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Activation of the hypoxia-inducible factor-pathway and stimulation of angiogenesis by application of prolyl hydroxylase inhibitors¹

CHRISTINA WARNECKE^*, WANJA GRIETHE^*,, ALEXANDER WEIDEMANN^*, JAN STEFFEN JÜRGENSEN¹, CARSTEN WILLAM¹, SEBASTIAN BACHMANN, YURI IVASHCHENKO, INGRID WAGNER, ULRICH FREI^*, MICHAEL WIESENER^* and KAI-UWE ECKARDT^*,1,2

^* Department of Nephrology and Medical Intensive Care and
Department of Anatomy, University Clinic Charité, Humboldt University Berlin, 13353 Berlin, Germany; and
Department of Cardiovascular Diseases, Aventis Pharma Deutschland GmbH, Industriepark Hoechst, H 825, 65926 Frankfurt am Main, Germany

²Correspondence: Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow Clinic, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: kai-uwe.eckardt{at}charite.de

SPECIFIC AIMS

The aim of this study was to investigate, whether protein hydroxylase inhibitors can be used to stabilize hypoxia-inducible factor (HIF) and to enhance HIF activity and its downstream effects, including the induction of angiogenesis, in vitro and in vivo. For this purpose we tested three known (procollagen) prolyl 4-hydroxylase inhibitors [L-mimosine (L-Mim), ethyl 3,4 dihydroxybenzoate (3,4 DHB) and 6-chlor-3-hydroxychinolin-2-carbonic acid–N-carboxymethylamid (S956711)] for their capability to inhibit the HIF/von Hippel-Lindau protein (pVHL) interaction, to increase HIF protein levels and to induce HIF target genes in cultured cells and in rat tissues, as well as for their effect on angiogenesis in a rat sponge model.

PRINCIPAL FINDINGS

1. Inhibition of HIF-1/pVHL interaction in vitro
All three compounds distinctly inhibited pVHL/HIF-1 interaction in a coimmunoprecipitation assay with ³⁵S-labeled in vitro transcribed and translated proteins when present during the HIF translation reaction (Fig. 1) . In the case of L-Mim and 3,4 DHB, this inhibition could be reversed by addition of FeCl₂, whereas the effect of S956711 was completely iron-independent and could be competed by 2-oxoglutarate.

View larger version (34K): [in this window] [in a new window]   Figure 1. Inhibition of the HIF-1/pVHL interaction by L-Mim, 3,4 DHB, and S956711 in vitro. A) Coimmunoprecipitation assays with radiolabeled HIF and pVHL. HIF was translated in vitro in the presence (+) or absence (–) of the inhibitors S956711 (S95), L-Mim, and 3,4 DHB and 100 µM FeCl2. Inhibitors were competed by further addition of FeCl2 (Fe2+) to a final concentration of 200 µM (2nd columns) or by addition of 5 mM 2-oxoglutarate (2-oxo; 3rd columns) to the HIF in vitro transcription/translation reaction (IVTT). Due to an internal translation start site, pVHL is expressed in two isoforms (p32/HA, p19/HA, HA=hemagglutinin tag). B) To demonstrate that the inhibitors had not impaired the IVTT, equal amounts of the HIF IVTTs were loaded on a separate gel.

2. Stabilization of HIF protein in cell cultures
L-Mim, 3,4 DHB, and S956711 induced HIF-1 and -2 protein under normoxic conditions in human fibrosarcoma HT1080, rat pheochromocytoma PC12W and mouse fibroblastoid 3T3L1 cells as demonstrated by immunoblot assays. The lowest effective concentrations were 100 µM for L-Mim and 3,4 DHB and 10 µM for S956711. At higher concentrations (800 µM L-Mim and 3,4 DHB, and 200 µM S95671, respectively) the effect equalled that of severe hypoxia (0.5% O₂). In agreement with the results of the coimmunoprecipitation assay, competition with iron eliminated the effect of L-Mim and 3,4 DHB, but not of S956711.

3. Induction of HIF target genes in cultured cells
RNase protection assays revealed that L-Mim, S956711, and (with lower efficiency) 3,4 DHB induced HIF target genes including glucose transporter 1, vascular endothelial growth factor, lactate dehydrogenase A, and carbonic anhydrase IX in PC12W, 3T3L1 and HT1080 cells, respectively. At high concentrations of L-Mim, target gene mRNA levels equalled or even exceeded those induced by hypoxia (0.5% O₂).

4. Stimulation of HIF-dependent luciferase activity
HIF-controlled luciferase expression was induced by the three compounds in HT1080 cells transiently transfected with a reporter construct containing six copies of a hypoxia-responsive element. L-Mim (800 µM) and S956711 (50 µM) increased luciferase activity 8.9- and 8.3-fold, respectively, 3,4 DHB 4.8-fold and hypoxia (0.5% O₂) 8.7-fold above control levels.

5. Anti-proliferative and cytotoxic side effects
Cell proliferation and release of lactate dehydrogenase (LDH) in the cell culture supernatant were monitored to detect potential antiproliferative and cytotoxic side effects of the inhibitors. Exposure of HT1080 cells to L-Mim for 72 h decreased proliferation by 27% (400 µM) as determined by a methyl tetrazolium salt (MTS) assay. Higher concentrations did not lead to a further decrease, indicating a saturation effect. 3,4 DHB decreased cell growth progressively by up to 54% (1000 µM) and S956711 by up to 23.7% (200 µM). L-Mim and 3,4 DHB incubation for 24 h led to a maximum LDH release of 5% (L-Mim) and 7.2% (3,4 DHB) compared with unstimulated controls, whereas S956711 had no significant effect up to a concentration of 200 µM.

6. Effects of the inhibitors after systemic administration in rats
To determine the efficiency of the compounds, in vivo single doses of 3,4 DHB (36 mg/200 g body weight) and S956711 (5.6 mg/200 g) were injected intraperitoneally in Sprague Dawley rats (n=3 for each group). L-Mim (120 mg/200 g) was administered as a suspension by oral gavage because of its limited solubility. After 6 h, rats were killed and brains, hearts, livers, and kidneys were removed and fixed. Immunohistochemistry was performed for HIF-1, HIF-2, and the HIF target gene heme oxygenase 1 (HO-1). Positive nuclear HIF-1 staining was detected in the kidneys of L-Mim- and S956711-treated rats, but not in the kidneys of 3,4 DHB- or vehicle-treated rats or any other organ under investigation. In the kidneys, HIF-1 protein expression was confined to tubular cells of the outer medulla. Nuclear HIF-2 signals were identified in interstitial cells of the same area but were scarce compared with HIF-1 signals. In spatial and quantitative association with HIF-1 accumulation, HO-1 protein expression was up-regulated in tubular cells of the outer medulla.

7. Stimulation of angiogenesis by the inhibitors
In a rat sponge model for angiogenesis, repeated local injection of L-Mim, 3,4 DHB or S956711 into s.c. implanted polyurethane sponges strongly increased invasion of highly vascularized fibrous tissue into the sponge centers as determined by hematoxylin-eosin (HE) staining and immunohistochemistry for the endothelial cell marker CD31 (Fig. 2)

View larger version (75K): [in this window] [in a new window]   Figure 2. Stimulation of angiogenesis by L-Mim, 3,4 DHB, and S956711 in the rat sponge model. Polyurethane sponges were implanted s.c. in rats and inhibitors were injected into the sponges four times every other day; 1st row: HE-stained cross sections of the sponges illustrating marked differences in the extent of tissue invasion. To assess the degree of vascularization, sections were stained for the endothelial cell marker CD31, using HRP- (2nd row) or fluorescence- (3rd row) coupled secondary antibodies; 2nd row: high-power views of areas in the center of each sponge (corresponding to black rectangles in the 1st row) illustrating that the inhibitors induced deep vascular invasion into the sponges. To estimate the degree of vascularization in the zone between the dense granulation tissue at the sponge margin and the center, CD31 staining in four rectangles of similar distance from the sponge margin (corresponding to green rectangles in the 1st row) was assessed using immunofluorescence (IF); 3rd row: representative high-power views of these areas. Columns indicate mean total fluorescence of all four rectangles related to controls.

Even at the transition of the granulation tissue, which had invaded the outer margin of all sponges, to the sponge centers, microvessel density was three- to fourfold higher in sponges injected with the inhibitors than in controls.

CONCLUSIONS AND SIGNIFICANCE

We demonstrate in this study that three structurally different inhibitors of prolyl 4-hydroxylase are capable of inducing HIF and HIF target genes in vitro and in vivo, thereby providing indirect proof in vivo of the concept of HIF regulation by oxygen-dependent prolyl hydroxylation (Fig. 3 ). On the other hand, it shows the potential of protein hydroxylase inhibitors as pharmacological activators of the HIF pathway and as therapeutic tools in treating ischemic diseases.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram of HIF regulation by enzymatic hydroxylation and proposed mechanism and consequences of protein hydroxylase inhibition. Similar to oxygen deprivation, inhibition of HIF prolyl hydroxylases (PHD) prevents HIF hydroxylation and thereby interaction with pVHL and proteasomal degradation. See also the structure of the catalytic center of PHD1, including cosubstrates 2-oxoglutarate and molecular oxygen, the cofactor iron, and chemical structure of the inhibitors used. ODD, oxygen-dependent degradation domain; TAD, transactivation domain.

The selective induction of HIF protein and the HIF target gene HO-1 in the outer medulla of the kidney after systemic application presumably reflects compound accumulation rather than confinement of the effect to specific cell types. Thus, application protocols need to be developed to enhance HIF-mediated gene expression in other tissues. The results in the rat sponge model illustrate that local application also has a great therapeutic potential.

Despite similar downstream effects, the molecular modes of action of the three inhibitors appeared to be different. L-Mim and 3,4 DHB acted primarily through iron chelation. In contrast, the effect of the structurally more complex S956711 was independent of iron but could be competed by excess 2-oxoglutarate in the HIF/pVHL interaction assay. S956711 activated HIF at markedly lower concentrations, indicating a higher potency and specificity for HIF hydroxylases compared with L-Mim and 3,4 DHB. The molecular basis of these different properties of the inhibitors remains to be clarified. All three compounds are known inhibitors of prolyl 4-hydroxylase and in the present work reduced proliferation of HT1080 cells. In general, when using compounds with residual activities on other cellular dioxygenases, side effects on extracellular collagen metabolism and growth in vivo have to be taken into account. Although we observed only little toxicity under the short-term conditions, development of inhibitors that are highly specific for HIF prolyl and asparagyl hydroxylation will facilitate their use in clinical practice.

While approaches to induce angiogenesis in vivo have so far focused on overexpression of single genes or application of their products, activation of the HIF system has the advantage of activating multiple genes that are (patho)physiologically activated by hypoxia in a coordinated fashion. This is likely to result in greater efficiency as well as in functional benefits for the newly formed vasculature.

In conclusion, we provide evidence for a significant biological effect of HIF activation in vivo by inhibitors of prolyl hydroxylases, and propose S956711 as a model compound for further development of PHD-specific inhibitors. Within the emerging array of complementary molecular tools for the treatment of ischemic diseases, pharmaceutical activators of the HIF system such as PHD inhibitors will probably play a pivotal role.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1062fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-1062fje

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