Cardiac and fibroblastic properties after HIF-1α stabilization

Cardiac and fibroblastic properties after HIF-1α stabilization

von Melanie Vogler
Kumulative Dissertation
Datum der mündl. Prüfung:2015-05-21
Erschienen:2015-06-01
Betreuer:Prof. Dr. Dörthe Katschinski
Gutachter:Prof. Dr. Susanne Lutz
Gutachter:Prof. Dr. Jörg Großhans
Gutachter:Prof. Dr. Hubertus Jarry
Gutachter:Prof. Dr. Jürgen Brockmöller
Gutachter:PD Dr. Kaomei Guan-Schmidt
Zum Verlinken/Zitieren: http://dx.doi.org/10.53846/goediss-5108

 

 

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The cellular oxygen sensing prolyl-4-hydroxylase domain proteins (PHDs) are essential regulators of the α-subunit of hypoxia-inducible transcription factors (HIFs) which are known to induce a variety of genes affecting cell survival, metabolism and angiogenesis. It was shown by the Institute of Cardiovascular Physiology, University Medical Center Göttingen, that stabilization of HIF-1α in cardiomyocyte-specific PHD2 knock out mice results in a tissue protective effect in acute myocardial infarction (MI) (Hölscher et al., 2011). Thus, pharmaceutical stabilization of HIF-1α with small molecule PHD inhibitors could be a suitable approach to induce cytoprotection in case of ischemia. Besides cardiomyocytes, fibroblasts are essentially involved in the organisation of the myocardium as well as in postischemic remodeling. In order to develop new cardio-protective therapeutic strategies, there is a strong need to further define fibroblast-specific functions in hypoxia and the role of HIF-1α in fibroblastic properties. Therefore, the aims of this thesis were (i) to evaluate the effect of a pharmaceutical PHD inhibition using the isoquinoline analog 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) for its applicability and protective properties in the heart, (ii) to analyze the role of PHD2 in cardiac fibroblasts via an inducible fibroblast-specific PHD2 knock out mouse, as well as (iii) to examine the morphological and functional responses of fibroblasts to hypoxia in vitro. I could show that L929 fibroblasts respond to hypoxia with striking morphological changes including an enlargement of cell area and volume, increased focal contact assembly and a dramatic reorganization of the actin cytoskeleton with prominent β-actin enriched circular stress fibers. These hypoxic adaptions are associated with enhanced cell spreading as well as reduction in migration properties. Furthermore, it could be shown that the maintenance of p-cofilin levels is dependent on HIF-1α which subsequently affects the actin filament arrangement and may cause the observed increase in cell area and reduction in migration under hypoxic conditions. To study the role of HIF-1α in fibroblastic properties in vivo a tamoxifen-inducible fibroblast-specific PHD2 knock out mouse was generated and Cre-mediated DNA recombination of Phd2 loxP sites could be detected, but lacks PHD2 knock out efficacy on mRNA and protein level. I could show that the PHD inhibitor ICA leads to a rapid and strong HIF-1α and HIF-2α accumulation in the heart and to the induction of HIF target genes which subsequently exert acute cardio-protective effects in MI. ICA treatment significantly reduced total infarct size after MI. This effect was seen not only after pre-conditional treatment, but also after post-conditional application of ICA. This indicates that there is a therapeutic time window for the treatment of ischemic diseases with systemic PHD inhibitors as a useful tool for clinical approaches. Summing up, I could show that hypoxia and specifically HIF-1α greatly affects fibroblastic properties and functions and that a transient HIF-1α stabilization via small molecule PHD inhibitors is a suitable approach to induce cytoprotection in case of ischemia.
Keywords: Hypoxia; HIF; PHD; ICA; MI
 
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