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Title: Prolyl hydroxylase domain proteins in venous thrombus resolution
Author: Grover, Steven Philip
ISNI:       0000 0004 5368 3263
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2015
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The prolyl hydroxylase domain (PHD) proteins serve as critical regulators of the cellular response to tissue hypoxia, like that present in the early venous thrombus, through regulation of hypoxia-inducible factor 1α (HIF1α) stability. This study sought to determine: (i) the expression of PHD1, PHD2 and PHD3 at the gene and protein level during natural thrombus resolution; (ii) the effect of gene specific deletion of Phd2 on thrombus resolution; (iii) the effect of pan- PHD inhibition on thrombus resolution; and (iv) the contribution of endogenous VEGFR signalling to thrombus resolution. All three PHD isoforms were expressed in the naturally resolving thrombus at the gene and protein level. Gene expression of Phd1 remained invariant while Phd2 and Phd3 expression demonstrated distinct temporal patterns. PHD isoforms were localised to the cellular component of the thrombus, with morphological analysis suggesting expression in both neutrophils and macrophages. Constitutive heterozygous Phd2 gene deletion failed to increase HIF1α stabilisation as was not associated with increased thrombus resolution. Inducible homozygous Phd2 gene deletion significantly enhanced HIF1α nuclear accumulation and transcriptional activity but thrombus resolution was unchanged. Pharmacological inhibition of PHD isoforms with novel small molecule inhibitor, AKB-4924 and JNJ-42041935, signficantly increased HIF1α nuclear accumulation and transcriptional activity. Treatment with these inhibitors significantly increased thrombus neovascularisation but thrombus resolution was unaffected. Blocking of endogenous VEGFR signalling using the pan- VEGFR inhibitor axitinib significantly impaired thrombus resolution. Axitinib treated thrombi remained larger and more occlusive for an extended period of time and this was associated with significant reductions in thrombus neovascularisation, macrophage recruitment and collagen deposition. Inhibition of PHD activity promotes thrombus neovascularisation, but other mechanisms are likely to regulate the removal of thrombus. Studies of thrombus resolution in Phd2 gene specific knockouts indicate that PHD2 activity does not play a major role in thrombus resolution. However, endogenous VEGFR signalling activity, downstream of HIF, is necessary for thrombus resolution.
Supervisor: Modarai, Bijan ; Smith, Alberto Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available