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Dystrophin deficiency associated with Duchenne muscular dystrophy (DMD) results in chronic inflammation and severe skeletal muscle degeneration, where the extent of muscle fibrosis contributes to disease severity. The microenvironment of dystrophic muscles is associated with variation in levels of markers of degeneration and regeneration. Since in dystrophic muscle apoptosis precedes necrosis, markers of apoptosis can be used as indicators of degeneration, while regeneration can be measured in terms of cytokines and growth factor expression”; and then throughout the text use “markers of apoptosis/degeneration. The present study is an attempt to evaluate the extent of degeneration and regeneration in DMD patient blood. Subjects were 24 boys with DMD diagnosed at the molecular level versus 20 age and socioeconomic matching healthy boys. In their blood, levels of Fas and FasL and Bax/Bcl-2 and plasma DNA fragmentation were measured as markers of apoptosis. The cytokine tumor necrosis factor alfa (TNF-α), and the growth factors: basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were measured as markers of regeneration. Plasma DNA fragmentation (0.38% ± 0.12 vs. 0.2% ± . 0.1.5) and Fas (9.9 ± 2.8 vs. 2 ± 0.1, p < 0.001) together with FasL mRNA expression in circulating lymphocytes (0.47 ± .09 vs. 0.24 ± .04, p < 0.001) were significantly increased in DMD patients compared to controls. There was a significant increase in Bax (0.19 ± 0.7 vs. 0.05 ± 0.1, p < 0.00001) expression and a significant decrease in Bcl-2 protein (6.4 ± 1.6 vs10 ± 2.8, p < 0.00001) as compared to controls. Among markers of regeneration, TNF- α (30.2 ± 9.5 vs. 3.6 ± 0.9) and bFGF (21.7 ± 10.3 vs. 4.75 ± 2.2) were significant increased while VEGF was significantly decreased (190 ± 115 vs. 210 ± 142.) in blood of DMD patients compared to controls. Our results indicate that Fas/FasL and Bax/Bcl-2 are involved in muscle atrophy and degeneration in DMD patients, while regeneration process does not cope with the degeneration.
DMD is an X-linked recessive disorder, primarily characterized by progressive muscle weakness and wasting. Mutations in dystrophin gene are the prime cause for muscle degeneration associated with DMD (1). Normally dystrophin interacts with several members of the dystrophin glycoprotein complex, which forms a mechanical as well as signaling link from the extracellular matrix to the cytoskeleton (2). Mutations in dystrophin result in membrane damage, allowing massive infiltration of immune cells, chronic inflammation, necrosis, and severe muscle degeneration (3). Normally, muscle cells possess the capacity to regenerate in response to injury signals (4), however, this ability is lost in DMD, presumably due to an exhaustion of satellite cells during ongoing degeneration and regeneration cycles (5). Although dystrophin mutations represent the primary cause of DMD, it is the secondary processes involving persistent inflammation and impaired regeneration that likely exacerbate disease progression (6). This results in chronic inflammation and severe skeletal muscle degeneration, where the extent of muscle fibrosis contributes to disease severity. Elevated numbers of inflammatory cells are known to be present at the sites of muscle injuries to interact with cytokine and growth factor signaling (7–9). It is evident that dystrophic muscles undergo increased oxidative stress and altered calcium homeostasis, which may contribute to myofiber loss by triggering both necrosis and apoptosis (10). In humans, DNA-fragmentation and expression of apoptosis-related proteins indicate that apoptosis plays a role in muscle degeneration and regeneration in muscular dystrophies (11).
Muscle tissue repair is a complex biological process that crucially involves activation of stem cells. Skeletal muscle contains two different stem cell types: 1) myogenic stem cells, so-called satellite cells (SCs), that reside beneath the basal lamina of muscle fibers (12) and 2) interstitial multipotent stem cells, which are extralaminal, exhibit fibroblastic morphology and do not express myogenic markers (13). Satellite cells could excrete growth factors including VEGF that would induce angiogenesis and improve cell survival (14). The VEGF is the prototypic member of a family of secreted, homodimeric glycoproteins with endothelial cell-specific mitogenic activity and the ability to stimulate angiogenesis in vivo (15). On the other hand, a number of growth factors such as fibroblast growth factor (FGF) can promote the activation and proliferation of skeletal satellite cell (16, 17).
TNF-α is an early and potent pro-inflammatory cytokine that stimulates the inflammatory response. Even minor trauma to muscle will increase levels of TNF-α by release from mast cells. It is also produced by neutrophils, macrophages and lymphocytes that accumulate rapidly at the site of injury. TNF-α increases rapidly within damaged myofibers and is expressed by myoblasts and myotubes (18–20). It is greatly elevated in injured normal damaged myofibers (18, 19) and myopathic skeletal muscle (21); is chemotactic for myoblasts in vitro (22) and mitogenic for satellite cells in vivo (20), suggesting a direct role in myogenesis of regenerating muscle (18).
The apoptosis cascade can be triggered by 2 main pathways, via an intrinsic, endogenous system such as the mitochondrial Bax/Bcl-2 or via an extrinsic system Fas and FasL involving transmembrane receptors of the death receptor family (23). FasL ligand induces apoptosis through cognate interaction with its receptor Fas (24). FasL is mainly present in activated T lymphocytes, natural killer cells, and macrophages (25, 26).
The aim of the present study is to investigate markers of degeneration and regeneration in blood of DMD patients compared to controls. Markers of degeneration are measured in terms of increased Fas/FasL and Bax/Bcl-2 and plasma DNA fragmentation. Markers of regeneration are the cytokine TNF-α and the growth factors: VEGF and bFGF.
Subjects were 24 boys diagnosed clinically and at the molecular level as having DMD (mean of age (8.1 ± 1.9), versus 20 age and socioeconomic matching healthy boys (mean of age 8.2 ± 2.2). Patients and controls were chosen to be free from any infection and receiving no therapeutic treatment known to increase the oxidative stress. Blood samples were drawn after their parents’ consent.
Total RNA was extracted from lymphocytes using QIAGEN RNA extraction kit (QIAGEN Inc, USA). The RNA samples were reverse transcribed using superscript reverse transcriptase, using QIAGEN OneStep RT-PCR kit (QIAGEN Inc USA, Clini Lab). Primer sequences were as follows: FasL, forward: 5’-CAA GTC CAA CTC AAG GTC CAT GCC-3’; FasL reverse: 5’-CAG AGA GAG CTC AGA TAC GTT- TGAC-3’ (27); β-actin (primers were synthesized simultaneously as an internal reference for all samples) (forward: 5’-GTG GGG CGC CCC AGG CAC CA-3’; β-actin reverse: 5’-CTC CTT AAT GTC ACG CAC GAT TTC-3’); Bax, forward: 5’-CAC CAG CTC TGA- GCA GAT G-3’; Bax reverse: 5’-GCG AGG CGG TGA- GCA CTC C-3’) (28). 5 µl of RT reaction of each cDNA were processed for PCR. Ten μL from each PCR reaction product were separated on a 2% agarose gel then stained with ethidium bromide. The appearance of specific bands (Bax 516 bp, β-actin 540 and FasL 345 bp) was evaluated under ultraviolet light and photographed. Photos were scanned and quantification of each band was carried out using GeneTools version 4 (Syngene, Cambridge, UK). Each quantified data point was related to its individual β-actin.
Soluble Fas protein was measured using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) (29).
DNA Fragmentation Assay: This is done according to the method of Ioannou and Chen 1996 (30). Separation of both fragmented and total DNA is carried out using DNA separating kit (Takara, Japan). DNA fragments were gradient separated from the intact DNA using polyethelene glycol (5% in Ethyl ether) and then quantified spectrophotometrically using Hoechst 33258 (0.2 µg/ml) as a chromophore.
ELISA Bcl-2: The amounts of Bcl2 in circulating lymphocytes were determined by a sandwich enzyme linked immunosorbent assay (ELISA) purchased from Cliniulab, using two anti-human BCL2 monoclonal murine antibodies (31).
Plasma analysis of the cytokine TNF-α was performed using ELISA R & D Kits (32), for the growth factor VEGF using the ACCUCYTE Human VEGF immunoassay kit (33) and for bFGF using human bFGF immunosorbant assay (ELISA) Quantitin kit (34).
Each experimental condition was performed and expressed as mean ± SD. Comparisons were made by Student’s t-test (two-tailed for independent samples).
Percentage of DNA fragmentation per total DNA in plasma showed a significant increase in DMD patients compared to controls (mean = 0.38% ± 0.12 vs. 0.2% ± 0.15, p < 0.001) as shown in Figure Figure44.
Fas protein in plasma showed a significant increase in DMD patients compared to controls (mean 9.9 ± 2.8 vs. 2 ± 0.1, p < 0.001) (Fig. (Fig.44).
FasL mRNA relative expression (Fig. (Fig.1)1) related to β-actin mRNA expression (Fig. (Fig.2)2) in circulating lymphocytes showed a significant increase in DMD patients compared to controls (mean 0.47 ± .09 vs. 0.24 ± .04, p < 0.001) (Fig. (Fig.44).
There is an inverse relationship between Bax and Bcl-2 gene expression. Bax mRNA relative expression (Fig. (Fig.3)3) in circulating lymphocytes related to β-actin mRNA expression (Fig. (Fig.2)2) showed a significant increase among DMD patients compared to controls (mean 0.19 ± 0.07 vs. 0.05 ± 0.01, p < 0.001) (Fig. (Fig.4).4). Bcl-2 protein in circulating lymphocytes (Fig. (Fig.5),5), showed a significant decrease among DMD patients compared to controls (mean 6.4 ± 1.6 vs. 10 ± 2.8, p < 0.001). TNF-α and bFGF were significantly higher in DMD patient blood compared to controls (TNF-α: 30.2 ± 9.5 vs. 3.6 ± 0.9 and bFGF: 21.7 ± 10.3 vs. 4.75 ± 2.2), while VEGF was lower in DMD patient blood compared to controls (190 ± 115 vs. 210 ± 142) (Fig. (Fig.55).
In normal skeletal muscle, damage due to contractile force is followed by an inflammatory response involving multiple cell types that subsides after several days. This transient inflammatory response is a normal homeostatic reaction to muscle damage that induces muscle repair. However in DMD patients a persistent inflammatory response in their skeletal muscles leads to an altered extracellular environment, including an increased presence of inflammatory cells (e.g., macrophages) and elevated levels of various inflammatory cytokines and growth factors. Unfortunately, the signals that lead to successful muscle repair in healthy muscle may promote muscle wasting and fibrosis in dystrophic muscle (34).
TNF-α is an important mediator of inflammatory and autoimmune diseases. It was reported that the mean serum TNF-α concentration in Duchenne muscular dystrophy patients was approximately 1,000 times higher than that in healthy subjects (18) and that TNF-α levels are upregulated in dystrophic muscles from animal models and DMD patients (21, 35). Our results are in agreement with such findings. Among its pleiotropic effects, TNF-α acts as a potent inducer of the inflammatory response transcription factor NF-κB (36). Although dystrophin mutations represent the primary cause of DMD, the secondary processes involving persistent inflammation and impaired regeneration are likely to exacerbate disease progression. The microenvironment of dystrophic muscles consists of elevated numbers of inflammatory cells that act as a complex interface for cytokine signaling (7–9).
Fas/FasL interaction is an important trigger for apoptosis in many cell types expressing Fas as a surface marker (26). In the present study plasma Fas has been shown to be significantly elevated in DMD patients compared to controls. Increased expression of death factor Fas was previously shown to be expressed in muscles of DMD patients compared to controls (37, 38).
A significant increase in Bax mRNA relative expression in blood mononuclear cells was associated with a significant decrease in Bcl-2 protein in the present study. It is a widely accepted view that Bax overexpression promotes cell death in response to apoptotic stimuli, whereas Bcl-2 protein inhibits it (39, 40). Increased Bax mRNA expression has been observed in aging human lymphocytes (41, 42). Exercise loading caused an increased expression in Bax localization in the muscular dystrophy animal model mdx (43). It was suggested that replicative aging of myogenic cells (satellite cells) owing to enhanced myofiber turnover is a common explanation of the progression of DMD pathogenesis (44). On the other hand epigenetics consumptions indicate that interactions between the primary genetic defect and disruptions in the production of free radicals contribute to DMD pathogenesis (45).
In the present study a significant increase in plasma DNA fragmentation percentage was observed in DMD patients compared to controls. DNA fragmentation, which is a marker of apoptosis, was measured in the blood stream, in order to eliminate the invasive technique of muscle biopsy, since it is difficult to identify necrosis in blood stream. DNA fragmentation detected in blood represents the DNA fragments that were released into the blood stream from body tissues and from circulating blood cells due to apoptosis. Apoptosis is a well-conserved cellular destructive process which has been implicated in a variety of diseases such as cancers and neurodegenerative diseases (24). Muscle exercise-induced apoptosis is a normal regulatory process that serves to remove certain damaged cells without a pronounced inflammatory response, thus ensuring optimal body performance (46). Lately, the activation of apoptotic machinery in different pathologic and physiologic muscle atrophic conditions including muscle disuse (47), hindlimb unloading (48), muscle dystrophy (37), sarcopenia (49), and neuromuscular diseases (50), has been demonstrated. Supporting our data, previous studies indicated that apoptotic morphology is increased in dystrophic (mdx mice) muscle and in cultured muscle cells (51). Recent studies suggest that cell death in mdx muscle may be initiated by apoptosis and followed by necrotic processes (52). Tissue sections of dystrophic muscle demonstrate apoptotic myonuclei in degenerating muscle fibers (10, 11, 53). Several groups have proposed that the intensity of the signal, such as intracellular ATP levels, hypoxia and/or reactive oxygen species can dictate whether a cell dies by a primarily necrotic, or an apoptotic, pathway (54–56).
Results of the present study showed increased levels of bFGF compared to controls. Growth factors, represent essential elements in the modulation of muscle cell regeneration and differentiation (57). Interestingly, many growth factors, including basic fibroblast growth factor (bFGF), have been shown to be upregulated in mdx mice (58) and serum levels have been shown to increase in DMD patients compared to controls. (59). DMD lack dystrophin and, as a result, their skeletal muscles show extensive muscle fiber damage and fibrosis, and regeneration (60). Mdx mice have a large number of degenerating and regenerative muscle fibers in the first 4 months of life, after which the number of degenerative and necrotic fibers declines (61). It has been suggested that bFGF participates in the degenerative and regenerative responses of striated muscle to dystrophic injury and is involved with the physiology of different striated muscles (61).
In the present study VEGF was lower in DMD patients compared to controls. Exercise is known to increase muscle VEGF mRNA (62, 63) and DMD patients usually have limited physical activity, which can explain the lower level of VEGF in our DMD patients. Data obtained from comparative studies on young and ageing muscle and on exercised and sedentary muscles, indicated that in aged compared with young men, muscle capillary contacts and capillary-to-fiber perimeter exchange index were lower and that VEGF muscle protein decreases with ageing (64, 65) Replicative aging of myogenic cells (satellite cells), owing to enhanced myofiber turnover, is an accepted common explanation of the progression of DMD pathogenesis (66). Supporting our finding can be obtained from a previous study that showed that intramuscular delivery of VEGF using recombinant adeno-associated virus vectors in mdx mice induced an increased forelimb strength and strength normalized to weight (67).
In the present study it can be speculated that the significant increase in tissue Fas detected in plasma as well as circulating lymphocytes’ FasL in DMD patients compared to controls contribute to the increased apoptosis in muscle cells and consequently to the DNA fragmentation detected in blood. Increase in Bax and decreased Bcl2 in circulating mononuclear cells of DMD patients compared to controls reflects the increase of oxidative stress in these patients (44, 45, 68). Our results indicate that apoptosis and its markers determined in blood of DMD patients can replace the invasive technique of tissue biopsy. Also, growth factors and cytokines are associated with DMD pathogenesis, where TNF-α, bFGF and VEGF can give a reflection of the severity of DMD pathology. Detecting such growth factors and cytokines biomarkers in blood of DMD patients represents for the first time a non invasive technique compared to the invasive technique of muscle biopsy previously used as an prognostic tool for of disease severity.