Regeneration of aged rat vocal folds using hepatocyte growth factor therapy

Abstract

Objectives/hypothesis: We investigated acute changes in extracellular matrix gene expression and histologic changes in the deposition of collagen and hyaluronan (HA) from hepatocyte growth factor (HGF) treatment of the aged rat vocal fold. We hypothesized that: 1) HGF induces matrix metalloproteinase gene expression, which might contribute to the downregulation of collagen; and 2) HGF induces hyaluronan synthase (HAS) gene expression, which might play a role in the upregulation of extracellular matrix HA.

Study design: Prospective animal study.

Methods: Fifteen, 18-month-old, Sprague-Dawley rats were involved in this study. For gene expression analyses, 10 rats were divided into two groups and received serial injections of sham (saline) or HGF (2 ng/microL) and sacrificed 2 weeks after the initial injection to investigate acute changes in extracellular matrix gene expression. A separate group of five animals received the above treatment and were sacrificed 4 weeks after the initial injection to investigate histologic changes in the deposition of collagen and HA.

Results: Real-time polymerase chain reaction revealed significantly upregulated matrix metalloproteinase(MMP)-2, -9, and HAS-3 messenger RNA (mRNA) expression and significantly downregulated procollagen type I mRNA expression in the HGF-treatment group, compared to the sham-treatment group. Histologic staining revealed significantly reduced collagen deposition and increased deposition of HA in the HGF-treated vocal fold, compared to the sham-treated vocal fold.

Conclusions: HGF induced the upregulation of MMP-2, -9, and HAS-3, and downregulated the expression of procollagen type I. Histologically, aged vocal folds treated with HGF revealed decreased collagen deposition, and increased deposition of HA, compared to sham-treated vocal folds.

Fig 1

Log-transformed mRNA expression ratios between the HGF-treatment group and the sham-treatment group for (A) matrix metalloproteinase (MMP)-2, (B) MMP-9, (C) procollagen type I, (D) hyaluronan synthase (HAS)-2, and (E) HAS-3, two-weeks after the initial treatment. The error bars represent 95.0% confidence intervals of the mean.

Fig 2

Coronal sections of the sham (A,C) and HGF treated vocal fold (B,D) stained with Verhoeff’s elastic stain for collagen, revealing decreased density of collagen staining in the lamina propria of the HGF-treated vocal fold, compared to the sham-treated vocal fold. Bars represent 100µm.

Fig 3

Coronal sections of the sham (A,C) and HGF treated vocal fold (B,D) stained with Alcian blue stain for HA, revealing increased density of HA staining in the lamina propria of the HGF-treated vocal fold, compared to the sham-treated vocal fold. Bars represent 100µm.

Fig 4

Percent density of collagen and HA staining between the sham and HGF-treated vocal fold. Data are shown as mean ± SD (n=5). Results revealed significantly lower density of collagen staining and increased density of HA staining in the HGF-treated vocal fold, compared to the sham-treated vocal fold.