Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Scleroderma (SSc) is a chronic autoimmune disease, characterized by excessive fibrosis of skin and internal organs due to uncontrolled proliferation of fibroblasts and deposition of extracellular matrix protein. Till date there is no promising therapy available for treatment of fibrosis in Scleroderma. Herein, we hypothesize that Phosphodiesterase5 inhibitors (PDE5i) attenuate fibrotic phenotype of cutaneous fibroblasts in patients with scleroderma.
Methods: Primary fibroblast cultures from midforearm skin of 13 Patients with scleroderma (as per ACR/LeRoy criteria) and 5 healthy individuals were established. To mimic disease condition fibroblasts were pre-stimulated with Transforming growth factor-beta1 (TGF-β1) (10 ng/ml) for 1 hour followed by further incubation with TGF-β1 and PDE5i; Sildenafil (10µM) and Zaprinast (10µM), for 24 hours (Strategy 1). In another strategy fibroblast were pre-treated with Sildenafil (10µM) and Zaprinast (10µM) for 1 hour followed by incubation with TGF-β1 (10 ng/ml) for 24 hours (Strategy 2). Expression of profibrotic genes; collagen 1 alpha 1 (COL1A1), collagen 1 alpha 2 (COL1A2), Fibronectin, alpha smooth muscle actin-1(ASMA1), connective tissue growth factor (CTGF), and antifibrotic gene; Matrix metalloproteinases 2/Tissue inhibitor of metalloproteinases (MMP2/TIMP) was analyzed with real time PCR and later their protein expressions were confirmed by Western Blot. Further, effect of PDE5i on TGF-β1 downstream signaling; canonical and non-canonical pathways, were analyzed with western blot. ANOVA and Student’s t-Test were used for statistical analysis in SPSS 13 software.
Results: TGF-β1 significantly increased the expression of COL1A1, COL1A2, ASMA1, fibronectin, and CTGF at mRNA and protein levels compared to untreated fibroblasts. TGF-β1 induced fibrotic genes expression were significantly higher in SSc fibroblasts compared to healthy fibroblasts. In both Healthy and SSc fibroblasts, Sildenafil and Zaprinast treatment in both the strategies, significantly reduced the mRNA and protein expression of type 1 collagen, ASMA-1, fibronectin and CTGF compared to treatment with TGF-β1. Moreover, Sildenafil and Zaprinast treatment restored the levels of MMP2/TIMP1 in fibroblasts which were suppressed by TGF-β1 treatment. MMP2 level was significantly increased in culture supernatant of Healthy and SSc fibroblasts treated with PDE5i. Sildenafil and Zaprinast both reduced the phosphorylation of Smad3 (canonical) and Erk1/2 (non-canonical) in SSc fibroblasts.
Conclusion: PDE5i, Sildenafil and Zaprinast, not only attenuate the pro-fibrotic phenotype but also increase anti-fibrotic response in TGF-β1 treated cutaneous fibroblasts in SSc patients. Antifibrotic effect of PDE5i is mediated by affecting both canonical as well non-canonical TGF-β1 mediated downstream signaling pathways.
To cite this abstract in AMA style:Agarwal V, Rai MK, Agrawal V, Singh H, Chaturvedi S. Phosphodiesterase-5 Inhibitors Attenuate Fibrotic Phenotype and Restore Anti-Fibrotic Resopnses of Cutaneous Fibroblasts in Patients with Scleroderma [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/phosphodiesterase-5-inhibitors-attenuate-fibrotic-phenotype-and-restore-anti-fibrotic-resopnses-of-cutaneous-fibroblasts-in-patients-with-scleroderma/. Accessed December 5, 2020.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/phosphodiesterase-5-inhibitors-attenuate-fibrotic-phenotype-and-restore-anti-fibrotic-resopnses-of-cutaneous-fibroblasts-in-patients-with-scleroderma/