Levels of adiponectin, a marker for PPAR-gamma activity, correlate with skin fibrosis in systemic sclerosis: potential utility as biomarker?
© Lakota et al.; licensee BioMed Central Ltd. 2012
Received: 7 December 2011
Accepted: 1 May 2012
Published: 1 May 2012
Progressive fibrosis in systemic sclerosis (SSc) is linked to aberrant transforming growth factor beta (TGF-beta) signaling. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) blocks fibrogenic TGF-beta responses in vitro and in vivo. Reduced expression and function of PPAR-gamma in patients with SSc may contribute to progression of fibrosis. Here we evaluated the levels of adiponectin, a sensitive and specific index of PPAR-gamma activity, as a potential fibrogenic biomarker in SSc.
Adiponectin levels were determined in the sera of 129 patients with SSc and 86 healthy controls, and serial determinations were performed in 27 patients. Levels of adiponectin mRNA in skin biopsies from SSc patients were assessed in an expression profiling microarray dataset. Regulation of adiponectin gene expression in explanted human subcutaneous preadipocytes and fibroblasts was examined by real-time quantitative PCR.
Patients with diffuse cutaneous SSc had reduced serum adiponectin levels. A significant inverse correlation between adiponectin levels and the modified Rodnan skin score was observed. In longitudinal studies changes in serum adiponectin levels were inversely correlated with changes in skin fibrosis. Skin biopsies from a subset of SSc patients showed reduced adiponectin mRNA expression which was inversely correlated with the skin score. An agonist ligand of PPAR-gamma potently induced adiponectin expression in explanted mesenchymal cells in vitro.
Levels of adiponectin, reflecting PPAR-gamma activity, are correlated with skin fibrosis and might have potential utility as a biomarker in SSc.
Systemic sclerosis (SSc) is a multisystem disorder with protean clinical manifestations and substantial patient-to-patient heterogeneity . Skin fibrosis characteristically shows rapid progression during early-stage disease but then reaches a plateau phase followed by slow regression. Transforming growth factor-beta (TGF-β) plays a key role in initiating and sustaining fibroblast activation and myofibroblast differentiation in SSc . Microarray-based expression profiling of SSc skin biopsies shows an association that a 'TGF-β-activated gene signature' is associated with extensive skin involvement . Multiple physiologic mechanisms regulate TGF-β signaling to prevent excessive tissue remodeling and fibrosis. One important endogenous anti-fibrotic defense mechanism involves the peroxisome proliferator-activated receptor-gamma (PPAR-γ) pathway, which blocks TGF-β responses .
The nuclear receptor PPAR-γ, initially identified in adipose tissue, plays key roles in regulation of adipogenesis, insulin sensitivity, and energy homeostasis . In addition to being expressed in adipocytes, PPAR-γ is expressed in endothelial cells, vascular smooth muscle cells, macrophages, and fibroblasts [4, 6]. Endogenous and diet-derived fatty acids and eicosanoids such as prostaglandin J2 (PGJ2) act as low-affinity natural PPAR-γ ligands, whereas the thiazolidenedione drugs are potent synthetic PPAR-γ agonists . Recent studies identified an entirely novel function for PPAR-γ in regulation of matrix homeostasis . Exposure of fibroblasts to pharmacological PPAR-γ ligands resulted in suppression of collagen synthesis, myofibroblast differentiation, and other TGF-β-induced fibrotic responses in vitro [4, 9, 10]. Moreover, treatment of mice with PPAR-γ agonists prevented and attenuated bleomycin-induced scleroderma in vivo . The significance of the anti-fibrotic activity of PPAR-γ is highlighted by genetic targeting experiments that demonstrate constitutive collagen overexpression and excessive fibrogenesis in PPAR-γ-null fibroblasts in vitro and in vivo . Importantly, PPAR-γ expression and activity are reduced in SSc skin biopsies and explanted fibroblasts and are inversely correlated with fibrogenic markers in the lesional skin [13, 14]. These observations suggest that impaired PPAR-γ expression or function underlies unopposed fibroblast activation and progression of fibrosis in SSc. Assessing PPAR-γ expression or activity therefore might be a novel approach for assessing fibrogenic activity in SSc.
Adiponectin is a 244-amino acid hormone secreted from white adipose tissue that regulates insulin sensitivity and energy balance. The adiponectin gene is located on chromosome 3q27, a susceptibility locus for diabetes and metabolic disorders. Adiponectin transcription is tightly regulated by PPAR-γ through direct binding to conserved cis-acting regulatory DNA elements. Circulating adiponectin is decreased in obesity and the metabolic syndrome, and levels are increased in mice and in humans after treatment with PPAR-γ agonist agents . Since serum adiponectin levels faithfully mirror PPAR-γ activity, adiponectin is now increasingly used as a biomarker of efficacy for PPAR-γ therapy . In view of the PPAR-γ defect seen in SSc, we hypothesized that circulating adiponectin might be reduced in some patients with SSc. We therefore determined adiponectin levels in 129 patients with well-characterized SSc and correlated levels with clinical and laboratory features of disease. The results reveal significantly reduced adiponectin levels in diffuse cutaneous SSc (dcSSc) patients with early-stage disease, inverse correlation with the extent of skin fibrosis, and rising levels that parallel improved skin scores over time.
Materials and methods
Patients with SSc
Patients with dcSSc
Patients with lcSSc
Age in years, median (range)
> 36 months
MRSS from 0 to 51, median (range)
BMI in kg/m2, median (range)
Determination of serum adiponectin levels
Serum samples were frozen at -80°C until assayed. Adiponectin levels were measured by using a multiplex assay kit (EMD Millipore, Billerica, MA, USA) in accordance with the instructions of the manufacturer. Briefly, serum samples (1:100 dilution) and standards were added to the wells, along with sonicated beads. After incubation and washing of the wells, antibodies were added, followed by streptavidin-phycoerythrin. Wells were then washed, and measurements on a Luminex 100 platform (Luminex Corporation, Austin, TX, USA) were performed. The assay detects all three (low-, medium-, and high-molecular weight) forms of adiponectin.
Regulation of adiponectin gene expression
Human subcutaneous preadipocytes (Zen-Bio, Inc., Research Triangle Park, NC, USA) and neonatal foreskin fibroblasts were maintained at 37°C in an atmosphere of 5% CO2 in preadipocyte medium (PM) (Zen-Bio, Inc.) or Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% vitamins, 1% penicillin/streptomycin, and 2 mM L-glutamine (all from BioWhittaker, Walkersville, MD, USA). Cells were studied between passages 4 and 8 at early confluence. Cultures were incubated in the presence or absence of pioglitazone (10 μM). After 48 hours of incubation, cultures were harvested, and mRNA levels were examined by real-time quantitative polymerase chain reaction (qPCR) by using primers for adiponectin (forward, 5'-TATCCCCAACATGCCCATTCG-3'; reverse, 5'-TAGGCAAAGTAGTACAGCCCA-3') and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5'-CATGAGAAGTATGACAACAGCCT-3'; reverse, 5'-AGTCCTTCCACGATACCAAAGT-3').
Adiponectin gene expression in skin biopsies
The generation and analysis of the genome-wide expression microarray dataset using skin biopsies from patients with SSc and healthy controls have been described (Gene Expression Omnibus accession number GSE9285) . The expression levels of adiponectin mRNA in each skin biopsy were extracted from this dataset and centered on its mean value across all arrays.
Mann-Whitney U tests or Kruskal-Wallis tests were used to compare adiponectin levels. The correlation between adiponectin levels and various values were analyzed by Spearman rank correlation test. Owing to non-normal distribution of the data, summary statistics are expressed as medians and interquartile ranges (IQRs). Data were analyzed by using SPSS Statistics 19 (SPSS Inc., Chicago, IL, USA). A P value of less than 0.05 was considered statistically significant.
Results and Discussion
The difference in adiponectin levels between patients with lcSSc and those with dcSSc suggested that adiponectin, as a marker of PPAR-γ signaling in the skin, might be correlated with skin fibrosis. Indeed, we demonstrated a weak but statistically significant inverse correlation between adiponectin levels and the extent of skin fibrosis (r = -0.261, n = 124, P = 0.003) (Figure 1b). This correlation persisted even when corrected for age, gender, ethnicity, and BMI. A more robust correlation between MRSS and adiponectin levels was seen in Scl-70-positive patients, suggesting the association of reduced PPAR-γ activity with active skin disease (Figure 1b). In contrast to the skin score, neither FVC nor DlCO or radiologic evidence of pulmonary fibrosis could be demonstrated to have a significant correlation with serum adiponectin levels (Additional files 1 and 2). The correlation of serum adiponectin levels with skin but not lung fibrosis points to potential differences in the pathomechanisms underlying fibrosis in these organs, and PPAR-γ has a more prominent role in skin fibrosis than in lung fibrosis.
Taken together, the present results provide support for the involvement of PPAR-γ in modulation of skin fibrogenesis in SSc and suggest that reduced adiponectin levels might reflect reduced PPAR-γ activity associated with active or progressive skin fibrosis. Defective PPAR-γ activity in SSc contributes to unopposed fibroblast activation and is implicated in pathogenesis . Adiponectin production is tightly regulated by PPAR-γ, and its levels in the serum represent a robust biomarker of PPAR-γ activity . We found significantly lower serum adiponectin levels in patients with dcSSc than in those with lcSSc, and levels were lowest in the early stage of disease, when fibrogenesis is presumed to be the most active. A gradual rise in the levels of adiponectin over time might parallel spontaneous or treatment-associated skin regression and may signal attenuation of fibrogenesis. The present studies did not compare adiponectin levels in SSc with those with other autoimune diseases. However, studies have shown that rheumatoid arthritis, systemic lupus erythematosus, and other immune and inflammatory disorders are generally associated with elevated serum adiponectin [20–24]. Thus, it is noteworthy that, in contrast, SSc is not associated with elevated adiponection, despite the chronic inflammation that is thought to be a hallmark of this disease .
The levels of adiponectin in the circulation reflect PPAR-γ signaling activity in mesenchymal cells . Thus, reduced adiponectin levels and adiponectin gene expression in lesional tissue in SSc indicate reduced adiponectin production that results from the impaired PPAR-γ activity. Adiponectin directly suppresses fibroblast migration and myofibroblast differentiation and blocks TGF-β-induced fibrotic responses, including collagen synthesis . Moreover, adiponectin-null mice show exaggerated fibrogenesis in the heart, liver, and kidneys, suggesting a potential anti-fibrotic role . Whether adiponectin is simply a marker for PPAR-γ signaling or, in fact, mediates its anti-fibrotic activities is currently unknown.
In summary, this cross-sectional analysis indicates that the levels of the PPAR-γ-regulated adiponectin are reduced in early-stage dcSSc and are inversely correlated with the skin score. Moreover, rising levels of adiponectin over time correlate with improvement in skin score. Reduced adiponectin reflecting impaired PPAR-γ activity might signify unopposed fibroblast activation and serve as a biomarker for ongoing fibrogenesis. Two recent studies with fewer patients reported reduced adiponectin levels in Japanese patients with SSc [20, 28]. These findings are consistent with our present results with a significantly larger patient cohort of a different ethnic background. Taken together, the results provide a compelling case for a role of PPAR-γ activity in skin fibrosis in SSc and support the prospective evaluation of adiponectin as a biomarker of fibrogenic activity.
body mass index
diffuse cutaneous systemic sclerosis
carbon monoxide diffusion capacity
forced vital capacity
limited cutaneous systemic sclerosis
modified Rodnan skin score
peroxisome proliferator-activated receptor gamma
quantitative polymerase chain reaction
transforming growth factor-beta.
We are grateful to Alexander Misharin for technical help. This work was supported by grants from the Association for the Development of Rheumatology (Slovenia), the Ministry of High Education, Science and Technology of Slovenia (grant P3-0314), the National Institutes of Health, the Scleroderma Research Foundation, and the Karen Brown Scleroderma Foundation.
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