Up-Regulation of Vascular Endothelial Growth Factor in Stromal Cells of Hemangioblastomas Is Correlated with Up-Regulation of the Transcription Factor HRF/HIF-2α

Hemangioblastomas, the most frequent manifestation of the hereditary von Hippel-Lindau disease (VHL), are highly vascularized tumors of the central nervous system. In previous studies, the endothelial-specific mitogen vascular endothelial growth factor (VEGF) was shown to be up-regulated in the stromal cells, the putative neoplastic cells in hemangioblastomas. Therefore, it was suggested that secretion of VEGF by stromal cells is the pathogenetic cause of the vascular lesions in hemangioblastomas. The novel basic helix loop helix transcription factor HRF/HIF-2α is a candidate regulator of VEGF expression during development. We therefore investigated expression of HRF/HIF-2α in hemangioblastomas and found the overexpression of VEGF mRNA in stromal cells to be highly correlated with elevated expression levels of HRF/HIF-2α mRNA. This finding is suggestive for a role of HRF in VEGF-dependent vascular growth in hemangioblastomas and could provide a link between transcriptional activation of the VEGF gene and loss of function of the VHL gene product. (Am J Pathol 1998, 153:25–29

FGF23 expression in rodents is directly induced via erythropoietin after inhibition of hypoxia inducible factor proline hydroxylase.

Plasma levels of FGF23 are increased in patients with chronic kidney disease. Beside its role in phosphate homeostasis, iron deficiency and anemia are associated with increased FGF23 plasma levels. Recently, FGF23 plasma levels were shown to be increased in mice after treatment with hypoxia inducible factor-proline hydroxylase (HIF-PH) inhibitors which are strong inducers of erythropoietin and erythropoiesis and are known to modulate iron uptake and availability. Therefore we investigated a potential context between expression of FGF23 and stimulation of erythropoiesis using a HIF-PH inhibitor and erythropoietin in rats. FGF23 plasma levels are induced at peak levels 2 h after intravenous injection of recombinant human Erythropoietin (rhEPO). Likewise induction of endogenous EPO using a HIF-PH inhibitor (BAY 85-3934) is followed by an increase of FGF23 plasma levels. In contrast to rhEPO the HIF-PH inhibitor induces lower peak levels of FGF23 applying equivalent hematopoietic doses. Bone and bone marrow were identified as sources of EPO-induced FGF23. Immediate induction of FGF23 mRNA was also detected in EPO receptor positive murine hematopoietic BAF3 cells after treatment with rhEPO but not after treatment with the HIF-PH inhibitor. Pretreatment of mice with a neutralizing anti-EPO antibody abrogated FGF23 induction by the HIF-PH inhibitor. Thus, direct impact on FGF23 expression by HIF-PH inhibition in vivo via hypoxia mimicking and modulation of iron metabolism appears unlikely. Collectively, the findings point to an EPO dependent regulation pathway of FGF23 gene expression which might be important in the context of erythropoiesis stimulating therapies in patients with renal anemia

Mimicking hypoxia to treat anemia: HIF-stabilizer BAY 85-3934 (Molidustat) stimulates erythropoietin production without hypertensive effects.

Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin-angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia

Effects of treatment with rhEPO and HIF-PH inhibitor on FGF23 plasma protein levels and mRNA expression in rats.

<p>(A) packed cell volume (PCV) in rats 9 days after once-daily dosing with ascending doses of BAY 85–3934 sodium (mg/kg as indicated) or after twice weekly dosing with 100 IU/kg rhEPO (EPO) s.c. (n = 6 animals per group; p<0.001 for all groups versus control). (B) levels of plasma EPO 6 h after first administration. (C) cFGF23 plasma levels 6 h and 24 h (D) after first dose and iFGF23 plasma levels 6 h (E) and 24 h (F) after first dose in the same animals as shown in A. (G) relative expression of FGF23 mRNA in the bone marrow and in the kidney (H) *p<0.05, **p<0.01 and ***p<0.001; t-test versus vehicle group.</p

Induction of FGF23 by EPO is not influenced by expansion of erythropoietic cell mass.

<p>Effects of single dose (1x) and twice weekly repeated dose administration for two weeks (5x) of 100 iU/kg rhEPO s.c. on erythropoiesis parameters and FGF23 mRNA expression in Wistar rats 4 h after last rhEPO administration. Experimental setup for treatment groups is shown in (A). Sham, control animals treated with saline only. (B) plasma levels of rhEPO 4 h after administration; (C) packed cell volume; (D) spleen weight; and (E) relative expression of EPO-receptor mRNA and FGF23 mRNA (F) in bone, bone marrow and spleen. **p<0.01, ***p<0.001; t-test (E,F, Mann-Whitney test) versus corresponding sham control group; nd, expression not detectable (CT>35).</p

Oligonucleotide primers and probes used for quantitative RT-PCR analysis of samples from rat and mouse tissues and murine cells.

<p>Oligonucleotide primers and probes used for quantitative RT-PCR analysis of samples from rat and mouse tissues and murine cells.</p