Молекулярно-генетические механизмы в основе процессов остеогенеза тема диссертации и автореферата по ВАК РФ 00.00.00, кандидат наук Семенова Дарья Сергеевна
Семенова Дарья Сергеевна
кандидат наук
2023
- Специальность ВАК РФ00.00.00
- Количество страниц 138
Оглавление диссертации кандидат наук Семенова Дарья Сергеевна
ВВЕДЕНИЕ
Актуальность исследования
Теоретическая и практическая значимость работы
Цель и задачи исследования
Научная новизна работы
Положения, выносимые на защиту
Публикации и апробация работы
Список статей, опубликованных в журналах, индексируемых системами WoS и/или Scopus:
1. ОБЗОР ЛИТЕРАТУРЫ
1.1. Механизмы патологической кальцификации тканей сходны с процессами физиологического формирования кости
1.2. Аортальный клапан
1.3. Внеклеточные везикулы являются инициаторами и медиаторами процессов оссификации тканей
1.4. Роль сигнального пути Notch в процессах остеогенной дифференцировки клеток
2. МАТЕРИАЛЫ И МЕТОДЫ
2.1. Получение клеточных культур
2.2. Культивирование клеток и индукция остеогенной дифференцировки
2.3. Сокультивирование клеточных культур
2.4. Производство лентивирусных частиц
2.5. Сортировка клеток
2.6. Окрашивание клеток на щелочную фосфатазу
2.7. Анализ активности промотора
2.8. Детекция апоптоза в клетках
2.9. ПЦР в реальном времени
3. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЯ
3.1. Исследование, направленное на определение, обладают ли MSC жировой ткани пациентов с тяжелой кальцификацией аортального клапана потенциалом и предрасположенностью к остеогенной дифференцировке
3.1.1. MSC жировой ткани пациентов с кальцинированным аортальным стенозом, в отличие от VIC, обладают достаточно низкой способностью к остеогенной дифференцировке
3.1.2. Обсуждение результатов, полученных при исследовании оценки остеогенного потенциала мезенхимных стволовых клеток, полученных из жировой ткани, у людей с кальцификацией аортального клапана по сравнению с aMSC здоровых людей
3.2. Исследование, направленное на изучение проостеогенных стимулов при сокультивировании эндотелиальных клеток аорты человека (HAEC) с гладкомышечными клетками аорты человека (SMC)
3.2.1. Эндотелиальные клетки индуцируют остеогенную дифференцировку в гладкомышечных клетках аорты при их сокультивировании даже без остеогенной среды
3.2.2. При сокультивировании SMC с HAEC происходит активация сигнального пути Notch
3.2.3. Активация сигнального пути Notch способствует остеогенной дифференцировке гладкомышечных клеток
3.2.4. Совместное культивирование SMC и HAEC усиливает экспрессию генов компонентов Notch именно в эндотелиальных клетках
3.2.5. Обсуждение результатов, полученных при исследовании того, какую роль играют межклеточные коммуникации между гладкомышечными клетками аорты и эндотелиальными клетками в процессах остеогенной дифференцировки
3.3. Исследование, направленное на изучение того, как различные компоненты сигнального пути Notch влияют на остеогенную дифференцировку и остео потенциал мезенхимных стволовых клеток, полученных из жировой ткани (aMSC)
3.3.1. Влияние различных компонентов сигнального пути Notch на остеогенный потенциал aMSC
3.3.2. Высокие дозы Notch снижают CSL-зависимую транскрипцию
3.3.3. Межклеточные коммуникации способствуют усилению и поддержанию процессов остеогенной дифференцировки в aMSC
3.3.4. Индукция генов Notch в монокультуре и в кокультуре
3.3.5. Высокие дозы экзогенных NICD и Jagl вызывают апоптоз в aMSC
3.3.6. Обсуждение результатов, полученных при исследовании того, как различные компоненты сигнального пути Notch влияют на остеогенный потенциал мезенхимных стволовых клеток, полученных из жировой ткани
ЗАКЛЮЧЕНИЕ
СПИСОК СОКРАЩЕНИЙ
СПИСОК ЛИТЕРАТУРЫ
Рекомендованный список диссертаций по специальности «Другие cпециальности», 00.00.00 шифр ВАК
Тканеспецифические особенности сигнального пути Notch в клетках сердечно-сосудистой системы2021 год, доктор наук Малашичева Анна Борисовна
Роль нарушений дифференцировки гладкомышечных клеток в патогенезе аневризмы грудной аорты2021 год, кандидат наук Костина Дарья Алексеевна
Влияние тканеспецифичных мутаций в гене LMNA на процесс дифференцировки клеток2023 год, кандидат наук Перепелина Ксения Игоревна
Регуляция мультипотентных мезенхимных стромальных клеток катехоламинами: сенситизация альфа1-адренорецепторов, управление фенотипом, возможное участие в развитии артериальной гипертензии, вызванной ожирением2024 год, кандидат наук Чечехин Вадим Игоревич
Морфофункциональные реакции стромальных стволовых клеток в условиях трехмерного моделирования регенерации костной ткани2021 год, кандидат наук Шунькин Егор Олегович
Введение диссертации (часть автореферата) на тему «Молекулярно-генетические механизмы в основе процессов остеогенеза»
ВВЕДЕНИЕ Актуальность исследования
Механизмы, являющиеся ключевыми и решающими в осуществлении патологических процессов, контролирующих развитие кальцификации сосудов и мягких тканей в организме человека, во многом имеют сходство с физиологическим формированием костной ткани скелета во время эмбрионального развития, а также в постнатальном периоде при регенерации. Оссификация - процесс создания и развития нового костного материала. Кальцификация тканей является процессом, посредством которого соли кальция накапливаются в тканях, что заставляет их затвердевать (Семенова Д.С. и др., 2021а). Многие из основных игроков, контролирующих процессы кальцификации сосудов (моноциты, факторы транскрипции, BMP и т. д.), также являются регуляторами формирования костной ткани скелета (Fuery et al., 2017).
На сегодняшний день мнение о пассивном процессе дегенерации, лежащем в основе развития кальцификации сосудов и оссификации мягких тканей, сменилось на наличие динамического сложно-регулируемого клеточно-молекулярного патологического процесса, который приводит к описываемому дефекту. Кальцификация сосудов развивается в ответ на хронические воспалительные стимулы, которые в изобилии присутствуют в организме людей, страдающих диабетом, гипертонией, гиперлипидемией и хронической болезнью почек (Hunt et al., 2002; Jeziorska et al., 1998; Möhler et al., 2001a; Shao et al., 2006; Soor et al., 2008; Zimmet et al., 2001; Семенова Д.С. и др. 2021а). Механизмы оссификации сосудов все еще находятся на стадии исследования, но, как было показано, они варьируют в зависимости от лежащих в основе данной патофизиологии процессов кальцификации, предшествующих оссификации тканей (Demer and Tintut, 2014a, 2008; Vattikuti and Towler, 2004; Семенова Д.С. и др. 2021а).
Существует два основных пути, посредством которых в организме осуществляется формирование костной ткани: формирование эндохондральной кости, которое включает хондрогенез и развитие хрящевого промежуточного продукта, а также внутримембранное формирование кости. Оно прогрессирует от прямой
дифференцировки мезенхимных стволовых клеток в остеобласты, которые формируют кость (Luo et al., 2019; Runyan and Gabrick, 2017; Семенова Д.С., 2021а).
Кальцификация артерий является активным процессом, контролируемым клетками, которые способствуют оссификации посредством экспрессии остеогенных факторов роста, белков матрикса и других молекул, ассоциированных с формированием кости, которые обычно экспрессируются остеобластами в костной ткани (Bostrom et al., 1993; Canfield et al., 1996; Proudfoot et al., 1998). Гипотезы происхождения остеобласто-подобных клеток при кальцификации сосудов весьма противоречивы, но одна из распространенных версий заключается в том, что эти клетки происходят из самой стенки сосуда. Кальцифицирующиеся клетки клапана инициируют образование ткани, подобной кости, посредством внутримембранной оссификации, эндохондральная оссификация также способствует этому, хоть и в меньшей степени (Egan et al., 2011). По-видимому, данный процесс опосредуется продукцией BMP2 и BMP4 наравне с остеопонтином, остеокальцином и остеонектином.
В местах оссификации также было установлено наличие резорбирующих кость остеокласто-подобных клеток (Mikhaylova et al., 2007). Поэтому, аналогично ортотопическому формированию кости, оссификация сосудов, вероятно, является активным процессом, который включает в себя непрерывное ремоделирование тканей. Таким образом, эти данные показывают, что ремоделирование клапана и его кальцификация происходят путем активации про-кальцифицирующих клеточных и молекулярных процессов, во многом сходных с процессами, протекающими во время физиологического образования костной ткани скелета.
Существует множество исследований, подтверждающих глобальную роль сигнального пути Notch в регуляции пролиферации, дифференцировки и самообновлении стволовых клеток во множестве типов тканей, в том числе и в костной. Notch принимает ключевое участи в развитии и регенерации костей скелета (Regan and Long, 2013). Кроме того, он особенно важен во время развития и поддержания гомеостаза сердечно-сосудистой системы (Krebs et al., 2000). Спектр прямых мишеней Notch является весьма широким и тканеспецифичным. Результат активации Notch зависит от типа клеток и контекста, обеспечиваемого множеством возможных вариантов комбинаций рецепторов и лигандов, которые трансдуцируют различные биологические эффекты (Briot et al., 2016). Существует множество свидетельств того, что сигнальный
путь Notch может играть важную роль именно во взрослом сердце, а также описано ключевое участие этого сигнального пути в патогенезе кальцификации аортального клапана (Garg et al., 2005; Irtyuga et al., 2017; Theodoris et al., 2015). Гены и кодируемые ими белки, принимающие участие в продвинутых стадиях остеогенной дифференцировки, как при эктопической оссификации, так при формировании костной ткани скелета относятся к семействам WNT, BMP и RUNX, которые в свою очередь, во многом, тем или иным образом находятся под контролем и регуляцией сигнального пути Notch.
Расшифровка механизмов, управляющих остеогенной дифференцировкой как в норме, так и при патологической кальцификации важна с точки зрения возможности управления этой дифференцировкой - для предотвращения эктопической остео-трансформации клеток и минерализации тканей при различных патологических состояниях, или же напротив - для возможности способствовать наиболее эффективной регенерации костной ткани.
Теоретическая и практическая значимость работы
С одной стороны, чрезвычайно важной задачей регенеративной медицины является нахождение решения эффективного наращивания и восстановления костной ткани, в том числе при помощи остеогенной дифференцировки мезенхимных стволовых клеток. Поэтому понимание того, какие молекулярные компоненты и каким образом они принимают участие на разных стадиях остеодифференцировки, является предпосылкой для возможности управлять процессами остеогенеза. С другой стороны, способность модулировать клеточные сигналы, управляя экспрессией остеогенов, возможно приблизит нас к умению предотвращать нежелательную остеогенную дифференцировку в местах эктопической оссификации.
В данной работе мы подошли с нескольких сторон к изучению процессов остеогенной трансформации клеток. Исследования проводились с использованием клеточных культур, полученных из биологического материала как пациентов с патологической оссификацией тканей, так и здоровых людей. Не многие лаборатории в
мире имеют столь широкий доступ к биологическому материалу, полученному от людей, поэтому данное исследование имеет преимущество и особый интерес.
Полученные в ходе выполнения работы результаты способны привнести не только научную ценность в мировое сообщество ученых, но также и стать важной предпосылкой для доклинических испытаний в будущем. Нахождение потенциальных терапевтических мишеней станет основой для разработки консервативных методов лечения кальцификации клапанов сердца и оссификации сосудов или же пациентов с аномалиями развития скелета и травмами, что поможет сохранить и улучшить качество жизни множества людей по всему миру.
Цель и задачи исследования
Цель настоящей работы: изучить молекулярно-генетические механизмы, лежащие в основе процессов остеогенной дифференцировки клеток в норме и при патологии
Задачи:
1. Провести оценку остеогенного потенциала мезенхимных стволовых клеток, полученных из жировой ткани людей с кальцификацией аортального клапана по сравнению с клетками здоровых людей
2. Изучить влияние сокультивирования эндотелиальных клеток аорты с гладкомышечными клетками на процессы остеогенной дифференцировки
3. Изучить роль сигнального пути Notch в процессах остеогенной дифференцировки кокультуры эндотелиальных клеток аорты и гладкомышечных клеток
4. Изучить роль сигнального пути Notch в процессах остеогенной дифференцировки гладкомышечных клеток
5. Изучить влияние различных компонентов сигнального пути Notch на остеогенный потенциал мезенхимных стволовых клеток жировой ткани
6. Изучить влияние и роль межклеточных коммуникаций на процессы остеогенной дифференцировки в мезенхимных стволовых клетках жировой ткани
Научная новизна работы Положения, выносимые на защиту
1. Было отмечено, что мезенхимные стволовые клетки жировой ткани пациентов с кальцинированным аортальным стенозом, в отличие от интерстициальных клеток аортального клапана, полученных от тех же пациентов, а также по сравнению с MSC здоровых доноров, обладают достаточно низкой способностью к остеогенной дифференцировке
2. Наблюдается, что эндотелиальные клетки индуцируют остеогенную дифференцировку в гладкомышечных клетках аорты при их сокультивировании даже в отсутствие остеогенной среды
3. Было показано, что при сокультивировании гладкомышечных клеток аорты с эндотелиальными клетками происходит активация сигнального пути Notch. Совместное культивирование SMC и HAEC усиливает экспрессию генов компонентов Notch именно в эндотелиальных клетках
4. Активация сигнального пути Notch способствует остеогенной дифференцировке гладкомышечных клеток
5. Активация сигнального пути Notch путем внесения в клетки лентивирусов, несущих NICD или Jag1 увеличивает способность мезенхимных стволовых клеток к остеогенной дифференцировке дозозависимым образом. Однако, чрезмерно сильная степень активации передачи сигналов Notch, вызывает апоптоз в культуре мезенхимных стволовых клеток, а также снижает остеогенную дифференцировку в клетках
6. Межклеточные коммуникации способствуют усилению и поддержанию процессов остеогенной дифференцировки в мезенхимных стволовых клетках жировой ткани
Публикации и апробация работы
По материалам диссертации опубликовано 11 работ: 3 научных статьи в журналах, индексируемых системами WoS и/или Scopus, и 8 публикаций в материалах международных и всероссийских конференций.
Основные положения и научные итоги диссертации были изложены в докладах на научных конференциях.
Список статей, опубликованных в журналах, индексируемых системами WoS и/или Scopus:
1. Semenova D, Bogdanova M, Kostina A, Golovkin A, Kostareva A, Malashicheva A. (2020) Dose-dependent mechanism of Notch action in promoting osteogenic differentiation of mesenchymal stem cells. Cell Tissue Res. 2020 Jan;379(1):169-179. DOI: 10.1007/s00441-019- 03130-7.
2. Kostina A, Semenova D, Kostina D, Uspensky V, Kostareva A, Malashicheva A. (2019) Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells. Biochem Biophys Res Commun. 2019 Jun 25;514(2):462- 468. doi: 10.1016/j.bbrc.2019.04.177
3. A Malashicheva, O Irtyuga, A Kostina, E Ignatieva, E Zhiduleva, D Semenova, A Golovkin, M Gordeev, O Moiseeva, A Kostareva. (2018). Osteogenic potential of adipose mesenchymal stem cells is not correlated with aortic valve calcification. Biological Communications. Vol. 63 issue 2, 117-122. DOI: 10.21638/spbu03.2018.204
Тезисы:
1. Семенова Д.С., Малашичева А.Б. (2017). Роль сигнального пути
Notch в остеогенной дифференцировке мезенхимных стволовых клеток.
Приложение 1 к журналу Трансляционная медицина 2017, с. 93 2.
2. Семенова Д.С., Малашичева А.Б. (2017). Дозо-зависимая роль различных компонентов сигнального пути Notch в остеогенной дифференцировке стволовых клеток. Гены и Клетки, том XII, № 3, 2017, с. 218219
3. Kostina A, Kiselev A, Semenova D, Irtyuga O, Kostareva A, Malashicheva A. Notch-dependent regulation of osteogenic differentiation of interstitial cells from human aortic valve. 2019. Сборник конференции The Notch Meeting 2019.
4. Semenova DS, Kostina AS, Irtyuga OB, Moiseeva OM, Malashicheva AB. Mechanisms of Notch-dependent intercellular communications in aortic valve calcification. 2019. Сборник конференции The Notch Meeting XI, 2019г.
5. Semenova D, Golovkin A, Malashicheva A. The role of Notch signaling pathway in osteogenic differentiation of mesenchymal stem cells. 2017. Сборник конференции The Notch Meeting X, 2017г.
6. Семенова Д.С., Малашичева А.Б. Роль сигнального пути Notch в кальцификации клапана аорты. 2018. XIX Зимняя молодежная школа по биофизике и молекулярной биологии. Тезисы напечатаны в сборнике конференции.
7. Семенова Д.С., Малашичева А.Б., Костина А.С. Остеогенная дифференцировка клеток при изучении патологий сердца и сосудов. StemCellBio 2018. Тезисы напечатаны в сборнике конференции «StemCellBio-2018: фундаментальная наука как основа клеточных технологий».
8. Семенова Д.С., Малашичева А.Б. Роль сигнального пути Notch в остеогенной дифференцировке мезенхимных стволовых клеток. 2017. XVIII Зимняя молодежная школа по биофизике и молекулярной биологии. Тезисы напечатаны в сборнике конференции.
1. ОБЗОР ЛИТЕРАТУРЫ
1.1. Механизмы патологической кальцификации тканей сходны с процессами физиологического формирования кости
Процессы, лежащие в основе физиологического образования костной ткани, обладают сходными признаками с механизмами, обуславливающими развитие патологической кальцификации тканей сердца и сосудов. Триггерные, инициирующие механизмы, приводящие к эктопической кальцификации сердца и сосудов, остаются в значительной степени неизученными. Кальцификация сосудов — частое сердечнососудистое осложнение, сопровождающее старение и различные патологические процессы в организме человека (Demer and Tintut, 2014b). Механизмы, участвующие в патогенезе сосудистой кальцификации, остаются в значительной степени неизвестными, и в настоящее время не существует терапии для предотвращения и устранения кальцификации, за исключением сложных инвазивных операций и дорогостоящих транскатетерных процедур, имеющих свои ограничения и недостатки. Детальное понимание механизмов кальцификации крайне необходимо для возможности создания других методов лечения заболевания.
Кальцификация сосудов может происходить в медиальном гладкомышечном слое и в интимальном слое стенки сосуда. Ранее кальцификацию сосудов считали пассивным процессом. Однако, за последние годы накопилось большое количество данных, свидетельствующих о том, что это активный и строго регулируемый процесс. Известно, что в этих процессах большая роль отведена сигнальным путям Notch/BMP/TGF-b. Было неоднократно показано, что сигнальный путь BMP играет важную роль в процессах остеогенной трансформации клеток (Семенова Д.С., 2021а). В экспериментальных моделях, активированные эндотелиальные клетки, как было показано, секретируют BMP2 и BMP4 в ответ на изменения паттерна ламинарного потока крови, также BMP2 был обнаружен в интерстициальных клетках, полученных из аортального клапана пожилых крыс (Seya et al., 2011). Белки BMP стимулируют кальцификацию посредством активации Smad и Wnt/p-катенин сигнальных путей, а также повышая экспрессию остеохондрогенного транскрипционного фактора Msx2. Эти сигнальные пути работают совместно, чтобы инициировать экспрессию транскрипционного фактора Runx2 (Bostrom et al., 2011). Когда экспрессируется Runx2,
клетки вступают на путь дифференцировки в остеобласты, повышается экспрессия связанных с кальцификацией белков, таких как остеопонтин, костный сиалопротеин II и остеокальцин, таким образом индуцируется кальцификация (Рис. 1) (Butcher et al., 2006).
Рисунок 1. Сигнальные пути, которые играют существенную роль в приобретении клетками остео-фенотипа (Leopold, 2012).
Сложные взаимодействия между молекулами этих основных сигнальных путей также подтверждают сходство между процессами патологической оссификации тканей и механизмами формирования кости. Кроме того, при патологической кальцификации наблюдается сходным образом отложения минералов во внеклеточном матриксе остеобластоподобными клетками (Bostrom et al., 2011). Происхождение остеобластоподобных клеток в сердечно-сосудистой системе является спорным вопросом, который требует дальнейшего изучения и уточнения. Предполагают, что остеогенные клетки в медиальном слое могут трансдифференцироваться из медиальных гладкомышечных клеток in situ (Speer et al., 2009). Другие исследования показали, что
клетки-предшественники мезенхимального происхождения в стенке сосуда могут быть вовлечены в кальцификацию сосудов (Farrington-Rock et al., 2004; Tintut et al., 2003).
Описано два основных типа формирования кости: формирование эндохондральной кости, которое включает хондрогенез и развитие хрящевого промежуточного продукта, а также внутримембранное формирование кости, которое прогрессирует от прямой дифференцировки мезенхимных стволовых клеток в остеобласты, формирующие кость. Патологическая кальцификация, происходящая а тканях клапанов или сосудов организма, по-видимому, включает как внутримембранные, так и эндохондральные процессы (Möhler et al., 2001b; Семенова Д.С. и др., 2021а).
Хорошо известно, что мезенхимные стволовые клетки (MSC), в том числе MSC костного мозга и MSC жировой ткани, обладают способностью дифференцироваться во многие типы клеток и, в том числе и в остеобластические и хондрогенные линии (Mushahary et al., 2018). Исследования, проведенные как на людях, так и на животных показали, что MSC могут попадать в кровоток. Было высказано предположение, что миграция отдаленных MSC из их исходной ниши с последующей активацией в сторону остеобластных клеток в пораженных сосудах также играет роль в процессе кальцификации сосудов (Otsuru et al., 2007; Rochefort et al., 2006; Wang et al., 2014). Так называемая «теория циркулирующих клеток» предполагает, что циркулирующие стволовые клетки / костно-мозговые остеопредшественники находят свое место в пораженных артериях, что способствует инициации и прогрессии кальцификации сосудов (Pal and Golledge, 2011). Кроме того, благодаря высокой пластичности гладкомышечные клетки сосудов обладают способностью переходить из своего нормального дифференцированного сократительного фенотипа во множество синтетических дедифференцированных состояний, демонстрирующих в некоторых случаях хондрогенную и остеогеную дифференцировки. Данные процессы также принимают ключевое участие в патогенезе сосудистых заболеваний (Boström, 2016).
Остеобласты, полученные из мезенхимных стволовых клеток (MSC) костного мозга, являются клетками, которые в первую очередь ответственны за образование кости путем кальцификации внеклеточного матрикса (Ortuno et al., 2010). Известно, что дифференцировка MSC в остеобласты находится под контролем многочисленных факторов транскрипции и сигнальных белков (Ortuno et al., 2010). RUNX2 и Osterix
(OSX) являются важными транскрипционными факторами, активация которых требуется для осуществления процессов остеогенной дифференцировки клеток (Yang et al., 2004). Хотя RUNX2 и OSX позиционируют как ключевые регуляторы остеогенеза, существуют также другие транскрипционные факторы, участвующие в дифференцировке остеобластов, в том числе DLX5, DLX3, FRA1, Twistl, ZBTB16 и ATF4 (Yang et al., 2004). Существуют данные, указывающие на то, что ZBTB16 является нижестоящим транскрипционным фактором, который участвует в дифференцировке остеобластов. Таким образом, ZBTB16 оказывает положительный регулирующий эффект на дифференцировку остеобластов за счет их близкого взаимодействия с ключевыми регуляторами остеогенеза OSX и RUNX2 (Семенова Д.С., 2021а; Семенова Д.С. 2021б).
ZBTB16/PLZF - высоко консервативный от нематоды Caenorhabditis elegans до человека ген. У человека ген PLZF содержит шесть экзонов и пять интронов. Размеры его экзонов варьируют от 87 до 1358 п.н. Экзоны распределяются по области примерно в 120 т.п.н. PLZF демонстрирует сложные паттерны сплайсинга в зависимости от ткани, в которой происходит экспрессия гена. Продукт имеет по крайней мере четыре изоформы обнаруживаемые с 1 экзоном. Белок PLZF включает девять мотивов Kruppel-подобных C2H2 цинковых пальцев на С-конце, менее известный домен RD2 и BTB / POZ (поксвирус, цинковый палец) на N-конце. Девять Kruppel-подобных C2H2 цинковых пальцев способствуют специфичному связыванию ДНК с генами-мишенями, что позволяет PLZF функционировать как фактор транскрипции. Домен BTB / POZ является эволюционно консервативным мотивом, который обеспечивает межбелковые взаимодействия и позволяет белкам POZ домена участвовать в различных процессах, включая гематопоэз, ангиогенез, нейрогенез, адипогенез, остеокластогенез и дифференцировку мышц (Liu et al., 2016).
Исследования показали важность функциональной активности ZBTB16 в дифференцировке стволовых клеток в остеобласты (Hemming et al., 2016; Onizuka et al., 2016). По всей видимости ZBTB16 работает как важный маркер на более поздних стадиях остеобластической дифференцировки стволовых клеток (Saugspier et al., 2010).
В литературе существуют свидетельства об участии ZBTB16 в развитии скелета (Fischer et al., 2008). Было показано, что ZBTB16 играет роль в спецификации паттернов
аксиального скелета и конечности. Кроме того, экспрессия ZBTB16 повышена в клетках пациентов, страдающих эктопическим формированием костной ткани (Inoue et al., 2006).
Одним из наиболее часто встречающихся заболеваний сердечно-сосудистой системы является развитие кальцификации клапана аорты. В настоящее время не существует медикаментозного лечения, чтобы предотвратить или остановить это заболевание. Основной особенностью заболевания является прогрессирующая минерализация ткани клапана. Во многом патологические процессы, обуславливающие минерализацию аортального клапана, объясняют также и механизмы, протекающие во время кальцификации сосудов. Обе патологические картины имеют сходство с процессами нормального физиологического образования костной ткани скелета. Как во время эмбрионального развития, так и в периоды регенерации костной ткани (Mathieu and Boulanger, 2014).
1.2. Аортальный клапан
Аортальный клапан (АК) является аваскулярным, тонким, гибким образованием. Он находится разграничивает собой левый желудочек сердца и аорту. Ключевой задачей АК является предотвращение обратного тока крови, которая во время сокращения желудочка сердца выходит в аорту.
Створки АК состоят из трех слоев: желудочкового, спонгиозного и аортального (Butcher et al., 2011). С аортальной и желудочковой сторон створки клапана покрыты эндотелиальными клетками (EC). Все слои в аортальном клапане содержат то или иное количество различных компонентов внеклеточного матрикса с преобладанием особых черт в каждом из слое. Аортальный преимущественно содержит коллагеновые волокна, ориентированные в круговом направлении в виде пучков и тяжей, и небольшого количества эластических волокон (Butcher et al., 2011). Желудочковый слой является более тонким и содержит гораздо большее количество эластических волокон по сравнению с аортальным. Спонгиозный слой содержит большое количество гликозамингликанов и протеогликанов. Одновременно с этим между всеми компонентами внеклеточного матрикса в трех слоях находятся интерстициальные клетки клапана (VIC) (Семенова Д.С., 2018, магистерская диссертация). Эта гетерогенная популяция клеток обладает уникальными характеристиками и необходима для поддержания функции и гомеостаза клапана посредством пролиферации клеток,
секреции матриксных металлопротеиназ и компонентов внеклеточного матрикса (Schoen, 2012).
Предполагается, что VIC являются основными функциональными единицами клапана, которые подвергаются минерализации (Rutkovskiy et al., 2017) и способны экспрессировать гены, ассоциированные с остеогенным фенотипом (Rabkin-Aikawa et al., 2004). Крайне важной задачей исследований, проводимых в настоящее время, является определение происхождения клеток, которые способствуют кальцификации клапана (Leszczynska and Mary Murphy, 2018). Обсуждалось то, что периферические MSC как раз и могут являться теми самыми клетками, которые способствуют кальцификации клапана аорты (Liu and Xu, 2016).
В частности в нашей лаборатории таже было показано, что VIC пациентов с кальцификацией аортального клапана являются более восприимчивыми к проостеогенной индукции и легче подвергаются остеогенной дифференцировке по сравнению с VIC, полученными из нормальных здоровых клапанов (A et al., 2018; Malashicheva et al., 2018).
1.3. Внеклеточные везикулы являются инициаторами и
медиаторами процессов оссификации тканей
Множество исследований в последние годы показали ключевую роль внеклеточных везикул, циркулирующих в ткани клапана, как в поддержании гомеостаза ткани, так и в развитии кальцинированного стеноза клапана, либо же в оссификации сосудов, в зависимости от содержимого везикул (Aikawa and Blaser, 2020; Blaser and Aikawa, 2018; Bouchareb et al., 2014; Jansen et al., 2017; Vito et al., 2021).
Внеклеточные везикулы обладают высокой гетерогенностью, однако их можно разделить на две основные категории: экзосомы и микровезикулы. Первоначально секреция внеклеточных везикул описывалась как способ удаления ненужных соединений из клетки (Pan and Johnstone, 1983). Однако теперь мы понимаем, что внеклеточные везикулы - это больше, чем просто переносчики отходов, и основной интерес в этой области сейчас сосредоточен на их способности обмениваться различными компонентами между клетками. Внеклеточные везикулы теперь рассматриваются как дополнительный механизм межклеточной коммуникации. Этот
способ позволяет клеткам обмениваться белками, липидами и генетическим материалом, и содержание везикул может варьировать в зависимости от клетки и условий. Однако в этой расширяющейся области многое остается неизвестным в отношении происхождения, биогенеза, секреции, таргетирования и судьбы этих пузырьков. Экзосомы обычно имеют диаметр 30-150 нм. Их образование происходит из эндосомальной системы. Экзосомы формируются во время созревания ранних эндосом, внутреннее почкование эндосомальной мембраны приводит к образованию мультивезикулярных эндосом, также обозначаемых как поздние эндосомы (McGough and Vincent, 2016). Во время этого процесса многие цитоплазматические компоненты могут быть встроены в эндосомы и затем направлены либо в лизосомы, либо на аутофагосому для деградации, либо на плазматическую мембрану для секреции (Liu et al., 2021). Микровезикулы в свою очередь имеют диаметр от 50 до 1000 нм. Их биогенез происходит посредством начального специфического перераспределения белков плазматической мембраны и липидных компонентов, направленного на регулирование жесткости мембраны. Затем прямое наружное образование пузырьков и отпочковывание этих участков плазматической мембраны приводит к высвобождению микровезикул во внеклеточное пространство (Schubert and Boutros, 2021). Немало достигнуто в понимании биологической роли внеклеточных пузырьков, происходящих из резидентных клеток сердца, в гомеостазе и восстановлении сердечной ткани (Mancuso et al., 2020). В частности, внеклеточные везикулы лежат в основе медиаторов паракринного действия сердечных стволовых клеток, широко известных своими регенеративными и репаративными функциями. Согласно большей части литературы, экзосомы могут служить инструментом для оценки и прогнозирования течения заболевания, а также могут предоставить новые клинические биомаркеры для разработки терапевтических подходов.
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Адренергическая регуляция постнатальных мультипотентных мезенхимных стромальных клеток человека: сенситизация рецепторов, активация стволовых клеток и управление их дифференцировкой2024 год, доктор наук Тюрин-Кузьмин Петр Алексеевич
Список литературы диссертационного исследования кандидат наук Семенова Дарья Сергеевна, 2023 год
СПИСОК ЛИТЕРАТУРЫ
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SAINT PETERSBURG STATE UNIVERSITY
Printed as a manuscript
Semenova Daria Sergeevna Molecular and genetic mechanisms underlying the processes of osteogenesis
1.5.22. Cell biology
Thesis for the degree of candidate biological sciences
Translation from Russian
Scientific adviser: Doctor of Biological Sciences, Malashicheva Anna Borisovna
Saint Petersburg, 2022
CONTENTS
INTRODUCTION...............................................................................................................................75
The relevance of the research........................................................................................................75
Theoretical and practical significance of the work......................................................................77
The aim and objectives of the study..............................................................................................77
Scientific novelty of the work.........................................................................................................78
Provisions for defense.....................................................................................................................78
Publications and approbation of work..........................................................................................79
1. LITERATURE REVIEW..............................................................................................................81
1.1. The mechanisms of pathological tissue calcification are similar to the processes of physiological bone formation.........................................................................................................81
1.2. Aortic valve...............................................................................................................................84
1.3. Extracellular vesicles are initiators and mediators of tissue ossification processes...........85
1.4. Role of the Notch signaling pathway in osteogenic cell differentiation...............................89
2. MATERIALS AND METHODS...................................................................................................93
2.1. Primary cell cultures................................................................................................................93
2.2. Cell cultivation and induction of osteogenic differentiation................................................94
2.3. Cocultivation of cell cultures...................................................................................................94
2.4. Production of lentiviruses........................................................................................................94
2.5. Magnetic cell separation..........................................................................................................95
2.6. Cell staining for alkaline phosphatase...................................................................................95
2.7. Promoter activity assay...........................................................................................................96
2.8. Detection of apoptosis in cells.................................................................................................96
2.9. Real-time PCR..........................................................................................................................96
3.1. A study to determine whether adipose tissue MSC from patients with severe aortic valve calcification have the potential and predisposition to osteogenic differentiation......................97
3.1.1. MSC of adipose tissue of patients with calcified aortic stenosis, in contrast to VIC, have a rather low ability for osteogenic differentiation............................................................................97
3.1.2. Discussion of the results obtained in the study of the evaluation of the osteogenic potential of mesenchymal adipose-derived stem cells in people with aortic valve calcification compared with aMSC of healthy people......................................................................................................100
3.2. A study aiming to investigate pro-osteogenic stimuli in coculture of human aortic endothelial cells (HAEC) with human aortic smooth muscle cells (SMC)...............................101
3.2.1. Endothelial cells induce osteogenic differentiation in aortic smooth muscle cells when they are co-cultured even without an osteogenic medium..................................................................102
3.2.2. Co-cultivation of SMC with HAEC activates Notch signaling pathway..........................104
3.2.3. Activation of the Notch signaling pathway promotes osteogenic differentiation of smooth muscle cells.................................................................................................................................105
3.2.4. Co-cultivation of SMC and HAEC enhances the expression of Notch component genes in
endothelial cells..........................................................................................................................106
3.2.5. Discussion of the results obtained in the study of the role played by intercellular communications between aortic smooth muscle cells and endothelial cells in the processes of osteogenic differentiation............................................................................................................109
3.3. A study to investigate how various components of the Notch signaling pathway influence osteogenic differentiation and osteopotential in adipose tissue-derived mesenchymal stem cells (aMSC)...........................................................................................................................................110
3.3.1. Effects of various components of Notch signaling pathway on the osteogenic potential of aMSC..........................................................................................................................................110
3.3.2. High doses of Notch reduce CSL-dependent transcription...............................................113
3.3.3. Intercellular communications contribute to the enhancement and maintenance of osteogenic differentiation processes in aMSC............................................................................114
3.3.4. Induction of Notch genes in monoculture and coculture of aMSC...................................117
3.3.5. High doses of exogenous NICD and Jagl induce apoptosis in aMSC..............................119
3.3.6. Discussion of the results obtained in the study of how various components of Notch signaling pathway affect the osteogenic potential of mesenchymal stem cells derived from adipose tissue..............................................................................................................................120
CONCLUSION.................................................................................................................................122
LIST OF ABBREVIATIONS..........................................................................................................124
REFERENCES..................................................................................................................................126
INTRODUCTION The relevance of the research
The mechanisms that are key and essential in the development of pathological processes that control the development of calcification of vessels and soft tissues in the human body are similar to the physiological formation of the bone tissue of the skeleton during embryonic development, as well as in the postnatal period during regeneration. Ossification is the process of creating and developing new bone material. Calcification of tissues is a process by which calcium salts accumulate in the tissues, which makes them harden (Семенова Д.С. и др., 2021а). Many of the main players controlling the processes of vessel calcification (monocytes, transcription factors, BMP, etc.) are also regulators of the formation of bone skeleton tissue (Fuery et al., 2017).
Nowadays, the opinion about the passive process of degeneration underlying the development of pathological vascular calcification and soft tissue ossification has changed to the presence of a dynamic, complexly regulated cellular and molecular pathological process that leads to the described defect. Vascular calcification develops in response to chronic inflammatory stimuli that are abundant in people with diabetes, hypertension, hyperlipidemia, and chronic kidney disease (Hunt et al., 2002; Jeziorska et al., 1998; Mohler et al., 2001a; Shao et al., 2006; Soor et al., 2008; Zimmet et al., 2001; Семенова Д.С., и др. 2021а). The mechanisms of vascular ossification are still under investigation, but have been shown to vary depending on the underlying pathophysiology of the calcification processes that precede tissue ossification (Demer and Tintut, 2014a, 2008; Vattikuti and Towler, 2004; Семенова Д.С., и др. 2021а).
There are two main ways by which bone tissue is formed in the body: the formation of an endochondral bone, which includes chondrogogenesis and the development of a cartilaginous intermediate product, as well as intraembrane bone formation. It progresses from the direct differentiation of mesenchymal stem cells to osteoblasts, which form the bone (Luo et al., 2019; Runyan and Gabrick, 2017; Семенова Д.С., 2021а).
Arterial calcification is an active cell-controlled process that promotes ossification through the expression of osteogenic growth factors, matrix proteins, and other bone formation-associated molecules that are normally expressed by osteoblasts in bone (Bostrom et al., 1993;
Canfield et al., 1996; Proudfoot et al., 1998). Hypotheses of the origin of osteoblast-like cells in vascular calcification are very controversial, but one of the most common versions is that these cells originate from the vessel wall itself. Calcifying valve cells initiate bone-like tissue formation through intramembrane ossification, and endochondral ossification also contributes to this, albeit to a lesser extent (Egan et al., 2011). Apparently, this process is mediated by the production of BMP2 and BMP4 along with osteopontin, osteocalcin, and osteonectin.
Ossification sites have also been found to contain bone-resorbing osteoclast-like cells (Mikhaylova et al., 2007). Therefore, similar to orthotopic bone formation, vascular ossification is likely an active process that involves continuous tissue remodeling. Thus, these data show that valve remodeling and calcification occur through the activation of pro-calcifying cellular and molecular processes, in many ways similar to the processes occurring during the physiological formation of bone tissue of the skeleton.
There are many studies confirming the global role of the Notch signaling pathway in the regulation of proliferation, differentiation, and self-renewal of stem cells in a variety of tissue types, including bone. Notch plays a key role in the development and regeneration of skeletal bones (Regan and Long, 2013). In addition, it is particularly important during the development and maintenance of cardiovascular homeostasis (Krebs et al., 2000). The spectrum of direct Notch targets is very broad and tissue specific. The outcome of Notch activation depends on the cell type and the context provided by the many possible combinations of receptors and ligands that transduce different biological effects (Briot et al., 2016). There is ample evidence that Notch signaling may play an important role specifically in the adult heart, and a key involvement of this signaling pathway in the pathogenesis of aortic valve calcification has been described (Garg et al., 2005; Irtyuga et al., 2017; Theodoris et al., 2015).
Genes and proteins encoded by them, which are involved in advanced stages of osteogenic differentiation, both in ectopic ossification and in the formation of skeletal bone tissue, belong to WNT, BMP, and RUNX families, which, in turn, are in many ways under control and regulation of the Notch signaling pathway.
To investigate the mechanisms that control osteogenic differentiation both in normal and pathological calcification is important from the point of view of the possibility of controlling this differentiation - to prevent ectopic osteotransformation of cells and tissue mineralization in various pathological conditions, or, on the contrary, to be able to promote the most effective bone regeneration.
Theoretical and practical significance of the work
On the one hand, an extremely important task of regenerative medicine is to find a solution for the effective growth and regeneration of bone tissue, including with the help of osteogenic differentiation of mesenchymal stem cells. Therefore, understanding which molecular components and how they are involved in different stages of osteodifferentiation is a prerequisite for being able to control the processes of osteogenesis. On the other hand, the ability to modulate cellular signals by controlling the expression of osteo gens may bring us closer to the ability to prevent unwanted osteogenic differentiation at sites of ectopic ossification.
In this work, we approached the study of the processes of osteogenic transformation of cells from several angles. The studies were carried out using cell cultures obtained from biological material of both patients with pathological tissue ossification and healthy people. Not many laboratories in the world have such wide access to biological material obtained from people, so this study has the advantage and special interest.
The results obtained in during this work can bring not only scientific value to the world community of scientists, but also become an important prerequisite for preclinical trials in the future. Finding potential therapeutic targets will be the basis for the development of conservative methods for the treatment of valvular calcification and vascular ossification or patients with skeletal anomalies, injuries, which will help maintain and improve the quality of life of many people around the world.
The aim and objectives of the study
The aim of this work: to study the molecular and genetic mechanisms underlying the processes of osteogenic cell differentiation in normal and pathological conditions.
Tasks:
1. To assess the osteogenic potential of mesenchymal stem cells derived from adipose tissue of people with aortic valve calcification compared with cells from healthy people
2. To study the effect of cocultivation of aortic endothelial cells with smooth muscle cells on the processes of osteogenic differentiation
3. To study the role of the Notch signaling pathway in the processes of osteogenic differentiation of a coculture of aortic endothelial cells and smooth muscle cells
4. To study the role of the Notch signaling pathway in the processes of osteogenic differentiation of smooth muscle cells
5. To study the effect of various components of the Notch signaling pathway on the osteogenic potential of adipose tissue mesenchymal stem cells
6. To study the influence and role of intercellular communications on the processes of osteogenic differentiation in mesenchymal stem cells of adipose tissue
Scientific novelty of the work Provisions for defense
1. It was noted that the mesenchymal stem cells of adipose tissue of patients with calcified aortic valve stenosis, in contrast to the interstitial cells of the aortic valve obtained from the same patients, as well as compared to the MSC of healthy donors, have a rather low ability for osteogenic differentiation
2. Endothelial cells are observed to induce osteogenic differentiation in aortic smooth muscle cells when they are cocultured even in the absence of an osteogenic medium.
3. It has been shown that when aortic smooth muscle cells are co-cultivated with endothelial cells, the Notch signaling pathway is activated. Co-cultivation of SMC and HAEC enhances the expression of Notch component genes in endothelial cells
4. Activation of the Notch signaling pathway promotes osteogenic differentiation of smooth muscle cells
5. Activation of the Notch signaling pathway by introducing lentiviruses carrying NICD or Jag1 into cells increases the ability of mesenchymal stem cells to osteogenic differentiation in a dose-dependent manner. However, excessive activation of Notch signaling induces
apoptosis in cultured mesenchymal stem cells and also reduces osteogenic differentiation in cells.
6. Intercellular communications contribute to the enhancement and maintenance of osteogenic differentiation processes in mesenchymal stem cells of adipose tissue
Publications and approbation of work
Based on the dissertation materials, 11 papers were published: 3 scientific articles in journals indexed by WoS and/or Scopus systems, and 8 publications in the materials of international and Russian conferences.
The main scientific results of the dissertation were presented in reports at scientific conferences.
List of articles published in journals indexed by WoS and/or Scopus:
1. Semenova D, Bogdanova M, Kostina A, Golovkin A, Kostareva A, Malashicheva A. (2020) Dose-dependent mechanism of Notch action in promoting osteogenic differentiation of mesenchymal stem cells. Cell Tissue Res. 2020 Jan;379(1):169-179. DOI: 10.1007/s00441-019-03130-7.
2. Kostina A, Semenova D, Kostina D, Uspensky V, Kostareva A, Malashicheva A. (2019) Human aortic endothelial cells have osteogenic Notch-dependent properties in co-culture with aortic smooth muscle cells. Biochem Biophys Res Commun. 2019 Jun 25;514(2):462-468. doi: 10.1016/j.bbrc.2019.04.177
3. A Malashicheva, O Irtyuga, A Kostina, E Ignatieva, E Zhiduleva, D Semenova, A Golovkin, M Gordeev, O Moiseeva, A Kostareva. (2018). Osteogenic potential of adipose mesenchymal stem cells is not correlated with aortic valve calcification. biological communications. Vol. 63 issue 2, 117-122. DOI: 10.21638/spbu03.2018.204
Theses:
1. Semenova D.S., Malashicheva A.B. (2017). The role of the Notch signaling pathway in osteogenic differentiation of mesenchymal stem cells. Appendix 1 to the journal Translational Medicine 2017, p. 93 2.
2. Semenova D.S., Malashicheva A.B. (2017). Dose-dependent role of various components of the Notch signaling pathway in osteogenic differentiation of stem cells. Genes and Cells, Volume XII, No. 3, 2017, p. 218-219
3. Kostina A, Kiselev A, Semenova D, Irtyuga O, Kostareva A, Malashicheva A. Notch-dependent regulation of osteogenic differentiation of interstitial cells from human aortic valve. 2019. Collection of The Notch Meeting 2019 conference.
4. Semenova DS, Kostina AS, Irtyuga OB, Moiseeva OM, Malashicheva AB. Mechanisms of Notch-dependent intercellular communications in aortic valve calcification. 2019. Collection of the conference The Notch Meeting XI, 2019
5. Semenova D, Golovkin A, Malashicheva A. The role of Notch signaling pathway in osteogenic differentiation of mesenchymal stem cells. 2017. Collection of the conference The Notch Meeting X, 2017.
6. Semenova D.S., Malashicheva A.B. The role of the Notch signaling pathway in aortic valve calcification. 2018. XIX Winter Youth School on Biophysics and Molecular Biology. The abstracts are published in the conference proceedings.
7. Semenova D.S., Malashicheva A.B., Kostina A.S. Osteogenic differentiation of cells in the study of pathologies of the heart and blood vessels. StemCellBio 2018. Theses are published in the collection of the conference "StemCellBio-2018: fundamental science as the basis of cellular technologies."
8. Semenova D.S., Malashicheva A.B. The role of the Notch signaling pathway in osteogenic differentiation of mesenchymal stem cells. 2017. XVIII Winter Youth School on Biophysics and Molecular Biology. The abstracts are published in the conference proceedings.
1. LITERATURE REVIEW
1.1. The mechanisms of pathological tissue calcification are similar to the processes of physiological bone formation
The processes underlying the physiological formation of bone tissue have similar features with the mechanisms that cause the development of pathological calcification of the tissues of the heart and blood vessels. Triggering, initiating mechanisms leading to ectopic calcification of the heart and blood vessels remain largely unexplored. Vascular calcification is a common cardiovascular complication that accompanies aging and various pathological processes in the human body (Demer and Tintut, 2014b). The mechanisms involved in the pathogenesis of vascular calcification remain largely unknown, and there are currently no therapies to prevent and reverse calcification, except for complex invasive surgeries and costly transcatheter procedures, which have their own limitations and disadvantages. A detailed understanding of the mechanisms of calcification is critical to the possibility of developing other treatments for the disease.
Vascular calcification can occur in the medial smooth muscle layer and in the intimal layer of the vessel wall. Previously, vascular calcification was considered a passive process. However, a large amount of evidence has accumulated in recent years, indicating that this is an active and highly regulated process. It is known that Notch/BMP/TGF-b signaling pathways play an important role in these processes. It has been repeatedly shown that the BMP signaling pathway plays an important role in the processes of osteogenic cell transformation (Semenova D. 2021a). In experimental models, activated endothelial cells have been shown to secrete BMP2 and BMP4 in response to changes in the laminar blood flow pattern, and BMP2 has also been found in interstitial cells derived from the aortic valve of elderly rats (Seya et al., 2011). BMP proteins stimulate calcification by activating the Smad and Wnt/p-catenin signaling pathways, as well as upregulating the expression of the osteochondrogenic transcription factor Msx2. These signaling pathways work together to initiate the expression of the Runx2 transcription factor (Bostrom et al., 2011). When Runx2 is expressed, cells enter the osteoblast differentiation pathway, and expression of calcification-associated proteins such as osteopontin, bone sialoprotein II, and osteocalcin is increased, thus calcification is induced (Fig. 1) (Butcher et al., 2006).
Figure 1. Signaling pathways that play a significant role in the acquisition of the osteophenotype by cells (Leopold, 2012).
The complex interactions between the molecules of these major signaling pathways also confirm the similarity between the processes of pathological tissue ossification and the mechanisms of bone formation. In addition, in pathological calcification, mineral deposits in the extracellular matrix by osteoblast-like cells are similarly observed (Bostrom et al., 2011). The origin of osteoblast-like cells in the cardiovascular system is a controversial issue that requires further study and clarification. It has been suggested that osteogenic cells in the medial layer can transdifferentiate from medial smooth muscle cells in situ (Speer et al., 2009). Other studies have shown that progenitor cells of mesenchymal origin in the vessel wall may be involved in vascular calcification (Farrington-Rock et al., 2004; Tintut et al., 2003).
Two main types of bone formation have been described: endochondral bone formation, which includes chondrogenesis and development of a cartilaginous intermediate, and intramembranous bone formation, which progresses from direct differentiation of mesenchymal stem cells into bone-forming osteoblasts. Pathological calcification occurring in
valvular or vascular tissues of the body appears to involve both intramembranous and endochondral processes (Mohler et al., 2001b; Семенова Д.С. и др., 2021а).
It is well known that mesenchymal stem cells (MSCs), including bone marrow and adipose tissue MSCs, have the ability to differentiate into many cell types, including osteoblastic and chondrogenic lines (Mushahary et al., 2018). Both human and animal studies have shown that MSCs can enter the bloodstream. It has been suggested that the migration of distant MSCs from their original niche, followed by activation towards osteoblastic cells in affected vessels, also plays a role in the process of vascular calcification (Otsuru et al., 2007; Rochefort et al., 2006; Wang et al., 2014). The so-called "circulating cell theory" suggests that circulating stem cells/bone marrow osteoprogenitors find their place in diseased arteries, which contributes to the initiation and progression of vascular calcification (Pal and Golledge, 2011). In addition, due to their high plasticity, vascular smooth muscle cells have the ability to transition from their normal differentiated contractile phenotype to a variety of synthetic dedifferentiated states, demonstrating in some cases chondrogenic and osteogenic differentiation. These processes also play a key role in the pathogenesis of vascular diseases (Bostrom, 2016).
Osteoblasts derived from bone marrow mesenchymal stem cells (MSCs) are the cells primarily responsible for bone formation by calcification of the extracellular matrix (Ortuno et al., 2010). It is known that the differentiation of MSCs into osteoblasts is under the control of numerous transcription factors and signaling proteins (Ortuno et al., 2010). RUNX2 and Osterix (OSX) are important transcription factors whose activation is required for the processes of osteogenic cell differentiation (Yang et al., 2004). Although RUNX2 and OSX have been positioned as key regulators of osteogenesis, there are also other transcription factors involved in osteoblast differentiation, including DLX5, DLX3, FRA1, Twistl, ZBTB16, and ATF4 (Yang et al., 2004). There is evidence that ZBTB16 is a downstream transcription factor that is involved in osteoblast differentiation. Thus, ZBTB16 has a positive regulatory effect on osteoblast differentiation due to their close interaction with the key osteogenesis regulators OSX and RUNX2 (Семенова Д.С., и др., 2021а; Семенова Д.С. и др., 20216).
ZBTB16/PLZF is a highly conserved gene from the nematode Caenorhabditis elegans to humans. In humans, the PLZF gene contains six exons and five introns. Its exon sizes vary from 87 to 1358 bp. Exons are distributed over a region of approximately 120 kb. PLZF exhibits complex splicing patterns depending on the tissue in which the gene is expressed. The product
has at least four isoforms detectable with 1 exon. The PLZF protein includes nine motifs of Kruppel-like C2H2 zinc fingers at the C-terminus, a lesser-known RD2 domain, and a BTB/POZ (poxvirus, zinc finger) at the N-terminus. Nine Kruppel-like C2H2 zinc fingers promote specific DNA binding to target genes, allowing PLZF to function as a transcription factor. The BTB/POZ domain is an evolutionarily conserved motif that mediates protein-to-protein interactions and allows POZ domain proteins to participate in various processes including hematopoiesis, angiogenesis, neurogenesis, adipogenesis, osteoclastogenesis, and muscle differentiation (Liu et al., 2016).
Studies have shown the importance of the functional activity of ZBTB16 in the differentiation of stem cells into osteoblasts (Hemming et al. 2016; Onizuka et al. 2016). ZBTB16 appears to function as an important marker in the later stages of osteoblastic stem cell differentiation (Saugspier et al., 2010).
There is evidence in the literature for the involvement of ZBTB16 in skeletal development (Fischer et al. 2008). ZBTB16 has been shown to play a role in the specification of axial and limb patterning. In addition, ZBTB16 expression is upregulated in cells from patients suffering from ectopic bone formation (Inoue et al., 2006).
One of the most common diseases of the cardiovascular system is the development of calcification of the aortic valve. Currently, there is no medical treatment to prevent or stop this disease. The main feature of the disease is the progressive mineralization of the valve tissue. In many ways, the pathological processes that cause the mineralization of the aortic valve also explain the mechanisms that occur during vascular calcification. Both pathological processes are similar to the processes of normal physiological formation of bone tissue of the skeleton. Both during embryonic development and during periods of bone regeneration (Mathieu and Boulanger, 2014).
1.2. Aortic valve
The aortic valve (AV) is an avascular, thin, flexible tissue formation. It is on the border between the left ventricle of the heart and aorta. The key function of the AV is to prevent the reverse flow of blood, which during the contraction of the ventricle of the heart goes into the aorta.
AV leaflets consist of three layers: ventricular, spongy and aortic (Butcher et al., 2011). On the aortic and ventricular sides of the valve leaflets are covered with endothelial cells (EC). All layers in the aortic valve leaflet contain one or another number of different components of the extracellular matrix with a predominance of special features in each of the layer. The aortic mainly contains collagen fibers oriented in a circular direction in the form of beams and heavy, and a small number of elastic fibers (Butcher et al., 2011). The ventricular layer is more thin and contains a much larger number of elastic fibers compared to the aortic. The spongiosis layer contains a large amount of glycosaminglycans and proteoglycans. At the same time, the interstitial valve cells (VIC) (Semenova D.S., 2018, master's dissertation) are between all components of the extracellular matrix in three layers. This heterogeneous population of cells has unique characteristics and is necessary to maintain the function and homeostasis of the valve by means of cell proliferation, the secretion of matrix metalloproteinase and components of extracellular matrix (Schoen, 2012).
It is assumed that VIC are the main functional units of the valve, which undergo mineralization (Rutkovskiy et al., 2017) and are able to express genes associated with the osteogenic phenotype (Rabkin-Aikawa et al., 2004). A critical task of ongoing research is to determine the origin of the cells that contribute to valve calcification (Leszczynska and Mary Murphy, 2018). It has been discussed that peripheral MSC may be the very cells that contribute to aortic valve calcification (Liu and Xu, 2016).
In particular, it has also been shown in our laboratory that VIC from patients with aortic valve calcification are more susceptible to pro-osteogenic induction and undergo osteogenic differentiation more easily compared to VIC derived from normal healthy valves (A et al., 2018; Malashicheva et al., 2018).
1.3. Extracellular vesicles are initiators and mediators of tissue ossification processes
Multiple studies in recent years have shown the key role of extracellular vesicles circulating in valve tissue in both maintaining tissue homeostasis and in the development of calcified valve stenosis or vessel ossification, depending on the contents of the vesicles (Aikawa and Blaser, 2020; Blaser and Aikawa, 2018; Bouchareb et al., 2014; Jansen et al., 2017; Vito et al., 2021).
Extracellular vesicles are highly heterogeneous, but they can be divided into two main categories: exosomes and microvesicles. The secretion of extracellular vesicles was originally described as a way to remove unwanted compounds from the cell (Pan and Johnstone, 1983). However, we now understand that extracellular vesicles are more than just waste carriers, and the main interest in this area is now focused on their ability to exchange various components between cells. Extracellular vesicles are now considered as an additional mechanism of intercellular communication. This method allows cells to exchange proteins, lipids and genetic material, and the content of vesicles may vary depending on the cell and conditions. However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting, and fate of these vesicles. Exosomes are typically 30-150 nm in diameter. Their formation comes from the endosomal system. Exosomes form during the maturation of early endosomes, internal budding of the endosomal membrane leads to the formation of multivesicular endosomes, also referred to as late endosomes (McGough and Vincent, 2016). During this process, many cytoplasmic components can be incorporated into endosomes and then targeted either to lysosomes, to the autophagosome for degradation, or to the plasma membrane for secretion (Liu et al., 2021). Microvesicles, in turn, have a diameter of 50 to 1000 nm. Their biogenesis occurs through an initial specific redistribution of plasma membrane proteins and lipid components aimed at regulating membrane stiffness. Direct external vesicle formation and budding of these plasma membrane regions then leads to the release of microvesicles into the extracellular space (Schubert and Boutros, 2021). Much has been achieved in understanding the biological role of extracellular vesicles derived from resident heart cells in homeostasis and repair of cardiac tissue (Mancuso et al., 2020). In particular, extracellular vesicles underlie the paracrine mediators of cardiac stem cells, widely known for their regenerative and reparative functions. According to much of the literature, exosomes can serve as a tool for assessing and predicting the course of a disease and may also provide new clinical biomarkers for developing therapeutic approaches.
In vitro experiments under physiological conditions have shown that extracellular vesicles derived from aortic valve cells contain high levels of calcification inhibitors (Jansen et al., 2017). Thus, under physiological, non-calcifying conditions, extracellular vesicles appear to maintain valve homeostasis and prevent calcification (Fig. 2).
Figure 2. Under physiological, non-calcifying conditions, extracellular vesicles maintain valve homeostasis and prevent tissue calcification.
In contrast, in pathological inflammatory conditions, extracellular vesicles begin to show the ability to stimulate calcification. They cause endothelial dysfunction, inflammation in valve tissues, and mediate transformation of valve endothelial cells through the endothelial-mesenchymal transition. EC differentiation is the result of several pathological conditions such as oscillatory shear stress and the presence of inflammatory cytokines (Xu et al., 2020). When stimulated, EC gradually lose their normal endothelial phenotype and activate myofibroblast gene regulation programs. After the cells acquire a mesenchymal or myofibroblasts phenotype, osteogenic differentiation of these cells can also occur. Furthermore, these cells, in turn, induce osteogenic differentiation of valve interstitial cells into osteoblast-like cells (Xu et al., 2020). Extracellular vesicles containing alkaline phosphatase, annexin, excess calcium and phosphate become primary calcification nuclei that aggregate and form microcalcifications,
and then macrocalcifications form. This eventually leads to calcified aortic valve stenosis (Jansen et al., 2017). (Fig. 3).
Figure 3. Role of extracellular vesicles in aortic valve tissue calcification under pathological conditions.
Matrix Gla protein (MGP), a y-rich carboxyglutamic acid and vitamin K dependent protein, prevents calcification by inhibiting BMP signaling (Yao et al., 2010). MGP levels have been shown to be significantly lower in patients with aortic valve calcification compared to healthy controls (Koos et al., 2009). These data suggest that a decrease in MGP expression or
activity may contribute to the progression of aortic valve calcification. MGP activity depends on carboxylation status and vitamin K availability (Semenova D.S., 2018, master's dissertation).
Fetuin-A is a liver-derived protein that is a potent circulating inhibitor of calcification (Jahnen-Dechent et al., 2011). Fetuin-A acts by binding calcium and phosphate deposits, stabilizing these ions and preventing them from being taken up by cells (Semenova D.S., 2018, master's dissertation). It is known that fetuin-A deficiency is associated with the development of soft tissue calcification in experimental models, and the level of fetuin-A in the blood serum of patients with aortic valve calcification is lower compared to the control group (Jahnen-Dechent et al., 2011; Kaden et al., 2007).
Thus, these data indicate that valve remodeling and calcification occur upon activation of pro-calcifying cellular and molecular processes. At the same time, the reduced activity of circulating inhibitors of calcification in this case becomes insufficient to prevent calcification of the aortic valve.
1.4. Role of the Notch signaling pathway in osteogenic cell differentiation
Osteogenic differentiation is a highly regulated process that occurs through the functional activity of specialized cells called osteoblasts. Activation factors for genes and cell signaling pathways in the early stages of osteogenic differentiation both in normal and pathological ossification remain the subject of active research. It is extremely important to investigate and understand the early mechanisms which trigger osteogenic differentiation, because it creates the potential opportunity to control and manage the processes of osteogenesis in the body. Induction of osteogenic differentiation may be necessary for bone tissue regeneration in complex fractures. Stopping the differentiation of cells in the osteogenic direction is required for prevention in various pathologies associated with pathological calcification of tissues (Semenova et al., 2019).
Notch1-4 are a family of transmembrane receptors. They play a critical role in cell fate decisions (Andersson et al., 2011). Notch signaling pathway controls skeletal development and maintenance of homeostasis, as well as cell differentiation towards osteoblasts and osteoclasts (Canalis, 2018; Zanotti and Canalis, 2016). Notch is activated by direct interaction between the receptor and the ligand. As a result of contact, the Notch receptor intracellular domain (NICD)
is released. During the implementation of the canonical signaling pathway, NICD is transferred to the nucleus, where it displaces transcriptional repressors and, interacting with proteins, initiates the transcription of signaling pathway target genes. The classical targets of the canonical Notch signaling pathway are the hairy enhancer of split (Hes)1, -5, -7, as well as the Hes related with YRPW motif (Hey)1, -2 and -L (Zhou et al., 2022).
Figure 4. Components and key participants in the Notch signaling pathway (Luxan et al., 2016).
Over the past decade, there has been a sharp increase in interest in the role played by the Notch signaling pathway in pro-osteogenic events during physiological development and bone healing. However, the interpretation of the results of studies conducted on various experimental models, for example, using transgenic mouse models and cell lines of different origins, simultaneously used different variants of activation of the Notch signaling pathway using different ligands. All these combinations of input data give conflicting results. Opinions regarding the specific role of Notch, consisting either in the induction and maintenance or, on the contrary, in the suppression of osteogenic cell differentiation, remain ambiguous to this day. There has been evidence that deletion or mutation of one or more Notch signaling pathway genes results in severe skeletal pathology in humans and in mouse models (Canalis, 2018). Mutations in the Jagged-1 (Jagl) or Notch2 genes cause Alagille syndrome. This autosomal dominant disease in humans is characterized by the development of severe abnormalities in the development and formation of skeletal tissue, including osteopenia and a high incidence of fractures. Dominant positive mutations activating Notch2 cause Hajdu-Cheney syndrome, which also has a highly pronounced skeletal phenotype (Canalis, 2018). Based on the results of a number of studies conducted on cell cultures, it became known about the ambiguous effect of Notch activation and signaling on the differentiation properties of cells. It turned out that Notch can both promote the induction of differentiation in cells (Doi et al., 2006; Shimizu et al., 2011; Tezuka et al., 2002; Ugarte et al., 2009) and have an inhibitory effect on osteogenesis processes (Bai et al., 2008; Hilton et al., 2008; Shindo et al., 2003). In mouse preosteoblast cells, Notch suppressed osteoblast maturation by binding NICD or Notch target genes HES1 and HEY1 to RUNX2 (Engin et al., 2008; Hilton et al., 2008; Salie et al., 2010). More recent work in cell models has shown that Notch promotes the induction of osteogenic differentiation (Cao et al., 2017; Cui et al., 2019; Liao et al., 2017; Liu and Xu, 2016). In a transgenic mouse model, loss of functional activity of the Notch signaling pathway resulted in bone osteoporosis, while Notch activation led mainly to the development of either osteoporosis or an osteosclerotic phenotype (Engin et al., 2008; Hilton et al., 2008; Zanotti et al., 2008; Zanotti and Canalis, 2016). Thus, the described controversial results still leave open the question of the role and influence of the Notch signaling pathway on the course of osteogenic differentiation in cells (Semenova et al., 2019). One of the main characterizing qualities of Notch is its tissue specificity, as well as the fine tuning of signaling due to the absence of an amplification step (Andersson et al., 2011; Yamamoto et al., 2014). The sensitivity of Notch to a specific degree of pathway activation depending on gene "dose" has been shown in some experimental systems
(Guruharsha et al., 2012). However, it was not previously known how exactly these mechanisms are carried out in the processes of osteogenic cell differentiation.
2. MATERIALS AND METHODS
2.1. Primary cell cultures
All procedures performed in human research comply with the ethical standards of the local and/or national research ethics committee and the 1964 Declaration of Helsinki and its subsequent amendments or comparable ethical standards.
1. 1. The study used adipose tissue mesenchymal stem cells (aMSC) obtained from healthy donors. The sampling was carried out by medical personnel. All patients signed an informed consent for the collection and use of the material in scientific research.
In addition, aMSC was also obtained from patients with aortic valve calcification.
The procedure for isolating aMSC from adipose tissue was described in detail in the bachelor's thesis (Semenova D.S., 2016, bachelor's dissertation). Under sterile conditions, 1 g of adipose tissue taken from the umbilical region was fragmented to a homogeneous mass by resuspension and transferred to 35 mm dishes with a solution of DMEM : type 1 collagenase (1:1). Spent incubation at 37°C for 30 minutes. After that, the suspension was centrifuged with 500g for 5-10 minutes, after which the supernatant was removed, the culture medium was added to the sediment and transferred to Petri dishes.
2. To obtain cultures of smooth muscle cells (SMCs), cells were isolated from the aortic wall by collagenase cleavage, as described previously (Kostina et al., 2019; Malashicheva et al., 2016). SMCs were cultured in a nutrient medium containing DMEM (Invitrogen) supplemented with 20% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine, sodium pyruvate, and penicillin/streptomycin (100 mg/L) (Invitrogen).
3. Human aortic endothelial cells (HAECs) were isolated from the aortic wall by collagenase digestion as previously described (Kostina et al., 2016). HAECs were cultured in endothelial cell medium ECM (ScienCell, USA).
4. Interstitial cells of the human aortic valve (VICs). Fragments of aortic valves in patients with calcified aortic stenosis were obtained during aortic valve replacement performed at the N.N. V.A. Almazov of the Ministry of Health of Russia. To obtain interstitial cells, the valve was incubated in a type IV collagenase solution at 37°C for 24 hours. The medium for culturing valve interstitial cells contained: DMEM (Gibco, USA), 15% FBS (Hyclone, USA),
1% penicillin/streptomycin (Invitrogene, USA), and 1% L-glutamine (Invitrogene, USA) (Semenova D.S., 2018, master's dissertation) (Семенова Д.С., и др., 2021а).
5. An immortalized human embryonic kidney cell line (HEK293T) was used to create lentiviral particles.
2.2. Cell cultivation and induction of osteogenic differentiation
Osteogenic differentiation in all cell types was induced by adding to the cultivation medium 50 цМ ascorbic acid, 0.1 цМ dexamethasone, and 10 mM beta-glycerophosphate (Sigma, USA).
The medium was changed twice a week. Differentiation was considered terminal after 20-21 days of induction.
After undergoing differentiation, the cells were stained with a specific dye that detects calcium deposits - alizarin red. Cells were washed with PBS, fixed in 70% ethanol for 60 min. Washed twice with distilled water and stained with alizarin red solution (Sigma) (Semenova et al., 2019).
2.3. Cocultivation of cell cultures
1. For experiments on the cocultivation of aMSCs, half of the required number of cells were seeded and transduced with lentiviral particles. The next day, the rest of the aMSCs were seeded onto the cell monolayer without any genetic modifications.
2. For experiments on co-cultivation of SMCs and HAECs, SMCs in the amount of 100x103 were seeded in 6-well plates coated with 0.2% gelatin. After 24 hours, the cell monolayer was seeded with HAECs in two different amounts: 100x103 or 500x103. The analysis was carried out on 5 and 10 days after the start of cocultivation.
2.4. Production of lentiviruses
The plasmids needed to package the lentiviral particles were a generous gift from Prof. Didier Trono (Switzerland). Plasmid pGa981-6 was donated by Professor Urban Lendahl (Sweden). 12XCSL-luciferase was cloned into the AscI and Spel restriction sites of pLVTHM-
T7-cm. Notch Intracellular Domain (NICD), Dll1, Dll4 and Jag1 viruses have been described previously (Kostina et al., 2016).
The production of lentiviruses was carried out according to the protocol described in details earlier (Malashicheva et al. 2007). Briefly, 293T cells were co-transfected with 15 p,g of plasmid of interest, 5.27 ^g of pMD2.G, and 9.73 ^g of pCMV-dR8.74psPAX2. Transfection was carried out using the calcium phosphate method or the method using PEI reagent. The lentivirus generated by the cells was concentrated from the supernatant by ultracentrifugation, then resuspended in 1% BSA/PBS and frozen in aliquots at -80°C. The virus titer was determined by GFP-expressing virus; cell transduction efficiency was 90-95%.
2.5. Magnetic cell separation
CD31, also known as PECAM-1, is a constitutively transmembrane glycoprotein that is expressed on the surface of endothelial cells. HAECs were sorted from SMCs in a magnetic cell separation (MACS) experiment with anti-CD31+ conjugated microbeads (Miltenyi Biotec, Germany) according to the manufacturer's instructions. After co-culture of the cells, cells were resuspended in medium, mixed with FcR Blocking Re agent and CD31 MicroBeads. The cells were then loaded onto a column placed in a magnetic field. CD31+ cells (endothelial cells) remained on the column, while cells negative for this marker (SMC) passed through it.
After removing the column from the magnetic field, the CD31+ cells retained by the magnet were eluted and analyzed (Kostina et al., 2019).
2.6. Cell staining for alkaline phosphatase
One of the methods for visualizing the passage of osteogenic differentiation in cells is the detection of alkaline phosphatase (ALP) activity. Staining for ALP was performed using Roche NBT/BCIP (Roche, Germany). 10 days after the start of cocultivation of SMCs and HAECs, the cells were washed with PBS, after which they were incubated with an alkaline phosphatase working solution for 15-20 min at room temperature. ALP activity was manifested as a blue staining.
2.7. Promoter activity assay
To assess the activity of the Notch signaling pathway, we transduced the cells with a lentivirus carrying the 12XCSL-luciferase reporter construction. Then the CSL activity was measured. Cell lysis was performed using a luciferase assay.
System (Promega) according to the manufacturer's recommendations. Luciferase activity was measured using Synergy2 (BioTek, USA). Samples were normalized for protein content using the Pierce BCA Protein Assay Kit (Thermo Scientific) (Semenova et al. 2019).
2.8. Detection of apoptosis in cells
On the 4th day after differentiation induction of aMSCs. Cells were removed from the plastic surface using trypsin. Cells were then diluted in Annexin Binding Buffer (Biolegend) and stained using AnnexinV-PE (Biolegend) according to the manufacturer's instructions. Flow cytometry was performed on a GuavaEasyCyte8 apparatus with the detection of annexin V + (apoptosis) cells in relative percentage. At least 10000 cells were analyzed per sample. All analyzes were performed in triplicate.
2.9. Real-time PCR
RNA was isolated from cell cultures using the ExtractRNA reagent (Evrogen, Russia). Total RNA was reverse transcribed using the MMLV RT kit (Evrogen, Russia). Real-time PCR was performed in an ABI 7500 thermal cycler (Applied Biosystems, USA). There were used specific forward and reverse primers for target genes (HEY1, HES, SPP1, RUNX2, POSTN, SOX5, ALP, NOTCH1-4, ZBTB16, BMP2, BMP4, COL1A1, IBSP, DLX2, PDK4). The expression levels of the analized genes were normalized according to the expression level of GAPDH housekeeping gene. The data were analyzed with 7500 Software v2.0.6 and Ct values were obtained for each gene. The change in gene expression levels (compared to controls) was calculated using the AACt method. All experiments were performed in three biological replicates, that is, in three independent experiments. (Семенова Д.С., и др., 2021а)
The information obtained using real-time PCR method was processed using Microsoft Excel and GraphPad Prism programs. The data in the results are presented as an average of all experiments performed.
3. RESULTS AND DISCUSSION
3.1. A study to determine whether adipose tissue MSC from patients with severe aortic valve calcification have the potential and predisposition to osteogenic differentiation
We have recently shown that aortic valve interstitial cells (VIC) derived from patients with severe aortic valve calcification are more susceptible to pro-osteogenic induction and more readily undergo osteogenic differentiation compared to VIC from normal healthy valves (A et al., 2018). Based on these data, we hypothesized that adipose tissue mesenchymal stem cells (aMSC) from patients with calcified valve stenosis may also be more prone to osteogenic transformation and may show the same responsiveness to pro-osteogenic stimuli as the VIC of patients with calcified valve stenosis (A Kostina et al. 2019).
3.1.1. MSC of adipose tissue of patients with calcified aortic stenosis, in contrast to VIC, have a rather low ability for osteogenic differentiation
In order to test the hypothesis that an increased potential for osteogenic differentiation may be a common characteristic of all stem cells from patients with pathological aortic valve ossification, we obtained MSC from the adipose tissue of patients with severe aortic valve calcification. VIC were isolated from the valves of the same patients. Adipose tissue MSC and VIC obtained from healthy donors were used as controls (Malashicheva et al., 2018). Immunophenotyping of MSC and VIC cells from patients and healthy donors was performed using commonly accepted MSC markers (Dmitrieva et al. 2015). As a result, no significant differences were observed either between the two cell types or between patients and healthy donors (Fig. 5).
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Figure 5. Determination of the phenotype of fat mesenchymal stem cells (MSC) and aortic valve interstitial cells (VIC) obtained from patients with calcification of the aortic valve (CAVD) and from healthy donors (C). Red indicated histograms with control isotypes; Green indicated histograms with appropriate stains (Malashicheva et al. 2018).
Then we compared aMSC-VIC pairs obtained from the same patients in terms of their ability to undergo osteogenic differentiation and acquire an osteo-like phenotype. After that, we compared them with aMSC and VIC of healthy donors, respectively (Fig. 6). We obtained evidence that patient's aMSC were completely devoid of the ability for osteogenic differentiation, while control aMSC were positive for calcium deposits. At the same time, VIC from calcified aortic valves showed a significantly more intense level of osteogenic differentiation compared to VIC from healthy donors when stained with alizarin red. We then compared the expression of one of the main osteogenic markers RUNX2 under control conditions and 21 days after the induction of osteogenic differentiation in cells (Fig. 7). As a result, we observed a significant increase in the level of RUNX2 expression after induction of
differentiation only in aMSC from healthy donors, as well as in the case of induction of osteogenic differentiation in VIC.
Thus, we can only speak of the existence of plasticity and sensitivity to osteogenic stimulation in VICs from patients with aortic valve calcification, but not in their aMSC.
Figure 6. Comparative sensitivity analysis to osteogenic stimuli of healthy donor VIC (C VIC), VIC from calcified aortic valves (VIC CAVD), healthy adipose tissue MSC (C MSC), and MSC from patients with aortic valve calcification (MSC CAVD). Stained with a specific dye, alizarin red. Groups were compared using the non-parametric Mann-Whitney test; the line represents the median. The bottom panel is osteogenic differentiation visualized by cell staining with alizarin red (Malashicheva et al. 2018).
Figure 7. Analysis of the expression of the osteogenic marker RUNX2. A comparison of sensitivity to osteogenic stimuli was made between healthy interstitial valve cells (C VIC), VIC from calcified aortic valves (VIC CAVD), healthy aMSC (C MSC), and aMSC from patients with aortic valve calcification (MSC CAVD). The cells were cultured in an osteogenic medium for 21 days and the expression level of RUNX2 was determined by real-time PCR. Groups were compared using the non-parametric Mann-Whitney test; the line represents the median. Asterisks indicate significant differences (p<0.05) in the content of RUNX2 mRNA between nondifferentiated and differentiated cells for this group (Malashicheva et al. 2018).
3.1.2. Discussion of the results obtained in the study of the evaluation of the osteogenic potential of mesenchymal adipose-derived stem cells in people with aortic valve calcification compared with aMSC of healthy people
We have shown that, despite the potential and ability of aortic valve interstitial cells from patients with severe aortic valve calcification to osteogenic differentiation, MSC derived from these same patients do not have the ability to osteogenic differentiation. At the same time, VIC of aortic valves from healthy donors had a significantly lower ability for osteogenic transformation compared to VIC of patients with calcification. MSC from healthy donors were
capable of osteogenic differentiation (Malashicheva et al., 2018). The difference between VIC of healthy and calcified valves in their differentiation ability was also recently shown in our laboratory (K. A et al. 2018). Surprisingly, the osteogenic potential of adipose MSC did not correlate with aortic valve calcification. Previously, it was reported that the osteogenic potential of MSC significantly decreases with age, as well as with cardiovascular disease (Dmitrieva et al. 2015). In this study, the group of patients with calcified aortic stenosis was indeed older than the control groups in both VIC and aMSC. To the best of our knowledge, no studies have yet been conducted that have examined different cells obtained from the same patients with calcified aortic stenosis in terms of analyzing their osteogenic potential. The "circulating stem cell theory" suggests that vascular calcification, and in particular valvular calcification, can be mediated and initiated by circulating MSC of various origins (K. I. Bostrom 2016; Bostrom, Rajamannan, and Towler 2011; Pal and Golledge 2011). Our data suggest that at least adipose tissue MSC are unlikely to be such mediators, since they are completely devoid of the ability for osteogenic differentiation in patients with calcified aortic stenosis (Malashicheva et al. 2018). This plasticity is most likely due to the embryological origin of these cells and reflects the tissue specificity of the aortic valve. Signals that promote osteogenic transformation of valve cells do not appear to initiate osteotransformation of adipose MSC in patients with aortic valve calcification. This issue of tissue specificity must be taken into account when modeling the disease with stem cells of mesenchymal origin.
3.2. A study aiming to investigate pro-osteogenic stimuli in coculture of human aortic endothelial cells (HAEC) with human aortic smooth muscle cells (SMC)
The role of intercellular signaling in the processes mediating cell differentiation, as well as determining their future fate, is significant. With the understanding of the general biological significance of intercellular signaling between different cell populations, the interest of scientists has now shifted from traditional cell monocultures to so-called cocultures, when two different types of cells are cultivated together. It has recently been shown in our laboratory that co-culture of human aortic valve interstitial cells with endothelial cells leads to a significant increase in the osteogenic potential of the cells (K. A et al. 2018). Thus, in this study, we investigate the coculture of human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (SMC) as a model system for studying pathological vascular calcification (A. Kostina et al. 2019).
3.2.1. Endothelial cells induce osteogenic differentiation in aortic smooth muscle cells when they are co-cultured even without an osteogenic medium
In order to find out whether there is an effect of intercellular communication between endothelial and smooth muscle cells on osteogenic differentiation in the absence of an osteogenic environment, we analyzed the expression levels of pro-osteogenic markers after co-cultivation of SMC with HAEC. Endothelial cells were added to SMC at two different doses 100x103 and 500x103 cells per well (Fig. 8). We have shown that RUNX2, POSTN, and COL1A1 expression levels are increased when two cell cultures are co-cultivated compared to SMC monoculture. At the same time, the degree of this increase depended on the dose of endothelial cells used in co-cultivation (Fig. 8, A). The mRNA of such osteo genes as BMP2, BMP4 and SOX5 significantly increases only when SMC were co-cultivated with a large number of endothelial cells (Fig. 8, A). Alkaline phosphatase staining 10 days after the start of coculture confirmed that endothelial cells induce an osteogenic phenotype in coculture with SMC. At the same time, we also noted that the intensity of staining correlates with the number of endothelial cells used in coculture (Fig. 8, B). No induction of osteogenic differentiation was found when SMC were co-cultivated with different amounts of SMC, which was used as a control (Fig. 8, B). Thus, we confirmed that it is not the increase in cell density itself that increases the osteogenic potential of SMC, but it is the specific effect of endothelial cells that has such an osteoinducing effect on SMC. Thus, endothelial cells are able to induce osteogenic phenotype in SMC without an osteogenic environment.
Figure 8. Aortic endothelial cells (HAEC) induce osteogenic differentiation in aortic smooth muscle cells (SMC) in a dose-dependent manner. (A) Activation of pro-osteogenic gene expression by co-cultivation of SMC with HAEC (n = 5) after 5 days of co-culture. mRNA levels were analyzed by real-time PCR and normalized for the level of GAPDH gene mRNA. (B) To assess the induction of the pro-osteogenic phenotype of cell culture, staining for alkaline
phosphatase activity was performed after 10 days from the start of co-cultivation. (Kostina et al., 2019).
3.2.2. Co-cultivation of SMC with HAEC activates Notch signaling pathway
The Notch signaling pathway is a key signaling pathway for cell-to-cell communication between neighboring cells. To find out whether Notch is involved in cell-to-cell communication during co-cultivation of SMC and HAEC cells, we examined the levels of mRNA expression of Notch component genes 5 days after the start of co-cultivation of cells. We found that the expression of HEY1 Notch target gene, as well as NOTCH1, NOTCH3, and NOTCH4 signaling pathway receptors, is upregulated when co-cultured with more endothelial cells compared to SMC monocultures. At the same time, NOTCH2 mRNA level only slightly decreased in the cell coculture (Fig. 9). Our results showed a possible correlation between the level of Notch activation and the efficiency of SMC osteogenic differentiation, which indicates the involvement of the signaling pathway in the processes of osteotransformation of SMC cells.
Figure 9 Co-cultivation of SMC and HAEC induces gene expression of Notch components in a dose-dependent manner. (Kostina et al., 2019)
3.2.3. Activation of the Notch signaling pathway promotes osteogenic differentiation of smooth muscle cells
To study the SMC response to Notch signaling pathway activation, we induced Notch signaling in smooth muscle cells by transduction with varying amounts lentiviruses bearing Notchl receptor intracellular domain (NICD). Different amounts of virus correspond to 20 and 100 multiplicity units of infection (MOI). Expression of HEY1 Notch target gene, SMC ACTA2 marker, MGP2 calcification inhibitor, and proosteogenic genes in the presence of various doses of NICD 5 days after lentiviral transduction. As a result, we observed a dose-dependent upregulation of expression of the Notch target genes HEY1 and ACTA2 in cultures that were further upregulated by NICD inoculation (Fig. 10). We analyzed the expression of the proosteogenic markers RUNX2, OPN, and POSTNand found that their expression levels increased in a dose-dependent manner in response to the degree of Notch activation. The literature describes thatMGP2 is presumably involved in the inhibition of osteoblast maturation and the development of osteogenic differentiation and calcification (Chiyoya et al. 2018). Accordingly, activation of the Notch signaling pathway led to a decrease in MGP2 expression in SMC (Fig. 10). Our data support that activation of Notch signaling promotes osteogenic differentiation in the SMC (A. Kostina et al. 2019).
Figure 10. Dose-dependent activation of the Notch signaling pathway induces the expression of pro-osteogenic genes in smooth muscle cells (SMC) (n = 10). mRNA levels were analyzed by real-time PCR and normalized to GAPDH. (Kostina et al., 2019).
3.2.4. Co-cultivation of SMC and HAEC enhances the expression of Notch component genes in endothelial cells
Intercellular communication in coculture causes changes in both cell types. In order to find out if there are any changes in gene expression caused by cell-to-cell communication in each cell type, we used anti-CD31+ conjugated microparticles to separate HAEC and SMC after they were co-cultured. SMC were co-cultured with various amounts of human aortic endothelial cells (HAEC). Different number of cells corresponds to 100x103 and 500x103 cells per well. HAEC and SMC were separated 5 days after the start of co-culture using anti-CD31+-conjugated microparticles. Gene expression analysis revealed a dose-dependent suppression of specific SMC markers ACTA2, CNN1 and SM22 in SMC after coculturing of HAEC cells
(Fig. 11, A). Expression levels of the HEY1 and NOTCH3 genes increased in a dose-dependent manner in both cell types, but to a large extent this occurred exclusively in endothelial cells. The level of NOTCH2 mRNA was increased in endothelial cells and decreased in SMC after co-culture of cell cultures. At the same time, NOTCH1 expression slightly decreased in HAEC and remained unchanged in SMC (Fig. 11, B). These results suggest that endothelial cells have an osteoinductive effect on SMC, and this effect depends on the activation of Notch signaling in endothelial cells (A. Kostina et al. 2019).
Figure 11. Co-cultivation of SMC with HAEC reduces the expression of mesenchymal markers in SMC and activates the Notch signaling pathway in endothelial cells. (A) Expression level of mesenchymal markers in SMC after 5 days of co-culture with HAEC. (B) Expression level of Notch component genes in SMC and HAEC after 5 days of co-culture. mRNA levels were analyzed by real-time PCR and normalized to GAPDH. (Kostina et al., 2019).
3.2.5. Discussion of the results obtained in the study of the role played by intercellular communications between aortic smooth muscle cells and endothelial cells in the processes of osteogenic differentiation
The functional integrity of the endothelial layer is an extremely important condition for maintaining homeostasis of the aortic wall tissue. Normal endothelial function prevents atherogenic/pro-osteogenic processes in the cardiovascular system, which plays a critical role in maintaining a normal SMC phenotype (Urbich and Dimmeler 2004). However, the specific role of endothelial cells in driving SMC phenotype remains unclear. The implementation of the individual functions of endothelial and smooth muscle cells depends on proper communication between these cell types. This intercellular communication begins in early embryogenesis, when blood vessels are just beginning to form. During development, endothelial cells differentiate from vascular progenitor cells. These cells migrate and multiply throughout the developing embryo. Endothelial cells initiate intercellular communication by sending a recruitment signal to smooth muscle cells and pericytes from the surrounding mesenchyme or neural crest tissue. Smooth muscle cells in turn respond with their own signals, and this complex multilayered communication begins and continues throughout postnatal life (Senger and Davis 2011). Our laboratory has provided evidence that endothelial cells can determine the pro-osteogenic response of mesenchymal interstitial cells in cocultures (K. A et al. 2018; R. A et al. 2019). The ability of endothelial cells to act on mesenchymal cells has also been shown in other laboratories (Hjortnaes, Camci-Unal, et al. 2015; Hjortnaes, Shapero, et al. 2015; Lilly 2014; Lilly and Kennard 2009). In this part of the work, we show the effect of aortic endothelial cells on the expression of proosteogenic genes in aortic smooth muscle cells. This finding highlights the importance of normal endothelial function in maintaining the integrity of the aortic wall (Kostina et al., 2019). We believe that incorrect signaling in endothelial cells may affect gene expression and the differentiation state of underlying mesenchymal cells. Notch is one of the most important signaling pathways for intercellular communication during development and postnatal life (Sjoqvist and Andersson 2019). In this study, we show the effect of Notch activation on the induction of the pro-osteogenic SMC and HAEC coculture phenotype. The question of the role of Notch in maintaining osteogenic differentiation still remains unresolved and controversial. We hypothesize that dysregulation of Notch signaling
in the endothelium may contribute to the development of an abnormal pro-osteogenic state of smooth muscle cells and interstitial cells in affected tissues (A. Kostina et al. 2019).
3.3. A study to investigate how various components of the Notch signaling pathway influence osteogenic differentiation and osteopotential in adipose tissue-derived mesenchymal stem cells (aMSC)
Interest in the role of the Notch signaling pathway in pro-osteogenic events during bone development and regeneration has continued to grow in recent years. However, the interpretation of different experimental models gives conflicting results. There are different opinions regarding the role of the influence of Notch on osteogenic differentiation. Adipose tissue mesenchymal stem cells (aMSC) are an excellent model system for studying the processes of osteogenic differentiation, so we took this particular cell culture for this study (Semenova et al. 2019).
3.3.1. Effects of various components of Notch signaling pathway on the osteogenic potential of aMSC
In order to analyze how various components of the Notch signaling pathway affect the osteogenic differentiation potential of mesenchymal stem cells, aMSC were transduced with lentiviruses bearing one of the following Notch ligands: Dll1, Dll4, Jag1, and Notch1 receptor intracellular domain (NICD) (A.S. Kostina et al. 2016). We believed that different doses of exogenously applied ligands and NICD could influence the process of osteogenic differentiation in aMSC. To test this hypothesis, we transduced aMSC with various amounts of the respective viruses and then induced osteogenic differentiation. We analyzed the final stage of cell culture calcification by staining cells with alizarin red 21 days after induction of differentiation (Fig. 12, A). As a result of the experiment, we found that Dll1 and Dll4 do not affect the osteogenic potential of aMSC (data not shown), while the introduction of viruses bearing NICD and Jag1 in both cases led to an increase in the intensity of staining of cultures with alizarin red at doses of 1 and 5 MOI (multiplicity units of infection). In addition, we noted that the higher dose of 15 MOI resulted in inhibition of osteogenic differentiation in both NICD and Jag1 (Fig. 12, A).
After that, we analyzed the activation of the main Notch target genes HEY1 and HES1 in response to the induction of osteogenic differentiation in the presence of different amounts of introduced viral particles bearing NICD and Jag1 (Fig. 12, B). We noted dose-dependent transcriptional activation of both HEY1 and HES1 upon cell stimulation with both NICD and Jag1. However, NICD activated a high level of HEY1 transcription, while Jag1 resulted in a lower increase in HEY1 transcription. Transcription of HES1 was also moderately activated both upon exogenous introduction of NICD and Jag1 into cells. The levels of activation of both Notch target genes depended on the dose of the applied NICD and Jag1. It is noteworthy that the introduction of high doses of NICD or Jag1 (20 MOI) into cells caused a decrease in the expression of both targets (Semenova et al., 2019).
We then analyzed the expression levels of SPP1 (bone sialoprotein I, also known as OPN osteopontin), a gene associated with osteoblast differentiation and maturation (Merry et al. 1993). Accordingly, SPP1 expression was upregulated upon induction of osteogenic differentiation in aMSC (Fig. 12, C). In addition, we noted that SPP1 transcription was dose-dependently enhanced when exogenous NICD and Jag1 were introduced into cells in 1 and 5 MOI doses. At the same time, we observed a decrease in SPP1 expression with a further increase in the introduced doses of NICD and Jag1 (Semenova et al. 2019).
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Figure 12. Exogenic NICD and Jag1 affects osteogenic differentiation in a dose-dependent manner. (A) Alizarin red staining of osteogenic differentiation and histogram representing the assessment of differentiated regions by the MosaiX program. (B) Expression analysis of Notch target genes HEY1 and HES1 in aMSC differentiated in the presence of various doses of NICD and Jag1. (C) Expression analysis of the pro-osteogenic SPP1 gene in MSC in which osteogenic differentiation was induced in the presence of different doses of NICD and Jag1. (Semenova et al., 2019).
The data presented here suggest that both NICD and Jag1 have the ability to stimulate and enhance osteogenic differentiation of MSC in a dose-dependent manner. Most likely, this is mediated by dose-dependent transcriptional activation of downstream targets. However, at the same time, an excessively high dose of Notch activation causes a suppression of the intensity of osteogenic differentiation (Semenova et al., 2019).
3.3.2. High doses of Notch reduce CSL-dependent transcription
We showed that a high dose of activation of the Notch signaling pathway led to a decrease in the expression of Notch target genes, as well as to a decrease in the intensity of osteogenic differentiation. To test how activated Notch acts in MSC at the CSL level, we co-transduced cells with varying amounts of NICD-bearing lentivirus and 12xCSL-luciferase and measured CSL luciferase activity with a luciferase assay system. An increase in the amount of NICD in cells caused an increase in CSL activity, and a further increase in the dose of NICD, at the same time, caused a decrease in CSL activity (Fig. 13). Thus, we believe that NICD-induced transcription depends on fine tuning the degree of Notch activation. High doses of Notch activation can result in downregulation of transcription of Notch target genes.
Figure 13. NICD affects CSL-dependent transcription in a dose-dependent manner. CSL activity was analyzed using the Luciferase Assay System. (Semenova et al., 2019).
3.3.3. Intercellular communications contribute to the enhancement and maintenance of osteogenic differentiation processes in aMSC
We noticed a lower level of HEY1 transcriptional activation upon MSC transduction with different doses of Jagl compared to NICD. We hypothesized that lateral regulatory mechanisms are involved in osteogenic induction processes, as is known for Notch signaling (Sjoqvist and Andersson 2019). Lateral regulation is especially important in the processes of intercellular communication and tissue-specific differentiation. Therefore, in order to elucidate exactly how communication between aMSC cells promotes osteogenic differentiation, we compared NICD and Jagl induced expression of target genes in monoculture and in coculture. In the first type of experiment, we directly injected viruses bearing NICD or Jag1 at different doses, which was a direct induction of Notch signaling pathway. After that, we induced
osteogenic differentiation in the cells. For induction in coculture, we first transduced half of aMSC cells with the appropriate viruses, and then added the second half of the aMSC without modification, after which we induced osteogenic differentiation (see "Materials and Methods"). We then analyzed the expression of the Notch target gene HEY1 and SPP1 osteo marker. In addition, we stained differentiated cells with alizarin red (Fig. 14). We noted that NICD activated HEY1 in a dose dependent manner in monoculture and to a lesser extent in co-cultured cells (Figure 14, A). However, the level of expression of the pro-osteogenic SPP1 gene decreased when a high dose of lentivirus bearing NICD was introduced (Fig. 14, B), and, accordingly, the intensity of osteogenic differentiation progression decreased with a high dose of NICD both in monoculture and in coculture (Fig. 14, C). Thus, both in monoculture and in co-culture of cells, the addition of NICD acts in a similar dose-dependent manner. Concurrently, we noted that high doses of NICD inhibit osteogenic cell differentiation. We then examined whether Jag1 had the same impact. In both aMSC monoculture and co-cultivation, Jag1 induces a moderate dose-dependent activation of HEY1 transcription (Fig. 14, A). Jag1 caused dose-dependent expression of pro-osteogenic gene SPP1 with a decrease at high concentrations of introduced Jag1 in monoculture, but not in co-cultivation of cells (Fig. 14, B). In coculture, the expression level of SPP1 was the same at all concentrations of introduced Jag1. In addition, osteogenic potential, as assessed by cell staining with Alizarin Red, remained more or less the same regardless of Jag1 dosage (Fig. 14, C). Thus, the dose-dependent effect of Jag1 in stimulating osteogenic differentiation is different in monoculture and in co-cultivation of cells. In contrast to the action of Jag1 in monoculture, intercellular communication is somehow able to regulate high doses of exogenous Jag1 in the case of co-cultivation and maintain osteogenic differentiation at the same level.
Figure 14. NICD and Jagl act differently in monoculture and coculture. Adipose tissue MSC were seeded at a density of 80x103/cm2 and the appropriate lentiviral concentrates were added to the culture medium. After 24 hours, osteogenic differentiation was induced. For co-cultivation experiments, cells were seeded at a density of 40x103/cm2, and appropriate
lentiviral concentrates were immediately added to the culture medium. After 24 hours, cells were seeded onto the cell monolayer at a density of 40x103/cm2, and after 24 hours osteogenic differentiation was induced. Different numbers of viruses correspond to multiplicity of infectious units (MOI) 1, 5, 10, 15 and 20. (A) Expression of Notch target gene HEY1 in aMSC was determined in the presence of different doses of NICD and Jag1 in monoculture and coculture. (B) Expression of the pro-osteogenic SPP1 gene in aMSC differentiated in the presence of different doses of NICD and Jag1 in monoculture and co-cultivation. (C) Alizarin red staining of aMSC cultures 21 days after induction of osteogenic differentiation. (Semenova et al., 2019).
3.3.4. Induction of Notch genes in monoculture and coculture of aMSC
In order to find out how Notch signaling pathway genes are induced by the introduction of exogenous NICD into monocultures and during co-cultivation of cells, we introduced different doses of NICD into monocultures and aMSC cocultures, respectively, after which we induced osteogenic differentiation in cells. The different numbers of viruses correspond to 1, 5, 10, and 15 multiplicity of infectious units (MOI). We analyzed the expression levels of NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4 and JAG1 3 days after differentiation induction (Fig. 15). The amount of introduced NICD, corresponding to 10 MOI, used in co-cultivation, led to a significant activation of transcription of the NOTCH1, NOTCH3, NOTCH4, DLL1 and DLL4 genes. Transcription of JAG1 was up-regulated in a dose-dependent manner, both in coculture and in monoculture. Together with the data presented in the previous sections on the effect of NICD dose on osteogenic differentiation, our results indicate that the "optimal" dose of NICD can be used to increase the efficiency of osteogenic differentiation, and this "optimal" dose is achieved through the coordinated action of several components of the Notch signaling pathway (Semenova et al., 2019).
DLL1
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Figure 15. Expression of Notch component genes in aMSC transduced with different doses of lentiviruses bearing NICD in coculture and monoculture. (Semenova et al., 2019).
3.3.5. High doses of exogenous NICD and Jag1 induce apoptosis in aMSC
As a result of our studies, we found that both NICD and Jag1 promoted aMSC osteogenic differentiation in a dose-dependent manner in monoculture. However, with a high dose of both NICD and Jag1, we observed a decrease in aMSC differentiation. We transduced aMSC with various doses of NICD and Jag1 and used a GFP-bearing virus as a control for the cytotoxicity of lentiviral infection. Three days after transduction, we checked the level of apoptosis by labeling the cells with annexin V using flow cytometry (Fig. 16). In nondifferentiated cells, the GFP-carrying virus caused a slight induction of aMSC apoptosis, these values were close to background values. A similar level of apoptosis was found when a virus carrying NICD was added to nondifferentiated cells. However, a high dose of Jag1 corresponding to 10 MOI induced a visible increase in the percentage of apoptotic cells (up to 10%). aMSCs in which osteogenic differentiation was induced were more sensitive to apoptosis induction in activated by NICD cells and even more sensitive when Jag1 was added to the cells. Our data suggest that high doses of both NICD and even, to a greater extent, Jag1 induce apoptosis in differentiating aMSC, which prevents further differentiation. This corresponds to the data obtained by staining cell cultures with alizarin red (Fig. 12, A).
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1 3 15 1 3 15 1 3 15 1 3 15 1 3 15 1 3 15 Cundif C dif GFP NICD JAG1 GFP NICD JAG1
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Figure 16. High doses of NICD and Jag1 induce apoptosis in aMSC upon induction of osteogenic differentiation. (Semenova et al., 2019).
3.3.6. Discussion of the results obtained in the study of how various components of Notch signaling pathway affect the osteogenic potential of mesenchymal stem cells derived from adipose tissue
In this study, we have shown that activation of Notch signaling pathway by introducing into cells lentiviruses bearing either NICD or Jag1 increases the ability of mesenchymal stem cells to osteogenic differentiation in a dose-dependent manner. However, we have also shown that an excessive degree of activation of Notch signaling, on the contrary, causes a decrease in osteogenic differentiation in cells. We have also shown differences in dose-dependent cell response to activation by exogenous Jag1 or NICD, in a co-culture system where only half of the cells carry the exogenous Notch signal. In some cellular contexts, the dose-dependent nature of the effect of Notch has already been described in the literature. Depending on the level of activation, Notch induced opposite responses in cells of the same type. (Guentchev and McKay 2006; Kristoffersen et al. 2013). However, to the best of our knowledge, a dose-dependent effect of Notch in osteogenic processes has not yet been shown. The role of Notch in maintaining osteogenic differentiation is still unsolved and controversial. A significant amount of data has been obtained confirming its stimulating role in osteogenic differentiation, at the same time, there are some data confirming the opposite point of view (Semenova et al., 2019). Both in vivo and in vitro experiments on Notch activation and inactivation provide conflicting data. However, another kind of experiment, such as analysis of Notch gene activation during osteogenic differentiation, has shown that Notch genes are activated during the differentiation process with well-defined activation and inactivation times (Bagheri et al. 2018; Ongaro et al. 2016; Urbanek et al. 2017). These data support an inducing role of Notch in osteogenic differentiation. Notch signaling is extremely dose sensitive due to the lack of a signal amplification step and no use of second messengers to signal from the cell surface to the nucleus (Yamamoto, Schulze, and Bellen 2014). By changing the number of ligands and receptors expressed in a cell, numerous scenarios of Notch activation patterns can be generated. We hypothesize that the initial level of Notch signaling may be critical for cell fate decisions regarding osteogenic differentiation. How this tight regulation is controlled at the molecular
level is the subject of future research. In our study, we observed differences in the activating effects of NICD and Jag1 in co-culture experiments. It turned out that Jag1 had more ability to regulate the strength and toxicity of the signal in the cell coculture system (Semenova et al., 2019). This finding is in good agreement with the recent data describe importance of Jag1 ligand in osteoblast cells and its direct effect on changes in bone geometry and bone mass (Youngstrom et al. 2016).
CONCLUSION
In this study, we comprehensively studied the mechanisms that mediate and regulate the processes of induction and maintenance of osteogenic differentiation, both in normal conditions and in various pathologies associated with the development of ectopic tissue calcification. Many molecular participants are common to the processes of osteogenesis in general, whether it is the processes of physiological formation of skeletal tissue in embryogenesis, or the development of aortic valve calcification or vascular ossification. For scientists, it is an extremely important task to find out and identify the main participants in the processes of osteogenic differentiation. Understanding of the main mechanisms that play a decisive role in these processes, give us a possibility to manage and regulate the processes of osteogenic differentiation in the human body.
A known clinical problem in the therapy and induction of bone regeneration is that methods based on overproduction of bone tissue by BMP proteins can result in the development of potentially fatal bone ectopia and malignant transformation. We show in the present study that fine-tuning of Notch activation intensity is an important factor and tool for stimulating physiologically appropriate osteogenic differentiation. At the same time, by targeting various components of Notch signaling pathway or other targets in this complex network of signaling pathways responsible for maintaining osteogenic differentiation in cells, we can achieve the opposite effect - suppression of cell osteotransformation. There is no doubt that further studies on the safety of Notch-activating therapy are needed. We believe that the extreme sensitivity of Notch signaling to dose determines that even minor disturbances in the strength of the transmitted signal can have a detrimental effect on the state of tissue differentiation. Thus, taking into consideration the strength of Notch activation, as well as remember about the tissue-specificity of signaling, is extremely important for research.
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