Abstract
Systemic sclerosis (SSc; scleroderma) is characterized by life-threatening progressive multiorgan fibrosis orchestrated by profibrotic myofibroblasts originating from different sources. Because recent data demonstrated that the majority of myofibroblasts in a murine scleroderma model arise from adipocytic progenitors through the adipocyte-myofibroblast transition process, we sought to determine whether the SSc microenvironment may affect the differentiation potential of adipose-derived stem cells (ADSC). Normal human ADSC from three donors were treated with serum from SSc patients (n = 6), serum from healthy individuals (n = 6), or recombinant human transforming growth factor-β1 (TGFβ1) as positive control of myofibroblastic phenotype induction. ADSC were subjected to in vitro adipogenic differentiation for up to 21 days in the presence of different stimuli followed by lipid content quantification. In selected experiments, adipocytic and mesenchymal/myofibroblast marker gene and protein expression levels were assessed by Real-Time PCR, immunoblotting and immunofluorescence after administration of different stimuli for 72 and 96 h, respectively. Cell contractile phenotype was assayed by collagen gel contraction assay. Likewise stimulation with TGFβ1, SSc serum was able to significantly inhibit the adipocyte differentiation of ADSC as testified by a strong decrease in red-colored lipid droplets after 21 days of adipogenic induction. Treatment of ADSC either with SSc serum or TGFβ1 resulted in the acquisition of a myofibroblast-like phenotype characterized by a reduced expression of the adipocytic markers perilipin and adiponectin, a significant upregulation of the mesenchymal/myofibroblast markers α-SMA+ stress fibers, S100A4 and type I collagen, and an ability to effectively contract collagen gels. In SSc, the pathologic environment may favor the differentiation of ADSC into profibrotic and contractile myofibroblast-like cells. These findings strengthen the notion that the generation of myofibroblasts from ADSC may be relevant in SSc pathophysiology potentially representing a new target for the prevention/treatment of multiorgan fibrosis.
Highlights
Systemic sclerosis (SSc; scleroderma) is a connective tissue disease characterized by dysregulation of innate and adaptive immunity, severe alterations in the microvasculature and progressive development of fibrotic lesions in the skin and internal organs [1,2,3]
It has been reported that stimulation of either undifferentiated human adipose-derived stem cells (ADSC) or ADSC committed to preadipocytes with profibrotic transforming growth factor-β (TGFβ) is able to induce the loss of adipocytic markers and the reciprocal acquisition of mesenchymal/myofibroblast markers [14,29]
At all three experimental time points, under the microscope, normal human ADSC from three donors challenged with recombinant human transforming growth factor-β1 (TGFβ1) and serum from patients with SSc showed decreased red-colored lipid droplets compared to both basal and healthy serum-treated ADSC (Figure 1A)
Summary
Systemic sclerosis (SSc; scleroderma) is a connective tissue disease characterized by dysregulation of innate and adaptive immunity, severe alterations in the microvasculature and progressive development of fibrotic lesions in the skin and internal organs [1,2,3]. Increasing evidence suggests that myofibroblasts may arise from different sources including expansion and activation of resident tissue fibroblasts and perivascular pericytes, recruitment of bone marrow-derived circulating precursors, and transdifferentiation of epithelial and endothelial cells [6,11,12,13]. By using a transgenic lineage tracing approach in experimental murine scleroderma, it was possible to demonstrate that the loss of intradermal adipose tissue precedes the onset of dermal fibrosis and is accompanied by early downregulation of adipocyte-associated genes followed by an acquisition of myofibroblast-associated genes [14]. It has been reported that stimulation of either undifferentiated human adipose-derived stem cells (ADSC) or ADSC committed to preadipocytes with profibrotic transforming growth factor-β (TGFβ) is able to induce the loss of adipocytic markers and the reciprocal acquisition of mesenchymal/myofibroblast markers [14,29]. In vitro experiments on different adipocyte-generating cultures further showed that myofibroblast differentiation may be driven by TGFβ [14], and by other pathways such as FIZZ1 and Wnt signaling [23,30,31]
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