Growth and organotypic branching of lung-specific microvascular cells on 2D and in 3D lung-derived matrices

Year 2024, , 6 - 14, 30.04.2024

Sena Nur Özkan , Ece Öztürk

https://doi.org/10.51753/flsrt.1351292

Abstract

Tissue-specific endothelial cells have vital roles in maintenance and functioning of native tissues with constant reciprocal crosstalk with resident cells. Three-dimensional (3D) physio-mimetic in vitro models which incorporate lung-specific microvasculature are needed to model lung-related diseases which involve modulation of endothelial cell behavior like cancer. In this study, we investigated the growth kinetics, morphological changes and responses to biological cues of lung microvasculature on two-dimensional (2D) and in lung matrix-derived 3D hydrogels. HUVEC and HULEC-5a cells were cultured on 2D and compared for their growth, morphologies, and responses to varying growth medium formulations. Brightfield and immunofluorescence imaging was performed to assess differences in morphology. For 3D cultures, native bovine lungs were decellularized, lyophilized, solubilized, and reconstituted into hydrogel form in which endothelial cells were embedded. Cell growth and organotypic branching was monitored in 3D hydrogels in the presence of varying biological cues including lung cancer cell secretome. HUVEC and HULEC-5a cells demonstrated comparable growth and morphology on 2D. However, in 3D lung-derived ECM hydrogels, tissue-specific HULEC-5a cells exhibited much better adaptation to their microenvironment, characterized by enhanced organotypic branching and longer branches. HULEC-5a growth was responsive to lung cancer cell-conditioned medium in both 2D and 3D conditions. In 3D, the concentration of ECM ligand significantly affected cell growth in long-term culture where molecular crowding had an inhibitory role. Our data reveals that HULEC-5a cells offer a reliable alternative to frequently pursued HUVECs with comparable growth and morphology. Due to their intrinsic program for cellular crosstalk with resident cells, the use of tissue-specific endothelium constitutes a vital aspect for modeling physiological and pathological processes. Furthermore, our study is the first demonstration of the synergy between lung-specific microvasculature with lung-specific ECM within a 3D in vitro model.

Keywords

Cancer, endothelial cells, extracellular matrix, lung microenvironment, tissue engineering

Supporting Institution

TÜBİTAK

Project Number

118C238

Thanks

This work was funded by the Scientific and Technological Research Council of Turkey (TÜBİTAK) (Grant No. 118C238). The entire responsibility of the publication/paper belongs to the owner of the publication. The financial support received from TÜBİTAK does not mean that the content of the publication is approved in a scientific sense by TÜBİTAK. The authors gratefully acknowledge the use of services and facilities of Koç University Research Center for Translational Medicine (KUTTAM). The authors declare no competing interests. Figure 1, Figure 4a and Figure 5a were created with BioRender.com.

References

• Andrée, B., Ichanti, H., Kalies, S., Heisterkamp, A., Strauß, S., Vogt, P.-M., Haverich, A., & Hilfiker, A. (2019). Formation of three-dimensional tubular endothelial cell networks under defined serum-free cell culture conditions in human collagen hydrogels. Scientific Reports, 9(1), 5437.
• Balzan, S., Del Carratore, R., Nardulli, C., Sabatino, L., Lubrano, V., & Iervasi, G. (2013). The stimulative effect of T3 and T4 on human myocardial endothelial cell proliferation, migration and angiogenesis. Journal of Clinical & Experimental Cardiolog, 4(280), 2.
• Barabutis, N., Verin, A., & Catravas, J. D. (2016). Regulation of pulmonary endothelial barrier function by kinases. American Journal of Physiology Lung Cellular and Molecular Physiology, 311(5), L832-l845.
• Bloom, S. I., Islam, M. T., Lesniewski, L. A., & Donato, A. J. (2023). Mechanisms and consequences of endothelial cell senescence. Nature Reviews Cardiology, 20(1), 38-51.
• Dejana, E., & Kuhl, M. (2010). The role of wnt signaling in physiological and pathological angiogenesis. Circulation Research, 107(8), 943-952.
• Fontana, F., Raimondi, M., Marzagalli, M., Sommariva, M., Gagliano, N., & Limonta, P. (2020). Three-dimensional cell cultures as an in vitro tool for prostate cancer modeling and drug discovery. International Journal of Molecular Sciences, 21(18), 6806.
• Goncharova, E. A., Chan, S. Y., Ventetuolo, C. E., Weissmann, N., Schermuly, R. T., Mullin, C. J., & Gladwin, M. T. (2020). Update in pulmonary vascular diseases and right ventricular dysfunction 2019. American Journal of Respiratory and Critical Care Medicine, 202(1), 22-28.
• Gordon, E., Schimmel, L., & Frye, M. (2020). The ımportance of mechanical forces for in vitro endothelial cell biology. Frontiers in Physiolgy, 11, 684.
• Han, H. X., & Geng, J. G. (2011). Over-expression of Slit2 induces vessel formation and changes blood vessel permeability in mouse brain. Acta Pharmacologica Sinica, 32(11), 1327-1336.
• Hennigs, J. K., Matuszcak, C., Trepel, M., & Körbelin, J. (2021). Vascular endothelial cells: Heterogeneity and targeting approaches. Cells, 10(10), 2712.
• Hida, K., Maishi, N., Annan, D. A., & Hida, Y. (2018). Contribution of tumor endothelial cells in cancer progression. International Journal of Moleculer Sciences, 19(5), 1272.
• Huttala, O., Vuorenpää, H., Toimela, T., Uotila, J., Kuokkanen, H., Ylikomi, T., Sarkanen, J. R., & Heinonen, T. (2015). Human vascular model with defined stimulation medium - a characterization study. Altex, 32(2), 125-136.
• Jang, J., Jung, Y., Kim, Y., Jho, E.-h., & Yoon, Y. (2017). LPS-induced inflammatory response is suppressed by Wnt inhibitors, Dickkopf-1 and LGK974. Scientific Reports, 7(1), 41612.
• Jensen, C., & Teng, Y. (2020). Is ıt time to start transitioning from 2D to 3D cell culture? Frontiers in Molecular Biosciences, 7, 33.
• Jourde-Chiche, N., Fakhouri, F., Dou, L., Bellien, J., Burtey, S., Frimat, M., ... & Roumenina, L. T. (2019). Endothelium structure and function in kidney health and disease. Nature Reviews Nephrology, 15(2), 87-108.
• Klein, D. (2018). The tumor vascular endothelium as decision maker in cancer therapy. Frontiers on Oncology, 8, 367.
• Kruger-Genge, A., Blocki, A., Franke, R. P., & Jung, F. (2019). Vascular endothelial cell biology: An update. International Journal of Molecular Sciences, 20(18), 4411.
• Kusoglu, A., Yangin, K., Ozkan, S. N., Sarica, S., Ornek, D., Solcan, N., ... & Ozturk, E. (2023). Different decellularization methods in bovine lung tissue reveals distinct biochemical composition, stiffness, and viscoelasticity in reconstituted hydrogels. ACS Applied Bio Materials, 6(2), 793-805.
• Lee, J. Y., Chang, J. K., Dominguez, A. A., Lee, H. P., Nam, S., Chang, J., ... & Chaudhuri, O. (2019). YAP-independent mechanotransduction drives breast cancer progression. Nature Communications, 10(1), 1848.
• Lin, S., Zhang, Q., Shao, X., Zhang, T., Xue, C., Shi, S., Zhao, D., & Lin, Y. (2017). IGF-1 promotes angiogenesis in endothelial cells/adipose-derived stem cells co-culture system with activation of PI3K/Akt signal pathway. Cell Proliferation, 50(6), e12390.
• Matsubara, M., & Bissell, M. J. (2016). Inhibitors of Rho kinase (ROCK) signaling revert the malignant phenotype of breast cancer cells in 3D context. Oncotarget, 7(22), 31602-31622.
• McHenry, P. R., & Prosperi, J. R. (2023). Proteins found in the triple-negative breast cancer secretome and their therapeutic potential. International Journal of Molecular Sciences, 24(3), 2100.
• Medina-Leyte, D. J., Domínguez-Pérez, M., Mercado, I., Villarreal-Molina, M. T., & Jacobo-Albavera, L. (2020). Use of human umbilical vein endothelial cells (HUVEC) as a model to study cardiovascular disease: A review. Applied Sciences, 10(3), 938.
• Mierke, C. T. (2023). Physical and biological advances in endothelial cell-based engineered co-culture model systems. Seminars in Cell & Developmental Biology, 147, 58-69.
• Nguyen, J., Lin, Y. Y., & Gerecht, S. (2021). The next generation of endothelial differentiation: Tissue-specific ECs. Cell Stem Cell, 28(7), 1188-1204.
• Paek, J., Park, S. E., Lu, Q., Park, K. T., Cho, M., Oh, J. M., ... & Huh, D. (2019). Microphysiological engineering of self-assembled and perfusable microvascular beds for the production of vascularized three-dimensional human microtissues. ACS Nano, 13(7), 7627-7643.
• Rafii, S., Butler, J. M., & Ding, B. S. (2016). Angiocrine functions of organ-specific endothelial cells. Nature, 529(7586), 316-325.
• Rayner, R. E., Makena, P., Prasad, G. L., & Cormet-Boyaka, E. (2019). Optimization of normal human bronchial epithelial (NHBE) cell 3D cultures for in vitro lung model studies. Scientific Reports, 9(1), 500.
• Ritchie, S., Reed, D. A., Pereira, B. A., & Timpson, P. (2021). The cancer cell secretome drives cooperative manipulation of the tumour microenvironment to accelerate tumourigenesis. Faculty Reviews, 10, 4.
• Rosen, R. S., Yang, J. H., Peña, J. S., Schloss, R., & Yarmush, M. L. (2023). An in vitro model of the macrophage-endothelial interface to characterize CAR T-cell induced cytokine storm. Scientific Reports, 13(1), 18835.
• Tatla, A. S., Justin, A. W., Watts, C., & Markaki, A. E. (2021). A vascularized tumoroid model for human glioblastoma angiogenesis. Scientific Reports, 11(1), 19550.
• Urbanczyk, M., Zbinden, A., & Schenke-Layland, K. (2022). Organ-specific endothelial cell heterogenicity and its impact on regenerative medicine and biomedical engineering applications. Advanced Drug Delivery Reviews, 186, 114323.
• Wakabayashi, T., & Naito, H. (2023). Cellular heterogeneity and stem cells of vascular endothelial cells in blood vessel formation and homeostasis: Insights from single-cell RNA sequencing. Frontiers in Cell and Developmental Biology, 11, 285.
• Zahari, S., Syafruddin, S. E., & Mohtar, M. A. (2023). Impact of the cancer cell secretome in driving breast cancer progression. Cancers, 15(9), 2653.