Dietary flavonoid apigenin inhibits endothelin-1-induced contraction of collagen gel

Abstract

Systemic sclerosis (SSc) is a connective tissue disease characterized by vasculopathy, excessive accumulation of extracellular matrix (ECM), and Wbrosis of the skin and internal organs [1]. Although the precise etiology of SSc is still unknown, it is generally accepted that many types of cells are involved in the development and progression of SSc. MyoWbroblasts, specialized contractile cells diVerentiated from Wbroblasts, are believed to be the major cells with great importance in all forms of Wbrotic diseases including SSc [2]. They have the morphological features intermediate between those of Wbroblasts and smooth muscle cells and characteristically express alpha-smooth muscle actin ( -SMA) [3]. Flavonoids, the largest class of polyphenols, have been well known to have a variety of biological properties [4]. Non-toxic and non-mutagenic apigenin is a common dietary Xavonoid [5], but it plays role in many cellular processes such as cell proliferation, migration, and tumor biology [4]. Recently, Ricupero et al. [6] reported that apigenin decreased the expression of myoWbroblast phenotype. We investigated the role of common Xavonoids, especially apigenin on endothelin-1 (ET-1)-induced contraction of collagen gel, in vitro model of extracellular matrix remodeling [7]. Collagen gel mixture was made with collagen stock solution (2 mg/ml; rat tail), 5£ free medium, 0.2 N NaOH, sterilized distilled water, and cell solution (2 £ 10/ml, CCD-1138 from ATCC, Manassas, VA and primary human skin Wbroblasts) on ice. Gel solution (500 l) was then cast into each well of a 24-well plate, which was pre-coated with 0.5% BSA for 90 min at 37°C. After gelation the gels were transferred into 6-well culture plate containing 5 ml of serum-free DMEM with ET-1 (100 nM) with or without pretreatment with its receptor-speciWc antagonists such as BQ-123, BQ-610, BQ-788, PD145065, and various Xavonoids (25 M), such as apigenin, kaempferol, quercetin, and naringenin for 24 h. The Xoating gels were incubated at 37°C and 5% CO2 for 4 days and the transverse diameter of gel were measured with screen ruler on JPEG image after taking pictures with digital camera. Culture with ET-1 and various concentrations (12.5, 25, 50, and 75 M) of Xavonoids for 24 h after starvation for 24 h did not inXuence the viability of primary human skin Wbroblasts (not shown). Incubation with ET-1 signiWcantly enhanced collagen gel contraction in skin Wbroblasts (Fig. 1a). ET-1-mediated gel contraction was signiWcantly blocked by pretreatment with the ETA receptor antagonist, BQ-123 and the ETA+B antagonist, PD145065, but not by the ETB receptor antagonists, BQ-610 or BQ-788 (Fig. 1b). Flavonoids (up to 75 M) do not inXuence the viability of skin Wbroblasts, but we used low concentration (25 M) of Xavonoid to show the in vivo eVect of dietary Xavonoids. All Xavonoids eYciently inhibited ET-1-induced contraction of collagen gel in human skin Wbroblast cell line (Fig. 2a) and human primary skin Wbroblast (Fig. 2b). Apigenin did not seem to be more eYcient in this inhibition compared to other Xavonoids. Inhibition of ET-1-induced contraction by apigenin showed dose-dependent manner (1.0, 3.0, 6.0, and 12.5 M) (Fig. 3). We demonstrated in this study that ET-1 induced collagen gel contraction through ETA receptor like in a previous report [7], and apigenin inhibited contraction of ET-1induced collagen gel. More importantly, the concentration of apigenin applied in this study included the physiologic concentration of apigenin in human plasma [8], and was J.-B. Jun (&) · Y.-I. Na · T.-H. Kim · D.-H. Yoo Hanyang University, Seoul, Korea e-mail: junjb@hanyang.ac.kr