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1 Department of Physiological Sciences, National Institute of Fitness and Sports, Shiromizu-cho 1, Kanoya, Kagoshima 891-2393, Japan
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Abstract |
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(HIF-1), vascular endothelial growth factor (VEGF), kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1), fms-like tyrosine kinase (Flt-1), angiopoietin-1 and tyrosine kinase with Ig and epidermal growth factor(EGF) homology domain 2 (Tie-2), was analysed using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The fibre cross-sectional area after denervation was about 20% of the control value, and the capillary to fibre ratio was significantly lower in denervated than in control muscles. The number of capillaries around each fibre also decreased to about 40% of the control value. These observations suggest that muscle capillarity decreases in response to chronic denervation. RT-PCR analysis showed that the expression of VEGF mRNA was lower in denervated than in control muscles, while the expression of HIF-1 mRNA remained unchanged. The expression levels of the KDR/Flk-1 and Flt-1 genes were decreased in the denervated muscle. The expression levels of angiopoietin-1 but not Tie-2 genes were decreased in the denervated muscle. These findings indicate that reduction in the expression of mRNAs in the VEGF/KDR/Flk-1 and Flt-1 as well as angiopoietin-1/Tie-2 signal pathways might be one of the reasons for the capillary regression during chronic denervation.
(Received 6 January 2005;
accepted after revision 28 January 2005; first published online 11 February 2005)
Corresponding author A. Wagatsuma: Department of Physiological Sciences, National Institute of Fitness and Sports, Shiromizu-cho 1, Kanoya, Kagoshima 891-2393, Japan. Email: waga{at}nifs-k.ac.jp
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Introduction |
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Vascular endothelial growth factor (VEGF), a mitogenic factor acting on endothelial cells, plays a crucial role in vasculogenesis and angiogenesis (Ferrara, 1999). VEGF exerts its biological effects through two tyrosine kinase receptors, KDR/Flk-1 and Flt-1, expressed predominantly on endothelial cells (Ferrara, 2001). These two receptors exhibit markedly different signalling and biological properties (Ferrara, 2001). VEGF signal pathways induce proliferation, differentiation, migration and survival of endothelial cells (Petrova et al. 1999). The angiopoietins (angiopoietin-1–4) are also angiogenic factors that make essential contributions to maturation, stabilization and remodelling of vasculature (Davis et al. 1996; Suri et al. 1996; Maisonpierre et al. 1997; Valenzuela et al. 1999). The four identified angiopoietins are ligands of the tyrosine kinase receptor, Tie-2 (Breier, 2000), which is specifically expressed in endothelial cells. Angiopoietin-1/Tie-2 signal plays a role as mediator in the process of maturation of vascular structure and maintenance of vascular integrity through the recruitment of pericytes and endothelial cells, whereas angiopoietin-2 interferes with the angiopoietin-1/Tie-2 signal, resulting in loosening vascular structure and exposing the endothelium to inducers of angiogenesis such as VEGF (Fam et al. 2003). In vivo experiments have shown an up-regulation of VEGF and its receptors as well as angiopoietin-1 and Tie-2 mRNAs in gastrocnemius muscle in response to exercise training (Lloyd et al. 2003), suggesting that increased muscle activity potentially increases the gene expression of angiogenesis-related factors which induce muscle capillarity. A synergistic effect of co-administration of angiopoietin-1 and VEGF has been shown to promote angiogenesis in acute ischaemic muscle (Shyu et al. 2003), indicating that angiopoietins act in concert with VEGF. Based on these observations, the down-regulation of these angiogenesis-related factors may be one reason why muscle capillarity is reduced after denervation.
To test this hypothesis, we characterized the expression of a variety of angiogenesis-related factors in mouse gastrocnemius muscle following denervation for 4 months. Our goals in this study were (1) to examine the changes in capillary supply following denervation and (2) to identify factors involved in denervation-induced capillary regression. We selected muscle denervation as a disuse model, because, during the first 4 months following denervation, capillaries are greatly lost in rat skeletal muscle (Borisov et al. 2000).
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Methods |
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Male ICR mice (CLEA Japan, Tokyo, Japan) were 5 weeks old and weighed between 26 and 31 g. The mice were housed in the animal facility under a 12–12 h light–dark cycle at room temperature (23 ± 2°C) and 55 ± 5% humidity. The mice were maintained on a diet of rodent chow (CE-2, CLEA Japan) and given water ad libitum.
Mice underwent mid-thigh sciatic nerve transection. A surgical level of anaesthesia was induced by intraperitoneal injection of pentobarbital sodium (30 mg kg–1). All surgical procedures were performed under aseptic conditions. The right sciatic nerve was isolated mid-thigh and transected 5–8 mm proximal to the trifurcation. Re-innervation was prevented by removing 5-mm segment of the nerve and placing the ends into nearby muscles. The left leg served as a control.
This study was approved by the Animal Committee of the National Institute of Fitness and Sports.
Capillary staining
Gastrocnemius muscle was dissected, and frozen in isopentane pre-cooled with liquid nitrogen. Capillary staining was performed according to a method reported previously (Ziada et al. 1984). Frozen transverse sections from the mid-belly region of gastrocnemius muscles were fixed for 10 min in acetone at –20°C, and air-dried before being stained for alkaline phosphatase to reveal the endogenous enzyme in the capillary endothelium. Staining was carried out for 1 h at 37–38°C using 0.1% nitro blue tetrazolium and 0.02% 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Roche, Mannheim, Germany) in 6.9 mmol MgSO4 and 27.5 mmol NaBO2 buffer adjusted to pH 9.2–9.4 with boric acid. After rinsing and a brief postfixation in sucrose-buffered formalin (4% formaldehyde, pH 7.3), slides were mounted in glycerol-based medium.
Capillaries and muscle fibres were counted in randomly selected fields (10 fields section–1) in the superficial region of the gastrocnemius muscle. We selected this region because the muscle capillarity had already decreased within 2 weeks, as previously described (Tyml et al. 1999). Morphometric measurements of fibre cross-sectional area and of capillarity (size of analysed field, 154 µm x 205 µm) were carried out by light microscopy with CCD camera. The capillary density was determined by counting the total number of capillaries in a muscle section and was expressed as the number of capillaries per square millimeter. To ensure that the analysis of capillary density was not subject to error from muscle atrophy or interstitial fibrosis, capillary density was also determined by dividing the number of capillaries by the number of muscle fibres to yield the capillary to fibre ratio. Therefore, the capillary density was again expressed both as capillaries per unit area and capillaries per muscle fibre (Waters et al. 2004). To determine capillary contacting, the number of capillaries in contact with a muscle fibre was counted.
RNA extraction and cDNA synthesis
Muscles from mice were collected and immediately frozen in liquid nitrogen. Tissues were then transferred to glass homogenizers on ice, and 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was added per 50 mg tissue. RNA integrity was confirmed by denaturing agarose gel electrophoresis, and the concentration was quantified by measuring the optical density (OD) at 260 nm. All samples had an optical density at 260 nm to optical density at 280 nm ratio (OD260/OD280) of at least 2.0.
After DNase I treatment to exclude DNA contamination, total RNA (1 µg) from the gastrocnemius was used to synthesize first-strand cDNA in a final volume of 10 µl containing 2 µl 4 x M-MLV buffer, 0.5 µl 10 mmol dNTP mixture, 0.25 µl 200 U ml–1 molony-murin leukemia virus (M-MLV) reverse transcriptase, 0.25 µl 40 U ml–1 RNase inhibitor, 0.5 µl 50 µmol oligo-(dT) primers, total RNA and RNase-free H2O. The reaction was incubated at 42°C for 10 min and then at 95°C for 2 min to inactivate the reverse transcriptase. The cDNA samples were divided into aliquots and stored at –80°C.
Semi-quantitative PCR
Semi-quantitative PCR was performed using an OPTICON DNA Engine (MJ Research Inc., Waltham, MA, USA) according to the manufacturer's instructions. Amplification was carried out in a total volume of 25 µl containing 6.25 µl 4 x RT-PCR buffer, 3 µl 3 mmol Mg2+, 0.75 µl 10 mmol dNTP mixture, 0.25 µl 20 µmol each primer, 0.25 µl 5 U ml–1 TaKaRa Ex Taq HS (TaKaRa, Japan), 3.75 µl RNase-free H2O and 10 µl of 1: 5 diluted cDNA. Primer pairs for ß-actin (Steuerwald et al. 2000), HIF-1 (Marti et al. 2000), VEGF (Simpson et al. 2000), KDR/Flk-1, Flt-1, angiopoietin-1 and Tie-2 (Bi et al. 1999) were used. Controls without reverse transcriptase were included in every analysis. Experiments were performed in duplicate for each sample. For each set of primers, PCR thermal cycle conditions were optimized to achieve a single-band PCR product in 2% agarose gel with ethidium bromide staining. We determined the optimal number of PCR cycles for each primer set in preliminary experiments so that the amplification process was conducted during the exponential phase of amplification. The PCR cycle for each set of primers was as follows: ß-actin (18 cycles), HIF-1 (28 cycles), VEGF (25 cycles), KDR/Flk-1 (22 cycles), Flt-1 (23 cycles), angiopoietin-1 (24 cycles) and Tie-2 (24 cycles). Aliquots of the PCR reaction were size-separated on 2% agarose gel in 0.04 M Tris-acetate plus 0.001 M EDTA (TAE). The agarose gels were stained with ethidium bromide and photographed under ultraviolet light. The density of each amplified band was measured using image analysis software, Gel-Pro Analyser 4.0 for Windows (Media Cybernetics, Silver Spring, MD, USA). The ß-actin gene is used as a control for the endogeneous RNA transcripts, and each sample was normalized by its ß-actin content.
Statistics
Values are means ±S.E.M. For analysis between control and denervation, Student's paired t test was used to determine significance. The level of significance was set at P < 0.01.
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Results |
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, VEGF, KDR/Flk-1, Flt-1, angiopoietin-1 and Tie-2. PCR products corresponding to ß-actin (244 bp), HIF-1 (187 bp), VEGF (189 bp), KDR/Flk-1 (382 bp), Flt-1 (505 bp), angiopoietin-1 (413 bp) and Tie-2 (258 bp) RNA transcripts are detected in skeletal muscle (Fig. 2). Figure 3 shows the expression levels of angiogenesis-related factors in control and chronically denervated muscles. HIF-1 mRNA levels remained unchanged in chronically denervated muscles compared with control muscles. VEGF mRNA levels were significantly lower in chronically denervated than in control muscles (P < 0.001). The expression levels of KDR/Flk-1 and Flt-1 genes decreased significantly (P < 0.001). The expression levels of angiopoietin-1 decreased significantly in the atrophic muscle (P < 0.001), while its receptor Tie-2 mRNA level remained unchanged.
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Discussion |
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In this study, the contralateral muscle of a chronically denervated muscle was used as a control muscle. It is possible that the expression of angiogenesis-related factors may be stimulated in contralateral muscle, which was overloaded more than in chronically denervated muscle. However, previous studies have shown that these factors are transiently increased in electrically stimulated or exercise-trained muscles and then return to nearly normal level within 2–3 weeks (Hang et al. 1995; Lloyd et al. 2003). Thus, in this study, the contralateral muscle is available as a control muscle.
It is generally assumed that prolonged imbalances between the perfusion capabilities of the blood vessels and the metabolic requirements of the tissue cells lead to modification of the vasculature to satisfy the tissue needs (Adair et al. 1990). This hypothesis suggests that the capillaries regress to meet the decreased metabolic demands of the denervated muscle. Our observations are generally consistent with this hypothesis. We used two parameters, capillary density and capillary to fibre ratio, which are widely used and accepted for muscle capillary quantification (Kano et al. 2002). In this study, the increased capillary density after denervation was observed, in agreement with previous observation of denervated rat gastrocnemius muscle (Chernukh & Alekseeva, 1975). The increase might be explained by the fact that the decrease in the number of capillaries is smaller than the decrease of muscle mass, resulting in an increase in capillary density in the atrophied muscle (Kano et al. 2000). Thus, this parameter does not reflect the change in capillary numbers of the atrophied muscle (Kano et al. 2002). Borisov et al. (2000) have demonstrated that capillary to fibre ratio decreased by 76%, from 1.55 to 0.37, after 4 months of denervation. The reduction rate is nearly consistent with our finding that it decreased by 70%. These observations could be interpreted to suggest that, in periods of muscle atrophy through decreases in fibre cross-sectional area, the capillaries are lost around a muscle fibre. To further examine this possibility, we used a more direct parameter for determining capillary supply to individual muscle fibre derived from counting the number of capillary contacts around a fibre (Plyley & Groom, 1975). The present study showed that the number of capillaries around each fibre decreased in the chronically denervated muscle. This observation suggests that a coupling between reduced functional demand and decreased muscle capillarity during denervation would be established.
Hang et al. (1995) have shown that VEGF mRNA levels are highest at the early stages of electric stimulation of skeletal muscle, then gradually decrease, and reach nearly control levels during prolonged stimulation. They suggest that VEGF expression is controlled by a negative feedback control system. If a feedback control system functions normally in the denervated muscle, the reduced expression levels of angiogenesis-related factors would be expected to return to normal levels because the capillaries regress to meet the decreased metabolic demands of the denervated muscle. However, unlike exercise-trained or electrically stimulated muscles (Hang et al. 1995; Lloyd et al. 2003), chronic denervation maintained reduced levels of angiogenesis-related factors in skeletal muscle after 4 months. The conclusion cannot be derived from the present study but this observation may be explained, at least in part, by changes in levels of nitric oxide (NO) produced by the muscle tissue. This hypothesis is supported by Kimura et al. (2000), who have shown that NO can up-regulate the VEGF promoter in human cells. The NO-responsive cis-elements in the VEGF promoter are the hypoxia-inducible factor-1 (HIF-1) binding site and an adjacent ancillary sequence that is located immediately downstream within the hypoxia-response element (HRE). Similar to the promoter region of VEGF, the Flt-1 promoter region also contains an HIF consensus binding site (Gerber et al. 1997). As a decreased neuronal nitric oxide synthase has been reported in the denervated muscle (Tew et al. 1997), it is likely that the reduction of NO-generating capacity during denervation has an effect on the expression levels of angiogenesis-related factors in muscle tissue. Besides NO involvement, we assume that other factors play a role in modulating expression of angiogenesis-related factors in chronically denervated muscle.
The present results did not show that expression of HIF-1 gene parallels expression of VEGF and Flt-1 genes which are mediated, at least in part, by the binding HIF-1 to an HIF-binding site located in the promoters of these genes (Levy et al. 1995; Gerber et al. 1997). However, the present result does not rule out the possible involvement of HIF-1 in activation of the VEGF and Flt-1 genes in skeletal muscle, because the regulation of HIF-1 expression and activity in vivo occurs at multiple levels (Semenza, 2000). Thus the stability of HIF-1 mRNA and/or DNA binding activity may decrease, affecting the expression of the VEGF and Flt-1 genes in chronically denervated muscle.
A decreased level of VEGF mRNA was found in chronically denervated muscle where muscle capillarity is decreased. This finding raises the possibility that a steady-state level of gene expression is required for the maintenance of capillarity in skeletal muscle. In support of this possibility, Tang et al. (2004) have demonstrated that capillary density and capillary to fibre ratio are decreased in VEGF-inactivated regions of skeletal muscle from VEGFloxP transgenic mice in which all three VEGF isoforms (VEGF120, VEGF168 and VEGF188) were inactivated in skeletal muscle through the viral delivery of cre recombinase to muscle fibres. The data further indicate that capillary regression is found accompanied by the appearance of TUNEL-positive apoptotic endothelial cells (Tang et al. 2004). Thus decreased VEGF levels may potentially initiate apoptotic pathway in endothelial cells and lead to regression of capillaries in chronically denervated muscle. However, it should be noted that VEGF-dependent stimulation of KDR/Flk-1 both up-regulates expression of KDR/Flk-1 gene and increases in cellular KDR/Flk-1 levels in endothelial cells (Shen et al. 1998). Therefore decreased expression levels of VEGF may affect expression levels of KDR/Flk-1 in skeletal muscle from VEGFloxP transgenic mice.
Chronic denervation decreased the expression levels of VEGF receptors (KDR/Flk-1 and Flt-1). KDR/Flk-1 is considered to be the major mediator of several physiological and pathological effects of VEGF on endothelial cells (Cross et al. 2003). This receptor is implicated in VEGF-dependent survival signals in endothelial cells through the phosphatidylinositol 3'-kinase (PI3-kinase)/Akt signal transduction pathway (Gerber et al. 1998). This pathway is thought to be important in protection from apoptosis (Yao & Cooper, 1995; Minshall et al. 1996; Dudek et al. 1997). Thus VEGF/KDR/Flk-1 signal pathway may be a factor leading to regression of muscle capillarity. Although the role of Flt-1 in the adult animal is less clearly defined compared to that of KDR/Flk-1, Flt-1 mRNA is expressed in both proliferating and quiescent endothelial cells, suggesting a role for this receptor in the maintenance of endothelial cells (Peters et al. 1993).
In this study, the expression levels of angiopoietin-1 but not Tie-2 decreased in the denervated muscle, and this study is, to our knowledge, the first to deal with the potential regulation of these factors. The observed denervation-mediated reduction of angiopoietin-1 expression suggests a destabilization of vascular structure that finally leads to capillary regression. Based on knockout experiments in mice, angiopietin-1-deficient mice have shown that endothelial cells appear to be poorly associated with the basement membrane and with perivascular cells (Suri et al. 1996). Thus, it can be deduced that a reduced coverage of pericytes surrounding the endothelium in rat skeletal muscle after long-term denervation (Dedkov et al. 2002) is due to the decreased expression levels of anigiopoietin-1. However, further studies are necessary before definitive conclusions can be drawn about the role of angiopoietin-1 and Tie-2 in the angiogenic response to chronic denervation, because changes in the ratio of angiopoietin-2 to angiopoietin-1 are thought to determine whether the net effect of the angiopoietins is to stabilize or destabilize the vasculature (Lloyd et al. 2003).
In conclusion, we have provided the first evidence that expression of the angiogenesis-related genes is altered in skeletal muscle when capillaries are lost because of chronic denervation. Chronic denervation induced a reduction of capillary supply to the gastrocnemius muscle. The expression of mRNAs related to the VEGF/KDR/Flk-1 and VEGF/Flt-1 as well as angiopietin-1/Tie-2 signal pathways declined following chronic denervation. Although the mechanism by which chronic denervation regulates the gene expression of angiogenesis-related factors remains to be identified, these findings may explain, at least partly, why capillaries are lost in chronically denervated muscle.
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