Inhibition of prolyl hydroxylase domain proteins selectively enhances venous thrombus neovascularisation.

BACKGROUND: Hypoxia within acute venous thrombi is thought to drive resolution through stabilisation of hypoxia inducible factor 1 alpha (HIF1α). Prolyl hydroxylase domain (PHD) isoforms are critical regulators of HIF1α stability. Non-selective inhibition of PHD isoforms with l-mimosine has been shown to increase HIF1α stabilisation and promote thrombus resolution. OBJECTIVE: The aim of this study was to investigate the therapeutic potential of PHD inhibition in venous thrombus resolution. METHODS: Thrombosis was induced in the inferior vena cava of mice using a combination of flow restriction and endothelial activation. Gene and protein expression of PHD isoforms in the resolving thrombus was measured by RT-PCR and immunohistochemistry. Thrombus resolution was quantified in mice treated with pan PHD inhibitors AKB-4924 and JNJ-42041935 or inducible all-cell Phd2 knockouts by micro-computed tomography, 3D high frequency ultrasound or endpoint histology. RESULTS: Resolving venous thrombi demonstrated significant temporal gene expression profiles for PHD2 and PHD3 (P < 0.05), but not for PHD1. PHD isoform protein expression was localised to early and late inflammatory cell infiltrates. Treatment with selective pan PHD inhibitors, AKB-4924 and JNJ-42041935, enhanced thrombus neovascularisation (P < 0.05), but had no significant effect on overall thrombus resolution. Thrombus resolution or its markers, macrophage accumulation and neovascularisation, did not differ significantly in inducible all-cell homozygous Phd2 knockouts compared with littermate controls (P > 0.05). CONCLUSIONS: This data suggests that PHD-mediated thrombus neovascularisation has a limited role in the resolution of venous thrombi. Directly targeting angiogenesis alone may not be a viable therapeutic strategy to enhance venous thrombus resolution.

Fungal pathogens research: novel and improved molecular approaches for the discovery of antifungal drug targets.

With the rise of fungal infection incidence amongst the patient population, the importance of developing new antifungal drug targets is higher than ever. This review mainly focuses on the three most prevalent fungal pathogens, Candida, Aspergillus and Cryptococcus, and on the most recent progresses in molecular research that contribute to a better understanding of the pathogen itself, but also its host and the interaction with its host. We consider the progress made in comparative genomics following the huge effort of fungal genome sequence projects undertaken in the last few years. We focus not only on currently used mammalian animal models such as mice, but also on novel non-mammalian models, such as the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, which offer useful tools in the area of the innate immune response to fungal infections. In addition we relate to the recent genomic and proteomic studies and focus on the use of these approaches in in vivo experiments in the pathogen itself as well as in the host. Finally, we describe the latest targeted mutagenesis strategy available in C. albicans and the use of RNA interference in both Cryptococcus neoformans and A. fumigatus. Our aim is not to give an exhaustive list of all new strategies but rather to give an overview of what will contribute most to the identification of new antifungal drug targets and the establishment of novel antifungal strategies.