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Toll-Like Receptor Signalling Pathways Regulate Hypoxic Stress Induced Fibroblast Growth Factor but Not Vascular Endothelial Growth Factor-A in Human Microvascular Endothelial Cells

Akhtar, Rukhsar; Tahir, Husain; Stewart, Elizabeth; Wei, Ruoxin; Mohammed, Imran; Amoaku, Winfried M.

Toll-Like Receptor Signalling Pathways Regulate Hypoxic Stress Induced Fibroblast Growth Factor but Not Vascular Endothelial Growth Factor-A in Human Microvascular Endothelial Cells Thumbnail


Authors

Rukhsar Akhtar

Husain Tahir

Elizabeth Stewart

Ruoxin Wei

Imran Mohammed

WINFRIED AMOAKU winfried.amoaku@nottingham.ac.uk
Clinical Assoc Prof & Reader in Ophthalmology & Visual Sciences



Abstract

Retinal diseases are the leading causes of irreversible blindness worldwide. The role of toll-like receptor (TLR) signalling mechanisms (MyD88 and TRIF) in the production of pro-angiogenic growth factors from human microvascular endothelial cells (HMEC-1) under hypoxic stress remains unexplored. HMEC-1 was incubated under normoxic (5% CO2 at 37 °C) and hypoxic (1% O2, 5% CO2, and 94% N2; at 37 °C) conditions for 2, 6, 24, and 48 h, respectively. For TLR pathway analysis, HMEC-1 was pre-treated with pharmacological inhibitors (Pepinh-MyD88 and Pepinh-TRIF) and subjected to normoxia and hypoxia conditions. Gene and protein expressions of vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor (FGF-2), hypoxia inducible factor 1-alpha (HIF1-α) were performed using quantitative polymerase chain reaction (qPCR), ELISA, and Western blot methodologies. Levels of TLR3 and TLR4 were analysed by flow cytometry. Under hypoxia, levels of VEGF-A and FGF-2 were elevated in a time-dependent fashion. Inhibition of MyD88 and TRIF signalling pathways decreased FGF-2 levels but failed to modulate the secretion of VEGF-A from HMEC-1. Blocking a known regulator, endothelin receptor (ETR), also had no effect on VEGF-A secretion from HMEC-1. Overall, this study provides the proof-of-concept to target TLR signalling pathways for the management of blinding retinal diseases.

Journal Article Type Article
Acceptance Date May 12, 2021
Online Publication Date May 27, 2021
Publication Date 2021-06
Deposit Date Nov 12, 2021
Publicly Available Date Nov 17, 2021
Journal International Journal of Translational Medicine
Print ISSN 2673-8937
Electronic ISSN 2673-8937
Publisher MDPI AG
Peer Reviewed Peer Reviewed
Volume 1
Issue 1
Pages 25-38
DOI https://doi.org/10.3390/ijtm1010003
Public URL https://nottingham-repository.worktribe.com/output/5619771
Publisher URL https://www.mdpi.com/2673-8937/1/1/3

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