Effect of hindlimb unweighting on expression of hypoxia-inducible factor-1alpha vascular endothelial growth factor, angiopoietin, and their receptors in mouse skeletal muscle.

Hindlimb unweighting (HU) leads to capillary regression in skeletal muscle. However, the molecular mechanism(s) remains to be elucidated. To gain insight into the regulation of this process, we investigated gene expression of hypoxia-inducible factor-1alpha (HIF-1alpha)vascular endothelial growth factor (VEGF), angiopoietin, and their receptors in the atrophied muscle induced by HU. The hindlimbs of mice were unweighted by tail-suspension and then the gastrocnemius muscles were isolated after 10 days. To assess the capillary distribution, the capillary endothelium in frozen transverse sections was identified by staining for alkaline phosphatase. The mRNA levels were analyzed using a real-time reverse transcription-polymerase chain reaction. After 10 days of HU, the number of capillaries around a muscle fiber was significantly decreased by 19.5 %, suggesting that capillary regression appears to occur. The expression of HIF-1alpha ?was significantly down-regulated after 10 days of HU. The expression of VEGF remained unchanged, whereas those of Flt-1, KDR/Flk-1, and neuropilin-1 were significantly down-regulated, suggesting that VEGF signaling through these receptors would be attenuated. The expression of angiopoietin-1, and -2, as well as their receptor, Tie-2 were also significantly down-regulated, suggesting that angiopoietin-1 signaling through Tie-2 would be attenuated. These findings suggest that alterations in expression of VEGF, angiopoietins, and their receptors may be associated with capillary regression after HU.

Time course of changes in angiogenesis-related factors in denervated muscle.

AIM: Denervation leads to capillary regression in skeletal muscle. To gain insight into the regulation of this process, we investigated the time course of changes in capillary supply and gene expression of angiogenesis-related factors during muscle denervation. METHOD: Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were isolated at 12 h, 1, 3, 5, 10, 20, or 30 days after surgery. The capillary supply was assessed by immunohistochemistry using anti-PECAM-1/CD31 antibody. The mRNA levels for angiogenesis-related factors were analysed using a real-time polymerase chain reaction. RESULTS: We found that the capillary-to-fibre ratio began to decrease 10 days after muscle denervation and decreased by 52% after 30 days. The levels of mRNA for vascular endothelial growth factor (VEGF), its receptors [fms-like tyrosine kinase (Flt-1) and a kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1)], angiopoietin-1 and angiopoietin-2 of denervated muscle were immediately down-regulated after 12 h and remained lower than control muscle until 30 days after muscle denervation. The levels of mRNA for the VEGF receptor, neuropilin-1, angiopoietin receptor and Tie-2 decreased within 12-24 h, but returned to near those of control muscle after 10-20 days, and again decreased after 30 days. CONCLUSIONS: These findings suggest that denervation-induced capillary regression may be associated with down-regulation of VEGF and angiopoietin signalling.

Upregulation of gene encoding adipogenic transcriptional factors C/EBPalpha and PPARgamma2 in denervated muscle.

Muscle denervation induces fatty degeneration in skeletal muscle. However, the possible mechanism(s) remains to be elucidated. To gain insight into the regulation of this process, this study was designed to characterize the expression pattern of genes encoding transcriptional factors that regulate adipogenesis and the terminal differentiation marker of adipocytes in denervated muscle. Female mice underwent surgery to transect the sciatic nerve, and then the gastrocnemius muscles were harvested 5, 10, 20 or 30 days after surgery. The extent of fatty degeneration was assessed as lipid accumulation by Oil Red O staining. The cellular localization of CCCAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma2 (PPARgamma2), which play an important role in the regulation of adipocyte differentiation, was assessed by immunohistochemistry. The mRNA levels were analysed using a real-time polymerase chain reaction. After muscle denervation, most muscle fibres atrophied pathologically, and lipid accumulation was observed in the superficial region of the gastrocnemius muscle, suggesting that fatty degeneration occurs in this model. Both C/EBPalpha and PPARgamma2 proteins were observed in the interstitial space of denervated muscle but detected in small amounts in normal muscle. The expression levels of C/EBPalpha and PPARgamma2 were significantly upregulated 30 days after muscle denervation. The expression levels of fatty acid binding protein 4 (FABP4), which reflects fatty acid metabolism, were decreased slightly at 5 and 10 days and then returned to control levels 30 days after muscle denervation. These findings suggest that muscle denervation-induced fatty degeneration may be mediated through C/EBPalpha and PPARgamma2.

Sequential expression of vascular endothelial growth factor, Flt-1, and KDR/Flk-1 in regenerating mouse skeletal muscle.

We investigated the expression of vascular endothelial growth factor (VEGF) and its receptors (Flt-1 and KDR/Flk-1) during muscle regeneration by immunohistochemistry and real-time RT-PCR. On days 5 and 7 after the induction of injury, VEGF and Flt-1 were detected in the cytoplasm and KDR/Flk-1 in the cytoplasm and on cell membranes of the same regenerating muscle fibers. The levels of these proteins in the regenerating muscle fibers gradually decreased until day 20. In contrast, these proteins were not detected in the fibers of normal muscle. This suggests that regenerating muscle fibers express VEGF and its receptors in response to injury. In addition, we found that the VEGF mRNA transcript transiently increased after 12 h of muscle injury and then returned to the basal levels observed in normal muscles on day 1. The expression of Flt-1 and KDR/Flk-1 mRNA transcripts also peaked on day 3 and then returned to the basal levels observed in normal muscles on day 10. These findings suggest that regenerating muscle fibers are an important source of VEGF and that VEGF signaling through Flt-1 and KDR/Flk-1 may be involved in the process of muscle regeneration in vivo.

Effect of aging on expression of angiogenesis-related factors in mouse skeletal muscle.

The molecular mechanisms by which capillary supply is maintained with advancing age remain to be elucidated. To help clarify these mechanisms, we investigated the gene expression levels of angiogenesis-related factors in young (2.5-month-old), adult (6-month-old), and old (22-month-old) mice. To assess the capillary supply, the capillary endothelium in frozen transverse sections was identified by staining for alkaline phosphatase. The mRNA levels for angiogenesis-related factors were analyzed using real-time RT-PCR. The capillary supply to individual muscle fibers, assessed as the number of capillaries around a muscle fiber, did not change with advancing age. Real-time RT-PCR analysis showed that (1) the level of mRNA for VEGF was lower in old animals than young animals; (2) the mRNA levels of Flt-1 and neuropilin-1 are lower in old animals than young animals, while that of KDR/Flk-1 remained unchanged with advancing age; and (3) the levels of mRNA for angiopoietin-1 and -2 remained unchanged, while the mRNA for Tie-2 was lower in old animals than young animals. These findings suggest that capillary supply is maintained irrespective of the down-regulation of several angiogenesis-related factors and that old animals possess the minimum levels of maintenance and reparative abilities needed to preserve the capillary supply.

Capillary supply and gene expression of angiogenesis-related factors in murine skeletal muscle following denervation.

Capillary supply of skeletal muscle decreases during denervation. To gain insight into the regulation of this process, we investigated capillary supply and gene expression of angiogenesis-related factors in mouse gastrocnemius muscle following denervation for 4 months. Frozen transverse sections were stained for alkaline phosphatase to detect endogenous enzyme in the capillary endothelium. The mRNA for angiogenesis-related factors, including hypoxia inducible factor-1alpha (HIF-1alpha), 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-1alpha 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.

Effect of treatment with nifedipine, an L-type calcium channel blocker, on muscular atrophy induced by hindlimb immobilization.

The purpose of this study was to investigate whether the prevention of calcium influx through L-type calcium channels contributed to the attenuation of muscular atrophy induced by hindlimb immobilization (HI) in a shortened position. Mice were divided into four groups (8 mice/group): control; nifedipine; HI; and HI with nifedipine. Mice received nifedipine at a dose of 5 mg/kg one day before and during the 8 days of HI. Quantitative alterations in the amount of myosin heavy chain (MyHC) and actin proteins in the soleus muscle were analyzed using SDS-PAGE. The weight of the soleus muscle decreased significantly by 40.8% (P<0.05) and 27.0% (P<0.05) after the hindlimb immobilization in the HI and HI with nifedipine groups, respectively, when compared to that of the control or nifedipine groups. Treatment with nifedipine alone appeared to have no effect on muscle mass or the amount of myofibrillar proteins. The level of MyHC proteins decreased significantly by 25.1% (P<0.001) and 17.4% (P<0.001) in the HI and HI with nifedipine groups, respectively. The level of MyHC protein in the HI with nifedipine group was significantly greater than that of the HI group (P<0.05), although there were no significant differences in the amount of actin protein. These findings suggest that nifedipine treatment may have a beneficial effect on muscular atrophy.

Molecular properties and gene expression of albumin in the skeletal muscle following hindlimb immobilization in a shortened position.

We investigated the effect of immobilization of hindlimb in a shortened position on proteins in the mouse soleus muscle and determined the molecular properties of a protein that had changed specifically in the atrophied muscle. A protein of 67.5 kDa was increased significantly (P<0.001) by 1.9-fold in the atrophied muscle compared to the control muscle as shown by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE). The isoelectric point of the protein was pH 6.3 in two-dimensional PAGE. We carried out N-terminal and peptide mapping analyses to identify the primary structure of the protein. A 17-amino acid sequence showed 100% homology with the mouse serum albumin precursor. A peptide map of the 67.5-kDa protein was similar to that of mouse serum albumin. Mass spectrometry also showed the protein to be most similar to mouse serum albumin. Thus, based on these criteria, the 67.5-kDa protein was indistinguishable from mouse serum albumin. An immunohistochemical study showed that the 67.5-kDa protein was located mainly in the endomysium of the control muscle and had accumulated in the widened interstitial spaces of the atrophied muscle. A reverse transcription-PCR assay revealed that the albumin gene was expressed in skeletal muscle tissue, but that its expression was significantly lower in the atrophied muscle than in the control muscle (P<0.001). We conclude that albumin is increased in atrophied muscle and suggest that this may be of clinical importance for analyzing the process of muscular atrophy.

Specific protein alteration in the soleus following immobilization-atrophy.

To investigate the effect of immobilization in a shortened position on protein alteration in the mouse soleus, total protein from immobilized and control muscle were analyzed by SDS-PAGE and two-dimensional polyacrylamide gel electrophoresis. Five-week-old male ddY mice (n=22) were used throughout this study. A 67.3 kDa protein was significantly increased by 1.7-fold in the immobilized muscles in SDS-PAGE compared to control muscle (P<0.001). This protein had the same mobility as serum albumin. The isoelectric point of the 67.3 kDa protein was pH 6.3, similar to serum albumin (range from pH 6.0 to 6.3). Specific antibody to the 67.3 kDa protein reacted with serum albumin by Western blot analysis. This protein was localized in the interstitial space in the control muscle and accumulated in pathologic areas in the immobilized muscle. It was presumed that this protein was a serum albumin-like protein.