Does Pepsin Play a Role in Etiology of Laryngeal Nodules?

OBJECTIVES: Vocal fold nodules (VFNs) are benign disorders affecting the superficial lamina propria of the true vocal folds. The etiology of VFNs still remains unclear but laryngeal trauma caused by vocal abuse, tobacco, alcohol, and laryngopharyngeal reflux (LPR) plays a crucial role on the pathogenesis. The aim of this study was to assess the presence of pepsin in formalin-fixed, paraffin-embedded (FEPE) specimens of VFNs to evaluate the role of LPR as a risk factor for VFNs. MATERIALS AND METHODS: A total of 28 pathology specimens of patients suffering from VFNs who had undergone laser microsurgery under general anesthesia were evaluated. The specimens were maintained in paraffin blocks in the pathology department. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) analyses were used to measure pepsin enzyme levels in the VFNs tissue specimens. Signs of LPR were assessed according to the reflux finding score. RESULTS: The mean reflux finding score of the patients was 13.6 ± 2.89 (8-21). According to WB and ELISA analyses, pepsin was detected with both the WB the ELISA tests in positive controls, but there was no pepsin enzyme in any of the 28 laryngeal FEPE VFNs specimens. CONCLUSION: The pepsin enzyme was not detected in any of the FEPE VFNs specimens, and it is concluded that further studies are needed to reveal the role of pepsin in the etiology of VFNs.

Detecting Laryngopharyngeal Reflux by Immunohistochemistry of Pepsin in the Biopsies of Vocal Fold Leukoplakia.

Laryngopharyngeal reflux (LPR) may contribute to the development of laryngeal diseases including vocal fold leukoplakia. Clinical methods of determining LPR are limited. Pepsin, as an exogenous protein, is considered as a biomarker of LPR. The aim of the current study was, therefore, to detect pepsin by immunohistochemistry in the biopsies from patients with vocal fold leukoplakia, and by which, to determine the potential association of LPR and vocal leukoplakia. A total of 26 biopsies from patients with vocal fold leukoplakia were examined in comparison with 20 vocal fold biopsies from control subjects. We found that 2 out of 26 patients (7.7%) were strongly positive, 4 of the 26 (15.4%) patients were positive, 11 of the 26 (42.3%) patients were weakly positive, and 9 of the 26 (34.6%) were negative staining for pepsin. In contrast, only 4 of the 20 (20.0%) control subjects were weakly positive and the rest (16; 80.0%) were negative staining for pepsin. There was significant difference between the two groups in terms of positivity of pepsin staining (χ2 = 24.181, P <0.001). These findings suggest that pepsin immunohistochemical staining could be a biomarker of LPR and that LPR may be a risk factor for the development of vocal fold leukoplakia.

Cyclooxygenase-2 expression and clinical parameters in laryngeal squamous cell carcinoma, vocal fold nodule, and laryngeal atypical hyperplasia.

BACKGROUND: The diagnostic role of cyclooxygenase-2 (COX-2) expression in laryngeal atypical hyperplasia, vocal fold nodule, and laryngeal squamous cell carcinoma was examined. METHODS: Specimens obtained from patients diagnosed with vocal fold nodule (n = 35), atypical hyperplasia (n = 35), laryngeal squamous cell carcinoma (n = 35), and clinical parameters were evaluated retrospectively. RESULTS: Although no staining was observed in patients with vocal fold nodules, staining was noted in laryngeal atypical hyperplasia and squamous cell carcinoma. The percentage of COX-2 staining was the highest in the carcinoma group. CONCLUSION: It was determined that COX-2 staining was significantly associated with laryngeal squamous cell carcinoma. It should be noted that overexpression of COX-2, a potentially important factor in the evolution of carcinogenesis in precancerous lesions, might be an indicator of the development of carcinoma.

532-nanometer potassium titanyl phosphate (KTP) laser-induced expression of selective matrix metalloproteinases (MMP) in the rat larynx.

OBJECTIVE/HYPOTHESIS: The 532-nm KTP laser is clinically useful to induce benign vocal fold lesion regression without a fibrotic response. Previously, we described an in vivo model for KTP-induced injury in the rat larynx. This study uses this model to correlate the KTP-induced histologic and biochemical changes with the absence of long-term vocal fold fibrosis seen in clinical scenarios. STUDY DESIGN: In vivo. METHODS: Unilateral vocal fold injury was induced via KTP laser at 10W (20mS pulse width) as described by our laboratory previously. Animals were subjected to serial endoscopic imaging from postoperative days 1 through 3. Animals were euthanized at 1 day, 4 weeks, and 12 weeks posttreatment and subjected to histologic analyses via hematoxylin and eosin and trichrome staining, as well as RT-PCR analyses for MMP-3, 9, transforming growth factor-beta (TGF-ß), and COX-2 mRNA expression. Uninjured vocal folds were used as controls. RESULTS: Our study revealed gross healing of the vocal fold mucosa by 3 days posttreatment, and an immediate, moderate inflammatory infiltrate with no subsequent ultrastructural changes on histology. MMP-3 and COX-2 expression increased transiently, although no changes were seen in expression of MMP-9, an MMP involved in extracellular matrix remodeling, or TGF-ß, a profibrotic cytokine. CONCLUSIONS: These data suggest that the KTP laser induces a modest inflammatory response, selective MMP expression, and no long-term fibrotic processes in a clinically relevant simulation.

Matrix Metalloproteinases (MMP-2 and MMP-9) and cyclooxygenase-2 (COX-2) expressions in vocal fold polyps.

The objective of this study was to investigate the roles of matrix metalloproteinases (MMP-2, MMP-9) and cyclooxygenase-2 (COX-2) in the pathogenesis of vocal fold polyps (VFPs). In this study, 20 VFPs and 8 normal vocal fold (VF) specimens were obtained from subjects undergoing surgery. Staining intensities were semiquantitatively assessed and statistically analyzed. Significant increases were observed in the expressions of MMP-2, MMP-9, and COX-2 in stromal spindle cells (P=0.00) and vascular wall (P=0.04, P=0.00, P=0.00) of VFPs compared with normal VFs. MMP-2 expression in surface epithelium basal cells (P=0.00) of VFPs showed enhancement compared with normal vocal folds, whereas MMP-9 and COX-2 expressions showed no significant differences. Our study showed that gelatinases and COX-2 may play a role in the development of VFPs. This is the first study to document the expression of gelatinases and COX-2 in VFPs.

[Possible involvement of hexosaminidase in vocal polyps--preliminary report]. / Rola heksozaminidazy w polipach faldów glosowych--doniesienie wstepne.

UNLABELLED: Vocal fold polyp is usually a result of the injury of the vocal cords. It often appears after vocal overuse or misuse causing trauma in vocal fold mid-membranous and wound formation. THE AIM OF THE STUDY was to assess the activity of hexosaminidase in vocal fold polyps in the comparison to the control tissue. MATERIAL AND METHODS: Vocal polyps (n=8) were collected from 8 patients during direct laryngoscopy. Specimens of normal vocal folds were obtained from 8 cadavers (n=8) served as controls. Specimens were immediately frozen in -80 degrees C. To assess hexosaminidase activity, release of p-nitrophenol from p-nitrophenol derivatives was used. RESULTS: We observed statistical differences between the activity of investigated enzyme in all vocal folds specimens compared with that in normal tissue homogenates. However the activity of HEX achieved a low level. Mean release of HEX from the activated cells in vocal fold specimens was 1.073 nkat/g wet tissue compared with 0.766 nkat/g wet tissue in normal cadaveric vocal fold homogenates. CONCLUSIONS: Low activity of HEX in vocal fold polyps suggests that inflammation may not be a primary factor in the development of the disease and other mechanism should likely be considered in the pathogenesis of vocal fold polyps.

Expression of matrix metalloproteinases (MMP-2 and MMP-9) in vocal fold polyps.

OBJECTIVE: Vocal fold polyps are the most common benign laryngeal lesions. Matrix metalloproteinases (MMP) play an important role in the physiological and pathological remodeling of tissues. The most important subgroup of MMP family consists of gelatinases A and B (MMP-2 and MMP-9). The objective of this study was investigation of the expression of MMP-2 and MMP-9 in vocal fold polyps and normal tissue of vocal folds. MATERIAL AND METHODS: The immunohistochemical expression of MMP-2 and MMP-9 was investigated in specimens taken by endolaryngeal microsurgery from vocal fold polyps (n=30) and normal tissue of vocal fold (n=13, control group). Expression of MMP-2 and MMP-9, both in epithelium and stroma cells, was graded on a semiquantitative scale, ranging from 0 (no expression) to 6 points (high expression). RESULTS: A statistically significant increase was observed in the expression of MMP-2 in stroma cells (P=0.0176) of vocal fold polyps compared to control vocal fold group, whereas no significant difference in the expression of MMP-2 was found in epithelium cells (P=0.1487). Comparison of expression of MMP-2 and MMP-9 in epithelium cells revealed a statistically significant increase in MMP-9 expression (P<0.01) in both groups. However, there was no statistically significant difference in the expression of MMP-9 between groups of vocal fold polyps and control vocal folds. CONCLUSION: Expression of MMP-2 in stroma was significantly higher in polyps than in normal tissue of vocal folds. Our data draw attention to the role of MMP-2 in the development of vocal fold polyps and necessity of further investigations to define its function in morphogenesis of laryngeal benign, premalignant, and malignant lesions.

Experimentally induced phonation increases matrix metalloproteinase-1 gene expression in normal rabbit vocal fold.

OBJECTIVES: An in vivo rabbit model was used to study the effect of 3 hours of experimentally induced phonation on messenger RNA expression of the normal vocal fold. STUDY DESIGN: Prospective; animal model. SUBJECTS AND METHODS: Ten rabbits received experimental phonation for 3 hours, followed by 1 hour of recovery. A separate group of 5 rabbits served as no-phonation controls. We measured messenger RNA expression of matrix metalloproteinase-1, MMP-9, and interleukin-1beta using real-time reverse-transcribed polymerase chain reaction. Gene expression ratios from phonation and control animals were assessed with the Mann-Whitney U test. RESULTS: Phonation (77 +/- 3 dB; 429 +/- 141 Hz) resulted in increased matrix metalloproteinase-1 gene expression from rabbits receiving experimental phonation compared with controls, and a nonsignificant increase in matrix metalloproteinase-9 and interleukin-1beta gene expression. CONCLUSION: Matrix metalloproteinases play a role in maintaining tissue homeostasis. Investigation of cellular responses to experimental phonation may provide insight into how matrix metalloproteinases and other extracellular matrices contribute to maintenance of the vocal fold and development of pathology.

Dynamic biomechanical strain inhibits IL-1beta-induced inflammation in vocal fold fibroblasts.

Despite the fact that vocal folds are subjected to extensive mechanical forces, the role of mechanical strain in vocal fold wound healing has been overlooked. Recent studies on other tissues have demonstrated that low physiological levels of mechanical forces are beneficial to injured tissues, reduce inflammation, and induce synthesis of matrix-associated proteins essential for enhanced wound healing. In this study, we speculated that mechanical strain of low magnitudes also attenuates the production of inflammatory mediators and alters the extracellular matrix synthesis to augment wound healing in cultured vocal fold fibroblasts. To test this hypothesis, fibroblasts from rabbit vocal folds were isolated and exposed to various magnitudes of cyclic tensile strain (CTS) in the presence or absence of interleukin-1beta (IL-1beta). Results suggest that IL-1beta activates proinflammatory gene transcription in vocal fold fibroblasts. Furthermore, CTS abrogates the IL-1beta-induced proinflammatory gene induction in a magnitude-dependent manner. In addition, CTS blocks IL-1beta-mediated inhibition of collagen type I synthesis, and thereby upregulates collagen synthesis in the presence of IL-1beta. These findings are the first to reveal the potential utility of low levels of mechanical signals in vocal fold wound healing, and support the emerging on vivo data suggesting beneficial effects of vocal exercise on acute phonotrauma.

[Superoxide dismutase (SOD) activity measured by ESR-spin trapping method in canine vocal cords].

Recently, superoxide dismutase (SOD) has been regarded as a scavenger of superoxide radical which is a important factor of 'reperfusion injury' in ischemic heart or in brain disorder. Its low activity means cell susceptibility to the superoxide radical in tissues. The SOD activity was examined in ten canine larynges after phonation by a electron spin resonance (ESR)-spin-trapping method. The values of mean SOD activity at the middle part of the membranous vocal cord, the vocal process of the arytenoid and the subglottal portion were 67.84 +/- 11.06 U/ml/g, 121.94 +/- 19.46U/ml/g and 115.46 +/- 19.18U/ml/g, respectively. There was a significant difference between the value of the membranous vocal cord and those of the other two (P less than 0.01). The result suggests that the low SOD activity at the part of the membranous vocal cord might relate to a pathogenesis of the laryngeal polyp and vocal nodule. The reperfusion injury may participate in the cause of such vocal cord disorders which grow commonly at the middle part of the membranous vocal cord.