fcmedicineФундаментальная и клиническая медицинаFundamental and Clinical Medicine2500-07642542-0941КемГМУ10.23946/2500-0764-2023-8-4-37-53fcmedicine-792Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL RESEARCHКолониеформирующие эндотелиальные клетки - кандидатная культура для тканевой сосудистой инженерии: паспорт генного и протеомного профиляColony-forming endothelial cells – candidate culture for tissue vascular engineering: the gene and proteomic profilehttps://orcid.org/0000-0002-8826-9244ХановаМ. Ю.KhanovaM. Yu.

Ханова Марьям Юрисовна - младший научный сотрудник лаборатории клеточных технологий.

650002, Кемерово, Сосновый бульвар, д. 6

Mariam Yu. Khanova - Junior Researcher, Laboratory of cells technologies, Research Institute for Complex Issues of Cardiovascular Diseases.

6, Sosnovy Boulevard, Kemerovo, 650002

khanovam@gmail.comhttps://orcid.org/0000-0001-8679-4857КутихинА. Г.KutikhinA. G.

Кутихин Антон Геннадьевич - доктор медицинских наук, заведующий лабораторией молекулярной, трансляционной и цифровой медицины.

650002, Кемерово, Сосновый бульвар, д. 6

Anton G. Kutikhin - MD, DSc, Head of Laboratory for Molecular, Translational and Digital Medicine, Research Institute for Complex Issues of Cardiovascular Diseases.

6, Sosnovy Boulevard, Kemerovo, 650002

https://orcid.org/0000-0002-4146-3373МатвееваВ. Г.MatveevaV. G.

Матвеева Вера Геннадьевна - кандидат медицинских наук, старший научный сотрудник лаборатории клеточных технологий.

650002, Кемерово, Сосновый бульвар, д. 6

Vera G. Matveeva - MD, PhD, Senior Researcher, Laboratory of cells technologies, Research Institute for Complex Issues of Cardiovascular Diseases.

6, Sosnovy Boulevard, Kemerovo, 650002

https://orcid.org/0000-0002-1079-1956ВеликановаЕ. A.VelikanovaE. A.

Великанова Елена Анатольевна - кандидат биологических наук, научный сотрудник лаборатории клеточных технологий.

650002, Кемерово, Сосновый бульвар, д. 6

Elena A. Velikanova - PhD (biology), Researcher, Laboratory of cells technologies, Research Institute for Complex Issues of Cardiovascular Diseases.

6, Sosnovy Boulevard, Kemerovo, 650002

https://orcid.org/0000-0002-2500-2147КривкинаЕ. О.KrivkinaE. O.

Кривкина Евгения Олеговна - младший научный сотрудник лаборатории клеточных технологий.

650002, Кемерово, Сосновый бульвар, д. 6

Evgeniya O. Krivkina - Junior Researcher, Laboratory of cells technologies, Research Institute for Complex Issues of Cardiovascular Diseases.

6, Sosnovy Boulevard, Kemerovo, 650002

https://orcid.org/0000-0002-8874-0788АнтоноваЛ. В.AntonovaL. V.

Антонова Лариса Валерьевна - доктор медицинских наук, заведующая лабораторией клеточных технологий.

650002, Кемерово, Сосновый бульвар, д. 6

Larisa V. Antonova - MD, DSc, Head of Lab of cells technologies, Research Institute for Complex Issues of Cardiovascular Diseases.

6, Sosnovy Boulevard, Kemerovo, 650002

ФГБНУ «Научно-исследовательский институт комплексных проблем сердечно-сосудистых заболеваний»РоссияResearch Institute for Complex Issues of Cardiovascular DiseasesRussian Federation202302012024843753Copyright © Ханова М.Ю., Кутихин А.Г., Матвеева В.Г., Великанова Е.A., Кривкина Е.О., Антонова Л.В., 20242024Ханова М.Ю., Кутихин А.Г., Матвеева В.Г., Великанова Е.A., Кривкина Е.О., Антонова Л.В.Khanova M.Y., Kutikhin A.G., Matveeva V.G., Velikanova E.A., Krivkina E.O., Antonova L.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://fcm.kemsmu.ru/jour/article/view/792Цель

Цель. Валидация культуры колониеформирующих эндотелиальных клеток (КФЭК) в качестве кандидатной культуры для тканевой сосудистой инженерии на основе сравнительного анализа протеомного профиля и профиля генной экспрессии с сравнении культурами эндотелиальных клеток пупочной вены человека (human umbilical vein endothelial cells HUVEC) и эндотелиальных клеток коронарной артерии человека (human coronary artery endothelial cells, HCAEC).

Материалы и методы

Материалы и методы. Культура КФЭК получена культивированием мононуклеаров периферической крови от пациентов с ишемической болезнью сердца. В качестве контроля взята коммерческая культура HCAEC, произведённая фирмой Cell Applications и культура HUVEC, полученная по адаптированному протоколу Jaffe.

Для выделения тотальной РНК клетки лизировали тризолом и обрабатывали ДНКазой. ДНК-библиотеки количественно определяли с помощью количественной полимеразной цепной реакции. Библиотеки ДНК эквимолярно смешивали и секвенировали на HiSeq 2000 (Illumina) с длиной парно-концевых прочтений 2x125 нуклеотидов.

Традиционный иммуноблотинг с применением панэндотелиальных маркеров CD31, vWF, VEGFR2/KDR, маркера эндотелиальных клеток-предшественников CD34, маркеров эндотелиально-мезенхимального перехода Snail+Slug, маркеров спецификации эндотелиальной дифференцировки: артериальной HEY2, венозной COUP-TFII и лимфатической LYVE1, VEGFR2. Дот-блоттинг 55 секретируемых белков, связанных с ангиогенезом, выполняли с использованием набора (Human Angiogenesis Array Kit, R&D Systems), в соответствии с протоколом производителя.

Результаты

Результаты. КФЭК сверхэкспрессирует маркеры всех трех линий эндотелиальной дифференцировки (KDR, VWF, CD34, NRP2, FLT4 и LYVE1 по сравнению с HCAEC; NOTCH4, DLL2) и LYVE1 по сравнению с HUVEC). Про-теомное профилирование подтверждает промежуточную эндотелиальную спецификацию КФЭК по сравнению с HCAEC и HUVEC в отношении экспрессии HEY2, LYVE1, VEGFR3, Snail и Slug. Выявлено 261 дифференциально экспрессируемых генов (ДЭГ) между КФЭК и HUVEC, и 470 ДЭГ между КФЭК и HCAEC.

Заключение

Заключение. Базовый профиль генной экспрессии колониеформирующих эндотелиальных клеток соответствует зрелым эндотелиальным клеткам и свидетельствует о промежуточной эндотелиальной спецификации КФЭК, может быть рекомендована для тканевой сосудистой инженерии.

Aim

Aim. To validate ECFC culture as a candidate culture for vascular tissue engineering using comparative analysis of the proteomic and gene expression profiles in comparison with cultures of human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC).

Materials and Methods

Materials and Methods. ECFC culture was obtained by cultivating peripheral blood mononuclear cells of patients with coronary artery disease. Commercial HCAECs produced by Cell Applications, and HUVECs cultured according to the modified protocol of Jaffe were used as controls.

The cells were lysed with TRIzol, and total RNA was isolated using a Purelink RNA Micro Scale Kit with concomitant DNase treatment. Next, rRNA depletion was carried out, followed by the creation of DNA libraries. DNA libraries were quantified using quantitative polymerase chain reaction on a CFX96 Touch Bio-Rad amplifier. DNA libraries were equimolarly mixed and sequenced on HiSeq 2000 (Illumina) with a paired-end reads of 2x125 nucleotides.

Conventional western blotting was performed using pan-endothelial markers CD31, vWF, VEG-FR2/KDR, marker of endothelial progenitor cells CD34, markers of epithelial-mesenchymal transition Snail and Slug, and markers of endothelial specification: arterial HEY2, venous COUP-TFII and lymphatic LYVE1, VEGFR2. Dot blotting against 55 angiogenesis-related proteins was performed using Proteome Profiler Human Angiogenesis Array Kit in accordance with the manufacturer's protocol.

Results

Results. ECFC overexpresses markers of all three endothelial lineages (KDR, VWF, CD34, NRP2, FLT4 and LYVE1 compared to HCAEC; NOTCH4, DLL2) and LYVE1 compared to HUVEC. Proteomic profiling indicated ECFC as an intermediate population between HCAEC and HU-VEC in term of the expression of HEY2, LYVE1, VEGFR3, Snail and Slug. 261 DEGs were detected between ECFC and HUVEC, and 470 DEGs between ECFC and HCAEC.

Conclusion

Conclusion. The gene expression profile of endothelial colony-forming cells corresponds to mature endothelial cells and indicates ECFC as an intermediate population between HCAEC and HUVEC. ECFC culture can be recommended for tissue vascular engineering.

колониеформирующие эндотелиальные клеткигенный профильпротеомный профильполнотранскриптомное секвенированиеendothelial colony-forming cellsgene profileproteomic profiletranscriptome profilingRNA-seqИсследование выполнено в рамках фундаментальной темы НИИ КПССЗ № 0419-20220001 «Молекулярные, клеточные и биомеханические механизмы патогенеза сердечно-сосудистых заболеваний в разработке новых методов лечения заболеваний сердечно-сосудистой системы на основе персонифицированной фармакотерапии, внедрения малоинвазивных медицинских изделий, биоматериалов и тканеинженерных имплантатов», № госрегистрации 122011900095-2 от 19.01.2022.This research was funded by the Complex Program of Basic Research under the Siberian Branch of the Russian Academy of Sciences within the Basic Research Topic of Research Institute for Complex Issues of Cardiovascular Diseases № 04192022-0001 «Discovering molecular, cellular and biomechanical mechanisms of cardiovascular diseases to develop novel approaches for their treatment, including personalised pharmacotherapy, minimally invasive surgery, composite biomaterials, and tissue-engineered cardiovascular implants».ReferencesCines D.B., Pollak E.S., Buck C.A., Loscalzo J., Zimmerman G.A., McEver R.P., Pober J.S., Wick T.M., Konkle B.A., Schwartz B.S., Barnathan E.S., McCrae K.R., Hug B.A., Schmidt A.M., Stern D.M. 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The authors declare that there are no conflicts of interest present.