Mechanisms of chronic complications of diabetes with focus on mitochondria and oxygen sensing

Chronic complications of diabetes represent a major medical and economical concern. It is an imperative need to establish the pathogenic mechanisms that contribute to development of chronic complications in order to design new therapeutic approaches. Several pathogenic pathways are activated in diabetes and have been proposed to be responsible for the development of long-term complications of diabetes including an overproduction of reactive oxygen species (ROS) by the mitochondria electron-transport chain, suggested to be a common mechanism for all the others. Hypoxia directly and through induction of ROS has been recently suggested to have an important role in the development of chronic complications of diabetes. Adaptive responses of cells to hypoxia are mediated by the hypoxia-inducible factor-1 (HIF-1), which is an ubiquitary heterodimeric transcription factor, regulated by oxygen at the degradation level of its alpha subunit. The oxygen regulation of HIF is complex and involves a family of hydroxylases (HIF hydroxylases), that need Fe2+ or alpha-ketoglutarate as cofactors and regulates both HIF stability and transactivation in hypoxia. Under hypoxic conditions, HIF-1alpha is stabilized against degradation and upregulates a series of genes (more than 70) involved in essential processes i.e. angiogenesis, glycolytic energy metabolism, cell proliferation and survival. ROS, produced in excess both in hyperglycemia and hypoxia can interact with different macromolecules including DNA. The close proximity of the mitochondrial DNA (mtDNA) to the ROS-generating sites makes mtDNA more vulnerable to oxidative damage. Different cellular specific antioxidants mechanisms are available but they are not always able to fully protect DNA from the oxidative damage. Beside mtDNA mutations that contribute to the maternal inherited diabetes (0.5-1%) several somatic mtDNA point mutations and deletions (classically associated with aging) have been described in patients with diabetes even though not consistent. A drawback of these studies is the heterogeneous genetic background of the patients with diabetes coupled with the lack of reliable information about the duration of the exposure to high glucose levels. The direct influence of chronic hyperglycaemia and/or hypoxia upon mtDNA stability and repair is not clear. Hyperglycemia impairs HIF-1alpha stability and function and it has been suggested that by this it contributes to the development of chronic complications of diabetes (wound healing, coronary heart disease etc). We have therefore hypothesized in the first paper that the defect in wound healing present in diabetes is a result of an inhibition of HIF-1 activity. We could first demonstrate that the repression of HIF in hyperglycemia is complex and implies not only the stability of the HIF-1alpha in hypoxia but also the transactivation of both N-terminal transactivation domain (NTAD) and C-terminal transactivation domain (CTAD). Furthermore we show that by blocking HIF-1alpha hydroxylation through chemical inhibition it is possible to reverse the negative regulatory effect of hyperglycemia on HIF-1 alpha both in vitro and in vivo in a mouse model (db/db) of diabetic wound. Local HIF-1 alpha induction was able to improve several processes essential for wound healing i.e. granulation, vascularisation, epidermal regeneration, and recruitment of endothelial precursors cells (EPC). Stabilisation of HIF-1alpha was necessary and sufficient for promoting wound healing in a diabetic environment. Thus, it is important to develop specific hydroxylase inhibitors as therapeutic agents for chronic diabetes wounds. In the second paper we investigated by long amplification QPCR the stability of mitochondrial DNA against ROS overproduction in diabetic condition. By using human dermal fibroblasts (HDF) we were able to confirm an increase of ROS production by hyperglycemia alone or in combination with hypoxia. However, mtDNA damage was observed only when the cells were exposed to both hyperglycemia and hypoxia, confirming the pathogenic role of the combination of these factors. The mtDNA damage was mediated through excess production of ROS by mitochondria as far as mtDNA was fully protected when the cells were treated with inhibitors of the electron transport chain. We have further studied by long amplification QPCR the effect of diabetes on mtDNA lesions in db/db mouse that is an inbred model of type 2 diabetes. We investigated the incidence of mtDNA lesions in heart and kidney of two groups of db/db mice: young prediabetic mice (before developing diabetes) and old diabetic mice (34 weeks) and compared with nondiabetic mice (heterozygotes) of the same age. Unexpectedly, the old diabetic mice had a lower incidence of mtDNA lesions in both tissues studied, compared with old non-diabetic and young prediabetic animals even though the tissues are exposed to an excess ROS (as shown by increased protein nitrosylation). This was explained by an increase in the antioxidant capacity (expression of Mn Superoxide Dismutase (SOD2) and Catalase) and of the mitochondrial base excision repair (mtBER) activity in old diabetic animals. In conclusion, we have shown that hypoxia plays together with hyperglycemia an important role in chronic complications of diabetes by activating several pathogenic pathways. Some mechanisms are fully compensated (i.e. antioxidant defence and reparatory capacity of mtDNA) while others (HIF repression) need to be addressed therapeutically in order to be able to improve the outcome