Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema

Abstract

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.

Keywords: Cigarette smoke; Clinical proteomics; Extracellular matrix*; Inflammation; Larynx; Oxidative stress; Protein Identification*; Reinke's edema; Vocal fold fibroblasts.

Graphical abstract

Fig. 1.

Proteomic profiling of vocal fold fibroblasts (VFF) exposed to cigarette smoke extract (CSE). Five near-primary VFF populations and an immortalized VFF line (VFF-E7) were either subjected to 5% CSE or air-bubbled control medium (ABC) and cultivated at 37 °C and 5% CO₂. Cells were harvested after 24 h to perform label-free quantitative proteomics. A, Number of unique and robustly detected (peptide PSM and protein FDR≤1% for identification, ≥2 peptide counts (razor + unique) for quantitation) proteins across distinct samples. B, Heat map of unique proteins that were detected in ≥3 sample pairs. Data is presented as log2-transformed fold change (log2FC; CSE versus ABC), sorted according to average log2FC. Clustering tree resulting from unsupervised clustering of samples is shown on top. C, heat map of the most upregulated 2% of proteins. D, heat map of the most downregulated 2% of proteins. In C and D, proteins are specified by official full name and official symbol.

Fig. 2.

Oxidative stress response, but no induction of vascular endothelial growth factors (VEGFs) in vocal fold fibroblasts (VFF) exposed to cigarette smoke extract (CSE). RNA was isolated from five near-primary VFF populations and an immortalized VFF line after the same experimental procedures as described in Fig. 1. A, analysis of NFE2L2 (also designated nuclear respiratory factor 2, NRF2) mRNA levels by RT-qPCR. B, analysis of VEGFA-D mRNA levels by RT-qPCR. *, p < 0.05. C, supernatants from immortalized VFF and one near-primary VFF population were collected after the same experimental procedures as described in Fig. 1., analysis of secreted protein levels of VEGF-A, VEGF-C and VEGF-D by Luminex Assay. *, p < 0.05; **, p < 0.01.

Fig. 3.

Cigarette smoke extract (CSE) decreases fibrillary collagen biosynthesis by vocal fold fibroblasts (VFF). VFF-E7 cells (n = 3–7) were either subjected to 5% CSE or air-bubbled control medium (ABC) and cultivated at 37 °C and 5% CO₂. Cells were analyzed after 4 days. A, analysis of COL1A1, COL1A2, and COL3 A1 mRNA levels by RT-qPCR. B, analysis of fibrillary collagen by SDS-PAGE and Silver stain of pepsin-digested cell lysates. Lanes 1–6 show 3 independent experimental replicates; human type I collagen standard was applied on lane 8. C, densitometric quantification of collagen α band intensities from samples shown in B. D, representative immunofluorescence microscopy images for cells stained for type I collagen (green) and nuclei (blue). NC = negative control (no primary antibody). Scale bar: 100 μm. *, p < 0.05; **, p < 0.01.

Fig. 4.

Cigarette smoke extract (CSE) increases hyaluronan biosynthesis by vocal fold fibroblasts (VFF). VFF-E7 cells (n = 3–7) were either subjected to 5% CSE or air-bubbled control medium (ABC) and cultivated at 37 °C and 5% CO₂. A, RT-qPCR analysis of hyaluronan synthase (HAS2, HAS3) and hyaluronidase (HYAL1, HYAL2) mRNA expression 4 days after treatments. B, analysis of hyaluronan biosynthesis by ELISA 1 and 4 days after treatments. C, representative fluorescence microscopy images for cells stained for hyaluronan (green) and nuclei (blue). HYAL = hyaluronidase treatment before cell fixation (staining specificity control). Scale bar: 100 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 5.

Cigarette smoke extract (CSE) modulates cytokine secretion in vocal fold fibroblasts (VFF). Immortalized VFF and one near-primary VFF population were either subjected to 5% CSE or air-bubbled control medium (ABC) and cultivated at 37 °C and 5% CO₂. Supernatants were harvested after 24 h to perform a Luminex Assay. A, secreted protein levels of cytokines: monocyte chemoattractant protein1 (MCP-1), interleukin 6 (IL-6) and interleukin 8 (IL-8). B, secreted protein levels of cytokines involved in tissue remodeling: hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF-basic). *, p < 0.05; **, p < 0.01.