Endothelial cells are an important source of BDNF in rat skeletal muscle

BDNF (brain-derived neurotrophic factor) is present in skeletal muscle, controlling muscular metabolism, strength and regeneration processes. However, there is no consensus on BDNF cellular source. Furthermore, while endothelial tissue expresses BDNF in large amount, whether endothelial cells inside muscle expressed BDNF has never been explored. The aim of the present study was to provide a comprehensive analysis of BDNF localization in rat skeletal muscle. Cellular localization of BDNF and activated Tropomyosin-related kinase B (TrkB) receptors was studied by immunohistochemical analysis on soleus (SOL) and gastrocnemius (GAS). BDNF and activated TrkB levels were also measured in muscle homogenates using Western blot analysis and/or Elisa tests. The results revealed BDNF immunostaining in all cell types examined with a prominent staining in endothelial cells and a stronger staining in type II than type I muscular fibers. Endothelial cells but not other cells displayed easily detectable activated TrkB receptor expression. Levels of BDNF and activated TrkB receptors were higher in SOL than GAS. In conclusion, endothelial cells are an important and still unexplored source of BDNF present in skeletal muscle. Endothelial BDNF expression likely explains why oxidative muscle exhibits higher BDNF levels than glycolytic muscle despite higher the BDNF expression by type II fibers.

Since its first identification in the brain in 1982 1 , neuronal BDNF (brain-derived neurotrophic factor) was the subject of a plethora of studies that have led to the consensus that neuronal-derived BDNF is crucial for neurogenesis, synaptogenesis, neuroplasticity and cognition 2,3 through phosphorylation at tyrosine 816 of full-length (FL) tropomyosin-related kinase B (p-TrkB Tyr816 ) 4,5 . However, we know now that BDNF is expressed in other cells than in neurons and therefore plays a more widespread role than initially thought. Skeletal muscle, which is the most abundant tissue in the body, was shown to synthesize BDNF in 1993 6 . The demonstration that physical exercise leads to an elevation of BDNF in active muscle 7 and the idea that BDNF originating from muscle might be involved in the beneficial effect of EX not only on muscles but also on distant organs explains the regain of interest for muscular BDNF.
Surprisingly, little is known about the cellular localization of BDNF. Available studies focused mainly on muscular fibers, satellite cells and neuromuscular junctions and led to controversial results. For certain authors, BDNF expression is restricted to the former 8,9 , while it is confined to the latter for others 10 . There is also no consensus concerning BDNF localization on the neuromuscular junction [11][12][13] . Although never explored, BDNF might be also expressed by endothelial cells present in muscle. Indeed, the cerebral and peripheral vasculatures including capillaries contain BDNF with a prominent expression by the endothelium [14][15][16] . To explore whether endothelial cells present in muscle express BDNF is of great interest as density of capillary network differs between oxidative and glycolytic muscles. In addition, if BDNF is present in endothelial cells, the elevation of circulating BDNF observed during or after exercise might possibly reflect the secretion of BDNF by endothelium and not by that of myofibers as generally thought according to the concept of myokines.
The present study tests the hypothesis that BDNF is expressed by endothelial cells in skeletal muscle and that this expression largely contributes to BDNF levels present within the muscle. For this purpose, we first provide a comprehensive analysis of BDNF localization in skeletal muscle in rats. Then, we compared BDNF content of oxidative muscle soleus (SOL) versus glycolytic muscle gastrocnemius (GAS) since capillary network was denser for the former.  Colocalization of BDNF with specific markers of satellite (MyoD-1, myoblast determination protein 1/PAX 7, Paired box 7) cells, axonal processes of motor neurons (SYN, synaptophysin) and endothelial (vWF, von Willebrand factor) cells in soleus (SOL) muscle. DAPI (blue) was used as nuclear marker. BDNF was stained either in green (a, b) or in red (c, d), MyoD-1 and PAX 7 were marked in red (a, b) and, SYN and vWF in green (c, d). Similar results were observed in all examinations (n = 4). The inserts show with higher magnification the colocalization of BDNF with each specific marker in presence of DAPI. Scale bar 50 µm (10 µm, for the insert). www.nature.com/scientificreports/ Experiments were then conducted on GAS. Figure 3 confirmed the high BDNF expression by type II fibers (Fig. 3a) as compared to type I fibers (Fig. 3b). As reported for SOL, BDNF staining was also detected in satellite cells, neurons and endothelial cells (data not shown).
BDNF levels in SOL versus GAS. The differential expression of BDNF in type II versus type I fibers combined with the strong expression of BDNF by endothelial cells led us to compare BDNF levels in SOL versus GAS muscle. Our hypothesis was that BDNF levels will be higher in SOL than GAS because capillaries and by extension endothelial cells are more abundant in SOL than GAS. BDNF levels were measured using ELISA test and Western blotting method (Fig. 4). As shown in Fig. 4a and b, BDNF levels were significantly higher (X2) in SOL than GAS irrespective of the method used. The cellular density of endothelial cells and of other cells were compared in SOL versus GAS (Fig. 5). The results showed that levels of vWF are threefold higher in SOL than GAS (Fig. 5a). Markers of satellite cells MyoD-1 (Fig. 5b) and PAX 7 (Fig. 5c) were also more abundant in SOL than GAS. In contrast, synaptophysin (SYN) levels ( Fig. 5d) did not differ between both muscles. Finally, as expected, marker of type I fibers was higher in SOL than GAS (Fig. 6a) while marker of type II fibers was higher in GAS than SOL (Fig. 6b).  . BDNF levels in the soleus (SOL) and gastrocnemius (GAS) muscles. BDNF protein levels in SOL and GAS muscles of control rats (n = 6 per group) were determined by both ELISA (a) and Western blotting (b) methods. BDNF results (mean ± SD) are expressed in pg/ml (for ELISA) or as percentage of SOL muscle values (for Western blotting). **p < 0.01, ***p < 0.001 versus SOL muscles. www.nature.com/scientificreports/ Activated TrkB levels in muscles. As shown in Fig. 7, levels of activated TrkB receptors (p-TrkB Tyr816 ) were higher in SOL than GAS and a correlation was found between BDNF and p-TrkB Tyr816 indicating that cellular BDNF secretion was in proportion with BDNF levels. We then explored localization of p-TrkB Tyr816 receptors. Immunohistochemical experiments were conducted using antibodies directed against activated TrkB receptors (i.e. FL TrkB receptors activated by BDNF) since antibodies directed against TrkB receptors detect both truncated (incomplete receptor devoid of tyrosine kinase activity) and FL TrkB receptors. As shown in Fig. 7c, these  www.nature.com/scientificreports/ receptors were easily detectable in endothelial cells. By contrast, it was rather difficult to detect their presence in other cell types thus making of endothelial cells a major recipient cell of secreted BDNF.

Discussion
The present study is the first to provide a comprehensive analysis of BDNF localization in skeletal muscle. The main results are that (i) muscular fibers, satellite cells, motor neurons (axonal processes) and endothelial cells simultaneously express BDNF, yet with a prominent expression by endothelial cells and a more pronounced BDNF expression by type II than type I muscular fibers, (ii) that the muscular expression of activated TrkB receptor is preponderantly attributable to endothelial cells as compared to other cells. BDNF localization in the skeletal muscle has not been extensively studied. This is surprising as the first detection of BDNF mRNA in skeletal muscle was reported in 1993 6 . In fact, we had to wait for the avaibility of anti-BDNF antibodies to advance research in this field. However, available studies on the localization of BDNF protein led to controversial data. The present study showed that myofibers, satellite cells and axonal processes constitutively expressed BDNF irrespective of the muscle type and that BDNF expression was higher in type II than type I fibers 17,18 . The new finding provided by the present study is the identification of endothelial cells as an important and still unidentified source of BDNF in muscles. Such endothelial BDNF expression in basal conditions likely explains why oxidative muscle exhibits higher BDNF levels than glycolytic muscles despite the higher expression of BDNF by type II fibers 19 . It is noteworthy that BDNF staining in endothelial cells contrasts with a previous study that fails to detect BDNF mRNA in endothelial cells within muscle 8 . In the same way, differential BDNF staining between type I versus type II myofibers contrast with the detection of a higher mRNA BDNF expression in the former. Such discrepancies between mRNA and proteins were previously reported in muscle 18,19 .
TrkB receptors including FL receptors that are equipped with kinase activity and truncated receptors that are devoid of kinase activity are both present in skeletal muscle. The binding of BDNF to FL TrkB receptors and their subsequent phosphorylation has been recently involved in the local effect of muscular BDNF [20][21][22] . The present study reported the presence of activated TrkB receptors in basal conditions, indicating that there is constitutive secretion of active BDNF by cells. Furthermore, the positive correlation that was identified between muscular BDNF levels and muscular activated TrkB receptors supports the notion that cellular BDNF secretion proportionate with cellular BDNF expression as previously reported for the brain 23 . The localization of activated TrkB receptors on muscular fibers, axonal processes and satellite cells are in line with reports on the role of BDNF on beta oxidation of fatty acids 7 and the switch from type I to type II fibers 17 , the stability of neuromuscular junction 11,24 and regulation of satellite cell differentiation 9,25 , respectively. However, the present study showed a prominent endothelial expression of activated TrkB receptors in basal conditions. The cellular source of BDNF involved in the activation of endothelial TrkB receptors might be endothelial cells themselves or neighboring cells including myofibers and satellite cells. The potential impact of activation of endothelial TrkB receptor activation is probably related to the production of nitric oxide (NO), identifying NO as an intermediate between BDNF and muscular function among which muscular metabolism and myogenic differentiation (for review [26][27][28] ).
Endothelial derived BDNF may also account for the elevation of blood BDNF levels in response to physical exercise. Indeed, we previously provide convincing arguments for a positive control of endothelial BDNF by shear stress and subsequent NO overproduction [14][15][16]29 . Supporting this hypothesis, studies reported lower blood BDNF levels in sedentary than in physically active people 30 and lower levels in patients at vascular risk, i.e. suffering from endothelial dysfunction [31][32][33] .
For authors that adhere to the notion that BDNF is a myokine considering that BDNF level elevation during or just after exercise corresponds to BDNF secreted by myofibers, the data presented in our study may lead to reinterpretation of changes in circulating BDNF observed in response to exercise.
In conclusion, our results identified for the first time endothelial cells as a main source of BDNF present in skeleton muscle. Further studies are needed to investigate whether BDNF derived from endothelial cells exerts autocrine, paracrine and/or endocrine role.

Methods
Animals. Experiments were carried out on 10 week-old Wistar rats (n = 14) according to the French Department of Agriculture guidelines (License 21-CAE-099) and approved by the local ethic committee (Ethics committee in animal experimentation, Grand campus Dijon, agreement number 105) and conform to ARRIVE guidelines. They conformed to the European convention for protection of vertebrate animals used for experimental and other scientific purposes. The animals were housed five per cage, kept under a 12-h/12-h light/dark cycle and allowed ad libitum access to food and water. Rats were purchased from Janvier (Le Genest Saint Isle, France). Elisa test. BDNF protein levels were determined with a commercial ELISA kit (BEK-2211-2P, Biosensis).
According to the manufacturer, BDNF antibodies do not cross react with nerve growth factor (NGF), neurotrophin-3 (NT-3), NT-4/5. The limit of sensitivity was fixed at 2 pg/ml. The measurements were performed according to the manufacturer's instructions. Supernatants of muscles homogenates were directly dropped on the plate without dilution. All assays were performed in duplicate. After antibody incubation, membranes were placed in Chemidoc imaging systems and a stain free image of the blot was captured to control the total protein loading and normalize data. Protein-antibody complexes were visualized using the enhanced chemiluminescence western blotting detection system (ECL 2, 1151-7371, Fisher Scientific) and Chemidoc imaging systems. Band densities were finally analyzed with ImageLab software (Bio-Rad) and standardized on total protein. The appropriate amounts of total proteins to be analyzed were previously determined from concentration (increasing amounts of proteins)/response (optical density of the band) curves from two rats both belonging to a particular group (on the same gel). All gels were run in triplicate.  . Expression and localisation of p-TrkB Tyr816 in SOL and GAS muscles. Correlation with muscular BDNF expression. Expression of p-TrkB Tyr816 (140 kDa) was determined in SOL and GAS muscles of control rats (n = 6 per group) by Western blotting method. Results are expressed as percentage of SOL muscle values (means ± SD). *p < 0.05 versus SOL muscle. Representative immunoblot is shown above bar chart (a). The correlation (scatter plot) between muscular BDNF and p-TrkB Tyr816 was evaluated in SOL and GAS muscles. Values are expressed as arbitrary units (A.U.) and the correlation coefficient (r) as well as the probability (p) are displayed on graph (n = 12) (b). Immunostainings of p-TrkB Tyr816 and vWF were shown in SOL muscle. DAPI (blue) was used as nuclear marker, red-labelled immunofluorescence represented p-TrkB Tyr816 (first line) and green-labelled immunofluorescence, vWF (second line). Similar results were observed in all examinations (n = 4), (c). The insert shows with higher magnification the colocalization of p-TrkB Tyr816 with vWF in presence of DAPI. Scale bar 50 µm (10 µm, for the insert). www.nature.com/scientificreports/ Probes, Invitrogen], 1/1000 in TBST with 1% of GS, for 60 min at RT. Finally, slides were mounted with DAPI (a nuclear marker)-containing mounting medium (Fluoro-gel with DAPI, FP-DT094A, Interchim, Montluçon, France). For vWF + BDNF and Pax7 + BDNF co-staining, slides were treated with an autofluorescence quenching kit (Vector® TrueVIEW®, SP-8500, Vector Labs, Burlingame, CA, USA) prior to mounting with the DAPIcontaining mounting medium of the kit. Slides were analyzed by using an epifluorescent microscope (Axioscop 40FL, Carl Zeiss, Oberkochen, Germany) and ProView v4.1 (Optika, Ponteranica, Italy). Concerning immunostaining directed against BDNF, it is also important to notice that regarding some unspecific bands obtained in WB assessment, the fluorescent signal may represent some unspecific labelling. However, data obtained in mice with tie-2 conditional deletion of BDNF showing that BDNF staining totally disappears in the endothelium of these animals (see Fig. 4 of supplemental data files) comfort us in the reliability of the antibody used in our study.

Histology. Experiments
Data and statistical analysis. SigmaPlot 14.0 was used for statistical analysis and all graphs. Data were expressed as means ± standard deviations (SD) for protein expression. Differences between two groups were assessed using parametric t-test or non-parametric Mann-Whitney test, depending on the normality and equal variance tests. T-test for Pearson's correlations were used to measure the strength of the relationship between paired data. A value of p < 0.05 was considered statistically significant.

Data availability
The data support the findings of this study are available from the corresponding author upon reasonable request.