SARS-Cov-2 Replication in a Blood–Brain Barrier Model Established with Human Brain Microvascular Endothelial Cells Induces Permeability and Disables ACE2-Dependent Regulation of Bradykinin B1 Receptor
Abstract
:1. Introduction
2. Results
2.1. Transformed HBMECs Do Not Sustain a SARS-CoV-2 Productive Infection
2.2. HBMECs Overexpressing ACE2 Are Permissive to SARS-CoV-2 Replication
2.3. Production of Inflammatory Mediators by HBMEC-ACE2 Infected with SARS-CoV-2
2.4. Infection of HBMEC-ACE2 with SARS-CoV-2 Induces Endothelial Permeability, with Virus and Mononuclear Cells Crossing
2.5. SARS-CoV-2 Infection Modulates ACE2-Dependent Regulation of DABK/B1R Pathway
3. Discussion
4. Materials and Methods
4.1. Cells and Viruses
4.2. Pseudovirus Construction and Transduction in HBMECs
4.3. HBMECs Infection with SARS-CoV-2
4.4. Analysis of Virus Replication, hACE2 and Cytokine Expression by RT-qPCR
4.5. Evaluation of Protein Expression by Western Blotting
4.6. Cell Viability Assay
4.7. Evaluation of dsRNA Staining by Immunofluorescence
4.8. Quantification of Nitric Oxide (NO) and Reactive Oxygen Species (ROS)
4.9. Assessment of ACE2 Enzymatic Activity
4.10. Ethical Statement and Isolation of PBMC and Human Primary Monocytes
4.11. Analysis of Monocyte Adhesion
4.12. Evaluation of Endothelial Permeability and of Virus and PBMC Extravasation
4.13. Analysis of Angiotensin II Degradation by ELISA
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequence (5′–3′) | |
---|---|---|
N genomic | Forward | TTACAAACATTGGCCGCAAA |
N genomic | Reverse | GCGCGACATTCCGAAGAA |
N subgenomic | Forward | CGATCTCTTGTAGATCTGTTCTCTAAACGAACAAATTAAAT |
N subgenomic | Reverse | TCTGGTTACTGCCAGTTGCCTCTG |
Gapdh | Forward | GTGGACCTGACCTGCCGTCT |
Gapdh | Reverse | GGAGGAGTGGGTGTCGCTGT |
Ace2 | Forward | GGGATCAGAGATCGGAAGAAGAAA |
Ace2 | Reverse | AGGAGGTCTGAACATCATCAGT |
Ccl5 | Forward | CCAGCAGTCGTCTTTGTCAC |
Ccl5 | Reverse | CTCTGGGTTGGCACACACTT |
Ccl2 | Forward | CAGCCAGATGCAATCAATGCC |
Ccl2 | Reverse | TGGAATCCTGAACCCACTTCT |
Il8 | Forward | CAGCCAAAACTCCACAGTCA |
Il8 | Reverse | TTGGAGAGCACATAAAAACATCT |
Il6 | Forward | TGTGAAAGCAGCAAAGAGGCACTG |
Il6 | Reverse | ACAGCTCTGGCTTGTTCCTCACTA |
Tnf | Forward | CAGAGGGAAGAGTTCCCCAGGGACC |
Tnf | Reverse | CCTTGGTCTGGTAGGAGACGGC |
Gene | Probe (5′–3′) | |
N | ACACTAGCCATCCTTACTGCGCTTCG |
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Coelho, S.V.A.; Souza, G.L.e.; Bezerra, B.B.; Lima, L.R.; Correa, I.A.; de Almeida, D.V.; Silva-Aguiar, R.P.d.; Pinheiro, A.A.S.; Sirois, P.; Caruso-Neves, C.; et al. SARS-Cov-2 Replication in a Blood–Brain Barrier Model Established with Human Brain Microvascular Endothelial Cells Induces Permeability and Disables ACE2-Dependent Regulation of Bradykinin B1 Receptor. Int. J. Mol. Sci. 2025, 26, 5540. https://doi.org/10.3390/ijms26125540
Coelho SVA, Souza GLe, Bezerra BB, Lima LR, Correa IA, de Almeida DV, Silva-Aguiar RPd, Pinheiro AAS, Sirois P, Caruso-Neves C, et al. SARS-Cov-2 Replication in a Blood–Brain Barrier Model Established with Human Brain Microvascular Endothelial Cells Induces Permeability and Disables ACE2-Dependent Regulation of Bradykinin B1 Receptor. International Journal of Molecular Sciences. 2025; 26(12):5540. https://doi.org/10.3390/ijms26125540
Chicago/Turabian StyleCoelho, Sharton Vinicius Antunes, Gabriela Lisboa e Souza, Bruno Braz Bezerra, Luan Rocha Lima, Isadora Alonso Correa, Dalziza Victalina de Almeida, Rodrigo Pacheco da Silva-Aguiar, Ana Acácia S. Pinheiro, Pierre Sirois, Celso Caruso-Neves, and et al. 2025. "SARS-Cov-2 Replication in a Blood–Brain Barrier Model Established with Human Brain Microvascular Endothelial Cells Induces Permeability and Disables ACE2-Dependent Regulation of Bradykinin B1 Receptor" International Journal of Molecular Sciences 26, no. 12: 5540. https://doi.org/10.3390/ijms26125540
APA StyleCoelho, S. V. A., Souza, G. L. e., Bezerra, B. B., Lima, L. R., Correa, I. A., de Almeida, D. V., Silva-Aguiar, R. P. d., Pinheiro, A. A. S., Sirois, P., Caruso-Neves, C., Costa, L. J. d., Scharfstein, J., & Arruda, L. B. d. (2025). SARS-Cov-2 Replication in a Blood–Brain Barrier Model Established with Human Brain Microvascular Endothelial Cells Induces Permeability and Disables ACE2-Dependent Regulation of Bradykinin B1 Receptor. International Journal of Molecular Sciences, 26(12), 5540. https://doi.org/10.3390/ijms26125540