Mechanism for PHD inhibition by DMOG in the model of cold

The main problem caused by cold ischaemia is that during warm reperfusion a number of damaging mechanisms lead to excessive endothelial injury. This injury is independent of the type of vessel and occurs during the first phase of vessel or organ transplantation. After implantation, the degree of endothelial injury is one of the factors that determine the functional integrity of the organ and the success of the transplantation.

The literature on organ preservation describes a number of new substances which could help to conserve endothelial function. These substances may contribute further to improving existing conservation protocols. The development process targets different pathways, including for example antioxidants, poly (ADP ribose) polymerase and NO-cGMP-PKG. The need for further research in the field of organ and vessel preservation is mandated by the declining number of organ donors and consequently the increasing distance on average between organ donor and recipient.

In our experimental model we isolated aortic rings from male rats and performed functional tests with the aim of establishing how vascular function is affected by the inhibition of PHDs by DMOG. DMOG was applied in the preservation solution during the cold ischaemic period. The role of the PHD-HIF system in I/R injury and other hypoxia-related disorders has already been shown in different animal models, such as myocardial, cerebral ischaemia, liver ischaemia–reperfusion and cancer models (Zebger-Gong et al. 2010).

The data presented in this thesis focus on the role of oxygen-sensing systems under pathophysiological conditions in a model of cold ischaemia–warm reperfusion. The prolyl hydoxylase inhibitor DMOG was used to modulate the oxygen-sensing system.

DMOG stabilises HIF under normoxic conditions. HIF-1 is a transcription factor that

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plays a central role in the response to hypoxia and ischaemia through the regulation of gene expression (inducing and suppressing, e.g. HO, NOS, CA, VEGF, EPO or GLUT).

Cold ischaemic storage is a common way to preserve tissues and organs against the loss of functional integrity during the ischaemic period. In vascular grafts and in transplanted organs the acute cold ischaemia and warm reperfusion injury leads to loss of the functional integrity of the vessels, which manifests as an acute endothelial dysfunction and later as transplant vasculopathy. Furthermore, vascular integrity of the graft is critically dependent on nitric oxide production by intact endothelial cells (Garbe et al. 2011). Endothelial integrity is crucial in the protection of vascular grafts because the vascular endothelium contributes to the prevention of platelet aggregation, to smooth muscle proliferation and to maintaining an adequate vascular tone. Therefore, the protection of endothelial integrity is essential. Experiments performed by He and colleagues have indicated that short-term storage of vessels in saline causes loss of endothelial function (He 2005). Intact endothelial and vascular smooth muscle function is particularly important for the prevention of postoperative graft thrombosis and stenosis (Garbe et al. 2011). Work by other groups has highlighted that the saline solution often used for cold storage was unable to prevent the loss of functional integrity of the vasculature. This is reflected in the reduced ability for endothelium-dependent relaxation and also the decreased development of smooth muscle tone to a high potassium concentration (Radovits et al. 2008). Previous published data from our research group showed that short-term storage was not able to induce a marked deficit in functional integrity (Radovits et al. 2009). Therefore, an in vitro or ex vivo model of cold ischaemic storage is not suitable for reliable pharmacological trials (Sand et al.

2003; Stocker et al. 2004; Zhang et al. 2004; Radovits et al. 2007).

It has been shown in various models of vascular diseases (e.g. for diabetes, atherosclerosis and ischaemia–reperfusion injury) that leukocyte-derived myelo-peroxidase plays an important role (because of the formation of ROS) in vascular injury (Zhang et al. 2004). Hydrogen peroxide is a substrate of MPO, which results in the generation of hypochlorous acid (Radovits et al. 2007). Hypochlorite was used to simulate reperfusion injury; acetylcholine-induced vasorelaxation was reduced by

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hypochlorite treatment by approximately 50% compared with the control animals, and was normalised in the group with DMOG-supplemented preservation. This type of experimental endothelium injury had previously been established in our laboratory (Hunter et al. 2005; Radovits et al. 2007).

The detection of DNA fragmentation using TUNEL staining is a widely used assay that acts as an indirect method for assessing apoptosis (Philip et al.). This study demon-strated that the exposure of aortic vascular segments to cold ischaemic storage followed by warm reperfusion resulted in the formation of DNA strand breaks in the vessel walls as evidenced by TUNEL staining; this was significantly reduced in the DMOG group.

To the best of our knowledge, this work shows for the first time the vascular effects of DMOG. The results presented clearly demonstrate that pharmacological inhibition of PHDs by DMOG results in significantly improved vasorelaxation after 24 h of cold ischaemia and hypochlorite-induced warm reperfusion injury. In the NaOCl group (injured by hypochlorite), we showed an endothelial function that was severely impaired.

The kinetics of expression of HO-1 of aortic rings in the NaOCl group was significantly lower compared with the DMOG group. The same trend was observed for vascular smooth muscle cells. It could be suggested that the phenomenon is caused by the early protective effects of HIF-stabilisation due to prolyl hydroxylase inhibition. This effect may protect the endothelium against I/R injury. The aortic rings without preconditioning probably suffer a stronger but delayed ischaemia–reperfusion injury. In the DMOG group, the observed ameliorated endothelial function was probably caused by HO-1-mediated CO release.

Bateman et al. found that during hypoxic conditions, mRNA level as well as in the protein level of HIF 1α was significantly rapidly elevated in the first 2 h followed by increased levels of the target genes (Bateman et al. 2007). This has also been reported by other authors (Czibik et al. 2009). Czibik et al. also reported that after gene therapy with HIF-1α in a murine model, the cardioprotective effect was associated with elevated serum bilirubin levels. This effect was mimicked by remote HO-1 treatment.

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CO has a physiological role in the regulation of vascular tone similar to that of nitric oxide. One mechanism for this may be through increased intracellular cGMP (Morita et al. 1995). CO produced by HO-1 has only local effects; therefore, only the same cell (autocrine) or a neighbouring cell (paracrine) can be affected. Lim et al. found HIF-1 activation after treatment with DMOG on a human microvascular endothelial cell line and also a highly activated HO-1 protein level particularly after 24 h in the cell culture media (Lim et al. 2011).

The in vitro measurements identified the role of the oxygen-sensing system in endothelium-dependent vasorelaxation but not in smooth muscle dependent relaxation.

The hypothesis for this was that either the smooth muscle layer is not as sensitive to changes of oxygen tension as the endothelial layer or the hypochlorite-induced in vitro injury could induce damage of endothelial cells but could not penetrate to the deeper tissue layers. The role of modulation of the oxygen-sensing system on isolated rat smooth muscle cell culture has also been investigated. In the NaCl group of vascular smooth muscle cells we were able to detect a significantly decreased level of HO-1, in contrast to the DMOG group.

Morita et al. found and suggested that endogenous accumulation of CO derived from smooth muscle cells suppresses the induction of HO under hypoxic conditions (Morita et al. 1995). This is a plausible explanation because HO-1 expression shows a biphasic characteristic with decreasing mRNA levels after long periods (48 h) of hypoxia (Ockaili et al. 2005).

This work indicated that the pharmacological modulation of the PHD-HIF pathway improved endothelium-dependent vasorelaxation through HIF stabilisation-induced HO-1 up-regulation after short-term storage. Based on our results, we concluded that the usage of prolyl hydroxylase inhibitors will be useful in targeting the prevention of vascular dysfunction of grafts. Research on the transcription factor HIF-1 and the identification of hypoxia-induced genes could lead to development of new treatments and pretreatments for hypoxia-related pathophysiological conditions such as myocardial ischaemic conditions, transplant vasculopathy or graft failure.

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6.2. THE EFFECTS OF Q50 IN A RODENT MODELS OF REGIONAL AND