Vascular Endothelial Growth Factor Overexpression in Ischemic Skeletal Muscle Enhances Myoglobin Expression In Vivo
Therapeutic angiogenesis using vascular endothelial growth factor (VEGF) is considered a promising new therapy for patients with arterial obstructive disease. Clinical improvements observed consist of improved muscle function and regression of rest pain or angina. However, direct evidence for improved vascularization, as evaluated by angiography, is weak. In this study, we report an angiogenesis-independent effect of VEGF on ischemic skeletal muscle, ie, upregulation of myoglobin after VEGF treatment. Mice received intramuscular injection with adenoviral VEGF-A or either adenoviral LacZ or PBS as control, followed by surgical induction of acute hindlimb ischemia at day 3. At day 6, capillary density was increased in calf muscle of Ad.VEGF-treated versus control mice (P<0.01). However, angiographic score of collateral arteries was unchanged between Ad.VEGF-treated and control mice. More interestingly, an increase in myoglobin was observed in Ad.VEGF-treated mice. Active myoglobin was 1.5-fold increased in calf muscle of Ad.VEGF-treated mice (P≤0.01). In addition, the number of myoglobin-stained myofibers was 2.6-fold increased in Ad.VEGF-treated mice (P=0.001). Furthermore, in ischemic muscle of 15 limb amputation patients, VEGF and myoglobin were coexpressed. Finally, in cultured C2C12 myotubes treated with rhVEGF, myoglobin mRNA was 2.8-fold raised as compared with PBS-treated cells (P=0.02). This effect could be blocked with the VEGF receptor tyrosine kinase inhibitor SU5416. In conclusion, we show that VEGF upregulates myoglobin in ischemic muscle both in vitro and in vivo. Increased myoglobin expression in VEGF-treated muscle implies an improved muscle oxygenation, which may, at least partly, explain observed clinical improvements in VEGF-treated patients, in the absence of improved vascularization.
Vascular endothelial growth factor-A (VEGF) is a major angiogenic growth factor.1 Several preclinical and clinical studies using VEGF (gene)therapy have shown promising results for the treatment of arterial obstructive disease.2–7 End-points in these clinical studies mainly consist of improved muscle function, as evaluated by exercise performance or angina classification, or regression of rest pain. It is assumed that these clinical improvements are induced by the induction of neovascularization. However, evidence for improved vascularization is scarcely reported. Moreover, intermittent claudication, implying a decreased function of limb musculature, does not correlate well with ankle brachial pressure index, a parameter of vascularization.8 In this study, we demonstrate an unanticipated effect after VEGF overexpression, namely an increase of myoglobin expression in skeletal muscle. Myoglobin is a cytoplasmic heme-containing protein, which is expressed in cardiomyocytes and skeletal myocytes. It functions as an intracellular oxygen reservoir in muscle cells and it facilitates oxygen supply, thereby maintaining cellular respiration during periods of high physiological demand, eg, during ischemia or exercise.9 In skeletal muscle, myoglobin is selectively expressed in oxidative muscle fibers, which mainly coexpress myosin heavy chain type I or IIa. Correspondingly, myoglobin is differentially upregulated in “red muscle,” which predominantly consists of oxidative fibers, as compared with “white muscle,” mainly consisting of glycolytic fibers.10 Studies of muscle composition in patients with limb ischemia showed a fiber-type switch in ischemic skeletal muscle from type IIb to IIa and subsequently to I fibers, thus toward myoglobin-containing, oxidative muscle fibers.11 Furthermore, myoglobin−/− mice demonstrate compensatory mechanisms, eg, increased expression of hypoxia-inducible transcription factors and VEGF.12 The necessity for compensatory mechanisms in myoglobin−/− mice underscores the important role of myoglobin in maintaining a physiological oxygenation state of skeletal muscle. VEGF-mediated enhancement of myoglobin expression may thus result in an angiogenesis-independent improvement of muscle oxygenation state and, thereby, muscle function. Indeed, recent data suggest that VEGF may induce angiogenesis-independent effects resulting in the recovery of damaged muscle. This was corroborated by the finding that administration of recombinant VEGF increases the number of regenerating cells in a skeletal muscle transplantation model.13 Furthermore, both VEGF and VEGF-receptor 2 are restrictedly expressed in atrophic and regenerating muscle cells of human ischemic skeletal muscle, suggesting a direct effect of VEGF on these specific cells.14 In addition, VEGF modulates skeletal myoblast function via VEGF receptor pathways in culture.15
In this study, we provide evidence that VEGF-A gene therapy acts on ischemic skeletal muscle by upregulating myoglobin expression in a mouse model of acute hindlimb ischemia. In addition, VEGF and myoglobin were coexpressed in ischemic muscle tissue of 15 limb amputation patients. Finally, an in vitro experiment in murine myotubes shows a direct stimulatory effect of VEGF on myoglobin mRNA expression in muscle cells. We propose that VEGF-mediated myoglobin expression might be beneficial for muscle function, explaining the observed clinical improvements in eg, exercise tests of VEGF-treated patients.
Materials and Methods
Gene Transfer in a Mouse Model of Acute Hindlimb Ischemia
An adenoviral vector carrying the VEGF gene (Ad.VEGF) was constructed using cDNA encoding the human VEGF165 isoform under control of the cytomegalovirus (CMV) promoter with SV40 polyadenylation signal. VEGF165 cDNA was cloned into plasmid CMV10, as previously described.16 An adenoviral vector carrying the Escherichia coli LacZ reporter gene (Ad.LacZ), under control of the CMV promoter was used as a control. Production of adenovirus stocks was performed as previously described.17
All animal experimental protocols were approved by the animal welfare committee of the Netherlands Organization for Applied Scientific Research (TNO, Leiden, The Netherlands). Male C57BL/6 mice (TNO), aged 10 weeks, were randomly allocated into two groups (n=6).
The effect of VEGF gene transfer on collateral vessel growth in the upper hindlimb was analyzed in one group (group A). For this, muscles of the left upper hindlimb surrounding the femoral artery were injected at T=0 with 40 μL of either Ad.VEGF or Ad.LacZ (1.5×1010 pfu/mL).
In the second group, the effect of VEGF gene transfer on VEGF and myoglobin expression and capillary growth was studied in the calf muscle (group B). For this, the gastrocnemius muscle of both limbs was injected at T=0 with either 40 μL of PBS, Ad.VEGF, or Ad.LacZ (1.5×1010 pfu/mL).
At day 3, ischemia of the left hindlimb was induced in animals of both groups by coagulation of the left femoral artery proximal to the bifurcation of deep and superficial femoral artery. At day 6, collateral vessel growth was studied by performing postmortem angiography in mice of group A. In mice of group B, the gastrocnemius muscles of both hindlimbs were dissected for further analysis.
To study collateral vessel growth, angiography of both hindlimbs was performed using polyacrylamide-bismuth contrast (Sigma), as previously described (for limited modifications, see the expanded Materials and Methods available in the online data supplement at http://circres.ahajournals.org).18 Grading of collateral filling was performed in a single blinded fashion and was based on the Rentrop classification.19 Grading was as follows: 0=no filling of collaterals, 1=filling of collaterals only, 2=partial filling of distal femoral artery, and 3=complete filling of distal femoral artery.
Blood flow was determined in the paws of Ad.VEGF- or Ad.LacZ-treated mice (n=6) using laser doppler perfusion imaging (LDPI) (Moor Instruments), as previously described.20
C2C12 Cells in Culture
C2C12 myoblasts, obtained from the American Type Culture Collection, Rockville, Md, were used to induce differentiation to myotubes by supplementing the growth medium with 2% horse serum, 1 μmol/L insulin, and 2.5 μmol/L dexamethasone. After 7 days of culture mature myotubes were incubated for 3, 6, 9, and 12 hours with 5 ng/mL human VEGF recombinant protein (RELIA Tech.) in differentiation medium (n=4). In a separate experiment (n=6), myotubes were incubated for 6 hours with 5 ng/mL human VEGF recombinant protein combined with the VEGF receptor tyrosine kinase inhibitor SU5416 (100 nmol/L) (kindly provided by Dr K Hoekman, Amsterdam). Subsequently, cells were washed with PBS and lysed in 1 mL GTC-βME buffer (4 mol/L Guanidinium-isothiocyanate, 25 mmol/L Na-citrate, 0.5% sarcosyl, and 0.1 mol/L 2-mercatoethanol), pH 7.0, and stored at −80°C for further RNA analysis.
Determination of Protein Expression
Immunohistochemistry was performed on paraffin-embedded muscle sections using antibodies for myoglobin (Santa Cruz Biotechnology), VEGF (Santa Cruz Biotechnology), CD31 (Pharmingen), and on cryosections for VEGFR-1 and VEGFR-2 (antibodies kindly provided by Dr HA Weich, Germany). In addition, cryosections were histochemically stained for nicotinamide dehydrogenase tetrazolium reductase (NADH-TR), as previously described.21 Stainings were quantified from randomly photographed sections (3 to 6 images per section) using image analysis (Qwin, Leica). Myoglobin expression in mice was expressed as follows: (area of muscle fibers stained for myoglobin/total area of muscle fibers)×100%. Intensity of VEGF and myoglobin staining of human skeletal muscle sections was studied at the different levels of each amputated limb in a single blinded fashion.
Analysis of Active Myoglobin
Concentration of active myoglobin in samples of murine gastrocnemius muscle was determined by peroxidase-based activity assay, as previously reported.22 Myoglobin was calibrated against a standard of horse myoglobin (Sigma).
Human VEGF165 levels in murine blood samples were assayed by ELISA (R&D systems) according to the manufacturer’s protocol.
Total RNA Isolation
Total RNA was extracted from frozen murine gastrocnemius muscle using the RNEasy isolation kit (QIAGen) according to the manufacturer’s protocol. RNA isolation from C2C12 cells was performed according to Chomczynski et al, as previously described.23
To prevent contamination of genomic DNA in PCR, RNA samples were treated with DNAse before cDNA synthesis using RQ1 RNAse-free DNAse (Promega) according to the manufacturer’s protocol.
Total RNA was reversed transcribed into cDNA as described in the expanded Materials and Methods available in the online data supplement at http://circres.ahajournals.org.
Primers pairs and probes for studying expression of human VEGF-A, mouse myoglobin, and mouse HPRT by real-time RT-PCR were designed using the specific criteria as described in the primer express software (Perkin Elmer). Mouse PCR primer sets for Troponin I (slow), cytochrome c oxidase IVa (Cox IVa), and the peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 (PGC-1α) were purchased (Applied Biosystems). Samples were normalized to housekeeping gene expression [GAPDH (Perkin Elmer) or HPRT for muscle or C2C12 samples, respectively]. PCR was performed as described in online data supplement. Myoglobin expression was calibrated against a construct containing myoglobin cDNA cloned into a pMOSBlue plasmid vector. Primers for mouse VEGFR1/FLT1 and VEGFR2/FLK1 were used for semiquantitative PCR as previously reported.24
Results are expressed as mean±SEM. Tests to compare means were performed as appropriate. A value of P<0.05 was considered statistically significant.
Effects of Ad.VEGF Gene Transfer in Mouse Model
Effect of Ad.VEGF Treatment on Collateral Vessel Growth
Three days after Ad.VEGF or Ad.LacZ gene transfer by intramuscular injection in one upper hindlimb, ischemia was induced by femoral artery occlusion in mice. All mice showed abnormal mobility of the occluded hindlimb until 3 days after occlusion. Spontaneous collateral vessel growth was already detectable within the first 3 days after occlusion, and a collateral vessel network was completely developed after 7 days (Figure 1A and 1B). No significant difference in collateral vessel growth between Ad.VEGF- and Ad.LacZ-treated mice 3 days after occlusion was observed (angiographic degrees 1.9±0.4 and 1.9±0.3, respectively; P=0.50, n=7) (Figure 1C through 1E). Correspondingly, there was no significant difference in blood flow recovery between ischemic limbs of Ad.VEGF- versus Ad.LacZ-treated mice, as measured with LDPI at various time points (n=6) (Figure 1F).
Effect of Ad.VEGF Treatment on Muscle Color and Capillary Formation
One day after Ad.VEGF injection in the gastrocnemius muscle of both hindlimbs, human VEGF (4.1±0.8 ng/mL) was detectable in the plasma. At day 6, VEGF concentration was below the detection limit of the assay. In addition, real-time PCR for human VEGF mRNA showed VEGF expression in the gastrocnemius muscle of Ad.VEGF-treated mice 6 days after injection, whereas the Ad.LacZ- and PBS-treated mice were negative (data not shown). This indicates that VEGF gene transfer resulted in a transient expression of VEGF protein.
Although no difference in collateral formation was observed, a striking red color of the treated muscle in the occluded limb was observed in all VEGF-treated mice, but not in Ad.LacZ-treated mice (Figure 2).
Moreover, the number and area of capillaries in gastrocnemius muscle of both occluded and nonoccluded limb was increased after Ad.VEGF treatment as compared with Ad.LacZ, as visualized by CD31 staining (Figure 3). Capillary density in Ad.VEGF-treated mice was 409±74 versus 185±24 capillaries/mm2 in the Ad.LacZ-treated mice for occluded limb (P=0.008, n=6), and 129±31 versus 39±11 capillaries/mm2 for nonoccluded limb, respectively (P=0.01). Moreover, a marked increase of area/capillary in both occluded and nonoccluded limb was noted in the Ad.VEGF versus Ad.LacZ group (149±11 versus 97±7 μm2 for occluded limb; P=0.001; and 147±11 versus 106±11 for nonoccluded limb, respectively; P=0.02). In addition, histological examination showed local extravasation of erythrocytes in some areas of muscle damage after femoral artery occlusion. The number of extravascular erythrocytes was not different between the two groups of mice (data not shown).
Ad.VEGF-Induced Myoglobin Expression in Ischemic Skeletal Muscle In Vivo
Activation of the myoglobin gene in transgenic mice results in a distinct red color of most muscles.25 Therefore, it was analyzed whether the VEGF-induced red color of muscle could be attributable to the upregulation of myoglobin after VEGF treatment. In Ad.VEGF-treated mice, indeed, an increase in the number of muscle fibers that positively stained for myoglobin was observed in sections of gastrocnemius muscle after femoral artery occlusion (Figure 4A and 4B).
The observed difference in myoglobin staining was quantified by image analysis (Figure 4C). The percentage of myoglobin-positive muscle fibers in gastrocnemius muscle was significantly higher in the Ad.VEGF group as compared with Ad.LacZ group (18±4.3 versus 7.0±4.1%; P=0.001, n=6). Moreover, Ad.VEGF treatment increased the number of mice positive for myoglobin as compared with Ad.LacZ (100% versus 50%). To determine the effect of Ad.VEGF treatment on functional myoglobin, active myoglobin content of gastrocnemius muscle was determined by a peroxidase-based activity assay (Figure 4D). Treatment with Ad.VEGF resulted in a significant increase of active myoglobin in gastrocnemius muscle after femoral artery occlusion as compared with Ad.LacZ or PBS treatment (64±6.8, 43±3.9, or 21±2.4 μg myoglobin/mg dry-weight, respectively; P≤0.01, n=5). In addition, Ad.LacZ treatment increased active myoglobin levels as compared with PBS (P<0.001). No significant difference in active myoglobin induction between Ad.VEGF and Ad.LacZ group was observed in the nonischemic limb.
Finally, to assess whether the increase of myoglobin was accompanied by an increase of mitochondrial content of the muscles, NADH-TR reactivity was determined in gastrocnemius muscle cryosections. Ad.VEGF treatment was accompanied by a significant increase in oxidative fibers in ischemic muscle as compared with Ad.LacZ (P=0.03, n=5) (Figure 4E).
Limb Amputation Patients: Coexpression of VEGF and Myoglobin in Ischemic Muscle of Limb Amputation Patients
To determine whether a similar correlation between VEGF and myoglobin also exists in patients with peripheral arterial obstructive disease, skeletal muscle samples were collected from amputated limbs of 15 patients. Patients consisted of 7 males and 8 females with a mean age of 67 years (range 52 to 88 years). In general, VEGF staining was absent in nonischemic gastrocnemius muscle near the amputation level (Figure 5A), whereas, more distally, cytoplasmic VEGF staining was evident in some muscle fibers of soleus muscle near the Achilles tendon. This was accompanied by histological signs of chronic ischemia, eg, disorganized muscle composition and adipose cells (Figure 5B). More distally, VEGF was present in a large number of muscle fibers in the severely ischemic interosseus muscle and was especially abundant in regenerating and atrophic muscle fibers (Figure 5C). In addition, CD31 staining of skeletal muscle revealed a significant increase in capillary density from the amputation level toward the distal interosseus muscle, suggesting an angiogenic response to ischemia (data not shown). Importantly, staining of sequential muscle cross-sections for VEGF and myoglobin showed colocalization of VEGF with myoglobin in individual muscle fibers of ischemic muscle (compare Figure 5C and 5D). Moreover, both VEGF and myoglobin expression in muscle were significantly elevated with an increased level of ischemia (P=0.018 and 0.047, respectively). Furthermore, an increased expression of VEGFR-1 and -2 was observed in cryosections of more distally located, ischemic muscle. Importantly, VEGFR-1 was not only expressed around vessels, but also around muscle fibers, suggesting muscle cell membrane-specific expression (Figure 5E). Staining for VEGFR-2 showed a similar expression pattern around muscle fibers of ischemic interosseus muscle (Figure 5F). Comparison of staining for VEGF receptors with anti-CD31 endothelial staining (Figure 5G) confirmed that VEGF receptors were additionally expressed on muscle fibers, thus not restrictedly expressed on the closely adjacent vascular cells. In both gastrocnemius and soleus muscle, however, VEGFR-2 was only expressed on vascular endothelial cells (data not shown).
Cultured Myotubes: Increased Myoglobin mRNA Levels by VEGF in Murine Myotubes in Culture
To more critically assess whether VEGF can directly act on muscle cells, the effect of VEGF was studied in vitro in differentiated murine myotubes (C2C12 cells). First, expression of VEGF receptor 1 and 2 mRNA in myotubes was confirmed by semiquantitative RT-PCR (Figure 6A). Secondly, the effect of human VEGF recombinant protein (rhVEGF) on myoglobin mRNA expression was studied at various time points. PBS-treated cells served as control. Myoglobin mRNA expression gradually increased, being 2.8-fold elevated at 9 hours (P=0.02, n=4), and decreased thereafter (Figure 6B). However, in PBS-treated cells, no increase in myoglobin mRNA expression was observed. Real-time RT-PCR measurements of myoglobin mRNA were within the linear range of a calibration curve using a construct containing myoglobin cDNA, indicating that the observed differences in PCR signal for myoglobin represent a true difference in mRNA levels (see online data supplement). To provide more evidence that the observed effects of VEGF are mediated through VEGF receptor pathways, we studied whether VEGF-induced myoglobin expression could be inhibited using the VEGF receptor tyrosine kinase inhibitor SU5416 in C2C12 cells. Induction of myoglobin mRNA expression with rhVEGF was completely inhibited when rhVEGF was combined with SU5416 (fold-induction versus PBS 2.91±0.94 or 0.71±0.13, respectively; P=0.05, n=6) (Figure 6C). To study whether VEGF induces muscle cell specification leading to more oxidative type I myotubes, expression of a set of genes characteristic of type I fibers, including Troponin I, Cox IVa, and PGC-1α, was studied. There was a significant 2-fold increased expression of mRNA for Troponin I (slow) 6 hours after VEGF treatment (P=0.04, n=3). In addition, Cox IVa and PGC-1α mRNA expressions were increased by 1.8- and 2.8-fold respectively, although this increase did not reach statistical significance for both genes (P=0.07 and 0.21, respectively, n=3) (Figure 6D).
Our data indicate that VEGF gene therapy results in enhanced myoglobin expression in ischemic skeletal muscle in mice. Furthermore, we show coexpression of VEGF and myoglobin in muscle biopsies from patients after limb amputation caused by peripheral arterial obstructive disease, which correlates with the degree of ischemia. In addition, we provide evidence for a direct regulation of myoglobin by VEGF in murine myotubes in culture. Importantly, these data may explain the puzzling inconsistencies shown in previous clinical trials with VEGF. VEGF-mediated increase of muscle myoglobin may clarify, at least partly, the observed clinical improvements of VEGF-treated patients, in particular the regression of intermittent claudication, rest pain, or angina, in the absence of improved vascular status.
Although VEGF is a well-known inducer of angiogenesis, its role in the induction and development of larger conductance collaterals via arteriogenesis is still a matter of debate.26–28 In this study, it was shown that collateral vessel growth was not enhanced by Ad.VEGF treatment in a mouse model. Nevertheless, a marked red color of gastrocnemius muscle in the ischemic limb was apparent in Ad.VEGF-treated mice. Indeed, capillary formation, a possible reason for this red color, was significantly increased in Ad.VEGF-treated muscle. Despite an increased capillary density, we also observed a marked enlargement of capillaries in Ad.VEGF-treated muscle, as previously described.29 Nevertheless, we were puzzled by the fact that an increase in capillary density was only correlated with a distinct redness of muscle in ischemic VEGF-treated limbs and not in the other groups.
A second possible explanation for the red color of muscle was that VEGF treatment induced extravasation of erythrocytes, because VEGF may increase vascular permeability.30 However, there was no evidence for an increased number of extravascular erythrocytes in histological sections of Ad.VEGF-treated muscle. Thus, we hypothesized that other factors affecting the color of muscle might play a role. Interestingly, activation of the myoglobin gene in transgenic mice results in a distinct red color of most muscles.25 In physiological conditions, the gastrocnemius muscle contains a relatively low percentage of myoglobin-containing oxidative fibers, ie, around 1% to 8% of type-I and 30% to 32% of type-IIa fibers in mice, and can be readily distinguished from dark red, type-I-enriched soleus muscle.31 However, color of gastrocnemius and soleus muscle was similar in limbs of Ad.VEGF-treated mice after femoral artery occlusion. Therefore, we analyzed the myoglobin content in the treated gastrocnemius muscles. Indeed, the number of myoglobin-stained muscle fibers and active myoglobin content were increased in calf muscle of Ad.VEGF-treated mice. Thus, VEGF-mediated myoglobin expression may, at least partly, explain the red color of gastrocnemius muscle in these mice.
VEGF-mediated myoglobin expression in vivo may be regulated by several molecular mechanisms. Firstly, VEGF may act directly on muscle fibers via membrane-bound VEGF receptors, resulting in stimulation of myoglobin expression. VEGF has previously been thought to be an endothelial cell specific mitogen. However, recent data demonstrate that it also has an effect on a large variety of cell types, eg, vascular smooth muscle cells, neural cells, endothelial precursor cells, and monocytes.32–35 In this study, we found abundant expression of VEGF receptor 1 and 2 at the margins of muscle fibers in ischemic muscle of the amputated limbs, suggesting an effect of VEGF on these specific cells via binding to its receptor. Upregulation of both VEGF-receptors in ischemic muscle may explain the observation that myoglobin expression was only increased in the ischemic limb of Ad.VEGF-treated mice and not in the nonischemic control limb.
To show that VEGF can directly act on skeletal muscle cells through classic VEGF receptor pathways, we cultured murine myoblasts (C2C12) that were differentiated into mature myotubes. First, we observed expression of VEGF receptor 1 and 2 mRNA in C2C12 cells, as previously described.15 Second, treatment with VEGF resulted in a significant 2.8-fold induction of myoglobin mRNA as compared with control. Third, the VEGF-induced myoglobin mRNA expression was completely blocked when VEGF was combined with the VEGF receptor tyrosine kinase inhibitor SU5416. Finally, VEGF-induced myoglobin expression was accompanied by an increased expression of genes characteristic of type I oxidative fibers, indicating activation of myofiber specialization.
We cannot exclude that VEGF may also act indirectly on myoglobin expression by stimulating the chemotaxis of leukocytes (eg, monocytes), which on their turn secrete factors that stimulate myoglobin expression. This hypothesis is supported by the observation that active myoglobin content was partially increased in the control virus-treated mice. Another explanation for an indirect effect of VEGF on myoglobin expression may lay in the protective properties of VEGF toward motor neurons against ischemic death.36 It has been shown that decreased neuromuscular activity elicits transitions from oxidative toward glycolytic muscle fiber phenotypes.37 VEGF may therefore, indirectly, induce a fiber type transition toward more oxidative, myoglobin-containing muscle fiber types.
Finally, studies of pathways downstream of neural activity have implicated NFAT and Mef2 transcription factors under the regulation of calcium signaling through calcineurin, a calcium/calmodulin-dependent protein phosphatase, in the control of myoglobin regulation.38,39 Interestingly, VEGF has been shown to activate the calcium-calcineurin pathway in endothelial cells.40 It remains to be determined whether VEGF is also able to activate the calcium-calcineurin pathway in skeletal muscle cells, and whether this can ultimately lead to activation of myoglobin transcription.
In conclusion, VEGF gene therapy offers a dual therapeutic effect for the improvement of ischemic muscle condition, first, via the induction of new capillaries, and second, via improved muscle oxygenation by increased myoglobin.
This study was sponsored by the TNO-LUMC-VUMC tripartite angiogenesis program and the NHS molecular cardiology program (M93.001). We would like to acknowledge Arjan van den Berg, Margreet de Vries, and Marten Engelse for their technical assistance, and Clemens Löwik for help with the C2C12 cells and VEGF receptor PCR.
Original received September 15, 2003; resubmission received April 26, 2004; revised resubmission received May 11, 2004; accepted May 11, 2004.
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989; 246: 1306–1309.
Baumgartner I, Pieczek A, Manor O, Blair R, Kearney M, Walsh K, Isner JM. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation. 1998; 97: 1114–1123.
Hendel RC, Henry TD, Rocha-Singh K, Isner JM, Kereiakes DJ, Giordano FJ, Simons M, Bonow RO. Effect of intracoronary recombinant human vascular endothelial growth factor on myocardial perfusion: evidence for a dose-dependent effect. Circulation. 2000; 101: 118–121.
Losordo DW, Vale PR, Symes JF, Dunnington CH, Esakof DD, Maysky M, Ashare AB, Lathi K, Isner JM. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation. 1998; 98: 2800–2804.
Losordo DW, Vale PR, Hendel RC, Milliken CE, Fortuin FD, Cummings N, Schatz RA, Asahara T, Isner JM, Kuntz RE. Phase 1/2 placebo-controlled, double-blind, dose-escalating trial of myocardial vascular endothelial growth factor 2 gene transfer by catheter delivery in patients with chronic myocardial ischemia. Circulation. 2002; 105: 2012–2018.
Tsurumi Y, Takeshita S, Chen D, Kearney M, Rossow ST, Passeri J, Horowitz JR, Symes JF, Isner JM. Direct intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor augments collateral development and tissue perfusion. Circulation. 1996; 94: 3281–3290.
Campbell WG, Gordon SE, Carlson CJ, Pattison JS, Hamilton MT, Booth FW. Differential global gene expression in red and white skeletal muscle. Am J Physiol Cell Physiol. 2001; 280: C763–C768.
Godecke A, Flogel U, Zanger K, Ding Z, Hirchenhain J, Decking UK, Schrader J. Disruption of myoglobin in mice induces multiple compensatory mechanisms. Proc Natl Acad Sci U S A. 1999; 96: 10495–10500.
Rissanen TT, Vajanto I, Hiltunen MO, Rutanen J, Kettunen MI, Niemi M, Leppanen P, Turunen MP, Markkanen JE, Arve K, Alhava E, Kauppinen RA, Yla-Herttuala S. Expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 (KDR/Flk-1) in ischemic skeletal muscle and its regeneration. Am J Pathol. 2002; 160: 1393–1403.
Quax PH, Lamfers ML, Lardenoye JH, Grimbergen JM, de Vries MR, Slomp J, de Ruiter MC, Kockx MM, Verheijen JH, van Hinsbergh VW. Adenoviral expression of a urokinase receptor-targeted protease inhibitor inhibits neointima formation in murine and human blood vessels. Circulation. 2001; 103: 562–569.
Cohen M, Rentrop KP. Limitation of myocardial ischemia by collateral circulation during sudden controlled coronary artery occlusion in human subjects: a prospective study. Circulation. 1986; 74: 469–476.
Dubowitz V, Brooke MH. Histological and histochemical stains and reactions. In: Dubowitz V, Brooke MH, eds. Muscle Biopsy: A Modern Approach. London: Saunders; 1973: 20–33.
Lee-de Groot MBE, Tombe AL, van der Laarse WJ. Calibrated histochemistry of myoglobin concentration in cardiomyocytes. J Histochem Cytochem. 1998; 46: 1077–1084.
Deindl E, Buschmann I, Hoefer IE, Podzuweit T, Boengler K, Vogel S, van Royen N, Fernandez B, Schaper W. Role of ischemia and of hypoxia-inducible genes in arteriogenesis after femoral artery occlusion in the rabbit. Circ Res. 2001; 89: 779–786.
Lindner V, Maciag T. The putative convergent and divergent natures of angiogenesis and arteriogenesis. Circ Res. 2001; 89: 747–749.
Mallat Z, Silvestre JS, Le Ricousse-Roussanne S, Lecomte-Raclet L, Corbaz A, Clergue M, Duriez M, Barateau V, Akira S, Tedgui A, Tobelem G, Chvatchko Y, Levy BI. Interleukin-18/interleukin-18 binding protein signaling modulates ischemia-induced neovascularization in mice hindlimb. Circ Res. 2002; 91: 441–448.
Rissanen TT, Markkanen JE, Gruchala M, Heikura T, Puranen A, Kettunen MI, Kholova I, Kauppinen RA, Achen MG, Stacker SA, Alitalo K, Yla-Herttuala S. VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ Res. 2003; 92: 1098–1106.
Kalka C, Masuda H, Takahashi T, Gordon R, Tepper O, Gravereaux E, Pieczek A, Iwaguro H, Hayashi SI, Isner JM, Asahara T. Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res. 2000; 86: 1198–1202.
Shen H, Clauss M, Ryan J, Schmidt AM, Tijburg P, Borden L, Connolly D, Stern D, Kao J. Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes. Blood. 1993; 81: 2767–2773.
Lambrechts D, Storkebaum E, Morimoto M, Del Favero J, Desmet F, Marklund SL, Wyns S, Thijs V, Andersson J, Van M, I, Al Chalabi A, Bornes S, Musson R, Hansen V, Beckman L, Adolfsson R, Pall HS, Prats H, Vermeire S, Rutgeerts P, Katayama S, Awata T, Leigh N, Lang-Lazdunski L, Dewerchin M, Shaw C, Moons L, Vlietinck R, Morrison KE, Robberecht W, Van Broeckhoven C, Collen D, Andersen PM, Carmeliet P. VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet. 2003; 34: 383–394.
Pette D. Historical perspectives: plasticity of mammalian skeletal muscle. J Appl Physiol. 2001; 90: 1119–1124.
Chin ER, Olson EN, Richardson JA, Yang Q, Humphries C, Shelton JM, Wu H, Zhu W, Bassel-Duby R, Williams RS. A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type. Genes Dev. 1998; 12: 2499–2509.
Wu H, Rothermel B, Kanatous S, Rosenberg P, Naya FJ, Shelton JM, Hutcheson KA, DiMaio JM, Olson EN, Bassel-Duby R, Williams RS. Activation of MEF2 by muscle activity is mediated through a calcineurin-dependent pathway. EMBO J. 2001; 20: 6414–6423.
Armesilla AL, Lorenzo E, Gomez dA, Martinez-Martinez S, Alfranca A, Redondo JM. Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression. Mol Cell Biol. 1999; 19: 2032–2043.