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Potential Roles of Vascular Endothelial Growth Factor During Skeletal Muscle Hypertrophy

Huey, Kimberly A.

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Exercise and Sport Sciences Reviews: July 2018 - Volume 46 - Issue 3 - p 195-202
doi: 10.1249/JES.0000000000000152
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Key Points

  • Vascular endothelial growth factor (VEGF) is a well-known angiogenic factor but also is likely involved in adult muscle growth, which involves both angiogenesis and fiber hypertrophy.
  • During the inflammatory response to muscle overload, VEGF acts as a chemoattractant for macrophages, which have been shown to be necessary for subsequent muscle hypertrophy.
  • Prominent muscle hypertrophic signaling pathways including IGF-1-Akt and Wnt-ß-catenin can activate VEGF transcription and secretion from muscle cells.
  • VEGF acts as a signaling molecule between satellite and endothelial cells, which is likely dependent on the close proximity of satellite cells and capillaries.
  • Inducible deletion of VEGF in adult muscle fibers demonstrated the necessity of VEGF for muscle hypertrophy and improved muscle force production in adult mouse muscle.

INTRODUCTION

Vascular endothelial growth factor, a well-known angiogenic factor, is a potent mitogen for endothelial cells, a prosurvival or antiapoptotic factor, and vascular permeability factor (1). Numerous studies have shown that VEGF is essential for capillary maintenance (2), endothelial cell and myofiber survival (2,3), exercise-induced angiogenesis (4), and muscular endurance (4). Taken together, the existing evidence suggests that chronic muscle use such as occurs with endurance exercise or chronic electrical stimulation requires VEGF-dependent angiogenesis, and the angiogenic stimuli include shear stress, increased contractile activity, muscle stretch, and hypoxia (5). The importance of VEGF in vascular remodeling is essentially conclusive in stimuli that promote muscular endurance; however, far less is known about VEGF’s angiogenic and myogenic roles during muscle hypertrophy.

Adult muscle hypertrophy involves coordinating several stages: injury/inflammation (6–8), satellite cell activation (8–12), myonuclear accretion (8,11,13), and contractile protein accumulation (14) and is associated with substantial angiogenesis. Accumulating evidence suggests myofiber VEGF is essential for both hypertrophy and angiogenesis. During adult muscle growth, VEGF may have roles within the inflammatory response, satellite-endothelial cell communication, and known hypertrophic signaling pathways, including insulin-like growth factor-1 (IGF-1)-Akt (15,16) and Wnt-ß-catenin (12,17). Initiation of the inflammatory response is evident within hours of the overload stimulus and generally peaks approximately 5 d later (6,18). The inflammatory response, including macrophage accumulation, is critical for subsequent overload-induced muscle hypertrophy (19). During the inflammatory response, VEGF acts as a chemoattractant for macrophages (20) that initiate the inflammatory response and release growth factors including IGF-1, a well-established anabolic hormone (15,21). The macrophage response includes a critical shift from proinflammatory to antiinflammatory states, which resolves inflammation, contributes to the activation of satellite cells, and supports fiber hypertrophy (22). The ability of macrophages to activate satellite cells is evidenced by the concurrent time frame of the inflammatory response and satellite cell activation. Significant increases in activated satellite cells are evident within 2–3 d after an overload stimulus (10,23). Although the necessity of satellite cells as a source of new myonuclei during adult muscle hypertrophy continues to be debated (24), recent evidence suggests their necessity is age dependent (13). Satellite cells are necessary for myogenesis, defined as processes involved in de novo fiber formation and regeneration (10,13), but these processes play a more minor role in adult muscle growth compared with muscle fiber hypertrophy. Interestingly, satellite cells may contribute to hypertrophy independent of myonuclear accretion or regeneration by regulating accumulation of extracellular matrix components (25,26).

Muscle cells produce and secrete VEGF, have VEGF receptors, and respond to VEGF stimulation (3,27,28). Because contractile activity increases muscle metabolism, angiogenesis could be considered a secondary consequence of the metabolic demands imposed by the increased muscle mass. Muscle fiber hypertrophy involves accumulation of protein mass, and angiogenesis is necessary to deliver oxygen and nutrients to and remove metabolic waste from the muscle fibers. Determining the temporal relation between angiogenesis and muscle fiber growth may inform whether angiogenesis facilitates hypertrophy by nutrient and growth factor delivery to muscle cells or is a response to metabolic stressors in active muscle. However, the mechanisms whereby VEGF contributes to muscle hypertrophy remains under investigation.

This review provides a brief description of the major processes during adult muscle hypertrophy and uses the following models to provide evidence for the potential roles of VEGF during muscle hypertrophy: 1) cultured muscle and endothelial cells, 2) animal studies using a mouse model with inducible VEGF deletion in adult muscle fibers, 3) animal models with manipulations of normal VEGF actions, 4) animal studies using various models of muscle hypertrophy, and 5) human studies using acute or chronic resistance exercise. The review concludes by providing the first direct in vivo evidence of the critical importance of VEGF in muscular and contractile adaptations to a hypertrophic stimulus (29). In a unique transgenic mouse model, mice developed a normal vascular system before specifically deleting VEGF in skeletal myofibers (skmVEGF−/−). VEGF loss in adult mouse muscle fibers resulted in muscular and vascular changes that contributed to an inadequate response to a hypertrophic stimulus as supported by impaired contractile adaptations, fiber hypertrophy, and angiogenesis compared with normal muscle (29). The VEGF-dependent mechanisms underlying these impaired responses to a potent growth stimulus may include the ability of VEGF to attract macrophages to the muscle, VEGF signaling between satellite and endothelial cells, and contributing to the growth promoting actions of IGF-1 and ß -catenin. Although the angiogenic actions of VEGF are well established, studies will be presented supporting the novel hypothesis that VEGF is essential for adult muscle hypertrophy by impacting inflammatory processes, satellite-endothelial cell interactions, and contractile protein accumulation by functioning within known hypertrophic signaling pathways (Fig. 1). An understanding of VEGF’s angiogenic and myogenic actions during adult muscle fiber hypertrophy is critical because inadequate responses lead to muscle dysfunction and weakness.

Figure 1
Figure 1:
Schematic illustration of potential roles of vascular endothelial growth factor (VEGF) during adult muscle hypertrophy. Myofibers produce and secrete VEGF, express VEGF receptors, and are responsive to VEGF stimulation. During the inflammatory response, VEGF acts as a chemoattractant for macrophages that release growth factors including insulin-like growth factor-1 (IGF-1). IGF-1 can act on myofibers, satellite cells, and promote the shift from proinflammatory to antiinflammatory phenotypes that support muscle growth. Prominent muscle hypertrophic signaling pathways, IGF-1-Akt-mammalian target of rapamycin (mTOR) and Wnt/ß-catenin can upregulate VEGF expression and secretion from myofibers. VEGF may act as a bidirectional signaling molecule between satellite and endothelial cells, which relies on close proximity between satellite cells and capillaries.

FUNCTIONAL OVERLOAD MODEL OF MUSCLE HYPERTROPHY

Adult muscle growth in response to a hypertrophic stimulus occurs through coordinated increases in the activation and differentiation of satellite cells (8–12), fiber repair and regeneration (8,10), and contractile protein accumulation (14). Functional overload (FO) is the most common in vivo animal model to study mechanisms of muscle growth. FO involves synergist muscle ablation, and plantaris overload is achieved by surgical removal of the soleus and gastrocnemius. FO is a powerful hypertrophic stimulus because 2 wk of overload results in a nearly doubling of muscle mass (30). FO of the extensor digitorum longus (EDL) or tibialis anterior (TA) also are used as experimental models; however, overload of these muscles is not associated with the robust hypertrophy seen in the plantaris.

Hypertrophy induced by FO of the plantaris provides an in vivo model to study muscle growth, and existing data show growth occurs in a time-dependent sequence of cellular events. There is an initial injury/inflammatory response, activation of muscle precursor cells (e.g., satellite cells), and a sustained increase in contractile protein accretion. Within a week after FO, there is some damage/injury (7,8), inflammation (6), and macrophage infiltration (18,31,32). During the initial few days, increases in muscle weight generally indicate inflammation (6) rather than muscle protein accumulation (14). In response to FO, contractile protein increases in response to factors including increased muscle IGF-1 (15,16), ribosome biogenesis (33), and increased translational efficiency (34).

HYPERTROPHIC STIMULI INCREASE VEGF EXPRESSION IN SKELETAL MUSCLE

The contribution of VEGF during loading-induced hypertrophy in animal and human (i.e., acute and chronic resistance exercise) models of FO is evidenced by increased VEGF mRNA and protein expression. VEGF is upregulated in response to both FO and resistance exercise in humans, but important differences exist between these models. In particular, an FO muscle is subjected to continuous increases in loading and activation during normal movement with body weight as the load. In contrast, during resistance training, the muscle is subjected to loads that are generally greater than body weight during discrete training sessions. In overloaded rat plantaris, VEGF mRNA and protein levels significantly increased after 3 or 7 d of FO (23). VEGF protein levels significantly increased 35% in mouse plantaris after 7 d of FO before returning to baseline levels after 14 d (29). In the rat plantaris overloaded by denervation rather than synergist ablation, VEGF mRNA and protein increased 1.8- and 8-fold, respectively, after 14 d (35).

Acute and chronic resistance training exercise also can upregulate VEGF expression in human skeletal muscle. For example, a single bout of high-intensity leg extensor exercise was associated with a significant increase in human skeletal muscle VEGF mRNA and protein (36). Ischemic muscle provides a strong stimulus for hypoxia-inducible factor-1 alpha (HIF-1α) upregulation, which activates genes with a hypoxia-responsive element, including VEGF. Although ischemia alone is not a sufficient stimulus for muscle growth, muscle contraction may produce local hypoxia because a single eccentric exercise bout in a normoxic environment significantly increased HIF-1α activation of the VEGF gene (37). In addition to acute responses, 8 wk of chronic resistance training resulted in significant increases in muscle cross-sectional area (CSA) and strength and a 51% increase in VEGF protein (38).

VEGF AND THE INFLAMMATORY RESPONSE

The observation that VEGF is upregulated during the inflammatory response to overload and interacts with inflammatory cells suggests its role in the initiation and resolution of inflammation (6), which is important for subsequent muscle repair and growth (31). Specifically, blocking the inflammatory response to FO with a COX-2 inhibitor decreased macrophage accumulation, cell proliferation, and urokinase-type plasminogen activator (uPA) activity and prevented the increases in plantaris mass and protein content (31). One stimulus for the early inflammatory response is some degree of muscle damage/injury because the force requirements of the muscle exceed normal demands, and myogenic adaptations have yet to fully occur (8,31,32). The inflammatory response to skeletal muscle FO is characterized by macrophage accumulation, which can initiate inflammatory actions and produce growth factors (18,22). VEGF is a chemoattractant for macrophages (20), suggesting that VEGF may recruit macrophages to hypertrophying skeletal muscle. During the early response to overload, VEGF may be released by activated satellite cells because VEGF inhibition in human muscle precursor cells reduced monocyte chemotaxis by 44% (20). Macrophages can be an important early source of IGF-1 in injured muscle (21) and increase muscle cell proliferation and differentiation, in vitro (39). Furthermore, macrophages recruited to skeletal muscle can be converted from an inflammatory profile to an antiinflammatory profile that supports myogenesis and muscle fiber growth (22). In vivo, depletion of antiinflammatory macrophages reduced the diameter of regenerating fibers (22). In addition, the importance of macrophages in normal muscle hypertrophy in response to FO comes from mice lacking uPA activity. uPA is necessary for macrophage accumulation, and in mice lacking uPA, the hypertrophic response to FO was significantly attenuated compared with wild-type (WT) mice (19). In addition, macrophage depletion with clodronate liposomes in WT mice blunted FO-associated muscle hypertrophy (19).

Cellular level changes during the inflammatory response to FO suggest an important role for VEGF as a chemoattractant for macrophages; however, functional responses also support a role for VEGF during FO-induced hypertrophy. First, macrophage accumulation requires adequate blood perfusion, and experiments using skmVEGF−/− mice demonstrated that VEGF was necessary for normal increases in perfusion to active muscles (40). Contraction-induced blood perfusion of the mouse gastrocnemius during 3 min of electrically stimulated muscle contractions was 85% lower in skmVEGF−/− than WT mice. Capillary loss did not cause this reduced perfusion because there was no evidence of capillary rarefaction in skmVEGF−/− muscles compared with WT. Reduced muscle perfusion to overloaded muscles likely impairs macrophage delivery to the muscle cells by the microcirculation. As the aforementioned evidence suggests, an impaired macrophage response in an overloaded muscle likely impairs long-term muscle growth. Functional changes in the microcirculation would impair the initiation and resolution of the inflammation, potentially contributing to reduced contractile function. Evidence suggesting greater muscle damage/injury and prolonged inflammation in the absence of VEGF is that muscle force fell significantly below control levels after 7 or 14 d of FO in skmVEGF−/− mice compared with sham (29). In contrast, WT mice maintained maximal force production at control levels and significantly increased force after 30 d of FO (29).

VEGF AND BIDIRECTIONAL SIGNALING BETWEEN SATELLITE AND CAPILLARY ENDOTHELIAL CELLS

Because macrophages can release factors such as IGF-1 (18), which can activate satellite cells (15), they likely contribute to satellite cell activation in response to FO in rodents (8–12) and resistance exercise in humans (41,42). Although the necessity of satellite cells as a source of new myonuclei during muscle growth is not entirely clear, satellite cells are critical for de novo fiber formation and fiber regeneration (10,13). The precise role of satellite cells remains controversial (24), and readers are referred to a recent review discussing the complexity of factors impacting satellite cell functions during adult muscle growth (43). Briefly, evidence from satellite cell–depleted mice suggests that satellite cells are not required for FO-induced hypertrophy and myonuclear accretion in mature mice (>4 months) for up to 8 wk (10). It also was reported that myonuclei in satellite cell-depleted mouse muscle were able to increase transcriptional output to meet the demands during the early stages of hypertrophy (44). However, long-term hypertrophy may require satellite cells (11). The apparent necessity of satellite cells for longer term hypertrophy may be related to a novel satellite cell function, regulation of the extracellular matrix (11,25,26). During prolonged overload, the reduced hypertrophic response in the absence of satellite cells was associated with excess extracellular matrix accumulation, which may have restricted fiber hypertrophy (11,25).

Satellite cells are located beneath the muscle fiber basal lamina, and the area surrounding satellite cells has been characterized as a vascular niche in which satellite cells are closely associated with capillary endothelial cells independent of the state of quiescence, proliferation, or differentiation (45,46). Using mice genetically engineered to visualize satellite cells in muscle cross sections (Myf5nlacZ/+ and Myf5GFP-P/+) it was demonstrated that 82% of satellite cells were less than 5 μm from endothelial cells in the TA muscle (45). Furthermore, in human deltoid muscle, satellite cells were co-localized with capillaries significantly more than myonuclei (88 ± 6% vs 54 ± 3%, respectively) (45). Interestingly, one of the factors that regulates satellite cell activity also may upregulate VEGF expression. MyoD is a member of the myogenic regulatory family of genes that direct the activation, proliferation, and differentiation of satellite cells. MyoD augments myonuclear number and cellular volume of muscle fibers and directly interacts with the VEGF promoter (27). In overloaded rat plantaris, MyoD protein increased 2 and 3 d after FO, which temporally matched increases in VEGF protein (23).

This close proximity of satellite and endothelial cells would be expected to have an important role in reciprocal VEGF signaling between these cells during angiogenesis and myogenesis (45). Myogenesis refers to processes contributing to muscle repair, regeneration, and de novo fiber formation and requires satellite cell activation (10,13). The majority of adult muscle growth is primarily due to the hypertrophy of existing fibers and associated increases in myonuclei, but myogenesis does occur. Consequently, satellite cells activated by overload can potentially contribute to myogenesis, myonuclear accretion, and extracellular matrix regulation in hypertrophying fibers (43). One mechanism to activate and sustain satellite cells is secretion of VEGF as well as other growth factors including basic fibroblast growth factor (bFGF), IGF-1, and hepatocyte growth factor (HGF) by capillary endothelial cells (45,47). The importance of endothelial cell–derived growth factors in satellite cell growth was directly evidenced by 41%–62.5% reductions in endothelial cell-sustained human myogenic precursor cell growth after IGF-1, HGF, bFGF, platelet-derived growth factor, or VEGF inhibition (45). Human muscle precursor cells treated with recombinant VEGF showed significantly greater increases in cell density than control over 8 d (45). Furthermore, although VEGF can activate satellite cells, they also can be a source of muscle VEGF as differentiating human satellite cells secreted VEGF (20). In a reciprocal signaling scenario, capillary loss in the absence of VEGF could reduce the satellite cells available for myogenesis, incorporation into mature myofibers, or extracellular matrix regulation during the growth process.

In addition to the in vitro evidence, animal and human models support an important role for VEGF signaling between endothelial and satellite cells. Changes in diseased or endurance-trained human muscle demonstrate the importance of a close physical relation between capillaries and satellite cells. In patients with amyopathic dermatomyositis, a form of dermatomyositis characterized by skin pathology without muscle weakness, there were respective 45% and 53% reductions in capillary number and satellite cell number per myofiber without any fiber damage or inflammation (45). On the other hand, in trained athletes, capillary and satellite cell numbers increased 33% and 56%, respectively, and satellite cells remained close to capillaries (45). Endothelial-satellite cell interactions also may have a role in the temporal relation between angiogenesis and fiber hypertrophy during chronic resistance training. A recent study reported that 12 wk of resistance training in adult men was associated with increases in type I and type II fiber CSA and capillary-to-fiber ratio (48). Over 12 wk, type I and type II fiber CSA increased approximately 15% and 28%, respectively, whereas capillary-to-fiber ratio increased approximately 6% in type I fibers and 9%–15% in type II fibers. Interestingly, although temporal associations between angiogenesis and hypertrophy were evident, the degree of hypertrophy was greater, suggesting angiogenic responses may not always be able to match myogenic responses.

Aging associated reductions in muscle mass and capillarization in human muscle also suggest that the close proximity of capillaries and satellite cells is important for growth (42,46). Finally, one could speculate that the significant reduction in capillary-to-fiber ratio in skmVEGF−/− mouse muscle may have led to fewer activated satellite cells, thus contributing to the blunted hypertrophic response to FO (29). Taken together, VEGF could directly impact satellite cell number and activation as a signaling molecule and indirectly by affecting muscle capillary density because close proximity of satellite and endothelial cells may be critical for satellite-endothelial cell interactions during muscle growth (45,46).

Age-Associated Changes in Muscle Mass and Capillarity

Changes in aged muscle indirectly suggest the importance of cooperative signaling between satellite and endothelial cells during muscle growth. Age-related declines in muscle mass are well established, and satellite cells in aged muscle are located farther from capillaries compared with young muscle (46). Thus, if close proximity of endothelial and satellite cells is a factor during normal muscle growth, morphometric changes associated with aging may compromise significant hypertrophy. The reduced proximity between satellite cells and capillaries may be due to a decreased ability of aged satellite cells to promote angiogenesis and maintain normal capillarity. Adult rat satellite cells cocultured with microvascular fragments elicited a greater angiogenic response compared with aged satellite cells (49). In addition, soluble proangiogenic factors (e.g., VEGF) secreted from aged satellite cells decreased compared with adult cells (49). Although satellite cells can promote angiogenesis, the loss of capillaries in an aged muscle could in turn negatively impact satellite cells because capillary endothelial cells secrete VEGF as well as other growth factors including bFGF, IGF-1, and HGF, which can activate and sustain satellite cells (45,47).

A recent study in aged individuals investigated the angiogenic and hypertrophic responses to 24 wk of whole-body resistance exercise (42). At the initiation of the training, subjects were classified as either low or high based on type II capillary-to-fiber perimeter exchange ratio. At the end of the training period, both type II fiber size and satellite cell content were significantly increased only in those with a high baseline type II muscle fiber capillarization. Neither fiber size nor satellite cell content was increased in those with initial low muscle fiber capillarization. Taken together, it was suggested that the spatial proximity of satellite cells and the microvascular may be important for satellite cell activation. Interestingly, no significant increases in capillarization were found. The authors concluded that type II muscle fiber capillarization at the initiation of resistance training was critical for significant fiber hypertrophy. Muscle VEGF was not measured in this study; however, it would be interesting to determine whether initial muscle fiber capillarization also impacts VEGF upregulation.

A rodent model also was recently used to investigate the angiogenic and hypertrophic response to denervation-induced overload of the plantaris in adult and aged mice (50). In both adult and aged mice, denervation‐induced overload induced increases in muscle mass and fiber cross-sectional area; however, the response was attenuated in old compared with young mice. Aging had a more dramatic effect on angiogenesis as evidenced by increases in capillary-to-fiber ratio that were significantly less in aged (9%) than adult (59%) muscle. The authors suggested that the attenuated angiogenesis in aged muscle likely contributed to the blunted hypertrophic response.

Muscle levels of VEGF can be reduced with aging, which would likely contribute to impaired angiogenesis and potentially muscle growth. VEGF mRNA expression was significantly lower in aged compared with adult rat satellite cells (49). This agrees with reports of lower VEGF levels in both resting and acutely exercised muscle in aged than young men and women (51,52). Muscle VEGF levels were 35% lower at rest and 50% lower after acute exercise in aged compared with young women (52). Muscle levels of the VEGF receptor, Flt-1, also are significantly lower in aged than adult mouse muscle, independent of overload status (50). Thus, angiogenesis and fiber growth in aged muscle may be due to both lower levels of VEGF and a reduced ability to respond to VEGF. Cooperative VEGF signaling between myogenic and angiogenic cells potentially contributes to impaired muscle hypertrophy in aged muscle and is an important area for future research.

VEGF DIRECTLY MODULATES HYPERTROPHIC SIGNALING IN MUSCLE CELLS

Akt-mTor

In addition to signaling between satellite and endothelial cells, VEGF may function within known muscle growth pathways including Akt-mTor and Wnt-β-catenin. Hypertrophic stimuli activate numerous signaling pathways, and the Akt-mTOR pathway and its downstream targets, p70S6K and 4E-BP1, are strongly implicated in muscle hypertrophy (16). FO increases Akt-mTOR signaling (12,16,34) and associated increases in muscle protein and DNA content. Several growth factors, including IGF-1, can activate Akt-mTOR signaling leading to phosphorylation and activation of Akt (15). The absence of myocyte-derived VEGF was associated with an exaggerated IGF-1 response to FO compared with WT (29). Seven or 14 d of FO significantly increased IGF-1 levels in both genotypes. However, IGF-1 responses in skmVEGF−/− mice were 45% and 40% higher after 7 or 14 d of FO, respectively, compared with WT. In spite of the more robust IGF-1 response in the skmVEGF−/− mice, FO-associated increases in plantaris mass and fiber cross-sectional area were significantly attenuated compared with WT. Interestingly, the exaggerated IGF-1 response in the skmVEGF−/− mice was not associated with greater increases in either total or phosphorylated Akt compared with WT mice after 7 d of FO. This suggests IGF-1 and VEGF have synergistic effects on Akt and that the mechanism of growth retardation lies downstream of Akt.

Akt activation can increase VEGF mRNA (28), and additional evidence suggests VEGF also may be a downstream factor within IGF-1-Akt signaling during muscle hypertrophy. For muscle-derived VEGF to have autocrine and paracrine effects on muscle and endothelial cells, it must be secreted into the extracellular space. Takahashi et al. (28) reported that addition of IGF-1 increased VEGF secretion from C2C12 cells 12.8-fold over control. Transfection of a dominant negative Akt blocked IGF-1-stimulated VEGF secretion whereas transfection of a constitutively active Akt caused the greatest increase in VEGF secretion (28). IGF-1 addition or transfection of active Akt induced similar myotube hypertrophy and leucine incorporation (28). Secreted VEGF can promote dose-dependent myoblast migration, which was blocked by VEGFR-1 (Flk-1) inhibitors (3). Furthermore, treatment with a soluble VEGF inhibitor, sFlt1, effectively reduced myogenesis and myotube hypertrophy in C2C12 cells (27). Constitutively active Akt also stimulated VEGF secretion from human skeletal muscle cells (28). In vitro evidence provides compelling evidence that VEGF signaling is necessary for myogenesis and increased myotube size, but additional verification is necessary.

More importantly, Akt signaling promoted VEGF synthesis and myofiber hypertrophy in vivo. Constitutively active Akt injected into mouse gastrocnemius increased VEGF in the injected portions and elevated levels of circulating VEGF 7 and 14 d after injection (28). It is probable based on existing data that Akt signaling is critical for VEGF secretion from myofibers, which serves to both maintain capillary density and promote growth in response to hypertrophic stimuli. VEGF also could act in an autocrine manner because C2C12 cells express Flt-1 and VEGFR-2 (KDR/flk-1) and directly respond to VEGF stimulation (27).

Wnt/β-catenin

FO can activate Wnt/β-catenin signaling, and evidence suggests that VEGF also functions within this pathway during adult muscle growth (17). The Wnt pathway contributes to skeletal muscle development and stem cell activation, and Wnt expression was significantly increased after 7 d of overload in the rat plantaris (12). β-catenin activity in the cytoplasm or as a nuclear transcription factor is a classic marker of Wnt activation. In vivo, 7 d of FO increased overall and nuclear β-catenin protein 301% and 434%, respectively, in mouse plantaris (17). This suggests hypertrophic stimuli promotes transcription of β-catenin-induced genes, which may include VEGF. The human VEGF gene promoter contains binding sites for β-catenin based on studies in nonmuscle cells (53). Potential hypertrophic actions of the β-catenin-VEGF pathway are supported by in vitro evidence. β-catenin overexpression in C2C12 myocytes promoted cellular hypertrophy, which was associated with increased VEGF expression and secretion (54). β-catenin transcriptional activity mediated increases in myocyte VEGF secretion because transfection of dominant negative N-cadherin, which prevents β-catenin-mediated transcription, prevented this increase (54). The β-catenin pathway also may stimulate VEGF signaling between endothelial and muscle cells. C2C12 cells transfected with β-catenin-secreted VEGF and addition of this supernatant to endothelial cells significantly increased endothelial cell proliferation (54). Importantly, addition of an anti-VEGF neutralizing antibody to the endothelial cells reversed this proliferative response. Taken together, β-catenin-associated increases in myocyte-derived VEGF may act in an autocrine fashion to stimulate hypertrophy and in a paracrine fashion to promote endothelial cell proliferation and capillary formation.

In addition to in vitro evidence supporting β-catenin-VEGF actions in muscle and endothelial cells, its importance has been demonstrated in a mouse hindlimb ischemia model (54). Hindlimb ischemia resulted in a significant, but transient, increase in β-catenin expression, demonstrating its role in angiogenesis. Furthermore, transgenic β-catenin overexpression in ischemic muscle significantly increased VEGF expression and angiogenesis compared with control tissues. Coupled with in vitro evidence, elevated muscle VEGF may accelerate endothelial cell proliferation to support fiber repair and hypertrophy.

VEGF IS NECESSARY FOR MUSCLE HYPERTROPHY AND INCREASED STRENGTH

The hypothesis that VEGF contributes to muscle hypertrophy has been directly tested in vivo using VEGF inhibitors or conditional deletion of skeletal muscle VEGF. In the overloaded mouse EDL, the presence of a VEGF receptor decoy (VEGF-Trap) impaired angiogenesis and hypertrophy. Fourteen days of FO increased EDL mass 20% or 14% under control conditions or with VEGF-Trap, respectively (55). Overloaded EDL muscles do not show the degree of hypertrophy observed with plantaris overload, which is likely related to normal usage patterns of ankle flexors versus extensors. In general, antigravity ankle extensors are used more than flexors, thus the overloaded plantaris experiences greater increases in loading than the overloaded EDL. Because EDL hypertrophy is modest and the VEGF-trap may have unknown and undesired adverse effects, conditionally deleting VEGF in the plantaris can more directly test VEGF actions during robust hypertrophy.

Our laboratory was the first to establish that VEGF is necessary for capillary maintenance that supports muscle hypertrophy and improved force production in hypertrophying adult mouse plantaris (29). SkmVEGF−/− mice developed a normal vascular system before specific targeting of the VEGF gene in skeletal myofibers using a tamoxifen-dependent inducible human skeletal muscle alpha-actin sequence. These experiments assessed muscular and vascular responses in skmVEGF−/− mice to 7, 14, or 30 d of FO. Hypertrophy of plantaris fibers and mass were significantly blunted compared with WT after 30 d of FO (Fig. 2), despite compensatory increases in IGF-1. Although FO did not induce angiogenesis in WT mice, capillary-to-fiber ratio was reduced below sham levels in skmVEGF−/− mice after 14 or 30 d of FO. The blunted hypertrophic response coupled with lack of capillary maintenance in skmVEGF−/− plantaris compromised functional adaptations as evidenced by reduced force production compared with WT after 14 or 30 d of FO (Fig. 3). Greater fiber hypertrophy and higher force production in WT mice despite the lack of angiogenesis suggests other critical roles for VEGF in response to a hypertrophic stimulus. Although angiogenesis may not always match muscle growth during rapid hypertrophy with normal VEGF levels, VEGF loss severely compromises functional adaptations due to capillary loss coupled with reduced fiber area.

Figure 2
Figure 2:
Changes in plantaris muscle mass and mean fiber area after 7, 14, or 30 d of functional overload (FO) in wild-type (WT) and skeletal muscle VEGF gene-deleted (skmVEGF-/-) mice. Average plantaris mass normalized to body mass (A) and mean fiber area (B) for sham and FO mice. Mean ± SE, *significant effect of FO (significantly different than sham within genotype), †significant difference between genotypes (P < 0.05), n = 8–12/FO group at each time point and n = 16–18 (mass) or n = 9 (fiber area) in sham groups. [Adapted from (29). Copyright © 2016 The American Physiological Society. Used with permission.]
Figure 3
Figure 3:
Changes in plantaris maximal isometric force after 7, 14, or 30 d of functional overload (FO) in wild-type (WT) and skeletal muscle VEGF gene-deleted (skmVEGF-/-) mice. Average maximal isometric force in torque relative to body mass (BM). Mean ± SE, *significant effect of FO (significantly different than sham within genotype), †significant difference between genotypes (P < 0.05), n = 8–12/FO group at each time point and 16–18 in sham groups. [Adapted from (29). Copyright © 2016 The American Physiological Society. Used with permission.]

CONCLUSIONS

In summary, both in vivo and in vitro evidence support the hypothesis that VEGF is critical for normal adult muscle growth in response to a hypertrophic stimulus. Early in the response to FO, VEGF may help maintain normal blood flow during the dramatic increases in muscle contractile activity, which would ensure macrophage and growth factor delivery to muscles. Subsequently, macrophages can secrete VEGF and other growth factors. An impaired growth response to FO in uPA-deficient mice or macrophage depletion in WT mice demonstrates the importance of macrophages during muscle hypertrophy. VEGF also acts as a signaling molecule between satellite and endothelial cells, which can promote satellite cell activation and angiogenesis, respectively. Furthermore, in vivo evidence in humans supports the premise that the close proximity of satellite cells and capillaries is important for normal adult muscle hypertrophy.

The skmVEGF−/− mouse model will be invaluable to further investigate the roles of VEGF in muscle hypertrophy. For example, it could be hypothesized that in skmVEGF−/− mice, the impaired angiogenic, hypertrophic, and contractile adaptations to FO could be due to factors including an impaired macrophage response, less activated satellite cells, and alterations in IGF-1-Akt and β-catenin signaling. This was evidenced by both reduced capillary density and fiber cross-sectional area in skmVEGF−/−compared with WT mice after 30 d of FO. Because activated IGF-1-Akt can lead to transcriptional upregulation and subsequent secretion of VEGF from muscle cells, it was interesting that the loss of muscle VEGF was associated with an exaggerated IGF-1 response, which was unable to overcome the attenuated growth response. This exaggerated IGF-1 response to FO in skmVEGF−/− mice should be investigated to understand interactions between VEGF and IGF-1 during muscle growth. Finally, the necessity of VEGF in macrophage accumulation and satellite cell activation and number in overloaded muscle is warranted in skmVEGF−/− mice based on their likely roles during hypertrophy.

Acknowledgment

This study was supported by a National Institute of Arthritis and Musculoskeletal and Skin Diseases grant R15AR060469 to Kimberly Huey.

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Keywords:

skeletal muscle hypertrophy; vascular endothelial growth factor; satellite cells; endothelial cells; Akt, ß-catenin

Copyright © 2018 by the American College of Sports Medicine