HIF and VEGF relationships in response to hypoxia and sciatic nerve stimulation in rat gastrocnemius

https://doi.org/10.1016/j.resp.2004.04.009Get rights and content

Abstract

To determine if hypoxia-inducible factor-1 (HIF-1) may regulate skeletal muscle vascular endothelial growth factor (VEGF) expression in response to exercise or hypoxia, rats underwent 1 h sciatic nerve electrical stimulation (ES), hypoxic exposure (H) or combined stimuli. HIF-1α protein levels increased six-fold with maximal (8 V) ES with or without H. Similar HIF-1α increases occurred with sub-maximal (6 V and 4 V) ES plus H, but not in sub-maximal ES or H alone. VEGF mRNA and protein levels increased three-fold in sub-maximal ES or H alone, six-fold in sub-maximal ES plus H, 6.3-fold with maximal ES, and 6.5-fold after maximal ES plus H. These data suggest: (1) intracellular hypoxia during normoxic exercise may exceed that during 8% oxygen breathing at rest and is more effective in stimulating HIF-1α; (2) HIF-1 may be an important regulator of exercise-induced VEGF transcription; and (3) breathing 8% O2 does not alter HIF-1α expression in skeletal muscle, implying that exercise-generated signals contribute to the regulation of HIF-1α and/or VEGF.

Introduction

In skeletal muscle, vascular endothelial growth factor (VEGF) has been found to be the most actively expressed angiogenic growth factor in response to exercise (Amaral et al., 2001, Breen et al., 1996, Gustafsson et al., 1999, Lloyd et al., 2003, Richardson et al., 1999). Furthermore, the musculature requires VEGF expression for the maintenance of capillary structures (Tang et al., 2004). We previously reported that rat skeletal muscle VEGF mRNA levels increase: (a) immediately just after a single bout of sub-maximal exercise in normoxia, (b) in resting rats kept hypoxic (12% inspired O2), and (c) to a higher level in rats that exercised under hypoxic stress (Breen et al., 1996). Similar studies with human subjects reported, elevated VEGF expression following an acute exercise bout that was not further increased by limiting oxygen supply (Gustafsson et al., 1999, Richardson et al., 1999).

One of the main signals generated during an acute exercise bout is a fall in intracellular PO2 (Richardson et al., 1995). VEGF is well known to be a hypoxia-responsive gene and the conserved consensus sequence for the hypoxic response element (HRE) is present in the VEGF promoter (Forsythe et al., 1996). The hypoxic inducible factor-1 (HIF-1), which recognizes the highly conserved HRE, is a heterodimer consisting of both α and β subunits (Wang et al., 1995), and HIF-1 transctivation regulates many hypoxic-responsive genes involved in glycogenesis, erythropoeisis, and angiogenesis. While the HIF-1β subunit is constitutively expressed, the oxygen-dependent HIF-1α subunit is not regulated at the mRNA level but through the rapid stabilization/destabilization of HIF-1α protein (Jewell et al., 2001). Thus, upon exposure to an hypoxic/anoxic gas mixture, the HIF-1 complex is instantaneously stabilized resulting in cellular HIF-1 accumulation, HIF-1–DNA binding and transactivation of target genes containing the HRE (Hofer et al., 2002). Upon re-exposure of intact cells in vitro or in vivo to 21% oxygen, the HIF-1α subunit is rapidly degraded via the ubiquitin–proteasome system (Ivan et al., 2002, Salceda and Caro, 1997) in which a prolyl hydroxylase modification of HIF-1α renders it a target for von Hippel–Landau interaction and proteasome degradation (Ivan et al., 2002). Based on the mechanism of action for HIF-1, this transcription factor has been hypothesized to be a key regulator of VEGF expression following an acute exercise session.

However, HIF-1 activity in several cell types has also been reported to be regulated by non-hypoxic stimuli including growth factors, cytokines, nitric oxide (NO), thrombin, angiotensin, and mechanical stress (Agani et al., 2002, Amaral et al., 2001, Kim et al., 2002, Kimura et al., 2001, Page et al., 2002, Richard et al., 2000, Sandau et al., 2001, Steensberg et al., 2002, Zelzer et al., 1998, Zhong et al., 2000). Non-hypoxic agents have the potential to increase HIF-1 activity to an equal or greater extent than that observed with hypoxia alone and, in contrast to the post-transcriptional control of HIF-1 stability, these non-hypoxic stimuli have been reported to regulate HIF-1α expression through transcriptional and translational mechanisms involving protein kinase c and phophatidylinositol 3-kinase (PI3K) signaling. (Kim et al., 2002, Kimura et al., 2001, Page et al., 2002, Sandau et al., 2001, Zelzer et al., 1998, Zhong et al., 2000). A subset of non-hypoxic stimuli (cobalt, desferroxamine, and NO) also have a mechanism of action similar to hypoxia-induced HIF-1α activation. Under normoxic conditions, NO has been reported to block ubiquitination, and stabilize the HIF-1α subunit via s-nitrosylation of the VGL E3 ligase component (Palmer et al., 2000). However, when cells are in a hypoxic environment, HIF-1α accumulation, DNA-binding, and transactivation were reported to be repressed by NO (Agani et al., 2002, Hagen et al., 2003). In addition, several potential non-hypoxic stimuli (NO, prostaglandins, and adensosine) have been reported to increase in skeletal muscle during an acute exercise session (Herbaczynska-Cedro et al., 1976, Hirai et al., 1994, Tominaga et al., 1980), and inhibiton of NO synthase or Ang II blunt the VEGF, and angiogenic responses to acute exercise. (Amaral et al., 2001, Gavin et al., 2000, Hudlicka et al., 2000). Evidence also exists for the role of mechanical stress and cytokines (IL-1β, IL-6, and TNF-α) to regulate HIF-α expression and these factors could potentially influence exercise-induced angiogenesis (Cannon et al., 1989, Fielding et al., 1993, Hudlicka et al., 2000, Lloyd et al., 2003, Steensberg et al., 2002, Stroka et al., 2001).

Few studies have examined the role of HIF-1 in response to acute exercise (Gustafsson et al., 1999). If VEGF gene activation in skeletal muscle were to occur predominantly via HIF-1 in response to exercise and/or hypoxia, we would expect a close relationship between VEGF expression and HIF-1α protein levels no matter whether induced by hypoxia, muscle contraction, or these two stimuli in combination. We sought to test this hypothesis by measuring VEGF and HIF-1α mRNA and protein responses to several levels of these stimuli alone and in combination in the gastrocnemius muscle of normal rats. This experimental design also allows us to determine if there is an interaction between muscle contraction-generated stimuli and hypoxia in regulating HIF-1 or VEGF expression.

Section snippets

Animal preparation

This experimental protocol was approved by the Animal Subjects Committee, University of California San Diego, CA, USA. Female Wistar rats, 8–12 weeks old, were used in this experiment. Every experimental or control group included six rats. For all groups, rats were anesthetized with pentobarbitol (40–60 mg/kg i.p.), a tracheal cannula was inserted, and connected to a ventilator set at a tidal volume of 2.5 ml, 1 cm H2O positive end-expiratory pressure, and breathing frequency of 50 times/min. The

ES and hypoxia

With maximal ES (8 V), the force of contraction of the gastrocnemius slowly decreased over the course of 1 h. Fatigue, therefore, developed progressively, indicating intense muscle contraction. After the hour of stimulation, force of contraction was 4 ± 1.5% of initial force. Fatigue developed much more slowly in sub-maximal stimulation. By the end of stimulation, force of contraction was 11 ± 3.5% (6 V), and 16 ± 3.2% (4 V) of initial. In the normoxic control group, the average arterial PO2 at the

Discussion

The principal outcome of this study is summarized in Fig. 4. It shows that: (1) in hypoxia at rest (FIO2=0.08) or submaximal ES in normoxia, increases in VEGF mRNA (<four-fold) were not accompanied by HIF-1 accumulation; (2) only in maximal stimulation during normoxic or H, or submaximal stimulation with H was HIF-1α protein increased. Under the latter conditions, VEGF mRNA levels increased in proportion to HIF-1α accumulation; (3) VEGF protein rose in proportion to VEGF mRNA levels under all

Acknowledgements

This research was supported by National Institutes of Health Grant HL17731 and UCSD Medicine Education and Research Foundation.

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