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The Regulation of Endothelial Cell Activation by the Extracellular Matrix after Acute Brain Injury

Kangwantas, Korakoch

[Thesis]. Manchester, UK: The University of Manchester; 2014.

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Abstract

Inflammation or injury of the central nervous system generally results in the activation of brain endothelial cells and a change in the composition and expression of the extracellular matrix (ECM) network of the basal lamina of the brain vasculature. The main contributors to brain damage are interleukin (IL)-1-mediated inflammatory processes. The main aim of this project is to investigate whether the ECM associated with brain endothelial cells is modified in response to ischaemic injury in vitro, and to test the hypothesis that alteration of ECM composition following injury is a critical regulator of IL-1-induced endothelial cell activation. The in vitro blood-brain barrier model used in this thesis was composed of brain endothelial cells and astrocytes and this model displayed classic BBB characteristics. Oxygen-glucose deprivation (OGD) for 2.5 hours followed by 4 hours of reperfusion ┬▒ 10 ng/ml IL-1╬▓ induced changes in rat brain endothelial cell (rBEC) morphology, occludin and ZO-1 distribution and cytokine-induced neutrophil chemoattractant (CINC)-1 release. Immunohistochemistry on brain sections from animals subjected to middle cerebral artery occlusion (MCAO) demonstrated upregulation of laminin ╬▒4 protein following 48 hours and 6 days of reperfusion. Fibronectin expression was also increased in the brain vessels of animals subjected to MCAO following 48 hours of reperfusion. Similarly, 2.5 hours OGD and 2 hours reperfusion ┬▒IL-1╬▓ induced changes in laminin ╬▒4, ╬▓1 and ╬│1, type IV collagen ╬▒1 chain and fibronectin mRNA expression in vitro. ECM molecules also influenced rBEC morphology, adhesion, proliferation, organisation and TEER. Integrin ╬▓1 mediated rBEC adhesion to type I collagen, type IV collagen and cellular fibronectin but not laminin-511 and the combination of laminin-411 and -511. Laminin-511 and the combination of laminin-411 and -511 significantly increased CINC-1 release compared to type I collagen. Laminin-411 and -511 also upregulated occludin protein expression and maintained occludin distribution following IL-1╬▓ treatment in rBECs. To conclude, ECM associated with brain endothelial cells was modified in response to ischaemic injury ┬▒IL-1╬▓ in vitro, and vascular ECM proteins altered rBEC activation in response to IL-1╬▓. Therefore, a change in ECM composition following injury is a critical regulator of IL-1-induced endothelial cell activation.

Bibliographic metadata

Type of resource:
Content type:
Form of thesis:
Type of submission:
Degree type:
Doctor of Philosophy
Degree programme:
PhD Pharmacy and Pharmaceutical Sciences
Publication date:
Location:
Manchester, UK
Total pages:
219
Abstract:
Inflammation or injury of the central nervous system generally results in the activation of brain endothelial cells and a change in the composition and expression of the extracellular matrix (ECM) network of the basal lamina of the brain vasculature. The main contributors to brain damage are interleukin (IL)-1-mediated inflammatory processes. The main aim of this project is to investigate whether the ECM associated with brain endothelial cells is modified in response to ischaemic injury in vitro, and to test the hypothesis that alteration of ECM composition following injury is a critical regulator of IL-1-induced endothelial cell activation. The in vitro blood-brain barrier model used in this thesis was composed of brain endothelial cells and astrocytes and this model displayed classic BBB characteristics. Oxygen-glucose deprivation (OGD) for 2.5 hours followed by 4 hours of reperfusion ┬▒ 10 ng/ml IL-1╬▓ induced changes in rat brain endothelial cell (rBEC) morphology, occludin and ZO-1 distribution and cytokine-induced neutrophil chemoattractant (CINC)-1 release. Immunohistochemistry on brain sections from animals subjected to middle cerebral artery occlusion (MCAO) demonstrated upregulation of laminin ╬▒4 protein following 48 hours and 6 days of reperfusion. Fibronectin expression was also increased in the brain vessels of animals subjected to MCAO following 48 hours of reperfusion. Similarly, 2.5 hours OGD and 2 hours reperfusion ┬▒IL-1╬▓ induced changes in laminin ╬▒4, ╬▓1 and ╬│1, type IV collagen ╬▒1 chain and fibronectin mRNA expression in vitro. ECM molecules also influenced rBEC morphology, adhesion, proliferation, organisation and TEER. Integrin ╬▓1 mediated rBEC adhesion to type I collagen, type IV collagen and cellular fibronectin but not laminin-511 and the combination of laminin-411 and -511. Laminin-511 and the combination of laminin-411 and -511 significantly increased CINC-1 release compared to type I collagen. Laminin-411 and -511 also upregulated occludin protein expression and maintained occludin distribution following IL-1╬▓ treatment in rBECs. To conclude, ECM associated with brain endothelial cells was modified in response to ischaemic injury ┬▒IL-1╬▓ in vitro, and vascular ECM proteins altered rBEC activation in response to IL-1╬▓. Therefore, a change in ECM composition following injury is a critical regulator of IL-1-induced endothelial cell activation.
Thesis main supervisor(s):
Thesis co-supervisor(s):
Thesis advisor(s):
Language:
en

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:228076
Created by:
Kangwantas, Korakoch
Created:
30th June, 2014, 02:45:08
Last modified by:
Kangwantas, Korakoch
Last modified:
1st August, 2014, 10:18:28

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