Connective tissue growth factor is overexpressed in muscles of human muscular dystrophy
Introduction
Recent studies have enhanced our understanding of the molecular basis of Duchenne muscular dystrophy (DMD) and other human muscular dystrophies [1], [2]. A deficiency of dystrophin does not invariably produce progressive muscle degeneration in all species [3], [4], suggesting that dystrophin deficiency is a condition for, rather than a determinant of, DMD [4]. Instead, secondary downstream mechanisms, such as inflammation [5], ischemia [6], or fibrosis [7], might have substantial roles in the muscle wasting and weakness of DMD.
Transforming growth factor (TGF)-beta1, which is the best-characterized fibrogenic mediator [8], is overexpressed in human dystrophic muscle, and increased TGF-beta1 mRNA levels are associated with the initial stage of tissue fibrosis [7]. In addition, the plasma TGF-beta1 level is elevated in patients with DMD and congenital muscular dystrophy [9]. These findings suggest that TGF-beta1 is involved in the fibrotic process of human muscular dystrophy. Recent studies demonstrated that connective tissue growth factor (CTGF) is the downstream autocrine mediator of TGF-beta1 [10]. CTGF is a secreted, extracellular matrix-associated protein expressed at high levels in fibroblasts [11] that modulates many cellular functions, including proliferation, migration, adhesion, and extracellular matrix production, and it is involved in many biological and pathological processes [12], [13], [14]. In addition to being a potent fibroblast mitogen and chemoattractant, CTGF stimulates the production of procollagen and fibronectin in fibroblasts [15] and plays an important role in promoting the formation of connective tissue after injury. Furthermore, there is growing evidence that CTGF is involved in the development of tissue fibrosis, based on the observation that CTGF expression is increased in skin and internal organ fibrosis, such as in scleroderma, idiopathic pulmonary fibrosis, chronic pancreatitis, liver cirrhosis, inflammatory bowel disease, renal fibrosis, and the fibrotic areas of atherosclerotic lesions [8], [13].
Interestingly, elevated levels of CTGF mRNA were recently reported in skeletal muscle in a canine model of DMD [16]. However, the precise immunolocalization of CTGF in dystrophic muscles and the role of CTGF in human muscular dystrophy have not been examined. In the present study, immunohistochemistry and the reverse transcription-polymerase chain reaction (RT-PCR) were used to examine whether CTGF is overexpressed in dystrophic muscle and whether TGF-beta1-CTGF pathway has a potential role in the progression of muscular dystrophy.
Section snippets
Immunohistochemistry
Diagnostic muscle biopsies were obtained from 26 patients, ages 6 months to 18 years. Written informed consent to use these specimens for research was obtained from all of the patients' families. The study was approved by the local ethical committee. Eight of the biopsies were from patients with DMD, six were from Fukuyama-type congenital muscular dystrophy (FCMD) patients, two were from Becker muscular dystrophy (BMD) patients, two were from spinal muscular atrophy patients, one was from a
Immunohistochemistry (Figs. 1–4, Table 2)
In normal muscle, immunoreactivity against anti-CTGF antibody was observed in the intramuscular capillaries (Fig. 1A), as reported in other tissues, and confirmed by double immunostaining with CTGF and CD31 (Fig. 2A–C). Double labeling with anti-CTGF antibody and alpha BT revealed that CTGF protein localized primarily to the NMJs (Fig. 2D–F).
In all biopsies of dystrophic muscle, there was a high level of CTGF immunoreactivity in the cytoplasm of regenerating muscle fibers that were identified
Discussion
The fibrotic process in dystrophic muscles might be a very complex process and might involve several growth factors, such as TGF-beta1 [7], CTGF in the present study, platelet-derived growth factor (PDGF) [20], and basic fibroblast growth factor (bFGF) [21], as well as various pathophysiological and biochemical pathways. The possible involvement of nitric oxide synthase [22], oxidative stress [23], the immune system [24], mast cell degranulation [25], phospholipase [26], and inflammation [5]
Acknowledgement
This work was supported partly by funding from the Japan–China Sasakawa Medical Fellowship and THE MORINAGA HOSHI-KAI, Japan.
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