Abstract

Suppression of Protein Kinase C-α Ameliorates Hyperglycaemia-Evoked In Vitro Cerebral Barrier Dysfunction

Title: Suppression of protein kinase C-α ameliorates hyperglycaemia-evoked in vitro cerebral barrier dysfunction.

Background: Ischaemic stroke is common in diabetes patients and occurs due to cerebral-barrier dysfunction. Although the pathological activation of protein kinase C (PKC) is linked with the disease progression, the PKC isoform-specific downstream signalling remains obscure and is the focus of this study which explored the clinical relevance of PKC-α inhibitor Ro-32-0432 under hyperglycaemia (HG).

Methods and Findings: Total PKC and RhoA activities were studied in human brain microvascular endothelial cells (HBMEC) exposed to normoglycaemia or HG. The integrity and function of an in vitro model of human BBB composed of HBMEC and human astrocytes were measured by transendothelial electrical resistance (TEER) and paracellular flux of Evans blue-labelled albumin (EBA), respectively. Exposure of HBMEC to HG for 72 hours led to significant increases in activities of total PKC and RhoA as well as in mono- and di-phosphorylation of MLC2 which concurred with substantial decreases in TEER and marked elevations in barrier permeability. Enhanced cellular contractility triggered by actin stress fibre formation appeared to further potentiate HG-mediated barrier dysfunction. Pre-treatment of HBMEC with Ro-32-0432 or transient PKC-α protein knockdown led to effective preservation of BBB integrity and function in hyperglycaemic settings by suppressing RhoA activity and subsequently normalising the MLC2 phosphorylation on Ser19 and Thr18- Ser19 residues. These observations were further supported by disappearance of stress fibres and subsequent restoration of the cortical actin staining.

Conclusions: Neutralisation of PKC-α activity may be of considerable therapeutic value in clinical settings accompanied by diabetes or stress HG.


Author(s):

Kirtiman Srivastava, Ulvi Bayraktutan



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