Cyclic Tensile Strain on Vocal Fold Fibroblasts Inhibits Cigarette Smoke-Induced Inflammation: Implications for Reinke Edema *,†,‡Haiyan Zhang, §Yawei Wang, *,†,‡Xiaohui Bai, *,†,‡Zhenghua Lv, *,†,‡Jidong Zou, *,†,‡Wei Xu, and *,†,‡Haibo Wang, *yzxJinan, P.R. China Summary: Objectives. To investigate whether patients with Reinke edema are more extrovertive than patients with carcinoma and, whether cyclic tensile strain (CTS) attenuates cigarette smoke condensate (CSC)-induced inflammation in human vocal fold fibroblasts (HVFF). Study Design. In vitro and ex vivo study. Methods. Clinical investigation and Eysenck personality questionnaire were performed to evaluate the personality and smoking status in individuals among groups. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was applied to test the viabilities of cultured HVFF exposed to CSC and/or CTS. The messenger RNA (mRNA) and protein expressions of cluster of differentiation 44 (CD44), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-9 (MMP-9) in tissues from Reinke edema, paracancerous site, normal vocal fold, and in HVFF with different treatments were measured by reverse transcriptase polymerase chain reaction or western blot, respectively. Results. Patients with either Reinke edema or carcinoma were moderate/heavy smokers; the extraversion score, however, was higher in Reinke edema patients than that in carcinoma patients. CD44 mRNA and protein expressions were significantly higher, whereas, COX-2 and MMP-9 expressions were significantly lower in Reinke edema tissues than those in paracancerous tissues. Treatment of HVFF with CSC led to the decrease in cell viability, the reduction in CD44 expression, but, the increase in COX-2 and MMP-9 expressions and, moreover, administration of CTS inhibited such effects of CSC on HVFF. Conclusions. Our results demonstrate that Reinke edema patients have more extrovert personality characteristics in comparison to carcinoma patients and, importantly, CTS attenuates CSC-induced inflammation in HVFF. Phonatory vibration may be a mechanism for lower expression of proinflammatory mediators in Reinke edema tissues in spite of cigarette smoke exposure. Key Words: Vocal fold–Reinke edema–Inflammation–Fibroblasts–Cigarette smoke condensate–Cyclic tensile strain.
INTRODUCTION Reinke edema, one of the cigarette smoke (CS)-associated laryngeal response diseases, characteristically presents with the edema of vocal folds due to the excessive production of extracellular matrix.1 Although it has been reported that the fibroblasts in lamina propria are important for the production of extracellular matrix molecules,2,3 the exact mechanism underpinning Reinke edema remains unclear. It has been documented that CS, one of the most detrimental factors to the human body, can elicit a significant inflammatory response in many tissues via regulation of certain key factors.4 Of these, cluster of differentiation 44 (CD44) is believed to be important in mediating many inflammatory reactions. As the receptor of hyaluronan, CD44 binding to hyaluronan can suppress proinflammatory cytokine-stimulated production of cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP) in synovial fibroblasts.5–7 CD44/ animals showed enhanced and prolonged infiltration of neutrophil and macrophage.8 Accepted for publication June 4, 2014. From the *Department of Otorhinolaryngology Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan, P.R. China; yShandong Provincial Key Laboratory of Otology, Jinan, P.R. China; zInstitute of Eye and ENT, Provincial Hospital affiliated to Shandong University, Jinan, P.R. China; and the xDepartment of Chemotherapy, Cancer Center, Qilu Hospital of Shandong University, Jinan, P.R. China. Address correspondence and reprint requests to Haibo Wang, Department of Otorhinolaryngology Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan 250021, P.R. China. E-mail: [email protected] Journal of Voice, Vol. 29, No. 1, pp. 13-21 0892-1997/$36.00 Ó 2015 The Voice Foundation http://dx.doi.org/10.1016/j.jvoice.2014.06.001
Through regulating these factors, CS is closely associated with numerous diseases, including vocal fold squamous cell carcinoma and Reinke edema.9,10 Recent studies showed that exposure to CS caused squamous metaplasia in the vocal folds of animals and the up-regulation of COX-2 and prostaglandin E2 (PGE2) expressions in human vocal fold fibroblasts, implying that CS may increase probability of neoplasia.11,12 On the contrary, one study demonstrated that, under CS-mediated oxidative stress, the fibroblasts in Reinke edema tissue exhibited a chemoprotective response, that is, the increased antioxidant gene activity in tissues, suggesting that Reinke edema may delay or even inhibit progression to malignancy.13 However, there have been no researches relevant to the etiology for eliciting this antioxidant mechanism. In this regard, it is important to know why the Reinke edema exhibits a chemoprotective reaction and why the two kinds of patients experience such different outcomes in spite of being exposed to similar condition of CS. Besides smoking, another essential etiologic factor of Reinke edema is excessive speaking.10,14 It has been reported that to be talkative is a way of being extroverted, and extraversion tends to be manifested in outgoing, talkative, and energetic behavior.15 In our long-term clinical practice, we have observed that most Reinke edema patients are more extroversive with excessive speaking, whereas, vocal fold carcinoma patients, it seems, are more introversive with much less communication. In view of this, it is speculated that vibrational stimulation may be one major cause for the different conditions of vocal fold. Available data have manifested that, during phonation, vocal
14 fold fibroblasts encounter various types of mechanical force, with tensile as the dominant form.16 A study has shown that tensile strain alters cell behavior and inhibits interleukin-1b (IL-1b)-induced inflammation in vocal fold fibroblasts.17 Moreover, recent studies have demonstrated that a proper physiological level of mechanical force benefits human fibroblasts of injured larynx and other tissues.18–20 We thus hypothesize that patients with Reinke edema have more extrovert personality characteristics in comparison to patients with carcinoma, that is, excessive phonation resulted from the extrovert personality involves in the pathogenesis of Reinke edema. In addition, like a double-edged sword, the same phonatory vibration may be the putative reason for protecting the vocal fold of Reinke edema patients from the CS-induced injuries via suppressing the inflammatory gene expression in fibroblasts. To test our hypothesis, we conducted both clinical and basic research. Specifically, smoking status of Reinke edema patients or vocal fold carcinoma patients was evaluated. In addition, Eysenck personality questionnaire (EPQ), an objective measure, was used to determine the personality of patients, which, in turn, may indirectly reflect the amount of vocal fold vibration. It has been shown that the differentiation of epithelial cell after injury is greatly influenced by the metabolism of fibroblasts in Reinke space and,21 also, the fibroblasts in paracancerous tissue may drive malignant tumorigenesis by providing the inflammatory microenvironment for tumor development.22 Accordingly, Reinke space tissues from Reinke edema and paracancerous site were collected for this study, then, the primary cultured human vocal fold fibroblasts (HVFF) were treated with cigarette smoke condensate (CSC) in the presence or absence of cyclic tensile strain (CTS) and, finally, the gene and protein expressions of CD44, COX-2, and MMP-9 were detected in tissues or cells. Through the aforementioned experiments, we sought to provide preliminary data regarding the effect of vibration on CS-induced responses in vocal folds. The investigation is a first step toward addressing the large question of why Reinke edema exhibits chemoprotective reaction. MATERIALS AND METHODS Participants Written informed consents were obtained from all subjects before their inclusion in the study, in accordance with the ethical standards of the Local Ethics Committee of Shandong Provincial Hospital. All subjects gave their written informed consent. In total, 55 patients (54 male, aged 42–71) with vocal fold squamous cell carcinoma, 67 patients (58 male, aged 38–69) with Reinke edema and 60 physically and mentally healthy control adults (52 male, aged 40–70) were included for the smoking status investigation and EPQ survey. All the participants possessed good understanding of the EPQ. The patients were hospitalized for the surgery in the Department of Otorhinolaryngology-Head and Neck Surgery of Shandong Provincial Hospital from September 2009 to December 2011. The diagnosis of the lesions was determined by at least two specialists on stroboscopic inspection and confirmed by the pathologist.
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For in vitro and ex vivo study, paracancerous and Reinke edema tissues were collected from 20 of the 55 vocal fold carcinoma patients and 20 of the 67 Reinke edema patients during routine surgical resection. Meanwhile, normal human vocal folds were obtained from 14 hypopharyngeal and six esophageal carcinoma patients, without smoking history or phonotrauma, who had undergone total laryngectomy as initial treatment. The vocal folds were judged to be without any evidence of disease by at least two specialists. Patients with neither systemic inflammatory disease nor current anti-inflammatory medications were selected for the study. Assessment of smoking status The association between cancer occurrence and cigarette smoking status was assessed via the classical method based on the duration of smoking and the number of cigarettes smoked per day.23 Both Reinke edema and carcinoma patients had prolonged duration of exposure to CS. Assessment of smoking status was based on the total smoke exposure (pack-years, PKY), which was calculated by multiplying the number of packs (1 pack ¼ 20 cigarettes) of cigarettes smoked per day by the number of years the person has smoked. The obtained PKY was then compared between Reinke edema and carcinoma patients. EPQ survey A Chinese version of EPQ modified by Gong et al24 was used in this study. It comprised 88 items to be answered in a binary mode of ‘‘yes/no.’’ The responses to these items define four independent personality dimensions: extraversion (E), neuroticism (N), psychoticism (P), and lie (L), among which E dimension, characterized by enjoyment of social interactions, talkative personality, novel activities, and a tendency toward impulsive behavior, and higher score on E dimensionality represents extroversion personality.25 The collection of vocal fold tissues Reinke space tissues were collected from vocal fold carcinoma patients and Reinke edema patients during routine surgical resection, and normal Reinke space tissues were obtained from hypopharyngeal or esophageal carcinoma patients who had undergone total laryngectomy. In detail, phonomicrosurgery via the direct endoscopic approach under general anesthesia was performed for Reinke edema, and the Reinke edema contents in Reinke space were removed using cup forceps by microflap approach. CO2 laser microlaryngoscopy was applied for vocal fold carcinoma, and Reinke space tissues of paracancerous site (5 mm away from cancer) were collected before the laser excision of lesions. Reinke space of normal vocal fold was dissected under microlaryngoscopy after total laryngectomy. Specifically, the epithelial layer was dissected using sickle knife and a small curved elevator, and then, Reinke space was directly visualized and collected using cup forceps under microlaryngoscope. All the tissues were used for reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analyses of CD44, COX-2, and MMP-9 expressions and, in addition, the normal vocal fold tissues were also used for primary culture of vocal fold fibroblasts.
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Primary culture of vocal fold fibroblasts HVFF were obtained respectively from 20 Reinke space tissues of normal vocal folds. The culture methodology used in this work followed the previous procedures.26 Each tissue was placed into basic Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco Invitrogen, Camarillo, CA) medium containing penicillin (50 U/mL), streptomycin (50 U/mL), and amphotericin B (12 ng/mL). After being rinsed twice, the tissue was placed in a Petri dish and minced into small pieces (1 mm3) using forceps and scalpel, then cultured on the six-well plates which were filled with 1–2 mL of 10% fetal bovine serum (FBS)-DMEM (Gibco Invitrogen, Camarillo, CA) complete medium containing antibiotics. The six-well plates were placed in a 37 C humidified incubator with 5% CO2. After 24 hours, extra 3–4 mL of 10% FBS-DMEM complete medium was gently added into the cultures. After 1–2 weeks of culture, the primary fibroblasts were grown into 100% confluence. The fourth to sixth passages were used in subsequent experiment.
MO) was added into each well, and the cells were cultured at 37 C for an additional 4 hours. The optical density (OD) values were measured at 570 nm using an enzyme-linked immunosorbant assay (ELISA) reader. The relative cell viability, which was defined relative to corresponding control cells, was calculated according to the following formula: relative cell viability (%) ¼ ODexperiment/ODcontrol 3 100% (ODblank was used to blank).29 As the results by MTT assay showed that median lethal dose of CSC for 24 hours was approximately 400 mg/mL, so optimal dosage of CSC (400 mg/mL) was established following the standard protocol.30 As for the mode of CTS, we selected CTS at a frequency of 0.5 Hz on the basis of previous investigation.17 Subsequently, the cells were cultured in 10% FBSDMEM containing CSC (400 mg/mL) and then treated with CTS (0.5 Hz, 24 h) at 2%, 4%, 8%, and 12%, respectively. Finally, the viabilities were tested. Also, an optimal mode of CTS (8%, 0.5 Hz, 24 h) was established and used in all subsequent experiments.
Immunofluorescence Cells were subcultured on six-well plates and grown in 10% FBS-DMEM complete medium. The major cell in Reinke space is fibroblasts, which contains intermediate filaments. As the antibody against vimentin is the marker for intermediate filaments, so, we stained for vimentin with the antibody in this work as previously reported.27,28 Primary cultured HVFF cells were washed twice in phosphate buffer solution (PBS) (Sigma, St. Louis, MO) and fixed in 10% formalin for 15 minutes. After washing in PBS for 5 minutes twice, cells were blocked with 10% normal goat-serum for 30 minutes at 37 C in a humidified atmosphere. The primary monoclonal anti-vimentin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added in PBS at 1:200 dilutions, incubated for 1 hour at 37 C, and then for 16–18 hours at 4 C in a humidified atmosphere. The replacement of the primary antibody with PBS served as negative control. After being washed three times in PBS, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated goat–anti-mouse secondary antibody (1:200) for 1 hour at room temperature. 40 , 6-diamidino-2-phenylindole dihydrochloride (DAPI) (2 mg/mL) was applied to mark nuclei. Finally, the slides were washed six times in PBS and photographed under Leica Optiphot photomicroscope (Leica Microsystems AG, Wetzlar, Germany).
Exposure of HVFF to CSC in the presence or absence of CTS To determine the effect of CTS, HVFF (8 3 104 cells/well) were plated onto rat tail collagen type I (BD Biosciences, San Jose, CA)-coated Bioflex II plates (Flexcell International, Hillsborough, NC) in the Flexcell loading station, which was equipped with a computer-regulated cyclic vacuum bioreactor that applied multiple modes of uniform equibiaxial strain to cells. There was a nearly linear relationship between vacuum level and strain.17 The cells reached 80% confluence within 4–5 days. Subsequently, cells were washed with PBS and incubated overnight in DMEM without FBS and divided into four groups: control cells, without any treatment; CTS-treated cells, treatment with only CTS (8%, 0.5 Hz) for 24 hours; CSCtreated cells, treatment with only CSC (400 mg/mL) for 24 hours; co-treated cells, treatment with both CSC (400 mg/ mL) and CTS (8%, 0.5 Hz) for 24 hours.
MTT assay MTT assay, an index of cell viability and cell growth, is based on the ability of viable cells to reduce MTT from a yellow watersoluble dye to a dark blue insoluble formazan product. In this work, MTT assay was applied to assess the viabilities of cultured HVFF in response to CSC (Murty Pharmaceuticals, Lexington, KY) and/or CTS and, accordingly, the optimal dosage of CSC and mode of CTS for all subsequent experiments were determined. Normal HVFF (8 3 104 cells/mL) were subcultured on a 96-well plate. Then the cells were cultured in 10% FBSDMEM containing different concentrations of CSC (0, 200, 300, 400, 500, and 600 mg/mL, respectively) for 24 hours. A total of 20 mL of MTT (5 mg/mL stock solution, Sigma, St. Louis,
RNA preparation and RT-PCR Total RNAs were extracted from the tissues and cultured HVFF using TRIzol reagent (Invitrogen, Camarillo, CA). The messenger RNA (mRNA) levels of CD44, COX-2, and MMP-9 were determined by RT-PCR. Anti-CD44 primary antibody was purchased from Abcam (Cambridge, United Kingdom). Primary antibodies against COX-2, MMP-9, and b-actin were all Santa Cruz products. The RT was performed using ExScript RT reagent kit (Takara Bio, Otsu, Shiga, Japan) in a final volume of 20 mL containing 1 mg total RNA, 4 mL 5 3 ExScript buffer, 1 mL dNTP mixture (Takara Bio, Otsu, Shiga, Japan), 1 mL Oligo(dT) primer (Takara Bio, Otsu, Shiga, Japan), 0.5 mL ExScript RTase, 0.5 mL RNase inhibitor (Takara Bio, Otsu, Shiga, Japan), with volume adjusted by RNase-free water. PCR was conducted according to the instructions of Takara Taq as following: predenaturation at 95 C for 3 minutes; degeneration at 95 C for 45 seconds, annealing at 60 C for 40 seconds, and elongation at 72 C for 50 seconds, for a total of 28 cycles. All of the produced PCR products were electrophoresed in a 2% agarose gel, and the agarose gel was stained with ethidium bromide and examined under ultraviolet
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TABLE 1. The Mean Scores on Extraversion (E) Dimension and Pack-Years (PKY) for Smoking Status Assessment in Individuals Among Reinke Edema (RE), Carcinoma and Control Group (n ¼ 182) EPQ-E
Smoking Status (PKY)
Factors Group
n
Mean (SD)
ANOVA
SNK Test
Mean (SD)
RE Carcinoma Control
67 55 60
62.61 (7.02) 41.35 (7.01) 49.20 (5.55)
F ¼ 165.02; P < 0.0001
RE > control Carcinoma < control RE > carcinoma; P < 0.05
45.69 (11.15) 46.38 (10.50)
Independent Sample t Test t ¼ 0.35; P ¼ 0.7257
Abbreviation: EPQ, Eysenck Personality Questionnaire.
light. The relative mRNA levels were quantified and calculated with the ImageJ software (National Institutes of Health, Bethesda, MD) after normalization against the internal control b-actin. For each gene, three independent PCRs from the same reverse transcription sample were performed. The primers used were as follows: 50 -AGCGGCTCCTCCAGTGAAA-30 and 50 TCTGTCTGTGCTGTCGGTGATC-30 for CD44; 50 -CAGGGTTG CTGGTGGTAGGA-30 and 50 -GAACACTGAATGAAGTAAAGG GACA-30 for COX-2; 50 -TCTTCCCTGGAGCCTGAGAAC-30 and 50 -GACACCAAACTGGATGACGATG-30 for MMP-9; 50 CATCGTGATGGACTCTGGTG-30 and 50 -AGGGCAACATAGCA CAGCTT-30 for b-actin. The expected amplification products of the target sequence with these primers were 116, 451, 427, 213 base pairs (bp) in length respectively.
quantified and calculated with the ImageJ software after normalization against the internal control b-actin. All experiments were performed in triplicate. Statistical analysis Statistical analysis was performed using the SAS 9.13 software (SAS Institute Inc., Cary, NC) package. The results of extraversion score and PKY were presented as means and standard deviation (SD) for measuring the donor differences, and the results of western blot and PCR analysis were presented either as digitized assessment of each band after densitometric analysis or as mean and SD of the mean from triplicate values. To determine whether significant differences in extraversion score, western blot values, and PCR values exist between groups, one-way analysis of variance (ANOVA) and the post hoc multiple comparison Student– Newman–Keuls (SNK) test were applied. Additionally, the constituent ratio of personality type among groups was analyzed with chi-square test. PKY was compared between Reinke edema and carcinoma groups using independent sample t test. To identify differences between CSC-treated cells in the absence or presence of CTS at various magnitudes, one-way ANOVA and the post hoc multiple comparison Dunnett test were used. Differences with P < 0.05 were considered significant.
Western blot analysis Total proteins were extracted from tissues and cultured cells using radio immunoprecipitation assay buffer according to manufacturer’s instruction. Briefly, the homogenized tissues or harvested cells were lysed with cold radioimmune precipitation buffer and protein lysis buffer for 30 minutes on ice. The lysates were transferred to Eppendorf tubes and clarified by centrifugation at 12 000g for 15 minutes at 4 C, and the supernatants were transferred for analysis. The Bradford method was used to determine the protein concentration in the tissue or cell lysates. A total of 40 mg of protein from each sample was used in western blot analysis with diluted primary antibodies as follows: CD44 (1:5000), COX-2 (1:200), MMP-9 (1:200), and b-actin (1:2000); and the corresponding specific protein bands were visualized at apparent molecular weights of 82, 72, 92, and 43 kDa, respectively. The relative protein expressions were
RESULTS Findings of the smoking status and personality Assessment of smoking status based on PKY showed that both Reinke edema and carcinoma patients were moderate/heavy smokers, and the comparison between them showed nonsignificant differences (t ¼ 0.35, P ¼ 0.7257, Table 1). Whereas, E score of Reinke edema patients was significantly higher than that of
TABLE 2. The Constituent Ratio of Personality Characteristics Among Reinke Edema (RE), Carcinoma, and Control Groups (n ¼ 182, c2 ¼ 158.7225, P < 0.001) Typical Introversion
Introversion
Intermediate
Extraversion
Typical Extraversion
Factors Group
n
(%)
n
(%)
n
(%)
n
(%)
n
(%)
Total
RE Carcinoma Control
0 18 0
0.00 32.73 0.00
0 17 13
0.00 30.91 21.67
13 20 39
19.40 36.36 65.00
12 0 8
17.91 0.00 13.33
42 0 0
62.69 0.00 0.00
67 55 60
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Cigarette Smoke-Induced Inflammation: Linked to Reinke Edema
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FIGURE 1. The results of Eysenck Personality Questionnaire (EPQ) survey in individuals among Reinke edema (RE), carcinoma, and control group. Extraversion (E) score of RE patients was significantly higher than that of carcinoma patients (*P < 0.05). RE patients were prone to be extroversive, whereas, vocal fold carcinoma patients were prone to be introversive. A. The analysis of E scores in individuals among groups. B. The constituent ratio of RE group. C. The constituent ratio of carcinoma group. D. The constituent ratio of control group. carcinoma patients, pairwise comparisons indicated significant differences among three groups (P < 0.05, Table 1). Furthermore, statistical analysis of constituent ratio demonstrated that Reinke edema patients were prone to be extroversive, whereas, vocal fold carcinoma patients were prone to be introversive (c2 ¼ 158.7225, P < 0.001, Table 2). In addition, the results of EPQ survey are also shown in Figure 1.
CTS attenuated the CSC-induced damages to HVFF MTT assay revealed that the HVFF survival rates were significantly reduced by the treatment with CSC at 300 mg/ mL, 400 mg/mL, 500 mg/mL, and 600 mg/mL (P < 0.05, Figure 5A). A total of 400 mg/mL of CSC optimally decreased cell survival rate, and then this dosage was used in all
The mRNA expression levels of CD44, COX-2, and MMP-9 were significantly different between Reinke edema and paracancerous tissues CD44 mRNA expression level was significantly higher in Reinke edema tissues than that in paracancerous tissues (P < 0.05); whereas COX-2 and MMP-9 mRNA expression levels in Reinke edema tissues were significantly downregulated compared with those in paracancerous tissues (P < 0.05) (Figure 2). The protein expression levels of CD44, COX-2, and MMP-9 were significantly different between Reinke edema and paracancerous tissues CD44 protein expression level was significantly higher in Reinke edema tissues than that in paracancerous tissues (P < 0.05), whereas COX-2 and MMP-9 protein expression levels were significantly down-regulated in Reinke edema compared with those in paracancerous tissues (P < 0.05) (Figure 3). Morphological observation and identification of HVFF HVFF were characterized by immunofluorescent staining. The respective blue (DAPI) and green (FITC) fluorescence of the nuclei and vimentin were observed in Figure 4. The results showed that almost 100% cells were vimentin-positive fibroblasts after four to six passages.
FIGURE 2. The mRNA expressions of CD44, COX-2, and MMP-9 in normal, Reinke edema (RE), and paracancerous tissues by RT-PCR. The expression of CD44 in RE tissues was higher than that in paracancerous tissues; COX-2 and MMP-9 expressions in RE tissues were significantly down-regulated compared with those in paracancerous tissues (*P < 0.05). A. Representative gel. B. Densitometric analysis (*P < 0.05).
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CTS inhibited CSC-induced COX-2 and MMP-9 mRNA expression in HVFF The effects of CTS and CSC on mRNA expressions of CD44, COX-2, and MMP-9 were examined. Compared with untreated cells, CD44 mRNA expression was down-regulated and COX2, MMP-9 mRNA expressions were up-regulated in CSCtreated group (P < 0.05). Furthermore, these effects of CSC were suppressed by treatment with CTS (Figure 6A). Statistical analysis revealed that, compared with CSC-treated group, CD44 mRNA expression was up-regulated, meanwhile, COX2, and MMP-9 mRNA expression were down-regulated in CTS and CSC-treated group (P < 0.05, Figure 6B). No significant difference was found between the CTS and control groups (P > 0.05).
FIGURE 3. The protein expressions of CD44, COX-2, and MMP-9 in normal, Reinke edema (RE), and paracancerous tissues by western blot. The protein expression of CD44 was higher, but, COX-2 and MMP-9 were lower in RE than those in paracancerous tissues (*P < 0.05). A. Representative gel. B. Densitometric analysis (*P < 0.05). subsequent experiments. Next, to examine the effects of CTS on the CSC-induced responses, HVFF were exposed to CTS of magnitudes between 0% and 12%. Figure 5B showed that the treatment of HVFF with CTS (8%, 0.5 Hz, 24 h) significantly increased the cell survival rate to 92.06 ± 5.54% (P < 0.05). Accordingly, CTS (8%, 0.5 Hz, 24 h) was used in the next experiments.
CTS inhibited CSC-induced COX-2 and MMP-9 protein expression in HVFF The effects of CTS on protein expressions of CD44, COX-2, and MMP-9 were determined after exposure of HVFF to CTS for 24 hours. Compared with untreated cells, in CSC-treated group, CD44 protein expression was significantly downregulated, whereas COX-2 and MMP-9 protein expressions were significantly up-regulated; however, such effects of CSC were significantly suppressed by treatment with CTS (P < 0.05, Figure 7). No significant difference in protein expression level was found between the CTS alone and control groups (P > 0.05). DISCUSSION Through vibration, the vocal folds transform aerodynamic energy of exhalation to acoustic energy.31,32 Their function is, however, not limited to voice production. Our present study shows that the
FIGURE 4. Identification of human vocal fold fibroblasts (HVFF) using immunohistochemical staining under fluorescence microscopy. A. Morphological observation of HVFF. B. Cell nuclei were stained by DAPI. C. Vimentin expressed in the cytoplasm of HVFF, not in nuclei. D. Merged image of nuclei and vimentin indicated that 100% cells were vimentin-positive fibroblasts.
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Cigarette Smoke-Induced Inflammation: Linked to Reinke Edema
FIGURE 5. Alteration of viabilities in HVFF exposed to different concentrations of cigarette smoke condensate (CSC) and/or different mode of cyclic tensile strain (CTS) by MTT assay. A. Cell viabilities were reduced by CSC in a dose-dependent manner, and significant difference could be found from 300 mg/mL (*P < 0.05). B. The combined effect of CTS and CSC on cell viabilities showed that CTS (8%, 0.5 Hz, 24 h) protected cells from CSC (400 mg/mL, 24 h)-induced injuries (*P < 0.05).
vocal folds vibration also has a potential function to suppress the CS-induced inflammation, which putatively explains the observation of the rare malignant transformation of Reinke edema. These results may provide valuable guidance to future clinical therapy of both Reinke edema and malignancy. Studies on vocal fold lesions reveal that vocal misuse is mainly associated with benign lesions, but, not with carcinoma,33 and that CS is closely associated with vocal fold carcinoma.13 Although CS is thought to be the most frequent etiologic factor for both Reinke edema and vocal fold carcinoma, it has also been proposed that Reinke edema does not carry an increased risk for malignant transformation.34 Interestingly, the significant difference in the amount of vocalization between patients with Reinke edema and vocal fold carcinoma was observed in our clinical practice. Such a clinical phenomenon also inspires the present study. In this work, all the Reinke edema patients reported excessive speaking, thereby confirming its important role in the pathogenesis of this laryngeal disease. Therefore, the precise pathophysiological mechanism underlying the actions of smoking and excess speaking on Reinke edema needs to be investigated further. EPQ, one of the most influential psychological scales with good reliability and validity, is generally used to determine the personality types by data analysis.35 In this work, patients with Reinke edema and carcinoma were both smokers, but, the patients in two groups exhibited different types of personality by the EPQ survey: Reinke edema patients are more extro-
19
FIGURE 6. The mRNA expressions of CD44, COX-2, and MMP-9 in cultured HVFF treated with CSC (400 mg/mL, 24 h) and/or CTS (8%, 0.5 Hz, 24 h). CD44 expression was lowest in CSC-treated group, which could be significantly up-regulated by the presence of CTS (*P < 0.05). COX-2 and MMP-9 expressions were highest in CSCtreated group, which could be significantly attenuated by CTS (*P < 0.05). Blots were representative of three separate experiments. A. Representative gel. B. Densitometric analysis (*P < 0.05). versive, whereas, carcinoma patients are more introversive. As well known, extroversive people talk more than the introversive ones, thus, more vibration happens in the vocal fold of extroverts. This means that the vocal folds of Reinke edema patients are inclined to be exposed to mechanical strain. Moreover, many studies have manifested that the mechanical force can benefit human fibroblasts of injured larynx and other tissues.17–20 The present study demonstrated that CD44 expressions were significantly higher, whereas, COX-2 and MMP-9 expressions were lower in Reinke edema tissues than those in paracancerous tissues. Then, we sought to determine whether the different amount of vocal fold vibration in individuals with different characters might explain distinct responses of vocal fold after similar CS exposure and the mechanical strain on vocal fold fibroblasts might have the potential function to suppress the CS-induced inflammation. In vitro study, treatment of HVFF with CSC led to the decrease in cell viability, which was increased by administration of CTS. This suggests that CTS protects the cells from CSC-induced damage. In addition, the proinflammatory effects of CSC exposing to the fibroblasts was mitigated by CTS. These results, corroborating our tissue data, indicated a stronger antiinflammatory response was triggered in CTS and CSC-treated group.
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of HVFF with CTS. The study provides preliminary data regarding that vocal fold vibration may be the potential reason for lower expression of proinflammatory mediators in Reinke edema tissues in spite of CS exposure. Actually, in vivo work is needed to comprehensively understand the pathophysiology of Reinke edema. Acknowledgments This work was supported by the National Natural Science Foundation of China (81000407); Natural Science Foundation of Shandong Province (Y2007C145); Natural Science Foundation of Shandong Province (ZR2011HQ018). REFERENCES
FIGURE 7. The protein expressions of CD44, COX-2 and MMP-9 in cultured HVFF treated with CSC (400 mg/mL, 24 h) and/or CTS (8%, 0.5 Hz, 24 h). CD44 protein expression was lowest in CSCtreated group, which could be significantly up-regulated by the presence of CTS (*P < 0.05), whereas, COX-2 and MMP-9 protein levels were highest in CSC-treated group, which could be significantly attenuated by CTS (*P < 0.05). Blots were representative of three separate experiments. A. Representative gel. B. Densitometric analysis (*P < 0.05). Although the system used for in vitro study can not exactly simulate physiologically-relevant forces exerting on fibroblasts in vivo, it is expected to reflect the principle effect of mechanical forces on CSC-stressed vocal fold fibroblasts. To the best of our knowledge, this is the first report that CTS-mediated activations of CD44, COX-2, and MMP-9 critically involve in the alleviation of CSC-induced inflammatory damages to HVFF, implying that mechanical strain on HVFF may be the reason for the reduced inflammatory responses to CS in Reinke edema tissues. Even so, in vivo experiments should be performed to decipher the precise pathophysiological mechanisms responsible for Reinke edema in the future study. CONCLUSION In summary, patients with Reinke edema have more extrovert personality characteristics in comparison to patients with carcinoma, implying that the vocal folds of Reinke edema patients are more inclined to be exposed to mechanical strain caused by phonation. We, for the first time, report that inflammatory reaction to CSC is dramatically reduced by treatment
1. Martins RH, Domingues MA, Fabro AT, Dias NH, Santana MF. Reinkes’ edema: immunoexpression study of fibronectin, laminin and collagen IV in 60 cases by immunohistochemical techniques. Braz J Otorhinolaryngol. 2009;75:821–825. 2. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteoglycans and their influence on biomechanics. Laryngoscope. 1999;109:845–854. 3. Moore J, Thibeault S. Insights into the role of elastin in vocal fold health and disease. J Voice. 2012;26:269–275. 4. Yan C, Wu W, Li H, Zhang G, Duerksen-Hughes PJ, Zhu X, Yang J. Benzo(a)pyrene treatment leads to changes in nuclear protein expression and alternative splicing. Mutat Res. 2010;686:47–56. 5. Shimizu M, Yasuda T, Nakagawa T, Yamashita E, Julovi SM, Hiramitsu T, Nakamura T. Hyaluronan inhibits matrix metalloproteinase-1 production by rheumatoid synovial fibroblasts stimulated by proinflammatory cytokines. J Rheumatol. 2003;30:1164–1172. 6. Mitsui Y, Gotoh M, Nakama K, Yamada T, Higuchi F, Nagata K. Hyaluronic acid inhibits mRNA expression of proinflammatory cytokines and cyclooxygenase-2/prostaglandin E(2) production via CD44 in interleukin1-stimulated subacromial synovial fibroblasts from patients with rotator cuff disease. J Orthop Res. 2008;26:1032–1037. 7. Reel B, Sala-Newby GB, Huang W-C, Newby AC. Diverse patterns of cyclooxygenase-independent metalloproteinase gene regulation in human monocytes. Br J Pharmacol. 2011;163:1679–1690. 8. Huebener P, Abou-Khamis T, Zymek P, et al. CD44 is critically involved in infarct healing by regulating the inflammatory and fibrotic response. J Immunol. 2008;180:2625–2633. 9. Liu C, Bronson RT, Russell RM, Wang XD. b-Cryptoxanthin supplementation prevents cigarette smoke-induced lung inflammation, oxidative damage, and squamous metaplasia in ferrets. Cancer Prev Res (Phila). 2011; 4:1255–1266. 10. Marcotullio D, Magliulo G, Pezone T. Reinke’s edema and risk factors: clinical and histopathologic aspects. Am J Otolaryngol. 2002;23:81–84. 11. Duarte JL, de Faria FA, Ceolin DS, Cestari TM, de Assis GF. Effects of passive smoke inhalation on the vocal cords of rats. Braz J Otorhinolaryngol. 2006;72:210–216. 12. Branski RC, Zhou H, Kraus DH, Sivasankar M. The effects of cigarette smoke condensate on vocal fold transepithelial resistance and inflammatory signaling in vocal fold fibroblasts. Laryngoscope. 2011;121:601–605. 13. Branski RC, Saltman B, Sulica L, Szeto H, Duflo S, Felsen D, Kraus DH. Cigarette smoke and reactive oxygen species metabolism: implications for the pathophysiology of Reinke’s edema. Laryngoscope. 2009;119: 2014–2018. 14. Garcia Alvarez CD, Campos Banales ME, Lopes Campos D, Rivero J, Perez Pinero B, Lopes Aguado D. Polyps, nodules and Reinke edema. An epidemiological and histopathological study. Acta Otorrinolaringol Esp. 1999;50:443–447. 15. Hampson SE, John OP, Goldberg LR. Category breadth and hierarchical structure in personality: studies of asymmetries in judgments of trait implications. J Pers Soc Psychol. 1986;51:37–54. 16. Meyer B, Candau P, Alcaras N, MacLeod P. Study of the mechanism of vocal fold vibration during phonation. Acta Otolaryngol. 1984;97:407–414.
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17. Branski RC, Perera P, Verdolini K, Rosen CA, Hebda PA, Agarwal S. Dynamic biomechanical strain inhibits IL-1beta-induced inflammation in vocal fold fibroblasts. J Voice. 2007;21:651–660. 18. Long P, Hu J, Piesco N, Buckley M, Agarwal S. Low magnitude of tensile strain inhibits IL-1beta-dependent induction of pro-inflammatory cytokines and induces synthesis of IL-10 in human periodontal ligament cells in vitro. J Dent Res. 2001;80:1416–1420. 19. Wolchok JC, Brokopp C, Underwood CJ, Tresco PA. The effect of bioreactor induced vibrational stimulation on extracellular matrix production from human derived fibroblasts. Biomaterials. 2009;30:327–335. 20. Kutty JK, Webb K. Vibration stimulates vocal mucosa-like matrix expression by hydrogel-encapsulated fibroblasts. J Tissue Eng Regen Med. 2010; 4:62–72. 21. Hirano M. Structure of the vocal fold in normal and disease states: anatomical and physical study. ASHA Rep. 1981;11:11–30. 22. Roxburgh CS, Salmond JM, Horgan PG, Oien KA, McMillan DC. Tumour inflammatory infiltrate predicts survival following curative resection for node-negative colorectal cancer. Eur J Cancer. 2009;45:2138–2145. 23. Leffondre K, Abrahamowicz M, Siemiatycki J, Rachet B. Modeling smoking history: a comparison of different approaches. Am J Epidemiol. 2002; 156:813–823. 24. Gong YX. Revised Eysenck Personality Questionnaire Manual. Changsha, China: Hunan Medical College; 1986. 25. Eysenck HJ, Eysenck SBG. Manual for the Eysenck Personality Questionnaire. London, UK: Hodder and Stoughton; 1975. 26. Thibeault SL, Li W, Bartley S. A method for identification of vocal fold lamina propria fibroblasts in culture. Otolaryngol Head Neck Surg. 2008; 139:816–822.
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27. Tillmann B, Rudert H, Schiinke M, Werner JA. Morphological studies on the pathogenesis of Reinke’s edema. Eur Arch Otorhinolaryngol. 1995; 252:469–474. 28. Du H-D, Wu C-P, Zhou L, Tian J. Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line. Acta Otolaryngol. 2013;133: 755–760. 29. Chuang JY, Wu CH, Lai MD, Chang WC, Hung JJ. Overexpression of Sp1 leads to p53-dependent apoptosis in cancer cells. Int J Cancer. 2009;125: 2066–2076. 30. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986;89:271–277. 31. Thomson SL, Mongeau L, Frankel SH. Aerodynamic transfer of energy to the vocal folds. J Acoust Soc Am. 2005;118:1689–1700. 32. Zhang Z, Neubauer J, Berry DA. Physical mechanisms of phonation onset: a linear stability analysis of an aeroelastic continuum model of phonation. J Acoust Soc Am. 2007;122:2279–2295. 33. Johns MM. Update on the etiology, diagnosis, and treatment of vocal fold nodules, polyps, and cysts. Curr Opin Otolaryngol Head Neck Surg. 2003; 11:456–461. 34. Martins RH, Fabro AT, Domingues MA, Chi AP, Gregorio EA. Is Reinke’s edema a precancerous lesion? Histological and electron microscopic aspects. J Voice. 2009;23:721–725. 35. Koutsopoulou V, Theodosopoulou E, Vantsi E, Kotrotsiou E, Kostandinou V, Dounousi E. Personality dimensions of haemodialysis patients related to initial renal disease. EDTNA ERCA J. 2002;28: 21–24.
INTRODUCTION Reinke edema, one of the cigarette smoke (CS)-associated laryngeal response diseases, characteristically presents with the edema of vocal folds due to the excessive production of extracellular matrix.1 Although it has been reported that the fibroblasts in lamina propria are important for the production of extracellular matrix molecules,2,3 the exact mechanism underpinning Reinke edema remains unclear. It has been documented that CS, one of the most detrimental factors to the human body, can elicit a significant inflammatory response in many tissues via regulation of certain key factors.4 Of these, cluster of differentiation 44 (CD44) is believed to be important in mediating many inflammatory reactions. As the receptor of hyaluronan, CD44 binding to hyaluronan can suppress proinflammatory cytokine-stimulated production of cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP) in synovial fibroblasts.5–7 CD44/ animals showed enhanced and prolonged infiltration of neutrophil and macrophage.8 Accepted for publication June 4, 2014. From the *Department of Otorhinolaryngology Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan, P.R. China; yShandong Provincial Key Laboratory of Otology, Jinan, P.R. China; zInstitute of Eye and ENT, Provincial Hospital affiliated to Shandong University, Jinan, P.R. China; and the xDepartment of Chemotherapy, Cancer Center, Qilu Hospital of Shandong University, Jinan, P.R. China. Address correspondence and reprint requests to Haibo Wang, Department of Otorhinolaryngology Head and Neck Surgery, Provincial Hospital affiliated to Shandong University, Jinan 250021, P.R. China. E-mail: [email protected] Journal of Voice, Vol. 29, No. 1, pp. 13-21 0892-1997/$36.00 Ó 2015 The Voice Foundation http://dx.doi.org/10.1016/j.jvoice.2014.06.001
Through regulating these factors, CS is closely associated with numerous diseases, including vocal fold squamous cell carcinoma and Reinke edema.9,10 Recent studies showed that exposure to CS caused squamous metaplasia in the vocal folds of animals and the up-regulation of COX-2 and prostaglandin E2 (PGE2) expressions in human vocal fold fibroblasts, implying that CS may increase probability of neoplasia.11,12 On the contrary, one study demonstrated that, under CS-mediated oxidative stress, the fibroblasts in Reinke edema tissue exhibited a chemoprotective response, that is, the increased antioxidant gene activity in tissues, suggesting that Reinke edema may delay or even inhibit progression to malignancy.13 However, there have been no researches relevant to the etiology for eliciting this antioxidant mechanism. In this regard, it is important to know why the Reinke edema exhibits a chemoprotective reaction and why the two kinds of patients experience such different outcomes in spite of being exposed to similar condition of CS. Besides smoking, another essential etiologic factor of Reinke edema is excessive speaking.10,14 It has been reported that to be talkative is a way of being extroverted, and extraversion tends to be manifested in outgoing, talkative, and energetic behavior.15 In our long-term clinical practice, we have observed that most Reinke edema patients are more extroversive with excessive speaking, whereas, vocal fold carcinoma patients, it seems, are more introversive with much less communication. In view of this, it is speculated that vibrational stimulation may be one major cause for the different conditions of vocal fold. Available data have manifested that, during phonation, vocal
14 fold fibroblasts encounter various types of mechanical force, with tensile as the dominant form.16 A study has shown that tensile strain alters cell behavior and inhibits interleukin-1b (IL-1b)-induced inflammation in vocal fold fibroblasts.17 Moreover, recent studies have demonstrated that a proper physiological level of mechanical force benefits human fibroblasts of injured larynx and other tissues.18–20 We thus hypothesize that patients with Reinke edema have more extrovert personality characteristics in comparison to patients with carcinoma, that is, excessive phonation resulted from the extrovert personality involves in the pathogenesis of Reinke edema. In addition, like a double-edged sword, the same phonatory vibration may be the putative reason for protecting the vocal fold of Reinke edema patients from the CS-induced injuries via suppressing the inflammatory gene expression in fibroblasts. To test our hypothesis, we conducted both clinical and basic research. Specifically, smoking status of Reinke edema patients or vocal fold carcinoma patients was evaluated. In addition, Eysenck personality questionnaire (EPQ), an objective measure, was used to determine the personality of patients, which, in turn, may indirectly reflect the amount of vocal fold vibration. It has been shown that the differentiation of epithelial cell after injury is greatly influenced by the metabolism of fibroblasts in Reinke space and,21 also, the fibroblasts in paracancerous tissue may drive malignant tumorigenesis by providing the inflammatory microenvironment for tumor development.22 Accordingly, Reinke space tissues from Reinke edema and paracancerous site were collected for this study, then, the primary cultured human vocal fold fibroblasts (HVFF) were treated with cigarette smoke condensate (CSC) in the presence or absence of cyclic tensile strain (CTS) and, finally, the gene and protein expressions of CD44, COX-2, and MMP-9 were detected in tissues or cells. Through the aforementioned experiments, we sought to provide preliminary data regarding the effect of vibration on CS-induced responses in vocal folds. The investigation is a first step toward addressing the large question of why Reinke edema exhibits chemoprotective reaction. MATERIALS AND METHODS Participants Written informed consents were obtained from all subjects before their inclusion in the study, in accordance with the ethical standards of the Local Ethics Committee of Shandong Provincial Hospital. All subjects gave their written informed consent. In total, 55 patients (54 male, aged 42–71) with vocal fold squamous cell carcinoma, 67 patients (58 male, aged 38–69) with Reinke edema and 60 physically and mentally healthy control adults (52 male, aged 40–70) were included for the smoking status investigation and EPQ survey. All the participants possessed good understanding of the EPQ. The patients were hospitalized for the surgery in the Department of Otorhinolaryngology-Head and Neck Surgery of Shandong Provincial Hospital from September 2009 to December 2011. The diagnosis of the lesions was determined by at least two specialists on stroboscopic inspection and confirmed by the pathologist.
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For in vitro and ex vivo study, paracancerous and Reinke edema tissues were collected from 20 of the 55 vocal fold carcinoma patients and 20 of the 67 Reinke edema patients during routine surgical resection. Meanwhile, normal human vocal folds were obtained from 14 hypopharyngeal and six esophageal carcinoma patients, without smoking history or phonotrauma, who had undergone total laryngectomy as initial treatment. The vocal folds were judged to be without any evidence of disease by at least two specialists. Patients with neither systemic inflammatory disease nor current anti-inflammatory medications were selected for the study. Assessment of smoking status The association between cancer occurrence and cigarette smoking status was assessed via the classical method based on the duration of smoking and the number of cigarettes smoked per day.23 Both Reinke edema and carcinoma patients had prolonged duration of exposure to CS. Assessment of smoking status was based on the total smoke exposure (pack-years, PKY), which was calculated by multiplying the number of packs (1 pack ¼ 20 cigarettes) of cigarettes smoked per day by the number of years the person has smoked. The obtained PKY was then compared between Reinke edema and carcinoma patients. EPQ survey A Chinese version of EPQ modified by Gong et al24 was used in this study. It comprised 88 items to be answered in a binary mode of ‘‘yes/no.’’ The responses to these items define four independent personality dimensions: extraversion (E), neuroticism (N), psychoticism (P), and lie (L), among which E dimension, characterized by enjoyment of social interactions, talkative personality, novel activities, and a tendency toward impulsive behavior, and higher score on E dimensionality represents extroversion personality.25 The collection of vocal fold tissues Reinke space tissues were collected from vocal fold carcinoma patients and Reinke edema patients during routine surgical resection, and normal Reinke space tissues were obtained from hypopharyngeal or esophageal carcinoma patients who had undergone total laryngectomy. In detail, phonomicrosurgery via the direct endoscopic approach under general anesthesia was performed for Reinke edema, and the Reinke edema contents in Reinke space were removed using cup forceps by microflap approach. CO2 laser microlaryngoscopy was applied for vocal fold carcinoma, and Reinke space tissues of paracancerous site (5 mm away from cancer) were collected before the laser excision of lesions. Reinke space of normal vocal fold was dissected under microlaryngoscopy after total laryngectomy. Specifically, the epithelial layer was dissected using sickle knife and a small curved elevator, and then, Reinke space was directly visualized and collected using cup forceps under microlaryngoscope. All the tissues were used for reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analyses of CD44, COX-2, and MMP-9 expressions and, in addition, the normal vocal fold tissues were also used for primary culture of vocal fold fibroblasts.
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Primary culture of vocal fold fibroblasts HVFF were obtained respectively from 20 Reinke space tissues of normal vocal folds. The culture methodology used in this work followed the previous procedures.26 Each tissue was placed into basic Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco Invitrogen, Camarillo, CA) medium containing penicillin (50 U/mL), streptomycin (50 U/mL), and amphotericin B (12 ng/mL). After being rinsed twice, the tissue was placed in a Petri dish and minced into small pieces (1 mm3) using forceps and scalpel, then cultured on the six-well plates which were filled with 1–2 mL of 10% fetal bovine serum (FBS)-DMEM (Gibco Invitrogen, Camarillo, CA) complete medium containing antibiotics. The six-well plates were placed in a 37 C humidified incubator with 5% CO2. After 24 hours, extra 3–4 mL of 10% FBS-DMEM complete medium was gently added into the cultures. After 1–2 weeks of culture, the primary fibroblasts were grown into 100% confluence. The fourth to sixth passages were used in subsequent experiment.
MO) was added into each well, and the cells were cultured at 37 C for an additional 4 hours. The optical density (OD) values were measured at 570 nm using an enzyme-linked immunosorbant assay (ELISA) reader. The relative cell viability, which was defined relative to corresponding control cells, was calculated according to the following formula: relative cell viability (%) ¼ ODexperiment/ODcontrol 3 100% (ODblank was used to blank).29 As the results by MTT assay showed that median lethal dose of CSC for 24 hours was approximately 400 mg/mL, so optimal dosage of CSC (400 mg/mL) was established following the standard protocol.30 As for the mode of CTS, we selected CTS at a frequency of 0.5 Hz on the basis of previous investigation.17 Subsequently, the cells were cultured in 10% FBSDMEM containing CSC (400 mg/mL) and then treated with CTS (0.5 Hz, 24 h) at 2%, 4%, 8%, and 12%, respectively. Finally, the viabilities were tested. Also, an optimal mode of CTS (8%, 0.5 Hz, 24 h) was established and used in all subsequent experiments.
Immunofluorescence Cells were subcultured on six-well plates and grown in 10% FBS-DMEM complete medium. The major cell in Reinke space is fibroblasts, which contains intermediate filaments. As the antibody against vimentin is the marker for intermediate filaments, so, we stained for vimentin with the antibody in this work as previously reported.27,28 Primary cultured HVFF cells were washed twice in phosphate buffer solution (PBS) (Sigma, St. Louis, MO) and fixed in 10% formalin for 15 minutes. After washing in PBS for 5 minutes twice, cells were blocked with 10% normal goat-serum for 30 minutes at 37 C in a humidified atmosphere. The primary monoclonal anti-vimentin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added in PBS at 1:200 dilutions, incubated for 1 hour at 37 C, and then for 16–18 hours at 4 C in a humidified atmosphere. The replacement of the primary antibody with PBS served as negative control. After being washed three times in PBS, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated goat–anti-mouse secondary antibody (1:200) for 1 hour at room temperature. 40 , 6-diamidino-2-phenylindole dihydrochloride (DAPI) (2 mg/mL) was applied to mark nuclei. Finally, the slides were washed six times in PBS and photographed under Leica Optiphot photomicroscope (Leica Microsystems AG, Wetzlar, Germany).
Exposure of HVFF to CSC in the presence or absence of CTS To determine the effect of CTS, HVFF (8 3 104 cells/well) were plated onto rat tail collagen type I (BD Biosciences, San Jose, CA)-coated Bioflex II plates (Flexcell International, Hillsborough, NC) in the Flexcell loading station, which was equipped with a computer-regulated cyclic vacuum bioreactor that applied multiple modes of uniform equibiaxial strain to cells. There was a nearly linear relationship between vacuum level and strain.17 The cells reached 80% confluence within 4–5 days. Subsequently, cells were washed with PBS and incubated overnight in DMEM without FBS and divided into four groups: control cells, without any treatment; CTS-treated cells, treatment with only CTS (8%, 0.5 Hz) for 24 hours; CSCtreated cells, treatment with only CSC (400 mg/mL) for 24 hours; co-treated cells, treatment with both CSC (400 mg/ mL) and CTS (8%, 0.5 Hz) for 24 hours.
MTT assay MTT assay, an index of cell viability and cell growth, is based on the ability of viable cells to reduce MTT from a yellow watersoluble dye to a dark blue insoluble formazan product. In this work, MTT assay was applied to assess the viabilities of cultured HVFF in response to CSC (Murty Pharmaceuticals, Lexington, KY) and/or CTS and, accordingly, the optimal dosage of CSC and mode of CTS for all subsequent experiments were determined. Normal HVFF (8 3 104 cells/mL) were subcultured on a 96-well plate. Then the cells were cultured in 10% FBSDMEM containing different concentrations of CSC (0, 200, 300, 400, 500, and 600 mg/mL, respectively) for 24 hours. A total of 20 mL of MTT (5 mg/mL stock solution, Sigma, St. Louis,
RNA preparation and RT-PCR Total RNAs were extracted from the tissues and cultured HVFF using TRIzol reagent (Invitrogen, Camarillo, CA). The messenger RNA (mRNA) levels of CD44, COX-2, and MMP-9 were determined by RT-PCR. Anti-CD44 primary antibody was purchased from Abcam (Cambridge, United Kingdom). Primary antibodies against COX-2, MMP-9, and b-actin were all Santa Cruz products. The RT was performed using ExScript RT reagent kit (Takara Bio, Otsu, Shiga, Japan) in a final volume of 20 mL containing 1 mg total RNA, 4 mL 5 3 ExScript buffer, 1 mL dNTP mixture (Takara Bio, Otsu, Shiga, Japan), 1 mL Oligo(dT) primer (Takara Bio, Otsu, Shiga, Japan), 0.5 mL ExScript RTase, 0.5 mL RNase inhibitor (Takara Bio, Otsu, Shiga, Japan), with volume adjusted by RNase-free water. PCR was conducted according to the instructions of Takara Taq as following: predenaturation at 95 C for 3 minutes; degeneration at 95 C for 45 seconds, annealing at 60 C for 40 seconds, and elongation at 72 C for 50 seconds, for a total of 28 cycles. All of the produced PCR products were electrophoresed in a 2% agarose gel, and the agarose gel was stained with ethidium bromide and examined under ultraviolet
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TABLE 1. The Mean Scores on Extraversion (E) Dimension and Pack-Years (PKY) for Smoking Status Assessment in Individuals Among Reinke Edema (RE), Carcinoma and Control Group (n ¼ 182) EPQ-E
Smoking Status (PKY)
Factors Group
n
Mean (SD)
ANOVA
SNK Test
Mean (SD)
RE Carcinoma Control
67 55 60
62.61 (7.02) 41.35 (7.01) 49.20 (5.55)
F ¼ 165.02; P < 0.0001
RE > control Carcinoma < control RE > carcinoma; P < 0.05
45.69 (11.15) 46.38 (10.50)
Independent Sample t Test t ¼ 0.35; P ¼ 0.7257
Abbreviation: EPQ, Eysenck Personality Questionnaire.
light. The relative mRNA levels were quantified and calculated with the ImageJ software (National Institutes of Health, Bethesda, MD) after normalization against the internal control b-actin. For each gene, three independent PCRs from the same reverse transcription sample were performed. The primers used were as follows: 50 -AGCGGCTCCTCCAGTGAAA-30 and 50 TCTGTCTGTGCTGTCGGTGATC-30 for CD44; 50 -CAGGGTTG CTGGTGGTAGGA-30 and 50 -GAACACTGAATGAAGTAAAGG GACA-30 for COX-2; 50 -TCTTCCCTGGAGCCTGAGAAC-30 and 50 -GACACCAAACTGGATGACGATG-30 for MMP-9; 50 CATCGTGATGGACTCTGGTG-30 and 50 -AGGGCAACATAGCA CAGCTT-30 for b-actin. The expected amplification products of the target sequence with these primers were 116, 451, 427, 213 base pairs (bp) in length respectively.
quantified and calculated with the ImageJ software after normalization against the internal control b-actin. All experiments were performed in triplicate. Statistical analysis Statistical analysis was performed using the SAS 9.13 software (SAS Institute Inc., Cary, NC) package. The results of extraversion score and PKY were presented as means and standard deviation (SD) for measuring the donor differences, and the results of western blot and PCR analysis were presented either as digitized assessment of each band after densitometric analysis or as mean and SD of the mean from triplicate values. To determine whether significant differences in extraversion score, western blot values, and PCR values exist between groups, one-way analysis of variance (ANOVA) and the post hoc multiple comparison Student– Newman–Keuls (SNK) test were applied. Additionally, the constituent ratio of personality type among groups was analyzed with chi-square test. PKY was compared between Reinke edema and carcinoma groups using independent sample t test. To identify differences between CSC-treated cells in the absence or presence of CTS at various magnitudes, one-way ANOVA and the post hoc multiple comparison Dunnett test were used. Differences with P < 0.05 were considered significant.
Western blot analysis Total proteins were extracted from tissues and cultured cells using radio immunoprecipitation assay buffer according to manufacturer’s instruction. Briefly, the homogenized tissues or harvested cells were lysed with cold radioimmune precipitation buffer and protein lysis buffer for 30 minutes on ice. The lysates were transferred to Eppendorf tubes and clarified by centrifugation at 12 000g for 15 minutes at 4 C, and the supernatants were transferred for analysis. The Bradford method was used to determine the protein concentration in the tissue or cell lysates. A total of 40 mg of protein from each sample was used in western blot analysis with diluted primary antibodies as follows: CD44 (1:5000), COX-2 (1:200), MMP-9 (1:200), and b-actin (1:2000); and the corresponding specific protein bands were visualized at apparent molecular weights of 82, 72, 92, and 43 kDa, respectively. The relative protein expressions were
RESULTS Findings of the smoking status and personality Assessment of smoking status based on PKY showed that both Reinke edema and carcinoma patients were moderate/heavy smokers, and the comparison between them showed nonsignificant differences (t ¼ 0.35, P ¼ 0.7257, Table 1). Whereas, E score of Reinke edema patients was significantly higher than that of
TABLE 2. The Constituent Ratio of Personality Characteristics Among Reinke Edema (RE), Carcinoma, and Control Groups (n ¼ 182, c2 ¼ 158.7225, P < 0.001) Typical Introversion
Introversion
Intermediate
Extraversion
Typical Extraversion
Factors Group
n
(%)
n
(%)
n
(%)
n
(%)
n
(%)
Total
RE Carcinoma Control
0 18 0
0.00 32.73 0.00
0 17 13
0.00 30.91 21.67
13 20 39
19.40 36.36 65.00
12 0 8
17.91 0.00 13.33
42 0 0
62.69 0.00 0.00
67 55 60
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Cigarette Smoke-Induced Inflammation: Linked to Reinke Edema
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FIGURE 1. The results of Eysenck Personality Questionnaire (EPQ) survey in individuals among Reinke edema (RE), carcinoma, and control group. Extraversion (E) score of RE patients was significantly higher than that of carcinoma patients (*P < 0.05). RE patients were prone to be extroversive, whereas, vocal fold carcinoma patients were prone to be introversive. A. The analysis of E scores in individuals among groups. B. The constituent ratio of RE group. C. The constituent ratio of carcinoma group. D. The constituent ratio of control group. carcinoma patients, pairwise comparisons indicated significant differences among three groups (P < 0.05, Table 1). Furthermore, statistical analysis of constituent ratio demonstrated that Reinke edema patients were prone to be extroversive, whereas, vocal fold carcinoma patients were prone to be introversive (c2 ¼ 158.7225, P < 0.001, Table 2). In addition, the results of EPQ survey are also shown in Figure 1.
CTS attenuated the CSC-induced damages to HVFF MTT assay revealed that the HVFF survival rates were significantly reduced by the treatment with CSC at 300 mg/ mL, 400 mg/mL, 500 mg/mL, and 600 mg/mL (P < 0.05, Figure 5A). A total of 400 mg/mL of CSC optimally decreased cell survival rate, and then this dosage was used in all
The mRNA expression levels of CD44, COX-2, and MMP-9 were significantly different between Reinke edema and paracancerous tissues CD44 mRNA expression level was significantly higher in Reinke edema tissues than that in paracancerous tissues (P < 0.05); whereas COX-2 and MMP-9 mRNA expression levels in Reinke edema tissues were significantly downregulated compared with those in paracancerous tissues (P < 0.05) (Figure 2). The protein expression levels of CD44, COX-2, and MMP-9 were significantly different between Reinke edema and paracancerous tissues CD44 protein expression level was significantly higher in Reinke edema tissues than that in paracancerous tissues (P < 0.05), whereas COX-2 and MMP-9 protein expression levels were significantly down-regulated in Reinke edema compared with those in paracancerous tissues (P < 0.05) (Figure 3). Morphological observation and identification of HVFF HVFF were characterized by immunofluorescent staining. The respective blue (DAPI) and green (FITC) fluorescence of the nuclei and vimentin were observed in Figure 4. The results showed that almost 100% cells were vimentin-positive fibroblasts after four to six passages.
FIGURE 2. The mRNA expressions of CD44, COX-2, and MMP-9 in normal, Reinke edema (RE), and paracancerous tissues by RT-PCR. The expression of CD44 in RE tissues was higher than that in paracancerous tissues; COX-2 and MMP-9 expressions in RE tissues were significantly down-regulated compared with those in paracancerous tissues (*P < 0.05). A. Representative gel. B. Densitometric analysis (*P < 0.05).
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CTS inhibited CSC-induced COX-2 and MMP-9 mRNA expression in HVFF The effects of CTS and CSC on mRNA expressions of CD44, COX-2, and MMP-9 were examined. Compared with untreated cells, CD44 mRNA expression was down-regulated and COX2, MMP-9 mRNA expressions were up-regulated in CSCtreated group (P < 0.05). Furthermore, these effects of CSC were suppressed by treatment with CTS (Figure 6A). Statistical analysis revealed that, compared with CSC-treated group, CD44 mRNA expression was up-regulated, meanwhile, COX2, and MMP-9 mRNA expression were down-regulated in CTS and CSC-treated group (P < 0.05, Figure 6B). No significant difference was found between the CTS and control groups (P > 0.05).
FIGURE 3. The protein expressions of CD44, COX-2, and MMP-9 in normal, Reinke edema (RE), and paracancerous tissues by western blot. The protein expression of CD44 was higher, but, COX-2 and MMP-9 were lower in RE than those in paracancerous tissues (*P < 0.05). A. Representative gel. B. Densitometric analysis (*P < 0.05). subsequent experiments. Next, to examine the effects of CTS on the CSC-induced responses, HVFF were exposed to CTS of magnitudes between 0% and 12%. Figure 5B showed that the treatment of HVFF with CTS (8%, 0.5 Hz, 24 h) significantly increased the cell survival rate to 92.06 ± 5.54% (P < 0.05). Accordingly, CTS (8%, 0.5 Hz, 24 h) was used in the next experiments.
CTS inhibited CSC-induced COX-2 and MMP-9 protein expression in HVFF The effects of CTS on protein expressions of CD44, COX-2, and MMP-9 were determined after exposure of HVFF to CTS for 24 hours. Compared with untreated cells, in CSC-treated group, CD44 protein expression was significantly downregulated, whereas COX-2 and MMP-9 protein expressions were significantly up-regulated; however, such effects of CSC were significantly suppressed by treatment with CTS (P < 0.05, Figure 7). No significant difference in protein expression level was found between the CTS alone and control groups (P > 0.05). DISCUSSION Through vibration, the vocal folds transform aerodynamic energy of exhalation to acoustic energy.31,32 Their function is, however, not limited to voice production. Our present study shows that the
FIGURE 4. Identification of human vocal fold fibroblasts (HVFF) using immunohistochemical staining under fluorescence microscopy. A. Morphological observation of HVFF. B. Cell nuclei were stained by DAPI. C. Vimentin expressed in the cytoplasm of HVFF, not in nuclei. D. Merged image of nuclei and vimentin indicated that 100% cells were vimentin-positive fibroblasts.
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Cigarette Smoke-Induced Inflammation: Linked to Reinke Edema
FIGURE 5. Alteration of viabilities in HVFF exposed to different concentrations of cigarette smoke condensate (CSC) and/or different mode of cyclic tensile strain (CTS) by MTT assay. A. Cell viabilities were reduced by CSC in a dose-dependent manner, and significant difference could be found from 300 mg/mL (*P < 0.05). B. The combined effect of CTS and CSC on cell viabilities showed that CTS (8%, 0.5 Hz, 24 h) protected cells from CSC (400 mg/mL, 24 h)-induced injuries (*P < 0.05).
vocal folds vibration also has a potential function to suppress the CS-induced inflammation, which putatively explains the observation of the rare malignant transformation of Reinke edema. These results may provide valuable guidance to future clinical therapy of both Reinke edema and malignancy. Studies on vocal fold lesions reveal that vocal misuse is mainly associated with benign lesions, but, not with carcinoma,33 and that CS is closely associated with vocal fold carcinoma.13 Although CS is thought to be the most frequent etiologic factor for both Reinke edema and vocal fold carcinoma, it has also been proposed that Reinke edema does not carry an increased risk for malignant transformation.34 Interestingly, the significant difference in the amount of vocalization between patients with Reinke edema and vocal fold carcinoma was observed in our clinical practice. Such a clinical phenomenon also inspires the present study. In this work, all the Reinke edema patients reported excessive speaking, thereby confirming its important role in the pathogenesis of this laryngeal disease. Therefore, the precise pathophysiological mechanism underlying the actions of smoking and excess speaking on Reinke edema needs to be investigated further. EPQ, one of the most influential psychological scales with good reliability and validity, is generally used to determine the personality types by data analysis.35 In this work, patients with Reinke edema and carcinoma were both smokers, but, the patients in two groups exhibited different types of personality by the EPQ survey: Reinke edema patients are more extro-
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FIGURE 6. The mRNA expressions of CD44, COX-2, and MMP-9 in cultured HVFF treated with CSC (400 mg/mL, 24 h) and/or CTS (8%, 0.5 Hz, 24 h). CD44 expression was lowest in CSC-treated group, which could be significantly up-regulated by the presence of CTS (*P < 0.05). COX-2 and MMP-9 expressions were highest in CSCtreated group, which could be significantly attenuated by CTS (*P < 0.05). Blots were representative of three separate experiments. A. Representative gel. B. Densitometric analysis (*P < 0.05). versive, whereas, carcinoma patients are more introversive. As well known, extroversive people talk more than the introversive ones, thus, more vibration happens in the vocal fold of extroverts. This means that the vocal folds of Reinke edema patients are inclined to be exposed to mechanical strain. Moreover, many studies have manifested that the mechanical force can benefit human fibroblasts of injured larynx and other tissues.17–20 The present study demonstrated that CD44 expressions were significantly higher, whereas, COX-2 and MMP-9 expressions were lower in Reinke edema tissues than those in paracancerous tissues. Then, we sought to determine whether the different amount of vocal fold vibration in individuals with different characters might explain distinct responses of vocal fold after similar CS exposure and the mechanical strain on vocal fold fibroblasts might have the potential function to suppress the CS-induced inflammation. In vitro study, treatment of HVFF with CSC led to the decrease in cell viability, which was increased by administration of CTS. This suggests that CTS protects the cells from CSC-induced damage. In addition, the proinflammatory effects of CSC exposing to the fibroblasts was mitigated by CTS. These results, corroborating our tissue data, indicated a stronger antiinflammatory response was triggered in CTS and CSC-treated group.
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Journal of Voice, Vol. 29, No. 1, 2015
of HVFF with CTS. The study provides preliminary data regarding that vocal fold vibration may be the potential reason for lower expression of proinflammatory mediators in Reinke edema tissues in spite of CS exposure. Actually, in vivo work is needed to comprehensively understand the pathophysiology of Reinke edema. Acknowledgments This work was supported by the National Natural Science Foundation of China (81000407); Natural Science Foundation of Shandong Province (Y2007C145); Natural Science Foundation of Shandong Province (ZR2011HQ018). REFERENCES
FIGURE 7. The protein expressions of CD44, COX-2 and MMP-9 in cultured HVFF treated with CSC (400 mg/mL, 24 h) and/or CTS (8%, 0.5 Hz, 24 h). CD44 protein expression was lowest in CSCtreated group, which could be significantly up-regulated by the presence of CTS (*P < 0.05), whereas, COX-2 and MMP-9 protein levels were highest in CSC-treated group, which could be significantly attenuated by CTS (*P < 0.05). Blots were representative of three separate experiments. A. Representative gel. B. Densitometric analysis (*P < 0.05). Although the system used for in vitro study can not exactly simulate physiologically-relevant forces exerting on fibroblasts in vivo, it is expected to reflect the principle effect of mechanical forces on CSC-stressed vocal fold fibroblasts. To the best of our knowledge, this is the first report that CTS-mediated activations of CD44, COX-2, and MMP-9 critically involve in the alleviation of CSC-induced inflammatory damages to HVFF, implying that mechanical strain on HVFF may be the reason for the reduced inflammatory responses to CS in Reinke edema tissues. Even so, in vivo experiments should be performed to decipher the precise pathophysiological mechanisms responsible for Reinke edema in the future study. CONCLUSION In summary, patients with Reinke edema have more extrovert personality characteristics in comparison to patients with carcinoma, implying that the vocal folds of Reinke edema patients are more inclined to be exposed to mechanical strain caused by phonation. We, for the first time, report that inflammatory reaction to CSC is dramatically reduced by treatment
1. Martins RH, Domingues MA, Fabro AT, Dias NH, Santana MF. Reinkes’ edema: immunoexpression study of fibronectin, laminin and collagen IV in 60 cases by immunohistochemical techniques. Braz J Otorhinolaryngol. 2009;75:821–825. 2. Gray SD, Titze IR, Chan R, Hammond TH. Vocal fold proteoglycans and their influence on biomechanics. Laryngoscope. 1999;109:845–854. 3. Moore J, Thibeault S. Insights into the role of elastin in vocal fold health and disease. J Voice. 2012;26:269–275. 4. Yan C, Wu W, Li H, Zhang G, Duerksen-Hughes PJ, Zhu X, Yang J. Benzo(a)pyrene treatment leads to changes in nuclear protein expression and alternative splicing. Mutat Res. 2010;686:47–56. 5. Shimizu M, Yasuda T, Nakagawa T, Yamashita E, Julovi SM, Hiramitsu T, Nakamura T. Hyaluronan inhibits matrix metalloproteinase-1 production by rheumatoid synovial fibroblasts stimulated by proinflammatory cytokines. J Rheumatol. 2003;30:1164–1172. 6. Mitsui Y, Gotoh M, Nakama K, Yamada T, Higuchi F, Nagata K. Hyaluronic acid inhibits mRNA expression of proinflammatory cytokines and cyclooxygenase-2/prostaglandin E(2) production via CD44 in interleukin1-stimulated subacromial synovial fibroblasts from patients with rotator cuff disease. J Orthop Res. 2008;26:1032–1037. 7. Reel B, Sala-Newby GB, Huang W-C, Newby AC. Diverse patterns of cyclooxygenase-independent metalloproteinase gene regulation in human monocytes. Br J Pharmacol. 2011;163:1679–1690. 8. Huebener P, Abou-Khamis T, Zymek P, et al. CD44 is critically involved in infarct healing by regulating the inflammatory and fibrotic response. J Immunol. 2008;180:2625–2633. 9. Liu C, Bronson RT, Russell RM, Wang XD. b-Cryptoxanthin supplementation prevents cigarette smoke-induced lung inflammation, oxidative damage, and squamous metaplasia in ferrets. Cancer Prev Res (Phila). 2011; 4:1255–1266. 10. Marcotullio D, Magliulo G, Pezone T. Reinke’s edema and risk factors: clinical and histopathologic aspects. Am J Otolaryngol. 2002;23:81–84. 11. Duarte JL, de Faria FA, Ceolin DS, Cestari TM, de Assis GF. Effects of passive smoke inhalation on the vocal cords of rats. Braz J Otorhinolaryngol. 2006;72:210–216. 12. Branski RC, Zhou H, Kraus DH, Sivasankar M. The effects of cigarette smoke condensate on vocal fold transepithelial resistance and inflammatory signaling in vocal fold fibroblasts. Laryngoscope. 2011;121:601–605. 13. Branski RC, Saltman B, Sulica L, Szeto H, Duflo S, Felsen D, Kraus DH. Cigarette smoke and reactive oxygen species metabolism: implications for the pathophysiology of Reinke’s edema. Laryngoscope. 2009;119: 2014–2018. 14. Garcia Alvarez CD, Campos Banales ME, Lopes Campos D, Rivero J, Perez Pinero B, Lopes Aguado D. Polyps, nodules and Reinke edema. An epidemiological and histopathological study. Acta Otorrinolaringol Esp. 1999;50:443–447. 15. Hampson SE, John OP, Goldberg LR. Category breadth and hierarchical structure in personality: studies of asymmetries in judgments of trait implications. J Pers Soc Psychol. 1986;51:37–54. 16. Meyer B, Candau P, Alcaras N, MacLeod P. Study of the mechanism of vocal fold vibration during phonation. Acta Otolaryngol. 1984;97:407–414.
Haiyan Zhang, et al
Cigarette Smoke-Induced Inflammation: Linked to Reinke Edema
17. Branski RC, Perera P, Verdolini K, Rosen CA, Hebda PA, Agarwal S. Dynamic biomechanical strain inhibits IL-1beta-induced inflammation in vocal fold fibroblasts. J Voice. 2007;21:651–660. 18. Long P, Hu J, Piesco N, Buckley M, Agarwal S. Low magnitude of tensile strain inhibits IL-1beta-dependent induction of pro-inflammatory cytokines and induces synthesis of IL-10 in human periodontal ligament cells in vitro. J Dent Res. 2001;80:1416–1420. 19. Wolchok JC, Brokopp C, Underwood CJ, Tresco PA. The effect of bioreactor induced vibrational stimulation on extracellular matrix production from human derived fibroblasts. Biomaterials. 2009;30:327–335. 20. Kutty JK, Webb K. Vibration stimulates vocal mucosa-like matrix expression by hydrogel-encapsulated fibroblasts. J Tissue Eng Regen Med. 2010; 4:62–72. 21. Hirano M. Structure of the vocal fold in normal and disease states: anatomical and physical study. ASHA Rep. 1981;11:11–30. 22. Roxburgh CS, Salmond JM, Horgan PG, Oien KA, McMillan DC. Tumour inflammatory infiltrate predicts survival following curative resection for node-negative colorectal cancer. Eur J Cancer. 2009;45:2138–2145. 23. Leffondre K, Abrahamowicz M, Siemiatycki J, Rachet B. Modeling smoking history: a comparison of different approaches. Am J Epidemiol. 2002; 156:813–823. 24. Gong YX. Revised Eysenck Personality Questionnaire Manual. Changsha, China: Hunan Medical College; 1986. 25. Eysenck HJ, Eysenck SBG. Manual for the Eysenck Personality Questionnaire. London, UK: Hodder and Stoughton; 1975. 26. Thibeault SL, Li W, Bartley S. A method for identification of vocal fold lamina propria fibroblasts in culture. Otolaryngol Head Neck Surg. 2008; 139:816–822.
21
27. Tillmann B, Rudert H, Schiinke M, Werner JA. Morphological studies on the pathogenesis of Reinke’s edema. Eur Arch Otorhinolaryngol. 1995; 252:469–474. 28. Du H-D, Wu C-P, Zhou L, Tian J. Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line. Acta Otolaryngol. 2013;133: 755–760. 29. Chuang JY, Wu CH, Lai MD, Chang WC, Hung JJ. Overexpression of Sp1 leads to p53-dependent apoptosis in cancer cells. Int J Cancer. 2009;125: 2066–2076. 30. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods. 1986;89:271–277. 31. Thomson SL, Mongeau L, Frankel SH. Aerodynamic transfer of energy to the vocal folds. J Acoust Soc Am. 2005;118:1689–1700. 32. Zhang Z, Neubauer J, Berry DA. Physical mechanisms of phonation onset: a linear stability analysis of an aeroelastic continuum model of phonation. J Acoust Soc Am. 2007;122:2279–2295. 33. Johns MM. Update on the etiology, diagnosis, and treatment of vocal fold nodules, polyps, and cysts. Curr Opin Otolaryngol Head Neck Surg. 2003; 11:456–461. 34. Martins RH, Fabro AT, Domingues MA, Chi AP, Gregorio EA. Is Reinke’s edema a precancerous lesion? Histological and electron microscopic aspects. J Voice. 2009;23:721–725. 35. Koutsopoulou V, Theodosopoulou E, Vantsi E, Kotrotsiou E, Kostandinou V, Dounousi E. Personality dimensions of haemodialysis patients related to initial renal disease. EDTNA ERCA J. 2002;28: 21–24.
