Exploring the signature of Reinke´s edema by proteomics

Reinkes edema (RE) is a uni- or bilateral swelling of the vocal folds, occurring in patients with nicotine and voice abuse. Voice of patients is characterized by decreased pitch and detrimental voice quality, making it often difficult for voice professionals who rely on their voices (teacher, call center agents etc.) So far treatment is only symptomatic, still the gold standard of therapy, microsurgery of the vocal folds, is far from being satisfactory. In the proposed project we seek to explore the cellular mechanisms of RE by protein analysis (proteomics), revealing the true pathophysiology of RE for the first time. During routine vocal fold surgery for RE we will take small mucosa specimens. From these we will extract vocal fold fibroblasts for subsequent analysis. Proteomic analysis will deliver a complete list of proteins produces specifically by these cells. These will be compared with vocal fold cells grown out of healthy laryngeal mucosa. In subsequent functional experiments we will explore certain proteins of interest (aka. candidate proteins) that are produced in a high number by RE fibroblasts for their biological relevance. By enhancing or blocking these candidate proteins we aim to draw conclusions on their role in the development of RE, which should pave to way to new (non-surgical) treatment options.

Reinkes edema (RE), a disease of the vocal fold, causes troubles with speaking and breathing and has a deep socioeconomical impact, especially in professions with a heavy vocal load. Main risk factors for RE are smoking and heavy voice use, but even after cessation of these factors, a normal, healthy state cannot be recovered. To better understand the underlying changes that cause this disease, the current study was aimed at investigating the molecular characteristics of RE. Considering the key role of vocal fold (VF) fibroblasts (VFF) in shaping the molecular constitution of the VF laminar propria, we hypothesized that changes in the properties of these cells could play a key role in the pathogenesis of this disease. To address this, we collected VF mucosa and isolated VFF from RE patients and healthy VFF from deceased individuals post-mortem. In one set of experiments, healthy VFF were treated with cigarette smoke extract (CSE) and compared to untreated cells. Further, immortalized VFF were exposed to CSE and vibration on a bioreactor, to evaluate the effect of a combination of the main risk factors on these cells. In a third experimental set-up, the gene expression of healthy VFF was compared to RE patient VFF by sequencing and follow-up experiments. Results from the first trial revealed that healthy VFF show different behaviour in terms of extracellular matrix (ECM) gene expression when cultured in cigarette smoke enriched medium compared to cells treated with standard medium. The combination of CSE and vibration as a treatment of immortalized VFF cells yielded alterations that were not found after one single treatment or the other alone. This finding is of importance to understand the interplay between the risk factors smoking and heavy voice use. Using sequencing, we found novel alterations in the gene expression of RE VFF samples, such as increases in mRNA levels of components of the ECM and factors that influence and interact with the ECM, changes that impact the signalling of inflammatory/ fibrotic mediators and a downregulation of pathways involved in cell cycle control, DNA repair and proliferation. In summary, we have detected distinct changes in VFF by CSE and vibration, as well as distinct alterations in RE VFF compared to healthy VFF. All of these changes most likely contribute to the development of the disease, but detailed effects of single factors and mechanisms involved remain to be determined.