ORIGINAL RESEARCH published: 11 June 2021 doi: 10.3389/fneur.2021.659922 Platelet Derived Growth Factor-AA Correlates With Muscle Function Tests and Quantitative Muscle Magnetic Resonance in Edited by: Jens Schmidt, Dystrophinopathies Universitätsklinikum Göttingen, Germany Alicia Alonso-Jiménez 1,2† , Esther Fernández-Simón 1,3,4† , Daniel Natera-de Benito 5 , Reviewed by: Carlos Ortez 5 , Carme García 6 , Elena Montiel 6 , Izaskun Belmonte 6 , Irene Pedrosa 6 , Melissa Hooijmans, Sonia Segovia 1,3 , Patricia Piñol-Jurado 1,3,4 , Ana Carrasco-Rozas 1,3 , VU University Medical Xavier Suárez-Calvet 1,3 , Cecilia Jimenez-Mallebrera 3,5,7 , Andrés Nascimento 3,5 , Center, Netherlands Jaume Llauger 8 , Claudia Nuñez-Peralta 8 , Paula Montesinos 9 , Jorge Alonso-Pérez 1,3 , John Vissing, Eduard Gallardo 1,3 , Isabel Illa 1,3 and Jordi Díaz-Manera 1,3,4* Rigshospitalet, Denmark 1 Neuromuscular Disorders Unit, Neurology Department, Hospital de la Santa Creu i Sant Pau, Departament de Medicina. *Correspondence: Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Neurology Department, Neuromuscular Reference Center, University Jordi Díaz-Manera Hospital of Antwerp, Antwerp, Belgium, 3 Biomedical Network Research Centre on Rare Diseases (CIBERER), Barcelona, jordi.diaz-manera@newcastle.ac.uk Spain, 4 John Walton Muscular Dystrophy Research Centre, International Centre for Life, Newcastle University, Newcastle † These authors have contributed upon Tyne, United Kingdom, 5 Neuromuscular Unit, Neuropediatrics Department, Institut de Recerca Sant Joan de Déu, equally to this work and share first Hospital Sant Joan de Déu, Barcelona, Spain, 6 Rehabilitation and Physiotherapy Department, Hospital de la Santa Creu i authorship Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain, 7 Departamento de Genética, Microbiología y Estadística, Universidad de Barcelona, Barcelona, Spain, 8 Radiology Department, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain, 9 Philips Healthcare Iberia, Madrid, Spain Specialty section: This article was submitted to Neuromuscular Disorders and Introduction: Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked Peripheral Neuropathies, a section of the journal muscular disorders produced by mutations in the DMD gene which encodes the protein Frontiers in Neurology dystrophin. Both diseases are characterized by progressive involvement of skeletal, Received: 02 February 2021 cardiac, and respiratory muscles. As new treatment strategies become available, reliable Accepted: 17 May 2021 Published: 11 June 2021 biomarkers and outcome measures that can monitor disease progression are needed Citation: for clinical trials. Alonso-Jiménez A, Methods: We collected clinical and functional data and blood samples from 19 DMD Fernández-Simón E, Natera-de Benito D, Ortez C, García C, patients, 13 BMD patients, and 66 healthy controls (8 pediatric and 58 adult controls), Montiel E, Belmonte I, Pedrosa I, and blood samples from 15 patients with dysferlinopathy (DYSF) and studied the Segovia S, Piñol-Jurado P, serum concentration of 4 growth factors involved in the process of muscle fibrosis. We Carrasco-Rozas A, Suárez-Calvet X, Jimenez-Mallebrera C, Nascimento A, correlated the serum concentration of these growth factors with several muscle function Llauger J, Nuñez-Peralta C, tests, spirometry results and fat fraction identified by quantitative Dixon muscle MRI. Montesinos P, Alonso-Pérez J, Gallardo E, Illa I and Díaz-Manera J Results: We found significant differences in the serum concentration of Platelet (2021) Platelet Derived Growth Derived Growth Factor-AA (PDGF-AA) between DMD patients and pediatric controls, Factor-AA Correlates With Muscle Function Tests and Quantitative in Connective Tissue Growth Factor (CTGF) between BMD patients and adult controls, Muscle Magnetic Resonance in and in and Transforming Growth Factor- β1 (TGF-β1) between BMD and DYSF patients. Dystrophinopathies. Front. Neurol. 12:659922. PDGF-AA showed a good correlation with several muscle function tests for both DMD doi: 10.3389/fneur.2021.659922 and BMD patients and with thigh fat fraction in BMD patients. Moreover, PDGF-AA levels Frontiers in Neurology | www.frontiersin.org 1 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD were increased in muscle biopsies of patients with DMD and BMD as was demonstrated by immunohistochemistry and Real-Time PCR studies. Conclusion: Our study suggests that PDGF-AA should be further investigated in a larger cohort of DMD and BMD patients because it might be a good biomarker candidate to monitor the progression of these diseases. Keywords: PDGF, MRI, biomarker, Duchenne, Becker, dystrophinopathy INTRODUCTION The main aim of this study was to analyze the serum concentration of a group of growth factors related to the process Duchenne (DMD) and Becker (BMD) muscular dystrophy are of muscle fibrosis in a cohort of 19 patients with DMD and 13 X-linked recessive disorders produced by mutations in the DMD patients with BMD and study whether there was a correlation gene which encodes the protein dystrophin (1). Dystrophin is with the results of muscle function tests and quantitative a cytoplasmic protein which links the intracellular cytoskeleton MRI (qMRI). network to transmembrane components of the dystrophin- glycoprotein complex, including dystroglycan, sarcoglycans, and sarcospan (2). Its role is to stabilize the membrane during muscle MATERIALS AND METHODS contraction to prevent contraction-induced damage (3). Study Design and Participants DMD has an homogeneous clinical picture characterized by We report the results of a transversal study of a cohort of 19 early onset of muscle weakness progressing during childhood DMD and 13 BMD patients seen at Hospital de la Santa Creu and leading to loss of ambulation during adolescence (4). i Sant Pau (HSCSP) and Hospital Sant Joan de Déu (HSJD) in All DMD patients invariably develop cardiac and respiratory Barcelona. All patients included in the study were assessed using involvement, which are the main causes of death. In contrast, muscle function tests and spirometry and filled out a daily life BMD is an heterogeneous disease characterized by muscle activities questionnaire. qMRI was not a mandatory test and weakness of variable severity that can start during childhood was performed in a subset of patients followed-up in HSCSP but also late in adulthood (5). BMD patients can also develop only. Blood samples were obtained before the muscle function respiratory and cardiac muscle involvement. Despite having assessment. The HSCSP and HSJD Ethics Committees approved substantial phenotypic differences, DMD and BMD share the study, and all participants signed an informed consent common pathological pathways. Mutations in the DMD gene form. All study procedures were performed in accordance with lead to abnormal dystrophin expression which results in a Spanish regulations. fragile muscle membrane susceptible to be damaged during Inclusion criteria for the study were: (1) Diagnosis of DMD or contraction (3, 6). Continuous muscle damage leads to persistent BMD confirmed by the identification of pathogenic variants in inflammation, muscle fiber necrosis and replacement by fat and the DMD gene and (2) willingness to complete muscle function fibrous tissue, producing permanent weakness and disability (6). tests, respiratory assessment, and daily life activities scale. In slow progressive diseases, the identification of functional changes over a short period is not an easy task. In recent years Functional Assessment several outcome measures such as the 6 Minutes-Walk Test Muscle function tests were performed by physiotherapists (6MWT), the Timed Up-and-Go Test (TuGo) or the North with considerable experience in neuromuscular disorders. We Star Ambulatory Assessment (NSAA) have been developed assessed patients using timed tests, functional ability scales, to monitor muscle function in natural history studies and and measurement of muscle strength. Timed tests included clinical trials (7–10). Additionally, specific tests have been the 6MWT, the 10 Meter Run/Walk test (10MWT), the Time developed to assess upper limb muscle function such as the to Climb Up (Tup4) and Down (Tdo4) Four Steps and Brooke Upper Extremity Functional Rating (Brooke scale) or the Time to Rise from the Floor (TRF). Functional ability the Performance of the Upper Limb (PUL) (11–13). However, included the NSAA and the Motor Function Measure 20-item as muscle fiber necrosis and replacement by fat is a slowly scale (MFM-20) (17). The MFM-20 was also analyzed based progressing process, changes in motor functional tests could on its 3 dimensions: D1 (standing and transfers), D2 (axial take time to be evident. In view of this limitation, muscle and proximal), and D3 (distal). PUL test was used to assess magnetic resonance imaging (MRI) and serum biomarkers have upper extremity function in both ambulant and non-ambulant been investigated as potential outcome measures that might individuals. Egen Klassification was used to assess mobility in complement muscle function tests (14–16). Although muscle wheelchair-bound patients (18). Muscle strength was studied MRI is becoming more widely accepted as a tool to monitor using the Medical Research Council scale (MRC). For analysis, 5- patients, there is not a serum biomarker accepted so far that point MRC power grades were converted to an 11-point scale as correlates with several muscle function tests and that could previously described (19). We assessed bilateral shoulder flexion, therefore be considered useful for clinical trials or natural shoulder abduction, elbow flexion, elbow flexion brachioradialis, history studies. wrist extension, wrist flexion, hip adduction, hip abduction, Frontiers in Neurology | www.frontiersin.org 2 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD hip flexion, hip extension, knee extension, knee flexion, ankle head, semitendinosus, and semimembranosus. We intended to eversion, ankle inversion, ankle plantar flexion, and ankle plantar include slices of the proximal, middle and distal part of the extension. Grip and pinch strength were studied using a hand thigh. To do so, we identified the head of the femur and set the dynamometer and the best of 3 attempts was used for analysis. first slice at 5 centimeters below the head. The remaining four Daily life activities were assessed using the ACTIVity LIMitations slices were distally distributed in the thighs with a separation scale for patients with upper and/or lower limb impairments of 20 slices between them as previously published (21). In (ACTIVLIM) (20). We also assessed respiratory function using patients in which individual muscles in the posterior or anterior the Carefusion Microlab ML 3500 MK8 spirometer (Carefusion, compartment of the thigh could not be analyzed due to a Yorba Linda, CA, USA). We analyzed the Forced Vital Capacity complete fat replacement, a ROI was manually drawn over the (FVC) while seating and lying and the Forced Expiratory Volume whole compartment. Neurovascular bundles were avoided. An in the first second (FEV1). average of the fat fraction of the 5 slices was calculated for each muscle, and weighted averages were calculated by normalizing Quantification of Growth Factors in Serum the FF by the area of the muscle (bigger muscles had higher Samples weight) to obtain the global thigh FF. We measured the serum concentration of the following growth factors related with muscle fibrosis: Serum Platelet-Derived RNA Extraction and Reverse Transcription Growth Factor BB (PDGF-BB), Transforming Growth Factor β1 Total RNA was extracted from triceps or biceps biopsy samples (TGF-β1), Platelet-Derived Growth Factor AA (PDGF-AA) and from 2 healthy controls and 2 DMD patients using RNeasy R connective tissue growth factor (CTGF). Blood was centrifuged Micro Kit (Qiagen, Hilden, Germany) following manufacturer’s at 1,600 g for 9 min at 4◦ C. Samples were aliquoted and stored instruction. RNA was quantified using a nanodrop ND-1000 at −80◦ C until analysis. PDGF-BB and TGF-β1 levels were spectrophotometer (Nanodrop Technologies Inc., Wil- mington, measured using commercial enzyme-linked immunosorbent DE, USA). One µg of total RNA was reverse-transcribed to assay (ELISA) kits (R&D, Minneapolis, MN, USA), according to complementary DNA (cDNA) using the High Capacity cDNA the manufacturer’s instructions. PDGF-AA human ELISA kit was Reverse Transcription Kit (Applied Biosystems, Foster City, provided by ThermoFisher (Thermo Fisher Scientific, Nepean, CA, USA). Canada) and CTGF by EIAAB Science Co (Wuhan, China). Minimum detectable cytokine concentrations for these assays Real-Time Quantitative PCR Analysis were measured to be 1.7 pg/ml for TGF-β1, 15 pg/ml for PDGF- Real-Time PCR (qPCR) was performed using the TaqMan R BB, 40 pg/ml for PDGF-AA and 0.18 ng/ml for CTGF. Samples Universal PCR Master Mix (Applied Biosystems, Foster City, were measured in duplicate and read on a microplate reader CA, USA) and a 7900HT Fast Real-Time PCR System (Applied Beckman Coulter AD 340 (Beckam-Coulter, Brea, CA, USA) with Biosystems, Foster City, CA). All mRNA- specific FAM- AD-LD software. labeled primers/probe were purchased from Applied Biosystems. These growth factors were determined in the 19 DMD Relative quantification was performed using the comparative Ct patients, 13 BMD patients, 15 patients with mutations in the method and all results were compared with the control samples. DYSF gene that were being visited in HSCSP (from now on GAPDH was used as endogenous control. DYSF patients) and in 66 healthy controls. The healthy controls were divided into a pediatric group (<15 years old) and adult Immunohistochemistry group (≥15 years old). Since the average age of DMD cohort is PDGF-AA immunohistochemistry was performed from significantly different from the DMB and DYSF cohort, the DMD paraffinized muscle. The samples were deparaffinized (xylol, group was compared to the pediatric control group while the absolute ethanol, 95◦ ethanol, and 70◦ ethanol), placed in DMB and DYSF group was compared to the adult control group. distilled water and pretreated with 10 mM citrate (pH 6) at 100◦ C. Then samples were washed in PBS and incubated in Muscle MRI blocking solution (4% bovine serum albumin). The primary anti- 7 DMD and 8 BMD patients underwent thigh muscle MRI PDGF-AA antibody (Merck Millipore, Darmstadt, Germany) in a 1.5T Ingenia MR system (Philips Healthcare, Best, the was then added at room temperature for 1 h. After 3 washes, it Netherlands) at HSCSP. Axial 3D FFE Dixon sequence was was incubated with biotinylated anti-rabbit antibody (Vector acquired with the following parameters: TR/TE = 5.78/1.84 ms, Laboratory Inc. Burlingame, CA) for 1 h at room temperature flip angle = 15◦ , voxel size = 1 × 1 × 3 mm and FOV 520 × 340 and then avidin-biotin peroxidase complex (Dako, Glostrup, × 300 mm. Water and fat images were automatically obtained Denmark) was added. Finally, the sections were soaked in from the Dixon acquisition using a single peak model. One Mayer’s hematoxylin for 10 s, washed under running water and investigator (A.A-J) estimated fat content in the images using the mounted in the aqueous mounting medium Aquatex (Merck PRIDE (Philips Research Image Development Environment) tool Millipore, Darmstadt, Germany). developed for this purpose. This tool provides the fat fraction (FF), defined as fat/(fat+water). Regions of interest (ROIs) were Data Analysis manually drawn on five slices on the right thigh of the following Non-parametric tests were used for the statistical analysis as muscles: rectus femoris, vastus intermedius, vastus lateralis, vastus we demonstrated that the distribution of the variables was not medialis, adductor magnus, sartorius, gracilis, biceps femoris long uniform as studied using Kolmogorov-Smirnov and did not Frontiers in Neurology | www.frontiersin.org 3 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD follow a normal distribution as studied using Shapiro-Willis and IQR of CTGF serum levels were 5.66 ng/ml (IQR: 3.82- test. The Mann-Whitney U test was used to identify significant 8.82) in BMD patients compared to 2.37 ng/ml (IQR: 1.35- differences in variables between 2 groups. Non-parametric 4.07) in adult controls (Kruskal-Wallis test/Dunn’s multiple Kruskal–Wallis test analysis was used to assess differences among comparison analysis, p = 0.004). The median and IQR of 3 groups followed by Dunn’s multiple comparison post-test. TGF-β1 serum levels were 59.10 ng/ml (IQR: 51.11-71.51) in We used Spearman’s rank correlation (coefficient reported as ρ) BMD patients compared to 40.42 ng/ml (IQR: 28.44-53.75) in to investigate any correlation between the serum concentration DYSF patients (Kruskal-Wallis test/Dunn’s multiple comparison of growth factors and the results of the muscle function tests, analysis, p = 0.01). Although PDGF-AA did not show statistical quality of life scales and the thigh FF obtained using Dixon differences, there was a tendency toward increased levels in imaging. Correlation coefficients higher than 0.6 were considered BMB patients 2415.60 ng/ml (IQR: 1228.59-3981.10) compared good. Because the objective of our research was to explore to adult controls 1859.62 (IQR: 1363.08-2477.52) (Kruskal-Wallis the possibilities of these growth factors for further studies, we test/Dunn’s multiple comparison analysis, p = 0.06). decided not to use multiple comparisons. The results of all statistical studies were considered significant if P-value was lower than 0.05. Statistical studies were performed using IBM Correlation of Growth Factor Serum SPSS R Statistics software version 21. Graphs were created using Samples With Muscle Function Test Graphpad software and R software. Results In a second step, we analyzed if there was a correlation between RESULTS growth factor serum concentration and the results of muscle function tests, spirometry, daily life activities scales and Dixon Cohort Description MRI in DMD and BMD patients. The growth factor that showed A total of 19 patients with DMD (mean age 10 ± 5.6 years), a larger number of significant correlations was PDGF-AA as 13 patients with BMD (mean age 31 ± 20.6 years), 15 DYSF it is shown in Table 2. In the case of DMD, PDGF-AA serum patients (mean age 46 ± 18.6 years), 8 pediatric controls (mean levels correlated with the results of the 10MWT, the 6MWT, the age 10 ± 2 years) and 58 adult controls (mean age 43 ± MRC scale. and the dimension 3 of the MFM scale. In the case 15.32 years) were enrolled in the study. There was no statistical of BMD, PDGF-AA correlated with the results of the 10MWT, difference in age between the DMD group and the pediatric the 6MWT, the Tup4, Tdo4, TRF and the NSAA, FVC sitting, controls (Mann-Whitney U-test, p = 0.979) and among the FEV1 and the thigh FF. Figure 2 shows graphs plotting these BMD, DYSF group and the adult control group (Kruskall-Wallis correlations. Moreover, we analyzed the distribution of PDGF- test, p = 0.052) We identified deletions in the DMD gene in AA values in relation to age and to the MRC total score and we 71.9% of patients. Duplications accounted for 12.5% of cases and observed a bell distribution both in DMD and in BMD patients 15.6% were due to point mutations. Among the DMD patients, (Figures 2E,F). Younger and stronger patients had low levels of 13 were ambulant (68.42%) and 6 non-ambulant (31.58%). PDGF-AA; patients in an intermediate stage that were losing In the BMD group, 8 patients were ambulant (61.5%) and 5 muscle strength had higher levels of PDGF-AA, while patients non-ambulant (38.5%). Five DYSF patients were non-ambulant in the end-stage of the disease, that were very weak or older, had (33.33%). Table 1 describes the mean demographic and clinical again low levels of PDGF-AA. Muscle strength measured by MRC data of all patients included in the cohort. significantly correlated with TGF- β1 levels in DMD patients (p = 0.026, Spearman’s rho = 0.523) and with CTGF levels in BMD Growth Factor Serum Levels Comparison patients (p = 0.012, Spearman’s rho = −0.669). We did not find Between DMD, BMD, and Age Matched statistically significant correlations between the PDGF-BB serum Controls levels and the function tests. Our first aim was to study whether there were differences in growth factor serum levels between DMD, BMD and DYSF patients compared to controls. We compared serum levels of PDGF-AA Expression in Skeletal Muscle PDGF-AA, PDGF-BB, CTGF, and TGF-β1 between DMD and Biopsies of DMD and BMD Patients pediatric healthy controls whereas BMD and DYSF patients were In a final step we studied PDGF-AA expression patterns in compared against adult healthy controls (Figure 1). In the case muscle biopsies of patients with DMD and BMD and in controls of DMD patients, we only observed significant differences in the using immunohistochemistry and Real Time PCR. As it has been serum levels of PDGF-AA as shown in Figure 1A. The median previously shown, PDGF-AA is expressed in the capillaries in and IQR of PDGF-AA serum levels were 2245.32 ng/ml (IQR: muscles of healthy controls, where it probably has a role in 1589.94-3832.68) in DMD patients compared to 1242.45 ng/ml binding the pericytes to the endothelial cell (Figure 3A) (22, 23). (IQR: 915.05-1373.13) in pediatric controls (Mann-Whitney U- In patients with DMD, we observed that muscle fibers expressed test, p = 0.002). In the case of BMD and DYSF patients we PDGF-AA as it is shown in Figure 3B. qPCR confirmed the observed significant differences in the serum levels of CTGF results of immunohistochemistry and showed an increased between BMD and adult controls and of TGF-β1 between BMD expression of PDGF-AA in muscles from DMD compared to and DYSF patients as shown in Figures 1C,D. The median healthy controls (Figure 3C). Frontiers in Neurology | www.frontiersin.org 4 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD TABLE 1 | Clinical features of the subjects included in the study. Patients DMD BMD DYSF Pediatric controls Adult controls Number of patients 19 13 15 8 58 Age (years) 10 ± 5.6 31 ± 20.6 46 ± 18.6 10 ± 2 43 ± 15.32 Non-ambulant 6 (31.58%) 5 (38.5%) 10 (66.7%) 0 0 Ventilator support 1 (5.3%) 2 (15.38%) 1 (6.66%) 0 0 Corticoid treatment 15 (78.94%) 0 0 0 0 qMRI 7 (36.8%) 8 (61.5%) 0 0 0 Age is given as mean ± standard deviation. Frequencies are given as absolute number followed by the percentage in brackets. DMD, Duchenne Muscular Dystrophy; BMD, Becker muscular dystrophy; DYSF, dysferlinopathy; qMRI, quantitative muscle magnetic resonance imaging. FIGURE 1 | Serum levels of different growth factors in DMD, BMD, DYSF and in pediatric and adult healthy controls. (A) PDGF-AA, (B) PDGF-BB, (C) TGF-β1, and (D) CTGF levels were measured. Variables are represented as median and interquartile range (IQR). Statistical significance of the results by Mann-Whitney test for the DMD compared against pediatric controls and Kruskal-Wallis test/Dunn’s multiple comparison analysis for the BMD, DYSF, and adult controls: *p ≤ 0.05 and **p ≤ 0.01. Frontiers in Neurology | www.frontiersin.org 5 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD TABLE 2 | Correlation between PDGF-AA serum levels and different muscle function tests, spirometry values, and muscle MRI. DMD BMD Test Correlation coefficient Spearman p-value Correlation coefficient Spearman p-value 10MWT 0.738 0.037 0.9 0.001 6MWT −0.637 0.026 −0.929 0.001 Tup4 0.287 0.365 0.9 0.001 Tdo4 0.105 0.746 −0.867 0.012 TRF 0.406 0.191 0.964 0.001 MRCT −0.537 0.022 −0.398 0.178 Pinch 0.574 0.082 0.452 0.260 Grip 0.676 0.152 0.476 0.233 PUL 0.741 0.205 0.467 0.302 MFM-D3 0.975 0.005 −0.61 0.885 Egen klassification −0.700 0.188 0.300 0.625 Activlim 0.521 0.231 −0.287 0.490 NSAA −0.124 0.717 −0.952 0.001 FVCs% 0.500 0.391 0.738 0.037 FVCl% 0.400 0.505 0.524 0.183 FEV1% 0.800 0.104 0.810 0.015 MRI Thigh FF −0.179 0.702 −0.786 0.021 Significance was analyzed using Spearman test. 10MWT, 10 meters run/walk test; 6MWT, 6 minutes walking test; Tup4, Time to climb up 4 steps; Tdo4, Time to go down 4 steps; TRF, Time to rise from floor; MRCT, Composite score of the muscle strength calculated using MRC score in both upper and lower limbs; PUL, Performance of the Upper Limb scale; MFM-D3, Motor Function Score 20 items (distal dimension); Activlim, ACTIVity LIMitations scale; NSAA, North Start Ambulatory Assessment; FVCs%, Force Vital Capacity Percentage Predicted while sitting; FVCl%, Force Vital Capacity Percentage Predicted while lying; FEV1%, Forced expiratory volume in the first second; MRI Thigh FF, fat fraction in thighs. DISCUSSION a good biomarker to study diseases’ progression (16). Other molecules present in muscle and involved in the pathophysiology The goal of the work behind this manuscript was to identify of muscular dystrophies have been studied. For example, Hathout serum candidate biomarkers to track progression in DMD et al. (26) found 44 proteins which were able to differentiate DMD and BMD. patients from healthy volunteers. Other studies aimed not only to The current development of new therapies for muscle differentiate between patients and controls, but also to correlate dystrophies, partly due to the progress in the field of genetics in with functional state and treatment (27–29) or muscle function the latest decades, raises the need for reliable outcome measures. tests (30). Fewer manuscripts aim to provide longitudinal data DMD and BMD are slow progressive diseases where weakness is on the suggested biomarkers (31–33). developed over years. However, clinical trials designed for these We were interested in investigating the serum concentration disorders must be able to prove presence or absence of effect in of PDGF-AA, PDGF-BB, CTGF, and TGF β1 in our patients a shorter period of time. Biomarkers that can closely track the because of their role in fibrosis. The process of muscle fibrosis pathophysiological mechanisms of these diseases, and therefore in patients with muscular dystrophies is highly complex and not be used as outcome measures, would be desirable. The research in completely understood (34). It is known that chronic muscle the field has been centered in two different measurements. On the degeneration leads to persistent inflammatory infiltration, one hand, qMRI has demonstrated to be a reliable tool to analyze muscle necrosis and activation of fibroadipogenic progenitor changes in muscle structure in patients with muscular diseases. cells (FAPs) and fibroblasts, which release proteins of the Fat replacement, assessed using Dixon imaging or spectroscopy extracellular matrix such as collagen-I, leading to the expansion could be a suitable outcome measure, because it correlates with of fibrotic tissue (34–36). The PDGF family in general and PDGF- muscle function and it is sensitive to changes over short periods AA in particular play an important role in this process (22, 37). in several disorders such as DMD, Pompe or LGMD-2I (7, PDGF-AA is produced by different cell types, such as platelets, 15, 24). On the other hand, blood biomarkers are especially muscle fibers or macrophages and acts via the PDGF receptor interesting because they are easy to obtain. Serum concentration alpha (PDGFRα) (38). When PDGF-AA binds to its receptor, of molecules such as cytokines, metalloproteinases or microRNAs several signaling pathways involved with cellular proliferation, have shown promising results in some neuromuscular diseases differentiation and motility are triggered (39). After an acute (16, 25). Creatine kinase (CK) is a widely accepted biomarker muscle injury, damaged fibers and inflammatory cells release for diagnosis of DMD, BMD and other muscular dystrophies. several cytokines, including PDGF-AA, which activate FAPs However, its levels fluctuate and are influenced by different inducing the release of extracellular matrix components (40). conditions such as physical activity, therefore it is not considered Extracellular matrix is needed for muscle regeneration as it acts Frontiers in Neurology | www.frontiersin.org 6 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD FIGURE 2 | Correlation between PDGF-AA levels and different muscle tests in patients with DMD and BMD. (A) Correlation between PDGF-AA and the results of the 6MWT; (B) Correlation between PDGF-AA 10 MWT; (C) Correlation between PDGF-AA and results of the NSAA scale; (D) Correlation between PDGF-AA and thigh fat fraction; (E) Distribution of PDGF-AA serum levels depending on the muscle strength quantified using MRC score; (F) distribution of PDGF-AA serum levels depending on the age of the patients. Spearman test was performed, R, Correlation coefficient; p, statistical significance. Red lines: BMD patients; blue lines: DMD patients. Frontiers in Neurology | www.frontiersin.org 7 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD FIGURE 3 | PDGF-AA expression is increased in muscles of patients with dystrophinopathy. (A) PDGF-AA expression in skeletal muscles of healthy controls; (B) PDGF-AA expression in a muscle biopsy of a DMD patient and (C) Real Time PCR showing PDGF-AA mRNA expression in skeletal muscle samples of controls and patients with dystrophinopathy. Bars show mean mRNA expression and standard deviation. as a scaffold for muscle fiber regeneration (41). When muscle BMD, and with the results of Dixon MRI in BMD. Because of the regeneration is completed, inflammatory cells and the new common underlying pathophysiologic mechanism of both DMD muscle fibers release TNF-α that promotes apoptosis of FAPs and and BMD, we speculate that serum levels of PDGF-AA should fibroblasts reducing their number and avoiding the development be further studied in a larger cohort of DMD/BMD patients and of fibrosis. At that stage, levels of PDGF-AA decrease (42). considered as a potential biomarker for the disease. However, in dystrophinopathies where there is chronic muscle Our results suggest that the expression of PDGF-AA in DMD damage, inflammatory cells permanently infiltrate the injured and BMD patients follows a bell curve distribution. In initial muscle continuously releasing PDGF-AA, which expand the stages of the disease, when histological changes are consistent number of activated FAPs leading to an increased non-controlled with acute muscle damage, PDGF-AA expression by muscle fibrotic response (40, 43). Consequently, treatment of murine fibers is still low, similarly to that observed in controls. As the models of DMD with tyrosine kinase inhibitors that block the disease progresses, muscle fibers degenerate leading to increased PDGFRα such as imatinib, crenolanib and nintedanib, decreased production of PDGF-AA that potentially activates FAPs involved fibrosis and improved muscle function of these animals (40, 42, in the expansion of fibrotic and adipogenic tissue (22). In 44). We think that due to this increased pro-fibrotic response advanced stages, when most of the muscle has been replaced in patients with DMD, the levels of PDGF-AA are high in by fibrotic and fatty tissue, levels drop again as there are no comparison to controls, as shown in our study. more muscle fibers releasing PDGF-AA. This hypothesis allows Other primary fibrotic growth factors including the TGF-β1 us to understand the results obtained in the correlations between family and CTGF have also been reported to be involved in PDGF-AA levels and the muscular function of the patients. the process of muscle fibrosis (36, 45, 46). TGF-β1 and PDGF- Patients in early stages of the disease, in whom muscle strength BB participate in mesenchymal cell proliferation and fibrotic assessed using the MRC scale is only mildly affected, have low remodeling (47, 48) and CTGF stimulates chemotaxis and levels of PDGF-AA. These patients in early stages are still able proliferation of fibroblasts and the production of extracellular to walk, have high scores on the NSAA, need little time to walk matrix components (45). 10 m and cover long distances in the 6MWT. Patients in an Despite its role in the physiopathology of muscle intermediate stage of the disease have high levels of PDGF-AA degeneration, there are only few reports studying the potential and are clearly weak as it is demonstrated using the MRC scales. utility of growth factors as biomarkers of the disease (49–51). These patients are able to cover 200–400 m in the 6MWT, but In our study, the serum concentration of PDGF-AA was they still have intact respiratory muscle strength. In contrast, increased in patients with DMD in comparison to pediatric patients in advanced stages of the disease have lower levels of controls. There was also an increase in PDGF-AA levels in serum PDGF-AA, are extremely weak as assessed with the MRC BMD patients compared to adult controls, although it did not scale and have higher fat replacement in the muscles. Moreover, reach statistical significance. Moreover, PDGF-AA serum levels these patients are not able to walk and have affected respiratory correlated with several muscle function tests both in DMD and muscles as shown using the spirometry. Although levels of CTGF Frontiers in Neurology | www.frontiersin.org 8 June 2021 | Volume 12 | Article 659922 Alonso-Jiménez et al. PDGF-AA in DMD and BMD were also increased in BMD patients in comparison to controls, DATA AVAILABILITY STATEMENT we only found significant correlations with the MRC scale. The process of fibrosis is not exclusive to muscular tissue. In The original contributions presented in the study are included fact, fibrotic tissue remodeling can affect virtually every system. in the article/supplementary material, further inquiries can be Both primary fibrotic diseases such as systemic sclerosis and directed to the corresponding author/s. idiopathic pulmonary fibrosis and secondary fibrotic responses in other organs share many commonalities. An initial injury triggers ETHICS STATEMENT the reparative process in which lymphocytes and macrophages release profibrotic mediators. These mediators promote the The studies involving human participants were reviewed and activation of myofibroblasts which liberate extracellular matrix approved by Hospital de la Santa Creu i Sant Pau Ethics proteins leading to structural changes. Therefore, it is not Committee and Hospital de Sant Joan de Deu Ethics Committee. surprising to find that the growth factors that we have studied Written informed consent to participate in this study was here are also implicated in other diseases such as atherosclerosis, provided by the participants’ legal guardian/next of kin. liver and pulmonary fibrosis, cirrhosis, myocardial infarction and neoplasms (52–58). However, the primary site of injury, the AUTHOR CONTRIBUTIONS cells that differentiate into myofibroblasts and the organization of the immune response differs among the different types of AA-J, EF-S, and JD-M contributed to the study conception disorders (52). Interestingly, in our study TGF-β1 showed a and design. Clinical evaluation of patients was performed by different behavior in BMD than in DYSF patients, since TGF- AA-J, JD-M, JA-P, DN-d, CO, and CJ-M. Blood samples and β1 levels were increased in BMD patients compared to DYSF data collection was obtained by SS. EG, JD-M, and EF-S patients. In our opinion this data suggests that, although the designed and performed the experiments. PP-J, AC-R, and final stage of the disease may be common among muscular XS-C also collaborated with the execution of the experiments. dystrophies, the process leading to replacement of muscle fibers PM, JD-M, CN-P, and JL designed the MRI protocol. by fibrotic and fatty tissue may have some particularities that The analysis of the MRI images was performed by AA-J. could be exclusive to each type of muscular dystrophy, such as Functional assessments were performed by IB, IP, CG, and the type of muscle injury or the molecular pathways that are EM. AA-J, EF-S, and JD-M analyzed the results and wrote the activated in response to this muscle injury. However, we did manuscript. All authors contributed with their comments to the not find significant differences in TGF- β1 between patients and final version. controls (only between BMD and DYSF patients) and therefore these results should be taken with caution. FUNDING The four growth factors that we have studied have been previously related with fibrosis and with muscle regeneration This work was supported by FIS PI15/01822 to JD-M and in muscular dystrophies (59, 60) but have also been studied in FIS (PI18/1525) to JD-M and XS-C, and FIS (PI19/00122) other neuromuscular diseases. For instance, our group identified to CJ-M (Fondo Europeo de Desarrollo Regional [FEDER], a decrease in PDGF-BB levels in patients with Pompe disease Instituto de Salud Carlos III, Spain). XS-C was supported suggesting that there is a deficit in muscle regeneration in this by Sara Borrell postdoctoral fellowship (CD18/00195), Fondo disease, and Sato et al. showed that CTGF levels were high in Social Europeo (FSE), Instituto de Salud Carlos III (Spain). patients with polymyositis and dermatomyositis (61, 62). Funders did not participate in the study design. This work This is a small study with several limitations: firstly, it is a is generated within the European Reference Network for transversal study without longitudinal data. Secondly, only half Neuromuscular diseases. of the patients underwent Dixon MRI, and the ones who did were already severely affected by the disease and therefore had high FF. ACKNOWLEDGMENTS Despite these limitations, our study suggests that serum levels of PDGF-AA can differentiate between DMD patients and We would like to thank all the patients and families who controls, and that they correlate with the results of different participated in the study for their support and patience. We are muscle tests. For BMD patients we have also found correlation indebted to the Biobanc de l’Hospital Infantil Sant Joan de Déu between PDGF-AA levels and muscle function tests, including fat per a la Investigació integrated in the Spanish Biobank Network muscle content assessed by Dixon MRI. Based on these results, we of ISCIII for the sample and data procurement. We thank the propose PDGF-AA as a growth factor to be further investigated MRI technician team for their help. We also thank Mr. John in in a larger cohort of patients and in longitudinal studies. Wilkos for his language advice. REFERENCES gastrointestinal and nutritional management. Lancet Neurol. 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