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The antimycotic ciclopirox olamine induces HIF-1 stability, VEGF expression, and angiogenesis¹

TOBIAS LINDEN^*, DÖRTHE M. KATSCHINSKI^*,2, KATRIN ECKHARDT, ANNETTE SCHEID, HORST PAGEL^* and ROLAND H. WENGER^*,,3

^* Institute of Physiology, Medical University of Lübeck, Lübeck, Germany;
Carl-Ludwig-Institute of Physiology, University of Leipzig, Leipzig, Germany; and
Department of Surgery, University Children’s Hospital of Zürich and Institute of Physiology, University of Zürich-Irchel, Zürich, Switzerland

³Correspondence: Carl-Ludwig-Institute of Physiology, University of Leipzig, Liebigstrasse 27, D-04103, Leipzig, Germany. E-mail: wenr{at}medizin.uni-leipzig.de

SPECIFIC AIMS

Therapeutic angiogenesis by recombinant angiogenic growth factors or by gene therapy has become an important treatment modality during the past few years. The hypoxia-inducible factor (HIF)-1 is a physiological regulator of vascular endothelial growth factor (VEGF) expression. Results obtained with transgenic mice expressing HIF-1 suggested a better angiogenic response than conditional VEGF overexpression, which is associated with edema, inflammation, and vascular leakage. We previously reported that the antimycotic ciclopirox olamine (CPX) acts as a bidentate iron chelator capable of stabilizing HIF-1. Here, we explored the possibility that CPX activates endogenous HIF-1 target genes, including VEGF, in in vitro cell culture and in vivo organ models.

PRINCIPAL FINDINGS

1. Evidence that CPX can induce angiogenesis in a mouse skin wound model
In early studies addressing the cutaneous effects of a 1% CPX solution applied on rabbit skin over 20 days, occasionally transient reddening of healthy skin and persistent reddening of experimentally wounded skin have been observed. We confirmed these results in experimentally induced wounds in mice. Therefore, punch-through ear holes of 2 mm diameter (a widespread way of marking mice in animal facilities) were treated daily with a commercially available dermal cream containing 1% CPX on one ear and a CPX-free but otherwise identical, common dermal cream on the other ear, respectively. CPX can cause reddening of the wound margin, which was never observed on the healthy skin area of the same ear. In this series, 4 of 10 animals showed similar effects, and none of the animals had more pronounced reddening on the placebo-treated wound margin than on the CPX-treated wound margin. As CPX is known for its anti-inflammatory properties, we suspected this reddening to represent enhanced angiogenesis during wound healing rather than inflammation. Thus, we were interested in whether CPX can cause induction of VEGF.

2. Effects of the iron chelators CPX, deferoxamine (DFX), and 2,2'-dipyridyl (DP) on HIF-1-dependent reporter gene expression and cell proliferation/viability
The effects of CPX on cell proliferation and on HIF-1 induction were compared with two other iron chelators, the hydrophilic DFX and the lipophilic DP, both well-known for their HIF-1-inducing capabilities. Chinese hamster ovary cells were stably transfected with a luciferase reporter gene under the control of the SV40 promoter and six transferrin-derived HIF-1 DNA-binding sites, allowing for the rapid determination of HIF-1 activity. All three iron chelators induced HIF-1-dependent reporter gene expression by 25- to 40-fold under normoxic conditions. It is interesting that whereas CPX was maximally active at 16 µM, the optimal concentrations of DFX and DP were approximately 10-fold higher at 150 µM in this cell line. Exposure to hypoxia did not further increase reporter gene activity at these concentrations. As assessed by parallel 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays, all three iron chelators similarly reduced cell proliferation/viability by 30–40% at concentrations that were below those optimally inducing reporter gene activity. No further decrease of proliferation/viability could then be observed within the range of iron chelator concentrations that induced reporter gene activity.

3. Induction of VEGF gene expression by CPX
We next investigated whether CPX also induces endogenous HIF-1 target genes, including VEGF. Consistent with the reporter gene studies, the same range of CPX concentrations strongly induced the HIF-1 protein in normoxic HepG2 hepatoma cells (Fig. 1 A). Although no HIF-1 protein could be detected in normoxic cells, hypoxia induced HIF-1 with a banding pattern and intensity resembling CPX. The combination of CPX and hypoxia did not further increase HIF-1 levels (Fig. 1A ). As determined by Northern blotting of mRNA derived from HepG2 cells treated with CPX, DFX, and DP, these iron chelators also induced the expression of the endogenous HIF-1 target genes VEGF, Glut-1, and aldolase but not of the control genes L28 and ß-actin (Fig. 1B ). The functionality of CPX-induced VEGF mRNA expression was further demonstrated by the accumulation of VEGF protein in the supernatant of the HepG2 cell culture (Fig. 1C ). Finally, CPX induced a luciferase reporter gene driven by a 1180-bp VEGF promoter fragment following transient transfection of HepG2 cells (Fig. 1D ).

View larger version (64K): [in this window] [in a new window]   Figure 1. Induction of VEGF gene expression by CPX. A) Immunoblot detection of HIF-1 in HepG2 cells treated with the indicated concentrations of CPX for 6 and 24 h under normoxic or hypoxic conditions. B) Northern blot analysis of steady-state mRNA levels in HepG2 cells treated with 10 µM CPX, 100 µM DFX, and 100 µM DP for 24, 48, or 72 h under normoxic or hypoxic conditions. The same blot was serially hybridized with cDNA probes for oxygen-regulated HIF-1 target genes [VEGF, glucose transporter-1 (Glut-1), and aldolase] and nonoxygen-regulated control genes (ribosomal protein L28 and ß-actin). C) Induction of VEGF protein levels in the supernatant of HepG2 cells as measured by enzyme-linked immunosorbent assay. CPX concentrations and incubation times are indicated. Duplicate measurements are shown. D) Induction of VEGF promoter activity in transiently transfected HepG2 cells treated with the indicated concentrations of CPX for 24 h. Luciferase activities were corrected for the values of a cotransfected renilla luciferase expression vector and normalized to the solvent-treated control. A representative example of three independent experiments is shown.

4. Estimation of the iron affinity of CPX
DFX has a very high affinity for Fe³⁺ with a stability constant of 10³¹. Apart from its higher lipophilicity, allowing better penetration of the cytoplasmic membrane, an unusually high iron affinity of CPX might contribute to the efficiency with which CPX can induce HIF-1. We thus estimated the iron affinity of CPX semiquantitatively, by comparing it with the iron affinity of DFX. Therefore, the fluorescence of fluorescein isothiocyanate-labeled DFX was partially quenched by adding increasing amounts of FeCl₃. Following addition of a three molar excess of CPX (1 mole of Fe³⁺ is chelated by 1 mole of DFX and 3 moles of CPX, respectively), the iron-dependent quenching of the fluorescence could be inhibited until an excess of iron was added. These data suggest that CPX binds iron with an even higher affinity than DFX.

5. Lack of HIF-1 target gene induction by CPX in the isolated, perfused rat kidney
It has been reported previously that systemically administered DFX can induce ubiquitous HIF-1 stability and kidney-specific erythropoietin expression in mice. Regarding a potential, topical application of CPX for therapeutic angiogenesis, it is conceivable that a fraction of the applied CPX might reach the bloodstream. To test the effects of CPX delivered by the bloodstream, isolated rat kidneys were perfused for 3 h under normoxic conditions with increasing concentrations of CPX. However, CPX did not induce the mRNA levels of the HIF-1 target genes VEGF, Glut-1, or aldolase.

6. Induction of angiogenesis in the chicken chorioallantoic membrane (CAM) by CPX
Based on our observations that CPX induced VEGF expression in cell culture, we analyzed its angiogenic capacity in an in vivo model of angiogenesis. Therefore, the chicken CAM at day 9 of development was overlaid with inert polymer discs containing various concentrations of CPX or solvent only. After 2 days of incubation, no signs of angiogenesis could be observed with the solvent control discs. In contrast, polymer discs containing 50 mM CPX consistently induced CAM angiogenesis, as evidenced by numerous, newly formed, radially arranged vessels (Fig. 2 ). A total of 10 CAMs treated with 50 mM CPX showed similar angiogenesis, and all 7 control CAMs lacked any signs of angiogenesis. CPX (5 mM) was indistinguishable from the controls, and 500 mM CPX was toxic to the chick embryo. Of note, the 1% CPX concentration in dermal cream preparations equals a molar concentration of 37 mM, which is close to the concentration found to be angiogenic in these CAM assays.

View larger version (80K): [in this window] [in a new window]   Figure 2. Schematic illustration of CPX function. By inhibiting the activity of the oxygen-sensing prolyl-hydroxylase-domain proteins (PHDs), CPX functionally stabilizes HIF-1, leading to a transcriptional up-regulation of VEGF and probably other angiogenic factors. The photomicrograph shows a CAM angiogenesis assay. Arrowheads denote some of the newly formed vessels radially arranged around an inert polymer disc containing CPX. CBP, cAMP-response element-binding protein (CREB)-binding protein; HBS, HIF-1 DNA-binding site.

CONCLUSIONS AND SIGNIFICANCE

HIF-1 gene therapy might be superior to VEGF gene therapy when applied for therapeutic angiogenesis, e.g., during wound healing. However, regarding safety, costs, and ease of application, a pharmaceutical approach would be beneficial compared with gene therapy. Our data demonstrated that CPX functionally activates HIF-1 and induces VEGF transcription, mRNA, and protein levels as well as angiogenesis (Fig. 2) . Wound healing in the skin may be complicated by microbial invasion, inflammation, and ischemia, leading to ulceration. As CPX is lipophilic, antimicrobiotic, anti-inflammatory, angiogenic, and not affecting healthy tissue, it might be beneficial for the topical treatment of skin wound tissues. Wenk and colleagues have also investigated the concept of iron chelation as a treatment modality in chronic wound healing. This group demonstrated that iron levels in exudates of chronic wounds are significantly increased when compared with wound fluids from acute wounds. Iron reacts with neutrophil-derived peroxide to produce the highly toxic hydroxyl radical (Fenton reaction), potentiating tissue damage and thus, iron release and continuous neutrophil infiltration leading to chronic wound disease, such as chronic venous leg ulcers. To interrupt this chronic inflammation, Wenk and colleagues developed a DFX-coupled wound dressing and demonstrated a reduction in iron concentration, matrix metalloproteinase-1 content, and lipid peroxidation. Our results suggest similar effects of CPX but with the additional benefits of ease of application, antimicrobial properties, and hypoxia adaptation, including angiogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0586fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0586fje

² Current address: Cell Physiology Group, Medical Faculty, Martin-Luther-University Halle, D-06112 Halle, Germany

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