Molecular analysis and computational modeling reveal temporally separable responses triggered by DENV-induced soluble factors in endothelial cells
Jenny Paola Alfaro-García, Julieta María Ramírez-Mejía, Paola Rojas-Estevez, Diego Alejandro Álvarez-Díaz, Geysson Javier Fernández, Carlos Alberto Orozco-Castaño, Boris Anghelo Rodríguez-Rey, Juan Carlos Gallego-Gómez, Miguel Vicente-Manzanares
Abstract
Dengue virus (DENV) represents a growing global health challenge with billions of people at risk. Severe Dengue (SD), a complication of DENV infection that involves generalized hemorrhage, is driven, at least in part, by endothelial dysfunction. Endothelial dysfunction refers to increased permeability due to inflammation, mechanical injury and/or modification of the genetic program of endothelial cells. Previous work showed that exposure of endothelial cells to conditioned media from DENV-infected cells (CMDV) increased permeability and cellular stiffness, repressed endothelial markers and induced mesenchymal genes.
Introduction
Dengue virus (DENV) is currently the most prevalent arbovirus in the world. The largest outbreak of dengue was recorded between 2023 and 2024, underscoring its growing prominence as a global threat to human health. Estimates suggest that half of the human population may be at risk [1–3]. Of particular concern is the progression of dengue to severe dengue (SD), a rare but life-threatening condition characterized by generalized hemorrhage. There is no specific treatment for SD, only palliative care [4,5].
Materials and method
4.1. Cell lines and viral infections
Human Microvascular Endothelial Cells (HMEC-1) (ATCC Cat# CRL-3243) were maintained at 37°C with 5% CO2 in RPMI supplemented with 10% FBS, 10mM L-glutamine, 100 U/mL penicillin/100 mg/mL streptomycin (P/S), 10ng/mL Epidermal Growth Factor (hEGF), and 1 µg/mL Hydrocortisone. Cells were used up to passage 10. Human Epithelial Kidney Cells (HEK-293) (ATCC Cat# CRL-1573) were maintained at 37°C with 5% CO2 in RPMI supplemented with 10% FBS, 10mM L-glutamine, 100 U/mL penicillin/100 mg/mL streptomycin (P/S), in the same way that HeLa Cells (ATCC Cat# CCL2), but in DMEM media. Aedes albopictus clone C6/36 HT cells (ATCC Cat# CRL-1660) were cultured at 34°C with 5% CO2 in L-15 media supplemented with 10% FBS and 1X P/S.
Results
2.1. CMDV induces expression of mesenchymal markers and morphological alterations in endothelial cells
To evaluate the effect of CMDV on endothelial trans-differentiation, HMEC-1 (endothelial) cells were exposed for 48 hours or 120 hours to CMDV or 5 ng/mL TGF-β1. The latter was used as a positive control that induces EndMT [30]. In agreement with previous results, CMDV had no cytopathic effect [16].
Discussion
Soluble factors released during DENV infection induce a transient and partial EndMT-like state in endothelial cells. In our model, exposure to CMDV caused morphological changes, intercellular junction disruption, and upregulation of some EndMT markers, leading to increased endothelial permeability [16]. Notably, this response was biphasic and reversible: at 48 hours, CMDV-exposed cells displayed a strong pro-inflammatory profile, whereas by 120 hours they had shifted toward an endothelial repair and angiogenic profile, similar to what has been reported for EMT [38].
Acknowledgments
The authors thank María Elena Peñaranda and Eva Harris (University of California, Berkeley) for their generous donation of reagents. We also thank Engineer Hernández Talero C.A. for consulting and peer review of the code and Ana I. García-Vega (Advanced Cellular Analysis Unit, CIC, Salamanca) for training and assistance with ImageJ code for image analysis.
Citation: Alfaro-García JP, Ramírez-Mejía JM, Rojas-Estevez P, Álvarez-Díaz DA, Fernández GJ, Orozco-Castaño CA, et al. (2026) Molecular analysis and computational modeling reveal temporally separable responses triggered by DENV-induced soluble factors in endothelial cells. PLoS One 21(7): e0354877. https://doi.org/10.1371/journal.pone.0354877
Editor: Victoria Pando-Robles, Instituto Nacional de Salud Publica Centro de Investigaciones sobre Enfermedades Infecciosas, MEXICO
Received: December 22, 2025; Accepted: July 14, 2026; Published: July 31, 2026
Copyright: © 2026 Alfaro-García et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All the original data, sequences and other materials not included within the paper and its Supporting Information files have been deposited in public repositories. Raw microscopy data for this study are publicly available from the figshare repository (https://doi.org/10.6084/m9.figshare.33025556). RNAseq sequence data for this study are publicly available from the NCBI BioProject repository under the accession number PRJNA1331842 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA1331842?reviewer=lu4r1nun9376f1c15sp7dekepv). CMDV non-directed network code for this study is publicly available from the GitHub repository (https://github.com/bioweaver/CMDV_nondirectModel_v.1.0).
Funding: This study was financially supported by the Ministerio de Ciencia, Innovación y Universidades (Spain) in the form of a grant (PID2023-153018NB-I00) received by MV-M. This study was also financially supported by the Fundación Científica Asociación Española Contra el Cáncer in the form of a grant (ECRIN-M3) received by MV-M. This study was also financially supported by the Ministerio de Ciencia (CO) in the form of a grant (CTO 917-2019) received by JCG-G. This study was also financially supported by the Universidad de Antioquia in the form of a grant (CODI-2020-34137) received by JCG-G.
Competing interests: The authors have declared that no competing interests exist.
