инфицированных клетках уменьшалась от 7,9% через 6 ч до 3,3% спустя 72 ч (контроль принят за 100%). Уровень экспрессии PAI-1 на протяжении исследования значительно варьировал: через 6 ч его показатель снижался до 49,6%, через 18 ч – возрастал до 116,3% с последующим резким падением до 18,9% спустя 24 ч. Через 48 ч и 72 ч выраженность экспрессии составляла 23,5 и 35% соответственно. В ряде белков исследуемого вируса обнаружены последовательности, гомологичные фрагментам eNOS и PAI-1.
Заключение. В ходе эксперимента с инфицированием клеток эндотелия ВГ А установлено, что вирус вызывает выраженное снижение экспрессии eNOS и модулирует экспрессию PAI-1. Описанное явление может быть использовано при дальнейшей разработке направлений патогенетической терапии сосудистых осложнений инфекции, вызываемой данным возбудителем.
Список литературы
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26. Digard P., Elton D., Bishop K., Medcalf E., Weeds A., Pope B. Modulation of nuclear localization of the influenza virus nucleoprotein through interaction with actin filaments. J. Virol. 1999; 73(3): 2222–31. https://doi.org/10.1128/jvi.73.3.2222-2231.1999
27. Wang S., Le T.Q., Kurihara N., Chida J., Cisse Y., Yano M., et al. Influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza. J. Infect. Dis. 2010; 202(7): 991–1001. https://doi.org/10.1086/656044
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32. Pritchard K.A. Jr., Ackerman A.W., Gross E.R., Stepp D.W., Shi Y., Fontana J.T., et al. Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase. J. Biol. Chem. 2001; 276(21): 17621–4. https://doi. org/10.1074/jbc.c100084200
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35. Natarajan M., Konopinski R., Krishnan M., Roman L., Bera A., Hongying Z., et al. Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells. Am. J. Physiol. Cell Physiol. 2015; 308(8): 673–83. https:// doi.org/10.1152/ajpcell.00367.2014
36. Yasar Yildiz S., Kuru P., Toksoy Oner E., Agirbasli M. Functional stability of plasminogen activator inhibitor-1. Scientific World Journal. 2014; 2014: 858293. https://doi.org/10.1155/2014/858293
37. Gando S., Levi M., Toh C. Disseminated intravascular coagulation. Nat. Rev. Dis. Primers. 2016; 2: 16037. https://doi.org/10.1038/ nrdp.2016.37
38. Hallberg P., Smedje H., Eriksson N., Kohnke H., Daniilidou M., Öhman I., et al. Pandemrix-induced narcolepsy is associated with genes related to immunity and neuronal survival. EBioMedicine. 2019; 40: 595–604. https://doi.org/10.1016/j.ebiom.2019.01.041
Problems of Virology. 2021; 66: 198-210
Modulation of endothelial factors activity in human endothelial cells in influenza A(H1N1)pdm09 virus infection
https://doi.org/10.36233/0507-4088-48Abstract
Introduction. Influenza A virus infection can lead to endothelial dysfunction (ED), including apoptosis of endothelial cells and modulation of endothelial factor activities. Affected biochemical factors may include those playing important roles in vascular homeostasis. However, the effect of this pathogen on the expression pattern of key endothelial factors is still unknown.
The aim of this work was to study the expression of endothelial nitric oxide synthase (eNOS) and plasminogen activator inhibitor-1 (PAI-1, serpin E1) in the EA.hy926 endothelial cells. Research objectives: to assess expression of eNOS and PAI-1 in endothelial cells infected with influenza virus A(H1N1)pdm09, and to identify homologous fragments in structure of viral proteins and endothelial factors.
Material and methods. Cells were infected with influenza virus A/St. Petersburg/48/16 (H1N1)pdm09 and analyzed in dynamics in 6, 12, 18, 24, 48, and 72 hrs post infection (hpi). Detection of endothelial factors expression levels was performed by immunocytochemical method (ICC) using antibodies for eNOS and PAI-1 while quantitative assessment of expression levels was carried out by program Nis-Elements F3.2 («Nikon», Japan). The search for homologous sequences between viral proteins and eNOS and PAI-1 was performed by computer comparison. Sequences were analyzed as fragments 12 amino acid residues (aar) in length.
Results and discussion. eNOS expression in infected cells had decreased to 7.9% by 6 hpi (control was taken as 100%) to 3.3% at 72 hpi. PAI-1 expression varied significantly over the course of the experiment: by 6 hpi it had decreased to 49.6%, and to 43.2% by 12 hpi. Later PAI-1 levels were: 116.3% (18 hpi); 18.9% (24 hpi); 23.5% (48 hpi), and 35% (72 hpi).
Conclusion. These results indicate that influenza A infection of endothelial cells causes a significant decrease in eNOS expression, while modulating PAI-1 one. The described phenomenon can be used in the further development of directions of pathogenetic therapy of vascular complications of infection caused by this pathogen.
References
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27. Wang S., Le T.Q., Kurihara N., Chida J., Cisse Y., Yano M., et al. Influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza. J. Infect. Dis. 2010; 202(7): 991–1001. https://doi.org/10.1086/656044
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33. Moncada S., Palmer R.M., Higgs E.A. Nitric oxide: physiology, pathophysiology, pharmacology. Pharm. Rev. 1991; 43(2): 109–42.
34. Ahmad R., Rasheed Z., Ahsan H. Biochemical and cellular toxicology of peroxynitrite: implications in cell death and autoimmune phenomenon. Immunopharmacol. Immunotoxicol. 2009; 31(3): 388–96. https://doi.org/10.1080/08923970802709197
35. Natarajan M., Konopinski R., Krishnan M., Roman L., Bera A., Hongying Z., et al. Inhibitor-κB kinase attenuates Hsp90-dependent endothelial nitric oxide synthase function in vascular endothelial cells. Am. J. Physiol. Cell Physiol. 2015; 308(8): 673–83. https:// doi.org/10.1152/ajpcell.00367.2014
36. Yasar Yildiz S., Kuru P., Toksoy Oner E., Agirbasli M. Functional stability of plasminogen activator inhibitor-1. Scientific World Journal. 2014; 2014: 858293. https://doi.org/10.1155/2014/858293
37. Gando S., Levi M., Toh C. Disseminated intravascular coagulation. Nat. Rev. Dis. Primers. 2016; 2: 16037. https://doi.org/10.1038/ nrdp.2016.37
38. Hallberg P., Smedje H., Eriksson N., Kohnke H., Daniilidou M., Öhman I., et al. Pandemrix-induced narcolepsy is associated with genes related to immunity and neuronal survival. EBioMedicine. 2019; 40: 595–604. https://doi.org/10.1016/j.ebiom.2019.01.041
