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Serving as the first barrier for circulating molecules and cells, ECs represent the main regulators of vascular homeostasis being able to respond to environmental changes, either physical or chemical signals, by producing several factors that regulate vascular tone and cellular adhesion. Healthy endothelium has anticoagulant properties that prevent the adhesion of leukocytes and platelets to the vessel walls, contributing to resistance to thrombus formation, and regulating inflammation, and vascular smooth muscle cell proliferation. Many risk factors of cardiovascular diseases (CVDs) promote the endothelial expression of chemokines, cytokines, and adhesion molecules. The resultant endothelial activation can lead to endothelial cell dysfunction (ECD). In vitro models of ECD allow the study of cellular and molecular mechanisms of disease and provide a research platform for screening potential therapeutic agents. Even though alternative models are available, such as animal models or ex vivo models, in vitro models offer higher experimental flexibility and reproducibility, making them a valuable tool for the understanding of pathophysiological mechanisms of several diseases, such as CVDs. Therefore, this review aims to synthesize the currently available in vitro models regarding ECD, emphasizing CVDs. This work will focus on 2D cell culture models (endothelial cell lines and primary ECs), 3D cell culture systems (scaffold-free and scaffold-based), and 3D cell culture models (such as organ-on-a-chip). We will dissect the role of external stimuli\u2014chemical and mechanical\u2014in triggering ECD.<\/jats:p>","DOI":"10.1007\/s11010-025-05289-w","type":"journal-article","created":{"date-parts":[[2025,4,21]],"date-time":"2025-04-21T11:40:24Z","timestamp":1745235624000},"page":"4671-4695","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Endothelial dysfunction in cardiovascular diseases: mechanisms and in vitro models"],"prefix":"10.1007","volume":"480","author":[{"given":"Ana","family":"Grego","sequence":"first","affiliation":[]},{"given":"Cristiana","family":"Fernandes","sequence":"additional","affiliation":[]},{"given":"Ivo","family":"Fonseca","sequence":"additional","affiliation":[]},{"given":"Marina","family":"Dias-Neto","sequence":"additional","affiliation":[]},{"given":"Raquel","family":"Costa","sequence":"additional","affiliation":[]},{"given":"Adelino","family":"Leite-Moreira","sequence":"additional","affiliation":[]},{"given":"Sandra Marisa","family":"Oliveira","sequence":"additional","affiliation":[]},{"given":"F\u00e1bio","family":"Trindade","sequence":"additional","affiliation":[]},{"given":"Rita","family":"Nogueira-Ferreira","sequence":"additional","affiliation":[]}],"member":"297","published-online":{"date-parts":[[2025,4,21]]},"reference":[{"key":"5289_CR1","doi-asserted-by":"publisher","DOI":"10.4199\/C00031ED1V01Y201105ISP019","volume-title":"Integrated systems physiology: from molecule to function to disease. 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