Regulators of angiogenesis in diabetes and tumors

Abstract

Angiogenesis is tightly regulated in order to provide adequate supply of nutrients and oxygen for normal cellular function. Tissue oxygenation represents a balance between the requirement for oxygen to maintain the cellular energy level and the potential risk of cellular oxidative damage. The master regulator of oxygen homeostasis is Hypoxia inducible factor-1 (HIF-1). Our basic hypothesis was therefore that HIF modulation might be relevant for diseases characterised by dysregulated angiogenesis, such as diabetes mellitus and tumors.

Chronic complications of diabetes are a major health problem, and a better understanding of their pathogenic mechanisms could offer a more rational therapy. Both hyperglycemia and hypoxia play important roles in the pathogenesis of diabetes complications and we propose that the interplay between these two factors is essential in determining the devellopement of chronic complications of diabetes. We demonstrated that high concentrations of glucose interfere with hypoxia-dependent stabilization of HIF-1alpha against proteasomal degradation in both primary human dermal fibroblasts and microvascular endothelial cells. The destabilizing effect of glucose was partially independent of prolyl hydroxylases (PHD). Hyperglycemia also inhibited transactivation and function of HIF-1 as demonstrated by a HRE (hypoxia responsive elements) reporter gene assay.

In agreement with our in vitro observations we detect lower expression of HIF-1 alpha in diabetic wounds then in venous ulcers that share the same hypoxic environment but not the chronic exposure to hyperglycemia. Based on these findings we further investigated the potential therapeutic effect of HIF-1 alpha up-regulation in diabetic woundsby using DMOG, a chemical inducer of HIF-1 alpha which showed virtually no toxic effect on primary dermal fibroblasts in vitro. Local treatment with DMOG normalized the healing rate of experimental wounds in db/db mice suggesting that HIF-1 alpha modulation is a promising therapeutical approach for diabetic wounds.

Dysregulated angiogenesis is also a hallmark of tumor diseases. Kaposi s sarcoma (KS) is a highly vascularised tumor, which depends strictly on angiogenetic stimuli. We have therefore investigated the HIF pathway in KS and its relation to the insulin like growth factor (IGF) system, known as a major factor in pathophysiology of different tumors. We demonstrated that KS cells express both HIF-1alpha and HIF-2 alpha even in normoxia and that IGF-I induced their expression, showing for the first time that a growth factor is able to induce accumulation of both HIFalpha congeners. HIF accumulation was followed by functional activation as demonstrated by a HRE reporter gene assay and by induction of HIF target genes (ex. VEGF). IGF-I induced accumulation of both HIF-1 alpha and HIF-2 alpha at a posttranscriptional level as demonstrated by qRT-PCR and cycloheximide chasing experiment and we suggest that IGF-I increased the translation of the alpha subunits.

The clinical relevance of our findings was confirmed by the identification of high levels of both HIF isofoms in tumor cells in biopsies from patients with AIDS-related KS. It was further demonstrated that IGF-I and IGF-II are potent growth factors for KS cells and have an additive effect with VEGF. IGF-I receptor (IGF-IR) was identified as the receptor mediating the IGFs effect and we described an autocrine loop in KS cells in which IGF-II is the endogenous ligand for IGF-IR. Finally, it was demonstrated that blocking the IGF-IR with picropodophyllin, a recently described selective IGF-IR tyrosine phosphorylation inhibitor, induced apoptosis and decreased HIF accumulation and function in KS cells, suggesting a potential future therapeutic approach.

In conclusion, HIF is an important factor both in chronic complications of diabetes and in vascular tumor KS. We have highlighted that glucose and IGF-I regulate HIF by other mechanisms than the classic hypoxic regulation. Finally it was demonstrated that modulation of HIF can be a successful approach for treatment of both diabetic wounds and KS.