Edited by: Jens Schmidt, Universitätsklinikum Göttingen, Germany
Reviewed by: Melissa Hooijmans, VU University Medical Center, Netherlands; John Vissing, Rigshospitalet, Denmark
This article was submitted to Neuromuscular Disorders and Peripheral Neuropathies, a section of the journal Frontiers in Neurology
†These authors have contributed equally to this work and share first authorship
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Duchenne (DMD) and Becker (BMD) muscular dystrophy are X-linked recessive disorders produced by mutations in the
DMD has an homogeneous clinical picture characterized by early onset of muscle weakness progressing during childhood and leading to loss of ambulation during adolescence (
In slow progressive diseases, the identification of functional changes over a short period is not an easy task. In recent years several outcome measures such as the 6 Minutes-Walk Test (6MWT), the Timed Up-and-Go Test (TuGo) or the North Star Ambulatory Assessment (NSAA) have been developed to monitor muscle function in natural history studies and clinical trials (
The main aim of this study was to analyze the serum concentration of a group of growth factors related to the process of muscle fibrosis in a cohort of 19 patients with DMD and 13 patients with BMD and study whether there was a correlation with the results of muscle function tests and quantitative MRI (qMRI).
We report the results of a transversal study of a cohort of 19 DMD and 13 BMD patients seen at Hospital de la Santa Creu i Sant Pau (HSCSP) and Hospital Sant Joan de Déu (HSJD) in Barcelona. All patients included in the study were assessed using muscle function tests and spirometry and filled out a daily life activities questionnaire. qMRI was not a mandatory test and was performed in a subset of patients followed-up in HSCSP only. Blood samples were obtained before the muscle function assessment. The HSCSP and HSJD Ethics Committees approved the study, and all participants signed an informed consent form. All study procedures were performed in accordance with Spanish regulations.
Inclusion criteria for the study were: (1) Diagnosis of DMD or BMD confirmed by the identification of pathogenic variants in the
Muscle function tests were performed by physiotherapists with considerable experience in neuromuscular disorders. We assessed patients using timed tests, functional ability scales, and measurement of muscle strength. Timed tests included the 6MWT, the 10 Meter Run/Walk test (10MWT), the Time to Climb Up (Tup4) and Down (Tdo4) Four Steps and the Time to Rise from the Floor (TRF). Functional ability included the NSAA and the Motor Function Measure 20-item scale (MFM-20) (
We measured the serum concentration of the following growth factors related with muscle fibrosis: Serum Platelet-Derived Growth Factor BB (PDGF-BB), Transforming Growth Factor β1 (TGF-β1), Platelet-Derived Growth Factor AA (PDGF-AA) and connective tissue growth factor (CTGF). Blood was centrifuged at 1,600 g for 9 min at 4°C. Samples were aliquoted and stored at −80°C until analysis. PDGF-BB and TGF-β1 levels were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D, Minneapolis, MN, USA), according to the manufacturer's instructions. PDGF-AA human ELISA kit was provided by ThermoFisher (Thermo Fisher Scientific, Nepean, Canada) and CTGF by EIAAB Science Co (Wuhan, China). Minimum detectable cytokine concentrations for these assays were measured to be 1.7 pg/ml for TGF-β1, 15 pg/ml for PDGF-BB, 40 pg/ml for PDGF-AA and 0.18 ng/ml for CTGF. Samples were measured in duplicate and read on a microplate reader Beckman Coulter AD 340 (Beckam-Coulter, Brea, CA, USA) with AD-LD software.
These growth factors were determined in the 19 DMD patients, 13 BMD patients, 15 patients with mutations in the
7 DMD and 8 BMD patients underwent thigh muscle MRI in a 1.5T Ingenia MR system (Philips Healthcare, Best, the Netherlands) at HSCSP. Axial 3D FFE Dixon sequence was acquired with the following parameters: TR/TE = 5.78/1.84 ms, flip angle = 15°, voxel size = 1 × 1 × 3 mm and FOV 520 × 340 × 300 mm. Water and fat images were automatically obtained from the Dixon acquisition using a single peak model. One investigator (A.A-J) estimated fat content in the images using the PRIDE (Philips Research Image Development Environment) tool developed for this purpose. This tool provides the fat fraction (FF), defined as fat/(fat+water). Regions of interest (ROIs) were manually drawn on five slices on the right thigh of the following muscles:
Total RNA was extracted from triceps or biceps biopsy samples from 2 healthy controls and 2 DMD patients using RNeasy® Micro Kit (Qiagen, Hilden, Germany) following manufacturer's instruction. RNA was quantified using a nanodrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wil- mington, DE, USA). One μg of total RNA was reverse-transcribed to complementary DNA (cDNA) using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA).
Real-Time PCR (qPCR) was performed using the TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). All mRNA- specific FAM-labeled primers/probe were purchased from Applied Biosystems. Relative quantification was performed using the comparative Ct method and all results were compared with the control samples. GAPDH was used as endogenous control.
PDGF-AA immunohistochemistry was performed from paraffinized muscle. The samples were deparaffinized (xylol, absolute ethanol, 95° ethanol, and 70° ethanol), placed in distilled water and pretreated with 10 mM citrate (pH 6) at 100°C. Then samples were washed in PBS and incubated in blocking solution (4% bovine serum albumin). The primary anti-PDGF-AA antibody (Merck Millipore, Darmstadt, Germany) was then added at room temperature for 1 h. After 3 washes, it was incubated with biotinylated anti-rabbit antibody (Vector Laboratory Inc. Burlingame, CA) for 1 h at room temperature and then avidin-biotin peroxidase complex (Dako, Glostrup, Denmark) was added. Finally, the sections were soaked in Mayer's hematoxylin for 10 s, washed under running water and mounted in the aqueous mounting medium Aquatex (Merck Millipore, Darmstadt, Germany).
Non-parametric tests were used for the statistical analysis as we demonstrated that the distribution of the variables was not uniform as studied using Kolmogorov-Smirnov and did not follow a normal distribution as studied using Shapiro-Willis test. The Mann-Whitney U test was used to identify significant differences in variables between 2 groups. Non-parametric Kruskal–Wallis test analysis was used to assess differences among 3 groups followed by Dunn's multiple comparison post-test. We used Spearman's rank correlation (coefficient reported as ρ) to investigate any correlation between the serum concentration of growth factors and the results of the muscle function tests, quality of life scales and the thigh FF obtained using Dixon imaging. Correlation coefficients higher than 0.6 were considered good. Because the objective of our research was to explore the possibilities of these growth factors for further studies, we decided not to use multiple comparisons. The results of all statistical studies were considered significant if
A total of 19 patients with DMD (mean age 10 ± 5.6 years), 13 patients with BMD (mean age 31 ± 20.6 years), 15 DYSF patients (mean age 46 ± 18.6 years), 8 pediatric controls (mean age 10 ± 2 years) and 58 adult controls (mean age 43 ± 15.32 years) were enrolled in the study. There was no statistical difference in age between the DMD group and the pediatric controls (Mann-Whitney
Clinical features of the subjects included in the study.
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 |
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, PDGF-BB, CTGF, and TGF-β1 between DMD and pediatric healthy controls whereas BMD and DYSF patients were compared against adult healthy controls (
Serum levels of different growth factors in DMD, BMD, DYSF and in pediatric and adult healthy controls.
In a second step, we analyzed if there was a correlation between growth factor serum concentration and the results of muscle function tests, spirometry, daily life activities scales and Dixon MRI in DMD and BMD patients. The growth factor that showed a larger number of significant correlations was PDGF-AA as it is shown in
Correlation between PDGF-AA serum levels and different muscle function tests, spirometry values, and muscle MRI.
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 |
Correlation between PDGF-AA levels and different muscle tests in patients with DMD and BMD.
In a final step we studied PDGF-AA expression patterns in muscle biopsies of patients with DMD and BMD and in controls using immunohistochemistry and Real Time PCR. As it has been previously shown, PDGF-AA is expressed in the capillaries in muscles of healthy controls, where it probably has a role in binding the pericytes to the endothelial cell (
PDGF-AA expression is increased in muscles of patients with dystrophinopathy.
The goal of the work behind this manuscript was to identify serum candidate biomarkers to track progression in DMD and BMD.
The current development of new therapies for muscle dystrophies, partly due to the progress in the field of genetics in the latest decades, raises the need for reliable outcome measures. DMD and BMD are slow progressive diseases where weakness is developed over years. However, clinical trials designed for these disorders must be able to prove presence or absence of effect in a shorter period of time. Biomarkers that can closely track the pathophysiological mechanisms of these diseases, and therefore be used as outcome measures, would be desirable. The research in the field has been centered in two different measurements. On the one hand, qMRI has demonstrated to be a reliable tool to analyze changes in muscle structure in patients with muscular diseases. Fat replacement, assessed using Dixon imaging or spectroscopy could be a suitable outcome measure, because it correlates with muscle function and it is sensitive to changes over short periods in several disorders such as DMD, Pompe or LGMD-2I (
We were interested in investigating the serum concentration of PDGF-AA, PDGF-BB, CTGF, and TGF β1 in our patients because of their role in fibrosis. The process of muscle fibrosis in patients with muscular dystrophies is highly complex and not completely understood (
Other primary fibrotic growth factors including the TGF-β1 family and CTGF have also been reported to be involved in the process of muscle fibrosis (
Despite its role in the physiopathology of muscle degeneration, there are only few reports studying the potential utility of growth factors as biomarkers of the disease (
In our study, the serum concentration of PDGF-AA was increased in patients with DMD in comparison to pediatric controls. There was also an increase in PDGF-AA levels in BMD patients compared to adult controls, although it did not reach statistical significance. Moreover, PDGF-AA serum levels correlated with several muscle function tests both in DMD and BMD, and with the results of Dixon MRI in BMD. Because of the common underlying pathophysiologic mechanism of both DMD and BMD, we speculate that serum levels of PDGF-AA should be further studied in a larger cohort of DMD/BMD patients and considered as a potential biomarker for the disease.
Our results suggest that the expression of PDGF-AA in DMD and BMD patients follows a bell curve distribution. In initial stages of the disease, when histological changes are consistent with acute muscle damage, PDGF-AA expression by muscle fibers is still low, similarly to that observed in controls. As the disease progresses, muscle fibers degenerate leading to increased production of PDGF-AA that potentially activates FAPs involved in the expansion of fibrotic and adipogenic tissue (
The process of fibrosis is not exclusive to muscular tissue. In fact, fibrotic tissue remodeling can affect virtually every system. Both primary fibrotic diseases such as systemic sclerosis and idiopathic pulmonary fibrosis and secondary fibrotic responses in other organs share many commonalities. An initial injury triggers the reparative process in which lymphocytes and macrophages release profibrotic mediators. These mediators promote the activation of myofibroblasts which liberate extracellular matrix proteins leading to structural changes. Therefore, it is not surprising to find that the growth factors that we have studied here are also implicated in other diseases such as atherosclerosis, liver and pulmonary fibrosis, cirrhosis, myocardial infarction and neoplasms (
The four growth factors that we have studied have been previously related with fibrosis and with muscle regeneration in muscular dystrophies (
This is a small study with several limitations: firstly, it is a transversal study without longitudinal data. Secondly, only half of the patients underwent Dixon MRI, and the ones who did were already severely affected by the disease and therefore had high FF.
Despite these limitations, our study suggests that serum levels of PDGF-AA can differentiate between DMD patients and controls, and that they correlate with the results of different muscle tests. For BMD patients we have also found correlation between PDGF-AA levels and muscle function tests, including fat muscle content assessed by Dixon MRI. Based on these results, we propose PDGF-AA as a growth factor to be further investigated in in a larger cohort of patients and in longitudinal studies.
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
The studies involving human participants were reviewed and approved by Hospital de la Santa Creu i Sant Pau Ethics Committee and Hospital de Sant Joan de Deu Ethics Committee. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.
AA-J, EF-S, and JD-M contributed to the study conception and design. Clinical evaluation of patients was performed by AA-J, JD-M, JA-P, DN-d, CO, and CJ-M. Blood samples and data collection was obtained by SS. EG, JD-M, and EF-S designed and performed the experiments. PP-J, AC-R, and XS-C also collaborated with the execution of the experiments. PM, JD-M, CN-P, and JL designed the MRI protocol. The analysis of the MRI images was performed by AA-J. Functional assessments were performed by IB, IP, CG, and EM. AA-J, EF-S, and JD-M analyzed the results and wrote the manuscript. All authors contributed with their comments to the final version.
PM is employed by the company Philips Healthcare Iberia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank all the patients and families who participated in the study for their support and patience. We are indebted to the Biobanc de l'Hospital Infantil Sant Joan de Déu per a la Investigació integrated in the Spanish Biobank Network of ISCIII for the sample and data procurement. We thank the MRI technician team for their help. We also thank Mr. John Wilkos for his language advice.