ABSTRACT
Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.
Subject(s)
Benzylisoquinolines/pharmacology , Fluorescent Dyes/pharmacology , Molecular Imaging/methods , Optical Imaging/methods , Prolyl Hydroxylases/metabolism , Prolyl-Hydroxylase Inhibitors/pharmacology , Benzylisoquinolines/chemical synthesis , Benzylisoquinolines/chemistry , Biocatalysis/drug effects , Dose-Response Relationship, Drug , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Molecular Structure , Prolyl-Hydroxylase Inhibitors/chemical synthesis , Prolyl-Hydroxylase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Chronic kidney disease (CKD) is associated with increased risk and worse prognosis of cardiovascular disease, including peripheral artery disease. An impaired angiogenic response to ischemia may contribute to poor outcomes of peripheral artery disease in patients with CKD. Hypoxia inducible factors (HIF) are master regulators of angiogenesis and therefore represent a promising target for therapeutic intervention. To test this we induced hind-limb ischemia in rats with CKD caused by 5/6 nephrectomy and administered two different treatments known to stabilize HIF protein in vivo: carbon monoxide and a pharmacological inhibitor of prolyl hydroxylation 2-(1-chloro-4- hydroxyisoquinoline-3-carboxamido) acetate (ICA). Expression levels of pro-angiogenic HIF target genes (Vegf, Vegf-r1, Vegf-r2, Ho-1) were measured by qRT-PCR. Capillary density was measured by CD31 immunofluorescence staining and HIF expression was evaluated by immunohistochemistry. Capillary density in ischemic skeletal muscle was significantly lower in CKD animals compared to sham controls. Rats with CKD showed significantly lower expression of HIF and all measured pro-angiogenic HIF target genes, including VEGF. Both HIF stabilizing treatments rescued HIF target gene expression in animals with CKD and led to significantly higher ischemia-induced capillary sprouting compared to untreated controls. ICA was effective regardless of whether it was administered before or after induction of ischemia and led to a HIF expression in skeletal muscle. Thus, impaired ischemia-induced angiogenesis in rats with CKD can be improved by HIF stabilization, even if started after onset of ischemia.
Subject(s)
Capillaries/drug effects , Carbon Monoxide/pharmacology , Glycine/analogs & derivatives , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ischemia/drug therapy , Isoquinolines/pharmacology , Muscle, Skeletal/blood supply , Neovascularization, Physiologic/drug effects , Renal Insufficiency, Chronic/metabolism , Signal Transduction/drug effects , Animals , Capillaries/metabolism , Capillaries/physiopathology , Cell Line , Disease Models, Animal , Gene Expression Regulation , Glycine/pharmacology , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase (Decyclizing)/metabolism , Hindlimb , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Ischemia/genetics , Ischemia/metabolism , Ischemia/physiopathology , Male , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Protein Stability , Rats, Sprague-Dawley , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/physiopathology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-1/genetics , Vascular Endothelial Growth Factor Receptor-1/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Reduced nephron number predisposes to hypertension and kidney disease. Interaction of the branching ureteric bud and surrounding mesenchymal cells determines nephron number. Since oxygen supply may be critical for intrauterine development, we tested whether hypoxia and hypoxia-inducible factor-1α (HIF-1α) influence nephrogenesis. We found that HIF-1α is required for branching of MDCK cells. In addition, culture of metanephric mouse kidneys with ureteric bud cell-specific stabilization or knockout of HIF-1α revealed a positive impact of HIF-1α on nephrogenesis. In contrast, widespread stabilization of HIF-1α in metanephric kidneys through hypoxia or HIF stabilizers impaired nephrogenesis, and pharmacological HIF inhibition enhanced nephrogenesis. Several lines of evidence suggest an inhibitory effect through the hypoxia response of mesenchymal cells. HIF-1α was expressed in mesenchymal cells during nephrogenesis. Expression of the anti-branching factors Bmp4 and Vegfa, secreted by mesenchymal cells, was increased upon HIF stabilization. The conditioned medium from hypoxic metanephric kidneys inhibited MDCK branching, which was partially rescued by Vegfa antibodies. Thus, the effect of HIF-1α on nephrogenesis appears context dependent. While HIF-1α in the ureteric bud is of importance for proper branching morphogenesis, the net effect of hypoxia-induced HIF activation in the embryonic kidney appears to be mesenchymal cell-dependent inhibition of ureter branching.
ABSTRACT
Several genetically modified mouse models implicated that prolyl-4-hydroxylase domain (PHD) enzymes are critical mediators for protecting tissues from an ischemic insult including myocardial infarction by affecting the stability and activation of hypoxia-inducible factor (HIF)-1 and HIF-2. Thus, the current efforts to develop small-molecule PHD inhibitors open a new therapeutic option for myocardial tissue protection during ischemia. Therefore, we aimed to investigate the applicability and efficacy of pharmacological HIFα stabilization by a small-molecule PHD inhibitor in the heart. We tested for protective effects in the acute phase of myocardial infarction after pre- or post-conditional application of the inhibitor. Application of the specific PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) resulted in HIF-1α and HIF-2α accumulation in heart muscle cells in vitro and in vivo. The rapid and robust responsiveness of cardiac tissue towards ICA was further confirmed by induction of the known HIF target genes heme oxygenase-1 and PHD3. Pre- and post-conditional treatment of mice undergoing myocardial infarction resulted in a significantly smaller infarct size. Tissue protection from ischemia after pre- or post-conditional ICA treatment demonstrates that there is a therapeutic time window for the application of the PHD inhibitor (PHI) post-myocardial infarction, which might be exploited for acute medical interventions.
