Abstract
The prolyl hydroxylase domain (PHD) proteins serve as critical regulators of thecellular 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.
| Date of Award | 2015 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Bijan Modarai (Supervisor) & Alberto Smith (Supervisor) |