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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., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Obliterative bronchiolitis (OB) is the major limiting factor for long-term survival after lung transplantation. As previously shown, donor treatment with a PHD-inhibitor activating hypoxia-inducible transcription factors (HIFs) prevents graft injury both in an allogenic kidney and aortic allograft transplant model. The aim of this study was to investigate the effect of HIF activation with a PHD-inhibitor on the development of OB., Methods: Fully MHC-mismatched C57BL/6 (H-2 b ) donor tracheas were orthotopically transplanted into CBA/J (H-2 k ) recipients. Donor animals received a single dose of PHD-inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40mg/kg i.p.) or vehicle control 4h before transplantation. Transplanted tracheas were harvested 14 or 30days after transplantation and were analyzed by histology, by immunofluorescence and by rtPCR for mRNA expression., Results: Donor pre-conditioning with ICA resulted in HIF accumulation and induction of HIF target genes: HO-1, VEGF, MIF, TGFβ, and EpoR, which persisted during different times of ischemia. Grafts of vehicle treated controls showed substantially more luminal obliteration on postoperative day 30 in contrast to groups pre-treated with ICA [luminal obliteration 29.2±5% (ICA) vs. 36.7±8% (control), p<0.01]. We found significantly lower expression of TNFα, PDGFß, MCP-1, E-selectin, and ICAM-1 14days after ICA premedication. In addition ICA pre-treated groups revealed decreased T-cell and macrophage infiltration in tracheal grafts on days 30 after transplantation (p<0.05)., Conclusion: Pre-treatment with ICA effectively reduced obliterative bronchiolitis. Our data suggest that activation of hypoxia-inducible transcription factors (HIFs) and thereby adaptation to low oxygen prevents the development of OB and allograft injury. Pharmaceutical inhibition of PHDs appears to be an attractive strategy for organ preservation that deserves further clinical evaluation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Objective: Many cellular responses to hypoxia are mediated by the transcription factor complex hypoxia-inducible factor (HIF). HIF stability is governed by a family of dioxygenases called HIF prolyl hydroxylases (PHDs). Isoquinolone-derived PHD inhibitors, like 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA), which stabilize the intracellular HIF-α have been suggested as a potentially beneficial therapeutic strategy for the treatment of disorders associated with ischemia. To stabilize HIF-α, ICA has to be taken up into proximal tubule cells (PCTs) across the basolateral membrane by one of the organic anion transporters 1, 2 or 3 (OAT1, OAT2 or OAT3). The release into the urine across the luminal membrane may be mediated by OAT4., Method: To demonstrate interaction of ICA with human OAT1, OAT2, OAT3 and OAT4, ICA was tested on these transporters stably transfected in HEK293 cells by using p-aminohippurate (PAH), cGMP and estrone-3-sulfate (ES) as reference substrates, respectively., Results: Uptakes of PAH and ES in OAT1- and OAT3-transfected HEK293 cells were inhibited by ICA with half-maximal inhibition values of 0.29 ± 0.05 and 2.58 ± 0.16 µM, respectively. OAT2 was less sensitive to ICA. Efflux experiments identified ICA as an OAT1 and OAT3 substrate. Preloading OAT4-transfected HEK293 cells with ICA stimulated ES uptake by 18.3 ± 3.8%., Conclusion: The uptake of ICA across the basolateral membrane of PCTs occurs mainly by OAT1 and the efflux into the tubular lumen by OAT4., (© 2015 S. Karger AG, Basel.)