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RAPID REPORT |
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Department of Physiology, University of Birmingham, Birmingham B15 2TT, UK
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Henry Wellcome Building of Molecular Physiology, Oxford, OX3 9 DU, UK
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Abstract |
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protein level, HIF-dependent gene expression and capillary-to-fibre ratio (C: F) in extensor digitorum longus and tibialis anterior muscles of mice. Immunohistochemical examination revealed that HIF-1 is present in non-ischaemic mouse skeletal muscles, but its amount increased profoundly in response to the combination of DMOG treatment and ischaemia. Combined treatment resulted in 39% increase in C: F in ischaemic muscles (P < 0.0001 versus controls) whereas individual treatments produced little effect under our conditions. Combined treatment led to a significant increase in endogenous HIF-1 protein (6.14 ± 1.1 versus 1.17 ± 0.2 in controls; P < 0.05) that was not apparent in mice treated with DMOG or femoral artery ligation alone. Ischaemia increased vascular endothelial growth factor (VEGF) protein production by 2.5-fold (P < 0.05 versus controls), irrespective of DMOG treatment. However, production of the VEGF receptor Flk-1 was more enhanced in ischaemic + DMOG-treated muscles (P < 0.001 and P < 0.05 compared with controls and untreated ischaemic muscles, respectively), which may explain the intensive growth of capillaries in those muscles. The findings indicate that treatment with DMOG has a potential therapeutic use in promoting angiogenesis in ischaemic diseases, and perhaps for improving muscle recovery after injury, exercise or training.
(Received 8 June 2004;
accepted after revision 13 August 2004;
first published online 19 August 2004)
Corresponding author S. Egginton: Department of Physiology, University of Birmingham, Birmingham B15 2TT, UK. Email: s.egginton{at}bham.ac.uk
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Introduction |
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More recently, attention was drawn to hypoxia inducible factor-1 (HIF) as regulator of a wide variety of oxygen-regulated genes including those controlling vascular growth such as VEGF, endothelial nitric oxide synthase (eNOS) and angiopoietin-2 (Semenza, 2001). HIF is a heterodimer of inducible and constitutively expressed ß basic helix–loop–helix (bHLH) PAS proteins. Activity of HIF is markedly increased when the intracellular oxygen tension falls, resulting in transactivation of genes containing a hypoxia response element (HRE). HIF activity is regulated by enzymatic oxygen-dependent hydroxylation of two specific prolyl residues and one critical asparaginyl residue by the oxoglutarate-dependent dioxygenases PHD 1–3 and a protein termed factor inhibiting HIF (FIH). Prolyl hydroxylation results in von Hippel-Lindau complex-mediated ubiquitylation of HIF and consequent degradation by the proteasome. Similarly, asparaginyl hydroxylation inhibits CBP/p300 coactivator recruitment by HIF chains (Bruick & McKnight, 2002).
Since HIF is a master regulator of tissue oxygen homeostasis, the modulation of its activity via pharmacological and DNA-based approaches has a potential therapeutic effect. HIF-1 gene therapy enhanced capillarization in both rabbit hindlimb ischaemic and rat myocardial infarction models, improving regional blood flow in ischaemic legs and reducing the infarct size (Vincent et al. 2000; Shyu et al. 2002). However, the systemic effects of these therapies have not been fully investigated. An alternative to gene therapy is promotion of HIF activity by administration of polypeptides that either interact with the proteasome (Li et al. 2000) or compete with VHL-binding/prolyl hydroxylation (Willam et al. 2002). Characterization of the hydroxylase family involved in normoxic HIF inactivation has led to the development of inhibitors capable of HIF activation (Mole et al. 2003; Schlemminger et al. 2003). One example is DMOG, a cell penetrant oxoglutarate analogue, expected to inhibit all enzymes of the oxoglutarate-dependent dioxygenase class, including collagen prolyl hydroxylases, PHD 1–3 and FIH. Experiments in tissue culture cells have shown that this molecule induces stabilization of both HIF-1 and HIF-2 proteins (Jaakkola et al. 2001), and also induces downstream gene expression. Toxicity has not been manifest, even when DMOG is used in the millimolar range, which is perhaps surprising given the importance of oxoglutarate in intermediary metabolism. Other HIF hydroxylase inhibitors have also been shown to affect the HIF system (Warnecke et al. 2003). However, there is a lack of data on the effects of these compounds on vascular growth induced after ischaemia, relevant to the use of this kind of inhibitor in clinical practice. Given the efficacy, and low toxicity, of DMOG in vitro, the aim of this study was to investigate whether this compound can activate endogenous HIF protein in vivo and thus promote angiogenesis in a mouse model of ischaemia.
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Methods |
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Two groups of mice (C57Bl6) were subjected to unilateral femoral artery ligation under fluothane inhalation anaesthesia (2% in O2). One group (n = 11) received dimethyloxalylglycine (DMOG) I.P. (8 mg in 0.5 ml saline) on days 1, 3, 5, 7 and 9 while the animals in the other group were injected with sterile saline (0.5 ml) at the same intervals (n = 6). A third group was treated with DMOG without ligation (n = 4) and four unoperated mice served as controls. After 11 days mice were terminally anaesthetized and the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles excised.
Immunohistochemistry
Vascularity was evaluated by estimation of the capillary-to-fibre ratio (C: F) based on alkaline phosphatase staining (Fast BCIP/NBT Tablets, Sigma, UK). An eyepiece graticule was used to count number of capillaries and fibres in an unbiased manner to estimate C: F, capillary density (CD) and fibre density (FD). For HIF-1 immunodetection the 6 µm cryosections of TA muscle were fixed in ethanol: acetone (1: 1). After blocking in a 10% normal horse serum sections were incubated with mouse monoclonal anti-HIF-1 for 3 h (1: 500; NOVUS Biologicals). Following a triple washing in 1% Tween in Tris-Buffered saline a biotinylated rabbit anti-mouse Ig (1: 200, Vector UK) was applied. Reactions were visualized using Vector Laboratories ABC Vectastain and DAB kits (Vector, UK).
Western blot analysis
The excised EDL muscles were homogenized in ice-cold buffer (8 M urea, 1/10 v/v of glycerol, 1/20 v/v of 20% SDS, 1/200 v/v of 1 MDTT, 1/100 v/v of 0.5 M Tris, pH 6.8) containing a protease inhibitor cocktail (Complete Mini, Roche) prior to PAGE and transfer onto PVDF membranes (Amersham, UK). Membranes were incubated with either mouse monoclonal anti-HIF-1 antibody (1: 500, NOVUS Biologicals) or rabbit polyclonal antibodies: anti-VEGF (1: 500, Santa Cruz), anti-Flk-1 (1: 250, Santa Cruz) or anti-ß-actin (1: 2000, Santa Cruz). Bound antibody was visualized with species-specific horseradish peroxidase-conjugated secondary antibody (Amersham, UK) and chemiluminescence system (Pierce). Signals were quantified using a Gel Doc 2000 scanner with Quantity One software (Bio-Rad, UK). The densities of bands were normalized to ß-actin.
Statistical analysis
Data are presented as means ± S.E.M., with samples from a minimum of four mice included in each experiment. C: F, CD, FD and protein levels were analysed using ANOVA with Fisher's PLSD post hoc test. Results were considered statistically significant at the 5% level.
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Results |
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To assess the effects of DMOG in a mouse model of hindlimb ischaemia HIF-1 protein was visualized in mouse tibialis anterior muscles by immunohistochemistry. In both control and DMOG-treated muscles without ligation a very faint signal was present (Fig. 1A and B). In ischaemic muscles treated with DMOG, there was widespread and strong nuclear staining of fibres and cells within the interstitium (Figs 1C and D), which was much more intense than in ischaemic muscles without DMOG treatment.
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protein and led to a 2.5-fold increase in its abundance (P < 0.05 versus control). Moreover, DMOG treatment elevated its abundance in ischaemic muscles to a greater extent, 2-fold and 5.5-fold increase in comparison to ischaemic and control muscles, respectively (both P < 0.05; Fig. 2A and B). The compound did not affect HIF-1 levels in muscles from non-operated mice.
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The activation of HIF target genes that are involved in angiogenesis was also analysed. DMOG treatment did not affect VEGF levels in muscles without arterial ligation (1.03 ± 0.02) compared with the control value of 0.98 ± 0.02 (Fig. 2A and C). However, the amount of VEGF protein present in response to femoral artery ligation increased significantly from control levels by 150% (P < 0.05). In ischaemic muscles treated with DMOG, VEGF levels increased to a similar extent. Muscle ischaemia led to enhanced production of one of the VEGF receptors, Flk-1 (2.5-fold increase versus controls, P < 0.05) but its expression in ischaemic muscles was further enhanced in response to DMOG treatment (greater than 40% increase compared with ischaemic muscles, P < 0.05; Fig. 2A and C). DMOG did not affect Flk-1 expression in animals without femoral artery ligation (Fig. 2A and C).
Angiogenesis
Eleven days after unilateral ligation of the femoral artery an increase in fibre density was noted, as expected from known effects of ischaemia on fibre size. Interestingly, although DMOG treatment in isolation was associated with an increase in FD the reduction in fibre size associated with ischaemia was partially ameliorated by DMOG treatment (Table 1). Any reduction in fibre size would lead to muscle shrinkage; if there was no capillary growth, capillary-to-fibre ratio (C: F) would be preserved (but not increased) whereas there would be an increase in capillary density (because less volume is occupied by muscle fibres). Unilateral ligation of the femoral artery or DMOG treatment alone caused no significant change in C: F. However, in DMOG-treated ischaemic muscles C: F was almost 40% higher than in controls (Table 1). Combined with increased FD, this implies a real increase in absolute capillary number, or perhaps tortuosity, either of which implies growth of capillaries.
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Discussion |
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An important challenge for therapeutic angiogenesis is the need to target the angiogenic response to ischaemic areas. One approach is local delivery of a therapeutic small molecule. Previous studies have demonstrated positive local effects of HIF degradation blockade on blood vessel growth in vivo in the artificial circumstances of polyethylene sponge assays (Willam et al. 2002; Warnecke et al. 2003). Local delivery to an ischaemic tissue is often technically demanding, requiring angiographically based delivery. Systemic administration is generally much simpler but would be expected to under supply the ischaemic area, which is by definition hypoperfused. In this study we demonstrate the bioavailability and physiological effectiveness of DMOG in a mouse model of hindlimb ischaemia.
We chose to study effects at an early time point in this work since angiogenesis is known to occur more rapidly in the mouse than the rat (Williams & Egginton, 2002). We used an alternate day dosing protocol because autoregulatory loops in the HIF system might mean that continuous treatment would be less effective than intermittent treatment, and it reduced stress for the animals.
HIF protein
Activity of DMOG was first assessed by staining and immunoblotting for HIF-1 protein in hindlimb skeletal muscle extracts. HIF-1 protein was detectable in normoxic mouse skeletal muscle, in agreement with a previous report (Stroka et al. 2001), suggesting that HIF-1 is necessary for a basal expression of genes responsible for cell homeostasis. In animals solely treated with DMOG our protocol resulted in HIF levels that were indistinguishable from control animals. Ischaemia induced by femoral artery ligation resulted in a small increase in level of the HIF-1 protein at the time point examined. Despite variability between animals the combination of DMOG treatment and femoral artery ligation produced a significant increase in HIF-1 protein, suggesting that in these circumstances DMOG is capable of inhibiting the relevant HIF prolyl hydroxylases and thereby stabilizing HIF-1 chains in vivo, although other modes of action are possible. The combined effect of DMOG and ischaemia was larger than that produced by the stimuli in isolation, suggesting a synergistic effect.
Intriguingly, we noticed a small increase in HIF-1 protein in the non-ischaemic, contralateral leg in mice treated with DMOG (data not shown), which contrasts with the lack of effect of DMOG in mice not subjected to an ischaemic insult. This may be due to the animals favouring the contralateral legs during locomotion after surgery, either producing relative local hypoxia or stretch-induced up-regulation of HIF, as has been demonstrated in cultured vascular smooth muscle cells and myocytes (Kim et al. 2002).
Angiogenesis
In addition to stabilizing HIF-1, the combination of ischaemia and DMOG treatment induced a significant effect on capillary supply in the muscles. Since gene therapy experiments expressing artificially activated HIF proteins (Vincent et al. 2000; Shyu et al. 2002) also induce angiogenesis it is likely that the DMOG-induced change in capillary density is HIF mediated. To explore directly what factors induce capillary growth under these conditions we examined the product of a candidate gene that is activated by HIF, VEGF, and its cognate receptor Flk-1. We observed a similar increase in VEGF protein level in both ischaemic EDL and in ischaemia + DMOG-treated muscles, yet C: F and CD were augmented only in the latter case, showing that activation of the VEGF gene alone did not determine the angiogenic outcome. Angiogenesis was successfully promoted only in experimental groups where both Flk-1 and HIF-1 proteins were augmented, suggesting the existence of a complex regulatory network. A similar observation was reported in rat ischaemic muscle where, even in the presence of increased VEGF protein, an enhanced level of Flk-1 was necessary for angiogenesis (Milkiewicz et al. 2003). VEGF receptor expression has been shown to induce endothelial cell proliferation in vitro (Takahashi & Shibuya, 1997), and in rat ischaemic muscles increased Flk-1 protein levels correlated with enhanced cell proliferation at the site of capillaries (Milkiewicz et al. 2003).
Endothelial cell-specific expression of Flk-1 is thought to be tightly regulated during development and crucial for initiation of vascular growth (Carmeliet, 2003). To our knowledge, there are no reports on activation of Flk-1 expression by HIF-1, but it has been reported to be activated by HIF-2 (Elvert et al. 2003). Unfortunately, we could not evaluate the level of HIF-2 in the samples from our experiments due to the lack of a reliable antibody to murine HIF-2. Nevertheless, based on the efficiency of DMOG in activation of both human HIF-1 and -2 it is likely that HIF-2 activity would also be increased in the ischaemic regions of mice after DMOG treatment.
Other responses
HIF-1 up-regulates expression of a number of genes that are involved not only in angiogenesis but in cellular metabolic adaptation to hypoxia such as erythropoiesis and glycolysis (De Muinck & Simons, 2004). It is to be anticipated that inducing such metabolic adaptation using a HIF hydroxylase inhibitor will synergise with the induction of angiogenesis in improving the functional outcome. This was not tested directly in this initial study, although the effect of DMOG in reducing the ischaemia-induced changes in fibre density are in keeping with this hypothesis. The relative roles of HIF-1 and HIF-2 in contributing to these responses and the relative importance of the different hydroxylases in determining HIF-1 and HIF-2 activity is a subject of active study. It will be interesting to dissect these phenomena further as inhibitors with specificity for individual hydroxylases become available.
Conclusions
The results reported here demonstrate HIF activation and concomitant capillary growth targeted to ischaemic muscles following systemic administration of a small molecule oxoglutarate-dependent dioxygenase inhibitor. This is an encouraging step towards achieving therapeutic angiogenesis in tissue otherwise refractory to capillary growth.
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References |
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