Inhibitory effect of angiogenin on the expression of type I collagen and fibronectin in dermal papilla cells from androgenetic alopecia areas

Abstract

Objective To evaluate effects of angiogenin on the expression of type Ⅰcollagen and fibronectin in dermal papilla cells from androgenetic alopecia areas, and to explore its possible mechanisms. Methods Dermal papilla cells were isolated from androgenetic alopecia areas and cultured. Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of androgen receptor in dermal papilla cells of different passages, and cell counting kit-8 (CCK-8) assay to evaluate the effect of angiogenin at different concentrations of 0, 10, 20, 40, 80, 160 μg/L on the proliferative activity of the dermal papilla cells cultured in a medium with or without 0.1 nmol/L dihydrotestosterone. The confluent first-passage dermal papilla cells were divided into 3 groups: control group receiving no treatment, dihydrotestosterone group treated with 0.1 nmol/L dihydrotestosterone, and dihydrotestosterone + angiogenin group treated with 0.1 nmol/L dihydrotestosterone and 80 μg/L angiogenin. After 48-hour treatment, real-time fluorescence-based quantitative PCR was conducted to measure the mRNA expression of type Ⅰ collagen gene, fibronectin and transforming growth factor-β1 (TGF-β1) , and Western blot analysis to determine the protein expression of type I collagen, fibronectin, TGF-β1, phosphorylated Smad2 (p-Smad2) and p-Smad3. Statistical analysis was done by one-way analysis of variance (ANOVA) , least significant difference (LSD) -t test and t test for two independent samples. Results The mRNA expression of androgen receptor significantly decreased during the subcultivation of in vitro cultured dermal papilla cells from androgenetic alopecia areas (P < 0.05) . Cell proliferation assay showed that 20 - 160 μg/L angiogenin could evidently antagonize the inhibitory effect of 0.1 nmol/L dihydrotestosterone on the proliferation of dermal papilla cells (all P < 0.05) . Compared with the control group, the dihydrotestosterone group showed significantly higher mRNA expression of type Ⅰ collagen gene, fibronectin and TGF-β1. However, the mRNA expression of type Ⅰ collagen gene, fibronectin and TGF-β1 was significantly lower in the dihydrotestosterone + angiogenin group than in the dihydrotestosterone group (type Ⅰcollagen gene: 1.563 ± 0.143 vs. 4.329 ± 0.165; fibronectin: 1.290 ± 0.063 vs. 2.156 ± 0.115; TGF-β1: 1.136 ± 0.098 vs. 1.707 ± 0.100; all P < 0.05) . Moreover, angiogenin could obviously suppress the expression of type Ⅰ collagen, fibronectin, TGF-β1, p-Smad2 and p-Smad3 protein by dihydrotestosterone-induced dermal papilla cells (all P < 0.05) . Conclusion Angiogenin can inhibit the expression of type Ⅰ collagen and fibronectin in dermal papilla cells from androgenetic alopecia areas in vitro, which may be associated with the down-regulated expression of TGF-β1 and inhibition of TGF-β1/Smad signaling pathway. Key words: Angiopoietins; Alopecia; Extracellular matrix; Transforming growth factor beta1; Dermal papilla cell