Stromal cell-derived factors in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by increased muscle damage and an abnormal blood flow after muscle contraction leading to a state of functional ischemia. Abundant evidence suggests that endothelial circulating progenitor cells (EPCs) play an important role in mediating vascular and muscle repair mechanisms and that the stromal cell-derived factor (SDF)-1 alpha chemokine is responsible for both progenitor cell mobilization from the bone marrow to peripheral blood and homing to the sites of vascular and tissue injury. Since normal neovascularization is disrupted in DMD pathogenesis and may contribute ultimately to heart failure and sudden death, the aim of the present study is to investigate whether the (SDF)-1 alpha, and EPCs surface receptors in terms of CD34, CD133 and kinase domain receptor (KDR) are involved in DMD pathophysiology. In the present study, peripheral blood concentrations of circulating CD34, CD133, and CD34/ CD 133 progenitor cells were measured by flow cytometry, together with serum levels of (SDF)-1alpha and hypoxia inducible factor (HIF-1alpha.), in 28 DMD patients vs. 20 healthy age and socioeconomic matching controls. Results showed a significant increase in the number of mononuclear cells bearing EPC markers, HIF-1alpha mRNA expression and serum (SDF)-1 alpha, indicating that regeneration is an ongoing process in these patients. However, this regeneration cannot counterbalance the damage induced by dystrophine mutation.

Introduction

Duchenne muscular dystrophy (DMD) represents an X-linked recessive disorder related to mutations in the dystrophin gene which is located on chromosome Xp21.1 (1). It is the most common and severe form of dystrophinopathies, characterized by progressive and disabling muscle weakness affecting approximately 1 in 3000 to 4000 male births (2). The disease is characterized by ongoing degeneration and regeneration of skeletal muscle that leads to replacement of muscle by connective tissue and fat (3). In addition to the profound skeletal muscle lesions, a distinctive cardiomyopathy has been recognized in DMD patients (4, 5). Cardiac lesions, characterized by subepicardial fibrosis, particularly of the posterobasal portion of the left ventricle (LV), occur in later stages of the disease and are apparently progressive (6-8), while severe cardiomyopathy develops in the later stages of the disease in a large percentage of patients (8).

Cardiomyopathy is a cardiocytic disease that could be followed by a vascular disease. Previous studies suggested that endothelium-mediated relaxation was attenuated in both coronary and peripheral vessels in cardiomyopathy patients (9, 10), which means that such patients suffer from endothelial dysfunction. Recent studies have identified a population of presumably bone marrow–derived cells, called circulating endothelial progenitor cells (EPCs) that can be isolated from bone marrow or circulating mononuclear cells (11). These EPCs express a variety of endothelial surface markers including CD34, Cd133 and KDR (12). They can incorporate into sites of neovascularization (13) and home to sites of endothelial denudation (14). Initial clinical studies demonstrated that risk factors for atherosclerosis are associated with reduced levels of circulating EPCs (15) and that the functional integrity of the endothelium correlates with the activities of EPCs (16). The number of circulating progenitor cells is thought to be a marker of vascular function and repair capacity and is known to decreases with age (17, 18).

Stromal cell-derived factor (SDF)-1α plays an important role in neovascularization. (SDF)-1α is an EPC chemokine known to be responsible for both progenitor cell mobilization from the bone marrow to peripheral blood and homing to the sites of vascular and tissue injury (19, 20). Recently, SDF-1α and its receptor were identified as essential in bone marrow retention of hematopoietic stem cells, cardiogenesis, angiogenesis, and recruitment of EPCs into ischemic tissue (21, 22). The production of SDF-1and other angiogenic factors is mediated by HIF-1α, a transcriptional activator that functions as a master regulator of responses to tissue hypoxia/ ischemia (23). HIF-1 is a heterodimer composed of a constitutively expressed HIF-1β subunit and an O2-regulated HIF-1α subunit (24).

Since it was suggested lately that normal neovascularization is disrupted in DMD pathogenesis (25), and it is well known that endothelial progenitor cells (EPC) play an important role in mediating vascular repair mechanisms, we assessed the number of EPCs with surface markers KDR, CD133, and CD34 progenitor cells in DMD patients vs. healthy control subjects. Plasma levels of SDF-1 and HIF-1α were additionally investigated.

Subjects and methods

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.

Methods

Peripheral blood mononuclear cell isolation and flow cytometry

Mononuclear cells were isolated using a Ficoll density gradient (Biocoll, Biochrom, Berlin, Germany) according to standard protocols as previously described (26). For FACS analysis, mononuclear cells were resuspended in 100 μl of PBS. Immunofluorescence cell staining was performed in duplicate with the use of the fluorescent conjugated antibodies CD34– FITC (Becton Dickinson, San Jose, USA; clone 8G12) and CD133-APC (Becton Dickinson, San Jose, USA; clone AC133). Cell fluorescence was measured immediately after staining, and data were analyzed with the help of CellQuest software (FACSCalibur, Becton Dickinson, Heidelberg, Germany). Units of all measured components are absolute cell counts obtained after the measurement of 250,000 events in a lymphocyte gate.

Reverse Transcriptase-polymerase Chain Reaction (RT-PCR) Analysis for HIF-1α

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: HIF-1α, forward: 5'- CTGTGATGAGGCTTACCATCAGC- 3'; reverse: 5'-CTCGGCTAGTTAGGGTACACTTC- 3'; β-actin forward: 5'-GTG GGG CGC CCC AGG CAC CA-3'; and reverse: 5'-CTC CTT AAT GTC ACG CAC GAT TTC-3'. 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 at 283 and 540 bp for HIF-1α and β-actin respectively were evaluated under ultraviolet light and photographed. Photoes were scanned and quantification of each band was carried out using gene tools version 4. Each quantified data point was related to its individual β-actin (27).

Enzyme-linked immunosorbent assay (ELISA)

Plasma levels of SDF-1 were measured using a commercially available ELISA kit according to the manufacturer's guidelines (R&D Systems, Minneapolis, USA). EDTA plasma probes were centrifuged for 15 min at 10,000 g within 30 min of collection. Probes were aliquoted and stored at −20°C before analysis. The lower detection limit of this assay is 18 pg/ml (28).

Statistical analysis

Each experimental condition was performed and expressed as mean ± SD. Comparisons were made by Student's t-test (two-tailed for independent samples).

Results

The results are listed in tables and figures. There was a significant increase in the number of mononuclear cells bearing EPC markers in DMD patients compared to controls. CD 34 (75 ± 6.2 vs. 60 ± 4.8), CD133 (86 ± 4.7 vs. 75 ± 5.3), KDR (61 ± 4.5 vs. 45 ± 5.6) (table 1 & figure 1). Also, cells bearing CD34 CD 133 (44 ± 7.2 vs. 34 ± 4.2), CD34 KDR (36.1 ± 6.5 vs. 21.5 ± 8.3) and CD 133 KDR (35.4 ± 5.7 vs. 25.5 ± 3.8) were significantly increased among DMD patients compared to controls (table 1 & figure 1). There was a significant increase in SDF-1 serum level (506.5 ± 75.9 vs. 435 ± 82.6) and HIF-ά mRNA relative expression (3.3 ± 1.2 vs. 1.8 ± 0.6) among DMD patients compared to controls.

Table 1.

Endothelial progenitor cells surface markers in blood of DMD patients compared to controls.

EPCs	DMD patients	Controls	t value	P value
CD34	75 ± 6.2	60 ± 4.8	9.1	P < 0.0001
CD133	86 ± 4.7	75 ± 5.3	7.2	P < 0.0001
KDR	61 ± 4.5	45 ± 5.6	10	P < 0.0001
CD34 /CD133	44 ± 7.2	34 ± 4.2	6	P < 0.001
CD 34 / KDR	36.1 ± 6.5	21.5 ± 8.3	6.5	P < 0.001
CD 133/ KDR	35.4 ± 5.7	25.5 ± 3.8	8.3	P < 0.0001

Figure 1.

Endothelial progenitor cells surface markers in blood of DMD patients compared to controls.

HIF-ά mRNA Relative Expression among DMD (3-7) compared to Controls (1-2).

SDF-1 and HIF-ά mRNA relative expression among DMD compared to controls.

Discussion

In the present study the number of mononuclear cells bearing EPC markers and HIF-ά together with serum SDF-1 were significantly increased indicating that regeneration is an ongoing process in DMD patients. CD34 has been shown to significantly higher among DMD patients compared to controls. To our knowledge circulating CD34 positive cells in blood of DMD patients has not been previously measured neither in blood of DMD patients nor in animal models. However it can be expected that CD34 cells are present in DMD patients for tissue regeneration, but their capacity for muscle regeneration is hindered. The latter assumption can be confirmed by the study of Li et al., 2008 (29), who reported different proliferative and myogenic abilities of mesenchymal stem cells (MSCs) in mdx (mice model for DMD compared to the normal C57BL/10 mice). mdx-MSCs exhibited increased heterochromatin, larger vacuoles, and more lysosomes under electron microscopy compared to C57BL/10-MSCs. C57BL/10-MSCs formed a few myotubes, while mdx-MSCs did not. They indicated that at passage 21, mdx-MSCs but not C57BL/10-MSCs had gradually lost their proliferative ability. In addition, there was a significant difference in the expression of CD34. They suggested that the changes in mouse MSC behaviour may be influenced by lack of dystrophin protein in mdx mouse. CD34 is a very interesting stem cell marker. It is an EPC/hematopoietic population, which is capable of differentiation into cardiomyocytes in vitro (30) and into cardiomyocytes and smooth muscle cells in vivo (31). Its pattern and level of expression in muscle stem cells change as these cells differentiate into myotubes (32).

In the present study CD133 level was also significantly higher among DMD patients compared to controls. This is in agreement with Marchesi et al (2008) (33), who found that there was an increase in CD133 cells in the blood of DMD patients compared with healthy controls and that the mean levels of CD133 cells in DMD subjects showed a tendency to decrease with advancing age. It was also suggested that the high level of CD133 cells in blood of DMD patients may indicate that these cells probably receive more specific signals for endothelial differentiation from DMD tissues such as a variety of inflammatory cytokines (34). CD133-positive cells fraction are mesennchymal stem cells (MSCs) with high proliferative potential. When placed in appropriate conditions, these cells proved their capacity to differentiate into adipocytes, osteocytes, chondrocytes, and neuronal/glial cells (35). Also blood-derived CD133- positive cells were shown to promote the repair of spinal cord injury and peripheral nerve defects (36). A subpopulation of human circulating stem cells expressing the CD133 antigen that can differentiate into endothelial and muscle cell types were identified (37).

KDR is a cell surface receptor and is also a term given to vascular endothelial growth factor receptor VEGFR-2. In the present study the mononuclear cells bearing KDR were significantly increased among DMD patients compared to controls. Data to support this finding are scarce. However, it has been recently indicated that there is an increased angiogenesis in the brain of dystrophin-deficient mdx mouse, and that this was related to increased levels of VEGF and VEGFR (38). In the present study there is also a significant increase in CD34 KDR and CD133 KDR mononuclear cells compared to controls. It ought to be mentioned that EPCs are mobilized from the bone marrow and contribute to postnatal vasculogenesis and vascular homeostasis, and that the number and the in vitro function of circulating EPCs are related to endothelial function (39). It is believed that EPCs are recruited at high frequency by the overexpression of SDF-1 (40), which has been shown to be elevated in the present study.

SDF-1 has been shown to be significantly increased in blood of DMD patients compared to controls. SDF-1 is known to play a key role in CD34-positive cell trafficking (41), and to mediate mobilization of haematopiotic stem cells and progenitor cells from the bone marrow by chemotaxis (42, 43). SDF-1 is known to be induced by hypoxia and to exert angiogenic effects (44-46). It has been demonstrated that SDF-1 is overexpressed in dystrophic muscle, enhances the extravasation of these cultured progenitor cells into skeletal muscle after intraarterial transplantation (47). This provides proof to the finding of the present study.

HIF-1α in the present study was also significantly higher among DMD patients compared to controls indicating that a hypoxic condition predominates in DMD. Induction of SDF-1 after ischemia is mediated by hypoxia- inducible factor-1 (HIF-1) as a transcription factor and the central mediator of cellular responses to hypoxic conditions (48). SDF-1 is readily degraded in normoxic cells and HIF-2 has been shown to be an important mediator of adaptive responses after ischemia (49). HIF-1 regulates the expression of hundreds of genes, including those encoding angiogenic cytokines such as stromal-derived factor (SDF)-1 (50-52). HIF-1 also mediates cell-autonomous responses to hypoxia in endothelial cells (53-55). These data suggest an activation of HIF-1alpha in the brain of dystrophic mice (38).

We conclude that hypoxic and/or ischemic conditions in muscle tissue of DMD patients initiate regenerative processes, which include secretion of (SDF)-1α that mobilizes EPC from the bone marrow to peripheral blood, homing it to the sites of vascular and tissue injury. However, the regeneration process does not balance the ongoing degeneration caused by dystrophin gene mutations in DMD patients. The variability in this balance may control the severity of phenotypic expression in DMD patients.

Table 2.

SDF-1 and HIF-ά mRNA relative expression in DMD patients compared to controls.

	DMD	Controls	t
SDF-1 pg/ml	506.5 ± 75.9	435 ± 82.6	3.1	P < 0.01
HIF-ά mRNA relative expression	3.3 ± 1.2	1.8 ± 0.6	5	P < 0.001