{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:18:36Z","timestamp":1785410316239,"version":"3.56.0"},"reference-count":38,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T00:00:00Z","timestamp":1783900800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004504","name":"Research Council of Lithuania","doi-asserted-by":"publisher","award":["The Role of Endothelial Transglutaminase-2 Signaling in Development of Neurovascular Dysfunction\u201d (S-MIP-23-94)"],"award-info":[{"award-number":["The Role of Endothelial Transglutaminase-2 Signaling in Development of Neurovascular Dysfunction\u201d (S-MIP-23-94)"]}],"id":[{"id":"10.13039\/501100004504","id-type":"DOI","asserted-by":"publisher"}]},{"award":["The Role of Endothelial Transglutaminase-2 Signaling in Development of Neurovascular Dysfunction\u201d (S-MIP-23-94)"],"award-info":[{"award-number":["The Role of Endothelial Transglutaminase-2 Signaling in Development of Neurovascular Dysfunction\u201d (S-MIP-23-94)"]}],"id":[{"id":"https:\/\/ror.org\/02gs16m83","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100009708","name":"Novo Nordisk Foundation","doi-asserted-by":"publisher","award":["NNF24OC0096782"],"award-info":[{"award-number":["NNF24OC0096782"]}],"id":[{"id":"10.13039\/501100009708","id-type":"DOI","asserted-by":"publisher"}]},{"award":["NNF24OC0096782"],"award-info":[{"award-number":["NNF24OC0096782"]}],"id":[{"id":"https:\/\/ror.org\/04txyc737","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Endothelial dysfunction is a defining feature of diabetic vascular disease and is characterized by impaired nitric oxide signaling, inflammatory activation, altered mechanotransduction, and disturbed angiogenic responses. The present study investigated whether hyperglycemia modulates endothelial phenotype in a model-dependent manner under distinct structural and mechanical culture conditions. EA.hy926 endothelial cells were cultured for 7 days under normoglycemic (5.5 mM) or high-glucose (25 mM) conditions as static monolayers (G-force = 1 g), adherent clinorotated cells, multicellular spheroids (MCSs) generated during clinorotation, and Matrigel-derived endothelial structures. Gene expression was assessed by qPCR using marker panels related to nitric oxide signaling, PI3K CA-AKT-mTOR signaling, inflammatory adhesion, angiogenesis, and structural adhesion, whereas protein abundance and spatial distribution of eNOS, AKT1, VCAM1, vinculin, and VEGFA together with CDH5\/VE-cadherin were analyzed by Western blotting and confocal microscopy. Clinorotation generated both adherent endothelial cells and MCSs. High glucose reduced eNOS-related expression in most models, with the strongest decreases in adherent clinorotated cells and MCSs, whereas Matrigel cultures showed a divergent transcriptional response. PI3K CA expression was markedly suppressed by high glucose in clinostat-derived populations, while mTOR was differentially regulated in spheroids and Matrigel cultures. VCAM1 expression was most prominent in MCSs, whereas ICAM1 was highest in Matrigel cultures. VEGF-related signaling differed substantially among models, and Matrigel cultures showed the strongest VEGFA-associated protein signal and the clearest angiogenic organization under normoglycemic conditions, which became less distinct under high glucose. Vinculin protein abundance was highest in MCSs and Matrigel cultures, reflecting pronounced differences in structural organization. Overall, these findings show that endothelial responses to hyperglycemia are strongly shaped by mechanical and structural context and support the use of complementary in vitro models for studying diabetic endothelial dysfunction.<\/jats:p>","DOI":"10.3390\/ijms27146233","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T02:34:44Z","timestamp":1783996484000},"page":"6233","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparative Analysis of Endothelial Cell Culture Models Under Altered Mechanical Conditions and Glucose Variations"],"prefix":"10.3390","volume":"27","author":[{"given":"Augusta","family":"Volkevi\u010di\u016bt\u0117","sequence":"first","affiliation":[{"name":"Preclinical Research Laboratory for Medicinal Products, Institute of Cardiology, Lithuanian University of Health Sciences, 50162 Kaunas, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jayashree","family":"Sahana","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7852-992X","authenticated-orcid":false,"given":"Est\u00e9fano","family":"Pinilla","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, 8000 Aarhus, Denmark"},{"name":"Hematopoiesis and Leukocyte Biology Lab, Baker Heart and Diabetes Institute, Melbourne 3004, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniela","family":"Melnik","sequence":"additional","affiliation":[{"name":"Department of Microgravity and Translational Regenerative Medicine, Otto von Guericke University, 39106 Magdeburg, Germany"},{"name":"Research Group \u201cMagdeburger Arbeitsgemeinschaft f\u00fcr Forschung unter Raumfahrt- und Schwerelosigkeitsbedingungen\u201d (MARS), Otto von Guericke University, 39106 Magdeburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3663-2953","authenticated-orcid":false,"given":"Luis Fernando","family":"Gonz\u00e1lez-Torres","sequence":"additional","affiliation":[{"name":"Department of Microgravity and Translational Regenerative Medicine, Otto von Guericke University, 39106 Magdeburg, Germany"},{"name":"Research Group \u201cMagdeburger Arbeitsgemeinschaft f\u00fcr Forschung unter Raumfahrt- und Schwerelosigkeitsbedingungen\u201d (MARS), Otto von Guericke University, 39106 Magdeburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8160-859X","authenticated-orcid":false,"given":"Markus","family":"Wehland","sequence":"additional","affiliation":[{"name":"Department of Microgravity and Translational Regenerative Medicine, Otto von Guericke University, 39106 Magdeburg, Germany"},{"name":"Research Group \u201cMagdeburger Arbeitsgemeinschaft f\u00fcr Forschung unter Raumfahrt- und Schwerelosigkeitsbedingungen\u201d (MARS), Otto von Guericke University, 39106 Magdeburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2210-708X","authenticated-orcid":false,"given":"Edgaras","family":"Stankevicius","sequence":"additional","affiliation":[{"name":"Preclinical Research Laboratory for Medicinal Products, Institute of Cardiology, Lithuanian University of Health Sciences, 50162 Kaunas, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4991-3105","authenticated-orcid":false,"given":"Daniela","family":"Grimm","sequence":"additional","affiliation":[{"name":"Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, 8000 Aarhus, Denmark"},{"name":"Department of Microgravity and Translational Regenerative Medicine, Otto von Guericke University, 39106 Magdeburg, Germany"},{"name":"Research Group \u201cMagdeburger Arbeitsgemeinschaft f\u00fcr Forschung unter Raumfahrt- und Schwerelosigkeitsbedingungen\u201d (MARS), Otto von Guericke University, 39106 Magdeburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2169-7666","authenticated-orcid":false,"given":"Ulf","family":"Simonsen","sequence":"additional","affiliation":[{"name":"Preclinical Research Laboratory for Medicinal Products, Institute of Cardiology, Lithuanian University of Health Sciences, 50162 Kaunas, Lithuania"},{"name":"Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Kr\u00fcger-Genge, A., Blocki, A., Franke, R.P., and Jung, F. (2019). Vascular endothelial cell biology: An update. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20184411"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1007\/s13239-020-00485-9","article-title":"The glycocalyx and its role in vascular physiology and vascular related diseases","volume":"12","author":"Weinbaum","year":"2021","journal-title":"Cardiovasc. Eng. Technol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Wakasugi, R., Suzuki, K., and Kaneko-Kawano, T. (2024). Molecular mechanisms regulating vascular endothelial permeability. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25126415"},{"key":"ref_4","first-page":"587","article-title":"Shear stress and endothelial function: Mechanisms and implications","volume":"11","author":"DeStefano","year":"2020","journal-title":"Front. Physiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"H989","DOI":"10.1152\/ajpheart.00431.2024","article-title":"Integrating molecular and cellular components of endothelial shear stress mechanotransduction","volume":"327","author":"Power","year":"2024","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1146\/annurev-physiol-042022-030946","article-title":"Mechanosensing by vascular endothelium","volume":"86","author":"Lim","year":"2024","journal-title":"Annu. Rev. Physiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2216","DOI":"10.1016\/j.bbadis.2013.08.006","article-title":"Endothelial dysfunction\u2014A major mediator of diabetic vascular disease","volume":"1832","author":"Sena","year":"2013","journal-title":"Biochim. Biophys. Acta Mol. Basis Dis."},{"key":"ref_8","first-page":"586","article-title":"Macro- and microvascular endothelial dysfunction in diabetes","volume":"8","author":"Shi","year":"2017","journal-title":"J. Diabetes Investig."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Yang, D.R., Wang, M.Y., Zhang, C.L., and Wang, Y. (2024). Endothelial dysfunction in vascular complications of diabetes: A comprehensive review of mechanisms and implications. Front. Endocrinol., 15.","DOI":"10.3389\/fendo.2024.1359255"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Dubsky, M., Veleba, J., Sojakova, D., Marhefkova, N., Fejfarova, V., and Jude, E.B. (2023). Endothelial dysfunction in diabetes mellitus: New insights. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241310705"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1038\/s41419-023-05935-5","article-title":"Vascular nitric oxide resistance in type 2 diabetes","volume":"14","author":"Bahadoran","year":"2023","journal-title":"Cell Death Dis."},{"key":"ref_12","first-page":"32727","article-title":"Vitexin suppresses high-glucose-upregulated adhesion molecule expression in endothelial cells through inhibiting NF-\u03baB signaling pathway","volume":"9","author":"Chen","year":"2024","journal-title":"ACS Omega"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.crbiot.2021.05.001","article-title":"Differences between common endothelial cell models (primary human aortic endothelial cells and EA.hy926 cells) revealed through transcriptomics, bioinformatics, and functional analysis","volume":"3","author":"Wang","year":"2021","journal-title":"Curr. Res. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"266","DOI":"10.1152\/physiol.00036.2016","article-title":"Modeling physiological events in 2D vs. 3D cell culture","volume":"32","author":"Duval","year":"2017","journal-title":"Physiology"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Beloglazova, I., Zubkova, E., Dergilev, K., Goltseva, Y., and Parfyonova, Y. (2022). New insight on 2D in vitro angiogenesis models: All that stretches is not a tube. Cells, 11.","DOI":"10.3390\/cells11203278"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Kopp, S., Kr\u00fcger, M., Bauer, J., Wehland, M., Corydon, T.J., Sahana, J., Nassef, M.Z., Melnik, D., Bauer, T.J., and Schulz, H. (2018). Microgravity Affects Thyroid Cancer Cells during the TEXUS-53 Mission Stronger than Hypergravity. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19124001"},{"key":"ref_17","first-page":"199","article-title":"Endothelial spheroid assay for in vitro angiogenesis","volume":"1464","author":"Lee","year":"2016","journal-title":"Methods Mol. Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1007\/s11033-023-09048-2","article-title":"Review: Human stem cell-based 3D in vitro angiogenesis models for preclinical drug screening applications","volume":"51","author":"Esparza","year":"2024","journal-title":"Mol. Biol. Rep."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/s41578-020-0199-8","article-title":"Synthetic alternatives to Matrigel","volume":"5","author":"Aisenbrey","year":"2020","journal-title":"Nat. Rev. Mater."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1007\/s12079-021-00643-1","article-title":"Matrigel: History\/background, uses, and future applications","volume":"16","author":"Passaniti","year":"2022","journal-title":"J. Cell Commun. Signal."},{"key":"ref_21","first-page":"639","article-title":"The Matrigel tube formation assay","volume":"4","author":"Arnaoutova","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Morbidelli, L., Genah, S., and Cialdai, F. (2021). Effect of microgravity on endothelial cell function, angiogenesis, and vessel remodeling during wound healing. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.720091"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/s41526-024-00442-z","article-title":"Cellular response in three-dimensional spheroids and tissues exposed to real and simulated microgravity: A narrative review","volume":"10","author":"Moroni","year":"2024","journal-title":"npj Microgravity"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Jok\u0161ien\u0117, J., Sahana, J., Wehland, M., Schulz, H., Cort\u00e9s-S\u00e1nchez, J.L., Prat-Duran, J., Grimm, D., and Simonsen, U. (2023). Effects of high glucose on human endothelial cells exposed to simulated microgravity. Biomolecules, 13.","DOI":"10.3390\/biom13020189"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"997","DOI":"10.1007\/s10557-022-07385-1","article-title":"Mechanobiology of the endothelium in vascular health and disease: In vitro shear stress models","volume":"37","author":"Jackson","year":"2023","journal-title":"Cardiovasc. Drugs Ther."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Mierke, C.T. (2024). Mechanosensory entities and functionality of endothelial cells. Front. Cell Dev. Biol., 12.","DOI":"10.3389\/fcell.2024.1446452"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Wang, X., Bove, A.M., Simone, G., and Ma, B. (2020). Molecular bases of VEGFR-2-mediated physiological function and pathological role. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.599281"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"102441","DOI":"10.1016\/j.ceb.2024.102441","article-title":"Endothelial cell mechanics and dynamics in angiogenesis","volume":"91","author":"Huveneers","year":"2024","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_29","first-page":"e220012","article-title":"Vinculin strengthens the endothelial barrier during vascular development","volume":"5","author":"Kotini","year":"2023","journal-title":"Vasc. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4677","DOI":"10.1016\/j.cub.2024.08.060","article-title":"Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions","volume":"34","author":"Iseler","year":"2024","journal-title":"Curr. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"C1073","DOI":"10.1152\/ajpcell.00605.2023","article-title":"Junctions at the crossroads: The impact of mechanical cues on endothelial cell-cell junction conformations and vascular permeability","volume":"327","author":"Brandon","year":"2024","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1096\/fj.00-0078com","article-title":"NOSIP, a novel modulator of endothelial nitric oxide synthase activity","volume":"15","author":"Dedio","year":"2001","journal-title":"FASEB J."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Nan, W., He, Y., Wang, S., and Zhang, Y. (2023). Molecular mechanism of VE-cadherin in regulating endothelial cell behaviour during angiogenesis. Front. Physiol., 14.","DOI":"10.3389\/fphys.2023.1234104"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.yjmcc.2021.06.002","article-title":"Organ-on-a-chip systems for vascular biology","volume":"159","author":"Mandrycky","year":"2021","journal-title":"J. Mol. Cell. Cardiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1080\/17460441.2023.2294947","article-title":"Vascular organs-on-chip made with patient-derived endothelial cells: Technologies to transform drug discovery and disease modeling","volume":"19","author":"Whitworth","year":"2024","journal-title":"Expert Opin. Drug Discov."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2241","DOI":"10.1161\/ATVBAHA.123.318233","article-title":"Engineering Organ-on-a-Chip Systems for Vascular Diseases","volume":"43","author":"Shakeri","year":"2023","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2620","DOI":"10.1039\/D4LC00968A","article-title":"Vascular architecture-on-chip: Engineering complex blood vessels for reproducing physiological and heterogeneous hemodynamics and endothelial function","volume":"25","author":"Lee","year":"2025","journal-title":"Lab Chip"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1186\/s12964-024-01503-2","article-title":"Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: Exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles","volume":"22","author":"Lisec","year":"2024","journal-title":"Cell Commun. Signal."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6233\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T04:12:54Z","timestamp":1784175174000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/14\/6233"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,13]]},"references-count":38,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["ijms27146233"],"URL":"https:\/\/doi.org\/10.3390\/ijms27146233","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,13]]}}}