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Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis

The aim of the study was to assess the suitability of endothelial colony-forming cells in the development of tissue engineering constructs based on the study of the gene expression profile compared to mature endothelial cells. MATERIALS AND METHODS: In the experiment, we used the endothelial colony-...

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Autores principales: Velikanova, E.A., Sinitsky, M.Yu., Sinitskaya, А.V., Matveeva, V.G., Khanova, М.Yu., Antonova, L.V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Privolzhsky Research Medical University 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10090920/
https://www.ncbi.nlm.nih.gov/pubmed/37064809
http://dx.doi.org/10.17691/stm2022.14.3.02
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author Velikanova, E.A.
Sinitsky, M.Yu.
Sinitskaya, А.V.
Matveeva, V.G.
Khanova, М.Yu.
Antonova, L.V.
author_facet Velikanova, E.A.
Sinitsky, M.Yu.
Sinitskaya, А.V.
Matveeva, V.G.
Khanova, М.Yu.
Antonova, L.V.
author_sort Velikanova, E.A.
collection PubMed
description The aim of the study was to assess the suitability of endothelial colony-forming cells in the development of tissue engineering constructs based on the study of the gene expression profile compared to mature endothelial cells. MATERIALS AND METHODS: In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer’s recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA). The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm(2). Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. RESULTS: In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. CONCLUSION: The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients’ peripheral blood can be used to develop personalized tissue-engineered constructs.
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spelling pubmed-100909202023-04-13 Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis Velikanova, E.A. Sinitsky, M.Yu. Sinitskaya, А.V. Matveeva, V.G. Khanova, М.Yu. Antonova, L.V. Sovrem Tekhnologii Med Biotechnologies The aim of the study was to assess the suitability of endothelial colony-forming cells in the development of tissue engineering constructs based on the study of the gene expression profile compared to mature endothelial cells. MATERIALS AND METHODS: In the experiment, we used the endothelial colony-forming cells (ECFC) obtained from the peripheral blood of patients who underwent percutaneous coronary intervention. The cells were isolated on a Histopaque 1077 density gradient (Sigma-Aldrich, USA), and then cultured in EGM-2MV culture medium (Lonza, Switzerland). A commercial culture of primary human coronary artery endothelial cells (HCAEC) was used as a control. The cells were unfrozen and cultured according to the manufacturer’s recommendations in MesoEndo Cell Growth Medium (Cell Applications, USA). The experiment was carried out in specialized μ-Luer plates in the perfusion system (IBIDI, Germany), which provided a continuous unidirectional flow of the culture medium with a shear stress of 5 dyn/cm(2). Control plates were cultured under standard conditions for a similar period of time. Total RNA was isolated from cell samples. The expression of the genes NOTCH4, NRP2, PLAT, PLAU, NOTCH1, FLT1, COL4A2, CD34, SERPINE1, HEY2, MKI67, KLF4, LYVE1, FLT4 was assessed using a quantitative real-time polymerase chain reaction. The expression of the genes was calculated by the ΔCt method and expressed on a logarithmic (log10) scale as a fold change relating to the control samples. RESULTS: In mature endothelial cells HCAEC when exposed to a laminar flow, only the transcription factor KLF4 and venous differentiation NRP2 marker values increased significantly. ECFC showed statistically significant growth in KLF4, NRP2, CD34, and LYVE1, as well as PLAU expression decrease. In addition, we observed the overexpression of FLT4, LYVE1, NOTCH4, and NRP2 in ECFC in relation to HCAEC and HEY2 hypoexpression. CD34 overexpression characteristic of progenitor cells was also found. An increase in COL4A2 expression associated with type IV collagen synthesis was a characteristic feature of ECFC. CONCLUSION: The gene expression profile of endothelial colony-forming cells is quite close to that of primary endothelial cells of the human coronary artery, and thus, the cells obtained from patients’ peripheral blood can be used to develop personalized tissue-engineered constructs. Privolzhsky Research Medical University 2022 2022-05-28 /pmc/articles/PMC10090920/ /pubmed/37064809 http://dx.doi.org/10.17691/stm2022.14.3.02 Text en https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Biotechnologies
Velikanova, E.A.
Sinitsky, M.Yu.
Sinitskaya, А.V.
Matveeva, V.G.
Khanova, М.Yu.
Antonova, L.V.
Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis
title Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis
title_full Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis
title_fullStr Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis
title_full_unstemmed Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis
title_short Evaluation of the Feasibility of Endothelial Colony-Forming Cells to Develop Tissue-Engineered Vascular Grafts Based on the Gene Expression Profile Analysis
title_sort evaluation of the feasibility of endothelial colony-forming cells to develop tissue-engineered vascular grafts based on the gene expression profile analysis
topic Biotechnologies
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10090920/
https://www.ncbi.nlm.nih.gov/pubmed/37064809
http://dx.doi.org/10.17691/stm2022.14.3.02
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