Abstract
Background/Aim: Angiogenesis induced in muscles or massaged tissue is thought to support their regeneration and performance. Therefore, different methods that could promote angiogenesis are investigated. The aim of this study was to examine whether the use of the foam roller massager for lower limb muscles affects VEGF-A and FGF-2 levels in young men. Materials and Methods: The study group included 60 healthy young men attending Military University of Land Forces, Wroclaw, Poland. The participants were randomly divided into two groups. The experimental group included 40 individuals who performed self-massage of the lower limbs using a foam roller. The control group comprised 20 individuals who did not perform massage. Massage was applied to lower limb muscles four times a week for seven weeks. Blood was collected before the experiment and after weeks 1, 3, 5, and 7. ELISA was used to determine changes in VEGF-A and FGF-2 levels in blood serum. Results: The results of the study demonstrated a significant increase in VEGF-A serum levels in the group of individuals who underwent massage each week compared to VEGF-A concentrations before the experiment. The increase in VEGF-A levels in the experimental group was observed throughout the experiment compared to the control group. No significant changes in serum FGF-2 levels were found. Conclusion: The use of a foam massage roller increased VEGF-A serum levels, which may indicate stimulation of angiogenesis.
Massage is commonly used in therapeutic and preventive treatments, cosmetology, and biological regeneration (1-3). Despite the widespread use of massage, only a few of the commonly accepted mechanisms of its action have been confirmed by objective and reliable studies. Unfortunately, knowledge about massage and its mechanisms means that its value, especially that related to the aspects of physiotherapy treatment, is not fully understood. Due to advanced research methods, including molecular biology techniques, it is possible to examine the mechanisms of action of massage (4-6). The probable mechanism of the action of massage is mechanotransduction, which transforms a mechanical factor into signals at the cellular level. It involves the cell cytoskeleton, which transfers the forces induced by the mechanical stimulus from the extracellular space to the cell interior via integrins. This leads to a targeted action that stimulates the expression of relevant genes, including those which may initiate angiogenesis (e.g., VEGF-A) (7-10).
Normal blood supply determined by the quality and quantity of blood vessels in tissues is one of the key determinants of their normal functioning. It is also important at the time of increased demand for nutrients and oxygen during exercise. The formation of new blood vessels may be essential for repair after injury and recovery after exercise and associated muscle strain (2, 11, 12). Stimulation of angiogenesis in the massaged tissue is one of the mechanisms of massage that has already been partially confirmed in animal studies. Recently, animal studies have shown that a mechanical factor in the form of massage applied to the tendon and belly of the long finger flexor muscle in rats simultaneously subjected to running training initiates angiogenesis. Through the expansion of the vascular network and better blood circulation, it can contribute to i) the efficiency of muscles and tendons; ii) promotion of structural remodelling of the tendon tissue; iii) creation of better conditions for regeneration and repair; and iv) stimulation of regenerative processes in the skin (13-17).
Secreted proangiogenic factors include cell mediators that stimulate proliferation and maturation of endothelial cells, e.g., vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (18-21).
VEGF is a key regulator of blood vessel formation (18, 22). It is synthesised by many cell types (23-25). The VEGF family consists of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and the placental growth factor (PlGF) (26, 27). These factors bind to specific receptors present on endothelial cells (27, 28). Activation of these receptors leads to proliferation of endothelial cells and promotes their growth, migration, and spatial organisation during vessel formation (26, 27, 29, 30). As presented in the reviews of Lal et al. (31) and Mukund and Subramaniam (32), VEGF-A regulates angiogenesis through promoting proliferation and migration of endothelial cells (31, 32). VEGF-B enhances cell survival and adaptive hypertrophy (31) and is supposed to increase capillary diameter and artery size (31). VEGF-B is considered to play an indirect role in angiogenesis (31). VEGF-C and VEGF-D are summarized as responsible for lymphoangiogenesis (31, 33). The production of VEGF is stimulated in an environment with reduced partial pressure of oxygen. Chronic hypoxia induces cellular production of hypoxia inducible factor (HIF), which is a transcription factor that stimulates the production of VEGF (23-25). The basic fibroblast growth factor (bFGF) is also one of the best-known modulators of angiogenesis. The FGF family comprises 22 proteins (34). FGF-2, also known as basic FGF or bFGF, is a paracrine fibroblast growth factor that can stimulate angiogenesis and vasculogenesis, promoting the growth of new blood vessels during embryogenesis and wound healing. bFGF is a potent inducer of angiogenesis in conditions such as limb ischemia and ischemic heart disease (35).
Recently, different methods that could improve body performance have been evaluated (2). These techniques might also be applied to support biological regeneration. This regeneration has become a commonly used method to support the regeneration of the body after physical activity. Massage with a roller is a type of treatment that is becoming more commonly used in biological regeneration and physiotherapy. So far, the effects of this form of massage have been poorly studied and objective research is warranted to confirm its effect on the body. However, it can be assumed that roller massage is a mechanical stimulus whose effects may be similar to those already proven by other forms of massage (36).
The systematic review of Guo et al. (37) concludes massage following strenuous exercise might act positively on muscle performance and relief from DOMS (delayed onset muscle soreness) (37). Foam roller and roller massager might improve pre- and post-exercise muscle performance (38). The study of Hodgson et al. (39) investigated the effects of four week-roller massage on muscle characteristics (39). However, the results showed this type of massage did not affect significantly muscle performance (39). The effect of massage on inflammatory markers levels and muscle performance was examined in nine male volunteers (40). It was demonstrated muscle soreness was not lessened by massage (40). Percussive massage could induce angiogenesis in type I skeletal muscle fibers (41). The pilot study of Best et al. (42) showed the positive effects of massage (42). The experiments performed on tibialis anterior muscles of rabbits demonstrated massage enhanced angiogenesis and regeneration of muscle fibers (42).
Based on animal studies which demonstrated that massage increases the secretion of proangiogenic growth factors such as VEGF-A and FGF-2, this study examined whether lower limb muscle massage performed with a foam roller four times a week for seven weeks could also increase the synthesis of these factors in young men (13, 15, 16). There have been no reports on the effects of a foam roller massage on VEGF-A and FGF-2 levels in humans. A possible change in their levels in humans would confirm the mechanism of action of foam roller massage in physiotherapy, biological regeneration, and cosmetology.
This effect would be consistent with the concept that massage (as a mechanical factor) causes cellular changes, converting mechanical energy into biochemical stimuli. The effect of these changes may include increased expression of proangiogenic factors. Therefore, effects of foam roller massage might lead not only to reflex changes in the form of normalisation of tension in the massaged tissues but also to the stimulation of their structural transformation.
Blood serum was used for the experiments due to the impossibility of collecting the skeletal muscle tissue from the massage area (ethical aspects). Blood serum concentrations of VEGF-A and FGF-2 indirectly reflect their synthesis and secretion by the massaged muscle fibers (43-45).
Materials and Methods
Study group. Sixty male undergraduate students at the Military University of Land Forces in Wroclaw were enrolled in the experiment. The study was approved by the Research Ethics Committee at the Wroclaw University of Health and Sport Sciences (consent No. 32/2018; 10th October 2018). Informed consent was obtained from all the subjects. The participants were 19-25 years of age. The body weight of the volunteers ranged from 75 kg to 85 kg. The participants were randomly divided into two groups. In the experimental group, including 40 participants, massage was performed with a foam roller, while in the control group (n=20), massage was not performed. The students were accommodated at the university facilities and their daily routine was strictly defined according to the daily schedule at the Military University of Land Forces. The physical activity schedule of the participants included: 20-min physical activity in the morning (from Monday to Friday) and Physical Education classes (6 h/week).
The exclusion criteria were as follows: injuries in the musculoskeletal system in the last six months, smoking or use of other stimulants, acute and chronic diseases, infections located in the massage area, recent surgical interventions, loss of skin integrity, inflammatory conditions, febrile states, tumours, aneurysms, osteoporosis, hypersensitivity of the skin, and diabetes. All participants underwent body composition analysis using a TANITA BC-418 MA analyser (Table I).
Characteristics of the study participants.
Experimental conditions. Self-massage was performed using a foam roller (33 cm in length and 14 cm in diameter) with an irregular surface (ridged). A hard roller made of EVA (Ethylene Vinyl Acetate) foam (Coolmed, Poland) was used in the study. The participants were instructed how to perform self-massage by a qualified staff. The procedure was performed in the following sequence: massage of the posterior aspect of the lower leg (triceps surae muscle), massage of the posterior aspect of the thigh (biceps femoris muscle, semitendinosus muscle, semimembranosus muscle), massage of the medial aspect of the thigh (adductor longus, brevis and magnus), massage of the lateral aspect of the thigh (biceps femoris and quadriceps femoris muscles), massage of the gluteal region (gluteus maximus muscle), massage of the anterior aspect of the thigh (quadriceps femoris muscle). The procedure was performed on each limb separately (Figure 1). The massage time was 9 min for each lower limb. The massage time for each muscle group was 1.5 min at a speed of 2.5 cm/s. It was performed under the supervision four times a week at around 1:00 p.m. for seven weeks (Figure 2). Blood samples were taken before the experiment and during the course of the experiment.
Self-massage performed by the study participants (A-C).
Timeline showing the duration and conditions of the experiment in each group.
Blood collection. Venous blood (9.8 ml) was collected into two S-Monovette serum tubes [4.9 ml, Sarstedt, (Nümbrecht, Germany)] with a coagulation activator. The blood was allowed to clot for 10-15 min in an upright position at room temperature. The samples were centrifuged at 4,000×g for 5 min at room temperature. The supernatant was partitioned into 1.5 ml tubes (Sarstedt) and stored at −80°C until further analysis. Blood samples from the study participants were collected before the experiment (week 0) and at weeks 1, 3, 5 and 7 of the experiment.
ELISA method. VEGF-A and FGF-2 concentrations were determined using ELISA kits for VEGF-A [ab119566, Abcam (Cambridge, UK)] and FGF-2 (ab99979, Abcam). VEGF-A and FGF-2 protein concentrations were determined according to the manufacturer’s instructions. Diluted blood serum (100 μl) or standard curve samples (100 μl) were added to the wells. To determine VEGF-A serum concentrations, plates were washed with wash buffer after 2 h of incubation at room temperature (RT). To assess FGF-2 concentrations, serum samples were incubated overnight at 4°C. Then, 100 μl of a biotin-conjugated polyclonal antibody (Biotin-conjugate anti-Human VEGF-A polyclonal antibody or Biotinylated anti-human FGF basic Detection antibody) was added and incubated for 1 h at room temperature. Next, streptavidin-HRP conjugate solution (100 μl) was added and incubated for 1 h at room temperature for VEGF-A and 45 min for FGF-2. Then, the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (100 μl) was applied and incubated for 30 min to induce the colour reaction, which was stopped by adding the Stop solution. The absorbance was measured using an Infinite 200 plate reader (TECAN, Männedorf, Switzerland) at 450 nm using a 620 nm correction filter. Protein concentrations were determined using Four Parameter Logistic (4-PL) standard curves.
Statistical analysis. Statistical analysis was performed using Prism 5.0 (GraphPad) and Statistica 10 (StatSoft) software. The distribution of data was determined using the Kolmogorov-Smirnov test. The results were statistically analysed using the Kruskal-Wallis test with the post-hoc Dunn’s multiple comparison test and the two-way ANOVA with Bonferroni post-test. The correlation between VEGF-A and FGF-2 levels was assessed using the Spearman correlation analysis. The results were considered statistically significant at p<0.05.
Results
The aim of this study was to evaluate VEGF-A and FGF-2 levels in blood serum after foam roller massage bouts. In the next step, the analysis of correlation between VEGF-A and FGF-2 serum concentrations was performed.
Serum VEGF-A levels in the study population. No significant changes in VEGF-A levels were observed in the control group except for week 5 when a significant increase compared to week 0 was found. A slight increase in VEGF-A levels was observed during the experiment with the exception of week 7 when a decrease in VEGF-A levels in relation to week 5 was noted (Figure 3A).
VEGF-A serum concentrations in the control group (A) and the experimental group (B) (*p<0.05, **p<0.01, ***p<0.001).
However, VEGF-A concentrations significantly increased in the experimental group after 1, 3, 5, and 7 weeks of massage compared to the concentrations assessed before the experiment (week 0). Furthermore, a slight decrease in VEGF-A levels was observed at week seven compared to week 5 (Figure 3B).
VEGF-A concentrations in the experimental group significantly increased after 1, 3, 5, and 7 weeks compared to the control group (Figure 4). No statistically significant difference was found between the group that underwent massage and the control group at the beginning of the experiment (week 0).
VEGF-A serum concentrations in the control group and the massaged group (*p<0.05, **p<0.01).
Serum FGF-2 levels in the study population. No statistically significant changes in FGF-2 concentrations were observed in the control (Figure 5A) and massaged (Figure 5B) groups. A statistically nonsignificant increase in FGF-2 concentrations was found in the massaged group during the course of the experiment.
FGF-2 serum concentrations in the control group (A) and the massaged group (B).
No statistically significant differences in FGF-2 levels were reported between the experimental group and the controls in individual weeks (Figure 6).
FGF-2 serum concentrations in the control group and the massaged group.
Correlation between serum levels of VEGF-A and FGF-2. To investigate the relationship between VEGF-A and FGF-2 serum levels, a correlation analysis was performed (Figure 7). No significant relationship between VEGF-A and FGF-2 levels evaluated for the indicated time frame (week 0-7) was shown (r=0.1283, p=0.1080).
Correlation analysis of the relationship between VEGF-A and FGF-2 serum levels (weeks 0-7).
Discussion
Angiogenesis is one of the processes involved in the normal functioning of the body and is activated via the increased production of proangiogenic factors (24, 35). The expansion of the blood vessel network may be important for recovery from exercise and associated muscle strain, as well as for repair processes after injury (2, 11, 12).
The primary role of massage is to change the characteristics of the connective tissue by applying force. The massaged tissue is stretched to the limit of its elasticity, which allows the activation of mechanoreceptors resulting in reflex changes. It also allows transmission of the acting force to the fibres forming the cell cytoskeleton. Through these actions, the mechanical factor reaches the inside of the cell, triggering a series of reactions at the cellular level (mechanotransduction). The effects of massage can include rapid changes in hormonal regulation (e.g., increase in endorphins) as well as long-term changes involving the stimulation of the expression of growth factors. An important difference is that changes in hormonal regulation occur in the body almost immediately after the massage, while the expression of growth factors requires a longer period and multiple repetitions of the stimulus. Most likely, the stimulation of such a reaction is facilitated by a non-specific factor (e.g., massage) that does not occur on a daily basis (4).
Skeletal muscle is one of the most plastic tissues that can adapt to changing conditions. Skeletal muscle adaptation is characterised by morphological, biochemical, and molecular changes. This adaptation varies and the extent of changes depends on many factors, including activity pattern, age, and muscle fibre type (46). The growth of the capillary network is also an example of the adaptation of skeletal muscles to intensive exercise (47, 48).
The formation of new blood vessels, leading to increased blood circulation in the muscles, may be an adaptation effect that results from physical training. An increased expression of VEGF-A in muscles in response to intensive exercise was observed (48-51).
The increase in serum VEGF-A concentrations following the use of foam massage roller may indicate the initiation of angiogenesis, which was particularly evident by week 5 of the experiment. After two weeks, a decrease in VEGF-A levels was observed, which may indicate habituation to the applied factor. By habituation effect, we mean the situation in which the maximum level of blood VEGF-A was achieved (week 5). Later, as the maximum effect of the massage was achieved, the VEGF-A blood level was not increasing and started to decrease. The foam roller massage used in this study is a form of massage that has become more commonly applied. However, there has been relatively little knowledge about the effects of this form of massage. Nevertheless, it is increasingly used in biological regeneration and physiotherapy (38, 52-54). One study showed that short self-massage using a roller before training did not affect the efficiency of the muscles (55).
However, our findings indicate that this form of massage can stimulate processes in the massaged tissues in the long term (increase in the VEGF-A concentration). It can indirectly contribute to the improvement of muscle working conditions, especially in situations of increased physical exercise load. Such effects of roller massage may be supported by the changes in VEGF-A concentrations, which increased during the massage cycle up to week 5. After this period, a decrease in the VEGF-A concentration was observed. This response may be due to the habituation process. Of note, similar changes were not found in the control group in which massage was not performed. Such a response is consistent with the concept that massage (as a mechanical factor) causes cellular changes that convert mechanical energy into molecular signals, which results in various cellular responses, such as increased expression of proangiogenic factors (e.g., VEGF-A). This phenomenon is known as mechanotransduction and is commonly described in the literature (4, 8, 9, 56-59).
Additionally, it has been noted that passive movement of the lower limbs in young people increases blood flow and VEGF levels in the muscles, producing results similar to those of exercise (60-62). This suggests that passive muscle stretching is a sufficient mechanical factor to initiate angiogenesis in skeletal muscles (63). Massage is an example of such an action. Stretching of the muscle tissue can provide a strong proangiogenic stimulus, which was confirmed by Rivilis et al. (64). Compression is another mechanical factor that stimulates angiogenesis (65). Roseguini et al. demonstrated that compression of rat limbs resulted in a significant increase in the VEGF expression (66).
The study of Thomas et al. (41) demonstrated positive effect of percussive massage on angiogenesis in type I skeletal muscle fibers (41). The pilot study of Best et al. (42), performed on tibialis anterior muscles of rabbits, showed enhancement of angiogenesis and regeneration of muscle fibers following massage (42). The systematic review of Guo et al. (37) summarizes the positive effects of massage following strenuous exercise on muscle performance and relief from DOMS (delayed onset muscle soreness) (37). The systematic review of Cheatham et al. (38) suggests amelioration of pre- and post-exercise muscle performance with the use of foam roller and roller massager (38). Four week-roller massage did not affect significantly muscle performance, as shown by Hodgson et al. (39). The meta-analysis performed by Davis et al. (67) suggests massage results in small reduction of DOMS (delayed onset muscle soreness) and small amelioration of flexibility (67).
Considering the above, it seems likely that massage can also be a mechanical factor that has a beneficial effect on regeneration and repair processes in tissues by stimulating angiogenesis. This is evidenced by studies showing that massage-like pressure could influence muscle recovery and its effect was stronger when applied immediately after exercise (68).
Roseguini et al. showed in animal studies that changes in VEGF expression were evident in the group where a high-frequency mechanical stimulus (compression) was applied (66). Such a mechanism of massage was confirmed by Hotta et al. (63). While investigating the role of the endothelium in skeletal muscle, Hotta et al. showed that extracellular mechanical stimuli, such as shear and tensile stresses, induced biochemical reactions inside vascular endothelial cells, thus stimulating the initiation of angiogenesis (63).
Our results show that a mechanical factor applied in the form of foam roller massage increased serum concentrations of VEGF-A and stimulated angiogenesis. This supports the hypothesis that mechanical stimulation results in gene activation, cell signalling, and the response of endothelial cells to the applied stimulus (64).
The review of Best et al. (69) suggested that during massage-based therapies, parameters such as frequency, magnification, and duration of tissue loading should be taken into consideration (69). Furthermore, it was suggested that the benefits of massage sessions can be limited (69). The Figure 3 in (69) concludes the massage-like mechanical stretching might elevate VEGF expression as well as regulate inflammation and stem cell activity (69).
As massage aims to press against muscles, it might be considered as a kind of exercise/mechanical strength applied to them. The study of Blocquiaux et al. (70) examined the effect of training on muscle methylome and transcriptome (70). The authors (70) verified the patterns of methylome and transcriptome in young and older adults (70). The review of Gorski and De Bock (43) describes the role of exercise in angiogenesis (43). Exercise training results in early adaptive events, including increased muscle vascularization (43). This is the reason why improved muscle angiogenesis and amelioration of symptoms of ischemia in peripheral artery disease might be achieved with exercise (43). Acute exercise bout leads to transient increase in skeletal muscle VEGF levels (43). This factor is indispensable for exercise-induced angiogenesis (43). It is suggested that skeletal muscle metabolism changes influence angiogenesis (43). Baseline muscle vascularization is controlled by estrogen-related receptor γ (ERR γ) direct binding to VEGF promoter and by ERRγ-mediated activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) (which induces VEGF expression) (43). Exercise results in the activation of peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC1α) which together with estrogen-related receptor α (ERRα) regulate VEGF and pro-angiogenic genes promoters activation (43). The stabilization of hypoxia inducible factor 1α (HIF1α) also leads to upregulation of pro-angiogenic factors in the muscles (43). All these circumstances result in the secretion of angiogenic molecules from the muscles, which results in muscle vascularization (43). The authors of the review (43) also proposed a model which could explain the effects of exercise on endothelial cell metabolism (43). According to Figure 2 of the review (43), in the resting stage, most of the energy in endothelial cells is produced glycolytically (43). Exercise triggers a series of cellular events that contribute to angiogenesis-promoting changes in endothelial cells. These processes might include the generation of hydrogen sulfide, which results in down-regulation of oxidative phosphorylation in endothelial cells (43). Increased glucose uptake could be noticed followed by increased migration and proliferation of endothelial cells (43). One of the important factors contributing to angiogenesis is Sirtuin 1 (SIRT1), which is involved in VEGF-induced angiogenesis (43). Best et al. (69) summarized the results of studies concerning after-massage regeneration (69). It was demonstrated that VEGFs expression was elevated after repetitive exercises of skeletal muscle, enhancing angiogenesis (69). The authors also presented other results, which indicated that the repair of skeletal muscles might depend on VEGF expression (69). Best et al. (69) mentioned a few studies indicating elevation of VEGF expression and increase in blood vessel density following electrical stimulation of rat hindlimb skeletal muscles (69). In addition, their analysis (69) included the statement of the American Massage Therapy Association, according to which therapeutic massage ameliorates muscle performance (69). The authors also present the results of the studies indicating advanced recovery of muscle function after 4 day-massage-like loading (69). Furthermore, their review demonstrated muscle recovery after controlled period of eccentric exercise was dependent on the magnitude and frequency of massage-like loading (69).
In this study, no significant increase in FGF-2 concentration was observed after roller massage. This may be due to the fact that we used peripheral blood to perform the examination as opposed to massaged tissue (tendon tissue) used in animal studies (15). We chose to perform our analysis in blood samples because it is considered unethical to obtain human muscle tissue samples. If we had performed our analysis directly on the massaged tissues or the study group had been larger, the changes in FGF-2 concentration could have been statistically significant (40, 71). This supposition is supported by the upward trend in FGF-2 concentration observed in the experimental group.
The lack of association between VEGF-A and FGF-2 levels might be explained by the fact the levels of VEGF-A and FGF-2 in each participant do not correlate with each other along with time. Therefore, the correlation between VEGF-A and FGF-2 was not observed.
The study of Jia et al. (72) investigated the effect of FGF-2 on endothelial cells, and therefore angiogenesis (72). A panel of experiments revealed proliferation, survival, and migration of endothelial cells were stimulated by FGF-2 through the SRSF1/SRSF3/SRPK1- pathway (72). This axis was shown to regulate VEGFR1 splicing in endothelial cells, leading to FGF-2 pro-angiogenic properties (72).
As reviewed by Yang et al. (73), endothelial and stromal cells might express VEGF in response to FGF (73). Moreover, the authors cited a study, which demonstrated the absence of Fgf2 in mice impaired wound healing (73, 74).
The role of VEGF and FGF in the aspect of other muscle components should also be considered. The review of Liu et al. (75) describes in detail the functions of VEGF in tendon repair. Tendon healing is a long process due to the characteristics of a tendon (75). Angiogenesis plays a key role in tendon regeneration (75). It was shown that VEGF levels in healthy tendons is low (75). But its levels increased at early stages of tendon healing after acute injury (75). As reviewed (75), tendon injury results in VEGF, FGF, IGF, PDGF, and TGF-β release from platelets (75). Therefore, VEGF is considered a pivotal angiogenic factor (75). As summarized in Figure 1 (75), VEGF roles include promotion of angiogenesis, enhancement of fibroblast proliferation, chemotactic for macrophages and granulocytes, and enhancement of production of other growth factors (75). However, it is also suggested that VEGF has a negative effect on tendon healing (75). In relation to native tendons, the results showed increased VEGF level and hypervascularity in degenerated Achilles tendons or chronic tendon tendinopathy (75). Due to the presence of active neovascularization in chronic tendinopathy, it is supposed tendon regeneration in degenerative tendon diseases can be impaired by VEGF-induced angiogenesis (75). VEGF was also shown to have an effect on endothelial cells and fibroblasts through promoting MMPs expression and discouraging TIMPs expression by these cells (75). This situation might lead to destruction of type I collagen, the main component of tendon ECM, therefore influencing biochemical characteristics of this structure (75). To conclude, in chronic tendon diseases, continuous VEGF expression and related vascularization might be negatively associated with biomechanics of tendons due to up-regulation of MMPs and down-regulation of TIMPs expression (75). The authors of the review (75) summarized tendon healing was executed by VEGF and appropriate angiogenesis at early stage (75). However, at later stage of tendon regeneration, continuous over-expression of VEGF and residence of blood vessels could harm tendon healing (75).
The importance of VEGF signaling in tendon cells is also reported by Tempfer et al. (76). Their experiments demonstrated the presence of VEGFR1, VEGFR2, and VEGFR3 in tendon cells characterized by Scleraxis expression (76). Moreover, temporarily elevated VEGFR1 and VEGFR3 expression was detected in injured rat Achilles tendons (76). VEGF-D temporal increase in the expression was observed in stumps and surrounding area (76). Incubation of 2D cultured-rat tendon cells with VEGF-D did not result in improved wound closure (76). However, VEGF-D incubation was observed to be followed by enhanced tendon cell proliferation (76). Furthermore, they showed that treatment of rat tendon cells with VEGF-D promoted the expression of selected MMPs (76).
The review of Lu et al. (77) collected the information concerning the role of bFGF in tendon healing (77). It was shown that exogenous bFGF could promote tendon regeneration through improving the biomechanics of the tendon and through enhancing cell proliferation, angiogenesis, and expression of collagens (77). bFGF was shown to participate in tendon healing through stimulating synthesis of type III collagen and type I collagen in the early and later phase of healing, respectively (77). The authors (77) summarized bFGF presence during tendon regeneration could result in promotion of angiogenesis, stimulation of tendon stem cells to differentiate into tendon cells, stimulation of collagen synthesis, and remodeling of ECM (77).
The study of Havis et al. (78) presented the contribution of FGF-4 to tendon differentiation (78). The authors (78) showed transduction with mouse Fgf4 using retroviral system led to induction of TNMD and THBS2 expression in chick limbs (78). TNMD and THBS2 are considered as late tendon markers (78). Therefore, the authors (78) stated that the muscle-dependent phase of chick limb tendon development is controlled by FGF4-induced TNMD and THBS2 expression (78). Also, FGF4 was demonstrated to enhance Scx (tendon marker) expression in early chick limb buds (78). Taking into consideration their results and findings of other authors, they suggested that FGF directs undifferentiated chick limb mesodermal cells towards the tendon lineage (78). Moreover, FGF4 was suggested to participate in muscle-independent and dependent phases of chick limb tendon development as a tenogenic factor (78).
The analyses performed by Chen et al. (79) aimed to verify the expression of selected genes in Achilles tendon rupture biopsies (79). The results presented increased FGF gene expression in injured tendon compared to intact area of the tendon, however, the difference was not statistically significant (79). Investigation demonstrated a positive correlation between total Achilles tendon total rupture score (ATRS) collected one year post-operation and FGF mRNA expression in the injured tendon (79). To conclude, these results suggest FGF mRNA level in injured Achilles tendons may be used as a prognostic biomarker for patient outcome (79).
Zhou et al. (80) presented interesting results indicating that bFGF and VEGFA loaded nanoparticle-coated sutures enhanced tendon regeneration (80).
The study of Tang et al. (81) reported tendon healing could be improved by gene therapy based on AAV-2 vector delivery of bFGF or VEGF to tendons (81). These experiments led to i) elevated expression of type I collagen, ii) down-regulation of MMPs expression and up-regulation of TIMPs expression, iii) increased proliferation and decreased apoptosis of tenocytes, iv) increase in healing strength of tendons (81).
There is a study that describes the effect of FGF-2 on skeletal muscle structure in aged humans (82). The experiments carried out by Mathes et al. (82) revealed an FGF-2-dependent signaling pathway that leads to intramuscular adipose tissue (IMAT) formation (82). FGF-2 promoted miR-29 expression in C2C12 myotubes, while the expression of SPARC (adipogenic inhibitor) was inhibited (82). Introduction of FGF2-over-expressing AAV9 vector into murine tibialis anterior muscle resulted in elevated muscle mass and increased IMAT formation (82). Moreover, aged mice were characterized by increase in Fgf2 and miR-29 expression, but decreased Sparc expression (82). The experiments conducted on human skeletal muscles also showed up-regulation of miR-29 and down-regulation of SPARC expression (82). The skeletal muscle FGF-2 level was shown to positively correlate with age (82). In addition, increased IMAT formation was noticed in aged human skeletal muscles (82).
The effect of FGF-2 on superior rectus muscle was examined in the study of Rudell and McLoon (83). It was shown short-term treatment with FGF-2 resulted in better muscle performance, while long-term treatment resulted in worse muscle performance (83).
The experiments carried out by Yamamoto et al. (84) showed that hyperbaric oxygen treatment resulted in elevated VEGF and bFGF levels in contused rat muscles (84). Moreover, increased muscle healing and blood vessel formation in rat skeletal muscles were noticed (84).
The effect of massage on structural changes in the body was confirmed in a study, which assessed the effect of massage combined with running training on the expression of proangiogenic factors in rat tendons and muscles. Massage combined with running training increased the expression of CD34, VEGF-A and FGF-2 proteins (13, 17). These results clearly demonstrated the effect of simultaneous massage and running training on angiogenesis in tendon and muscle tissue.
Therefore, it can be assumed that the effect of roller massage on the body is not limited only to reflex changes in the form of normalisation of tension in the massaged tissues but can also stimulate their structural reconstruction. These both facts advocate for the use of foam roller in biological regeneration and physiotherapy. In addition, this form of massage can also be applied in broadly defined preventive health care, e.g., in the elderly to prevent the adverse effects of ageing and the associated atrophic changes. Moreover, it could be employed in cosmetology to maintain the optimal function of tissues (e.g., the skin). The beneficial effect of massage can be evidenced by the studies which confirmed the effect of massage on the increase of proangiogenic factors in the skin (16).
Based on our results and literature data, we hypothesize that mechanotransduction would be responsible for the observed outcomes. This phenomenon relies on transformation of mechanical force into cellular signals. It results in cellular responses to the applied factor, which includes the regulation of the gene expression (e.g., VEGF-A) (7-10).
The studies and reviews present different conditions of applying massage (37-42). The variations include different types of massage (foam roller, western massage, Swedish massage, Chinese massage, massage with tennis ball), massage area, time and frequency of massage, applied pressure, and applied actions/variables (37-42). This suggests diverse parameters of massage determine various responses to employed force.
While considering muscle performance, other factors that affect the functioning of muscles should be taken into account. The editorial of Dobrowolny and Scicchitano (85) suggests ageing can contribute to functional and morphological changes of neuromuscular junctions (85). This might lead to the loss of strength and mass of skeletal muscles (85). The relationship between neuromuscular modifications and the skeletal tissue might be explained by neuromuscular diseases, which are reviewed by Iolascon et al. (86). The first example of this kind of diseases may be myasthenia gravis, which causes the weakness of muscles (86). This disease affects neuromuscular junction (86). Rapid fatigue is observed first in extraocular muscles, proximal limbs, and facial muscles (87). According to the presented studies, in early stages of myasthenia gravis progression, human muscle atrophy was demonstrated (87). In addition, type II fibers were characterized by increased atrophy in relation to type I fibers (87). Duchenne muscular dystrophy is the next condition that concerns skeletal muscle structure (86). The outcomes of this disease, including reduced muscle strength and loss of ambulation, influence bones (86). It is also hypothesized that muscular contraction is affected by myotendinous junction structural changes that lead to decrease in force transmission and mechanical stimulation of bones (86). Loss of dystrophin in mature myofibers results in many defects, including muscle degeneration (87). Dystrophin was shown to be a key regulator of the stability of the neuromuscular junction (87). The third instance is the case of Pompe disease which influences skeletal muscle metabolism (86). It is characterized by glycogen accumulation within lysosome, resulting in its later abundant accumulation in myofibrils (86, 87). As reviewed (87), the excessive amount of glycogen within skeletal muscles contributes to many cellular alterations leading to muscle weakness (87). It is also suggested that muscle weakness in this disease might be caused by neurological disfunction (87).
A condition such as sarcopenia should also be mentioned. The definition of sarcopenia includes a state in which there is decline in muscle mass and function in older adults (88, 89). The review of Clark (88) presents studies indicating that age-related muscle weakness is strongly associated with neurological and non-muscle mass related factors (88, 89). Furthermore, it was concluded that elderly-related weakening of the muscle and physical function are related to neural control of skeletal muscle (88).
The review by Weerapong et al. (3) indicates that massage is believed by many people to, inter alia, lower neuromuscular excitability leading to decreased muscle tension. In addition, the other effect of massage might include the change of the level of neurological activation, which might result in active muscle stiffness alteration (3).
Of note, this study was conducted on a very homogenous group of individuals in terms of sex and age who were trained and had a similar lifestyle, diet, and level of physical activity, which increases its credibility and value.
The results of this study are the first to show the changes in VEGF-A and FGF-2 serum levels after the use of foam roller massage in humans. These results showed that the massage with a foam roller could affect serum VEGF-A levels, which is the main factor stimulating the formation of new blood vessels. The increase in serum VEGF-A concentration under the influence of muscle foam roller may extend the knowledge of the mechanism of action of massage and the possibilities of its use (90).
Acknowledgements
The content presented in this manuscript constitutes the PhD thesis of Adam Rosłanowski (2021) entitled “Wpływ masażu mięśni szkieletowych na stężenie wybranych czynników wzrostu we krwi u młodych mężczyzn” Wroclaw University of Health and Sport Sciences, Wroclaw, Poland. Available at: https://bip.awf.wroc.pl/artykul/208/1713/publiczna-obrona-rozprawy-doktorskiej-mgr-adama-roslanowskiego (Last accessed on November 8, 2022).
Footnotes
Authors’ Contributions
Conceptualization: AR and WA; Methodology: AR, WA, PD, KRW and AK; Resources: AR, AJ, and DL; Data curation: AR, AJ, and DL; Investigation: KRW, AK, AR and AP; Project administration: AR, WA, PD, KRW, AK and AP; Formal analysis: JG; Supervision: WA and PD; Validation: KRW and AK; Visualization: AP, AR, JG; Writing - original draft: AR and WA; Writing - review & editing: PD, KRW and AP; Funding acquisition: AR and WA.
Funding
This study was financially supported by the Wroclaw University of Health and Sport Sciences grant, grant no 02/2018/2019.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received June 2, 2023.
- Revision received July 12, 2023.
- Accepted July 13, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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