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Hypoxia-Induced miR-101 Impairs Endothelial Barrier Integrity Through Altering VE-Cadherin and Claudin-5

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Abstract

Stroke is a life-threatening medical condition across the world that adversely affects the integrity of the blood–brain barrier (BBB). The brain microvascular endothelial cells are the important constituent of the BBB. These cells line the blood vessels and form a semipermeable barrier. Disruptions in adherens junction and tight junction proteins of brain microvascular endothelial cells compromise the integrity of BBB. The Vascular Endothelial (VE)-cadherin is an integral adherens junction protein required for the establishment and maintenance of the endothelial barrier integrity. This study aims to investigate the role of miRNA in hypoxia-induced endothelial barrier disruption. In this study, brain endothelial cells were exposed to hypoxic conditions for different time points. Western blotting, overexpression and knockdown of miRNA, real-time PCR, TEER, and sodium fluorescein assay were used to examine the effect of hypoxic conditions on brain endothelial cells. Hypoxic exposure was validated using HIF-1α protein. Exposure to hypoxic conditions resulted to a significant decrease in endothelial barrier resistance and an increase in sodium fluorescein migration across the endothelial barrier. Reduction in endothelial barrier resistance demonstrated compromised barrier integrity, whereas the increase in migration of sodium fluorescein across the barrier indicated the increase in barrier permeability. The present study revealed microRNA-101 decreases the expression of VE-cadherin and claudin-5 in brain endothelial cells exposed to the hypoxic conditions.

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Data Availability

All data generated in this study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors acknowledge the funding support provided through the Institutions of Eminence (IoE-6031) Scheme of Banaras Hindu University, Varanasi, and Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Astha Shukla and Apoorva acknowledge the financial assistance provided by UGC, India, through NON-NET fellowship. Utkarsh Bhardwaj is a recipient of the Indian Council of Medical Research (ICMR)-Senior Research Fellowship. The authors also acknowledge Ms. Neha Pandey for her critical help during manuscript drafting.

Funding

This work was financially supported by the funding received by the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, (BT/PR23625/MED/122/77/2017).

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Authors and Affiliations

  1. Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, 221005, UP, India

    Astha Shukla, Utkarsh Bhardwaj,  Apoorva & Sunit K. Singh

  2. Molecular and Cellular Neurosciences, National Brain Research Centre, Manesar, 122052, Haryana, India

    Pankaj Seth

  3. Dr. B R Ambedkar Center for Biomedical Research, University of Delhi (North Campus), New Delhi, 110007, India

    Sunit K. Singh

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  1. Astha Shukla
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Contributions

AS and UB did the experiments and analyzed the data, and SKS conceptualized the hypothesis and experimental design. AS, UB and Apoorva drafted the manuscript. PS provided the hCMEC/D3 cells and shared his knowledge as a Co-Investigator. SKS supervised the work and reviewed the manuscript. All authors read and approved the final manuscript.

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Correspondence to Sunit K. Singh.

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Shukla, A., Bhardwaj, U., Apoorva et al. Hypoxia-Induced miR-101 Impairs Endothelial Barrier Integrity Through Altering VE-Cadherin and Claudin-5. Mol Neurobiol 61, 1807–1817 (2024). https://doi.org/10.1007/s12035-023-03662-8

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