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

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.


Introduction
Laryngeal polyps are the most common benign lesions of the vocal folds. They account for approximately 39 to 70% of all nodular laryngeal lesions (1)(2)(3)(4)(5). Polyps occur only in the vocal fold area, most often on one side. Often the first step to the disease is phonotrauma (brusque movements of vocal fold membrane during phonation). It could be possible that latter leads to the injury of the vessels, hemorrhage, leakage of fibrin, thrombosis, and proliferation of capillaries (6). This pathology can be caused by aggravated tension of vocal folds and by some other factors (dust, smoke, etc.), which affect the mucous membrane of the vocal folds, thus damaging the walls of blood vessel (1).
Recent studies indicate that matrix metallopro-teinases (MMPs) play an important role in the physiological and pathological remodeling of tissues (7). MMPs are a family of proteolytic zinc-and calciumcontaining enzymes, which are responsible for degrading most components of extracellular matrix and basement membrane. There are six subgroups of MMP described; however, the most important subgroup includes gelatinases A and B (MMP-2 and MMP-9) (8,9). These gelatinases have the capacity to degrade the triple helix type IV and V collagen of basal lamina of the basement membrane (10), thus leading to tissue edema and cell migration through vascular basement membrane (11,12). MMP-2 and MMP-9 can be produced by keratinocytes, endothelial cells, fibroblasts, osteoblasts, inflammatory, tumor, and other cells (8).
Most studies have focused on the prognostic value of MMP expression in laryngeal cancer growth and spreading to the lymph nodes (9,13,14). However, until now just several studies investigated expression of MMP in benign vocal fold lesions (10,15). To our knowledge, only one study was devoted to investigate expression of MMP-2 and MMP-9 in vocal fold polyps (VFPs), so far (15).
In the present study, we compared the expression of MMP-2 and MMP-9 in VFPs and normal tissue of vocal folds -control vocal folds (CVFs).

Patients and samples
The VFP group consisted of 30 patients who underwent removal of vocal fold polyps by endolaryngeal microsurgery at the Department of Otorhinolaryngology, Kaunas University of Medicine Hospital (KUMH). The age of the patients ranged from 21 to 69 years (median age, 42 years). There were 18 (60%) males and 12 (40%) females in the VFP group.
Only the cases with typical clinical and histological findings were included in the VFP group. Clinically, nodular lesions localized on the free margin or upper surface of the vocal fold, visually appearing as single lesion of various sizes with a smooth, regular surface, grayish-red to dark-red in color, and distinct margins surrounded by a normal tissue of the vocal fold were considered as a polyp (1)(2)(3)(4)(5). Small blood vessels and hemorrhages sometimes were seen through the thin epithelial layer (Fig. 1). Pathohistological diagnosis of VFPs was made at the Department of Pathological Anatomy, KUMH.
Formalin-fixed and paraffin-embedded samples of normal tissues from vocal folds were retrieved from the archives of the Department of Pathological Anatomy, KUMH. They served as a CVF group. This group consisted of 13 women which age ranged from 23 to 71 years (median, 38 years).
Protocol of the study was approved by Ethics Committee for Biomedical Research of Kaunas region.

Immunohistochemical detection of MMP
Immunohistochemical detection of MMPs was carried out at the Institute of Cardiology, KUMH. Three-micrometer-thick sections were deparaffinized and rehydrated by slide stainer Varistain Gemini (ThermoShendon). Then sections were washed with distilled water and heated in TRIS/EDTA buffer (pH 9.0) for 8 min at 110°C in Microwave Histoprocessor RHS-1 (Milestone, Microwave Laboratory Systems). Shandon Coverplate system was used for immunohistochemical labeling. After blocking the activity of endogenous peroxidases, all slides were incubated in the primary antibody buffer for 1 h at a dilution of 1:100 for MMP-2 (clone 17B11, Novocastra) and MMP-9 (clone 15W2, Novocastra) in antibody diluent (DakoCytomation), followed by sequential 30-min incubations with Advance™ HRP Link and Advance™ HRP Enzyme (DakoCytomation). The binding of antibodies was detected by Liquid DAB+ Substrate-Chromogen System (DakoCytomation). Finally, the sections were counterstained with Mayer's hematoxylin (J. T. Baker) and mounted using xylenebased mounting medium Consul-Mount TM (Shandon).
Expression of MMP-2 and MMP-9 was graded on a semiquantitative scale, ranging from 0 (which represented no expression) to 3 points (which represented the highest expression). Two investigators independently of each other carried out grading, and expression of MMPs was calculated as a score composed of the sum of grades defined by both investigators. Final score ranged from 0 to 6 points: 0, no expression; 1-2, low; 3-4, moderate; and 5-6, high expression (16). The expression of MMP-2 and MMP-9 was graded in epithelium and stroma.

Statistical analysis
The nonparametric Kruskal-Wallis test was used for comparison between VFP and CVF groups. The nonparametric sign test was used to compare measurements in the VFP or CVF group. The results were considered statistically significant when P£0.05.

Results
Immunostaining for MMP-9 was detected in all VFP and CVF specimens (100%), both in epithelium and stroma. Nearly two-thirds of the specimens (19 of the 30 cases, 63.3%) taken from VFPs showed cytoplasmic immunostaining for MMP-2 in epithelium. It did not differ (P=0.0585) from the prevalence of immunostaining for MMP-2 in the epithelium of CVF specimens (12 of the 13 specimens, 92.3%). All specimens (100%) from VFP showed immunostaining for MMP-2 in stroma. Its prevalence in VFPs was higher (P=0.0335) in comparison to CVF group (11 of the 13 cases, 84.6%). Expression of MMP-2 and MMP-9 in specimens from VFPs and CVFs is presented in Table 1. According to the semiquantitative grading, expression of MMP-9 in epithelial cells of VFPs (range 4 to 6, median score 5) was much higher than MMP-2 (range 0 to 2, median score 1.5; P=0.000001) (Fig. 2). The same regularity was valid for expression of MMP-9 and MMP-2 in an epithelium covering normal vocal folds (range 3 to 6, median score 5 and range 0 to 2, median score 2, respectively; P<0.0015). However, there was no significant difference in epithelial expression of MMP-9 and MMP-2 between VFP and CVF samples ( Table 2). Expression of MMP-2 in stroma was significantly higher in samples from VFPs (range 1 to 6, median 3) than from CVFs (range 0 to 4, median 2, P=0.0176) (Fig. 3). Expression of MMP-9 in stroma did not differ between VFP and control groups.

Discussion
In the present study, we compared expression of MMP-2 and MMP-9 in VFPs and normal vocal folds. VFPs have been attributed to vocal abuse that has resulted in a hemorrhagic event or increased vascular permeability (1,6,17). The combination of the presence of fibrin, iron pigment in macrophages and signs of recent hemorrhage appeared mostly in vocal fold polyps (6). All these pathological factors can lead to edema and hyaline degeneration of VFP stroma. On the other hand, such area can undergo some organization, including neovascularization and following growth of the polyp (4).
Numerous studies were dedicated to investigate the multiple actions of MMPs in the physiological and pathological remodeling of tissues (10,11,14,(18)(19)(20)23). Majority of them analyzed the role of MMPs in morphogenesis of tumors (10,11,13,14,23). The review dedicated to the mechanisms of invasion and metastatic development of malignant tumors showed that MMPs had ability to degrade extracellular matrices, basement membranes, and other proteins, as well as to decrease cellular immunity (7). Only several studies investigated MMP-2 and MMP-9 expression in laryngeal carcinoma (9,10,(21)(22)(23). It was demonstrated that high expression of MMP-2 in Expression is presented as the number of cases with final scores representing the sum of the grades of two investigators (0, none; 1-2, low; 3-4, medium; 5-6, high). VFP -vocal fold polyp; CVF -control vocal fold; MMP -matrix metalloproteinases. laryngeal squamous cell carcinoma indicated poor survival prognosis for patients (9,23). According to Christopoulos et al. (21), immunostaining for MMP-2 and MMP-9 in the tumor cells from laryngeal carcinoma was decreased with increasing stage of cancer. It is of interest that expression level of mRNR of both gelatinases increased as the stage of cancer increased. Only few studies have been performed to investigate the role of MMPs in morphogenesis of benign tumors in nasopharynx or larynx (15,18,19). Chen et al. (18) found that MMP-9 was associated with the development of nasal polyps, while other study (19) indicated that MMP-2 might play an important role in the remodeling of nasal polyps. Bhandari et al. (19) observed no MMP-9-positive cells in nasal polyps; however, MMP-2 positivity was present in basal cells in the surface epithelium and in subepithelial tissues (19). These data are in agreement with the results presented in our study. We observed a statistically significant increase in the expression of MMP-2 in stroma cells of VFP as compared to the CVF group. This may suggest an impact of MMP-2 on remodeling and neovascularization in VFP stroma tissue. Similar data were presented by Karahan et al. (15). To our knowledge, until now, the latter study was the first to investigate MMP-2 and MMP-9 expression in VFPs and  normal tissue of vocal folds. Peschos et al. (10) draw attention to the expression of MMP-9 in epithelial cells of the larynx. They showed that expression of MMP-9 in carcinoma in situ was lower than in invasive carcinoma, although this difference was not statistically significant (10). Our results indicate that expression of MMP-9 in epithelial cells is significantly higher than the expression of MMP-2 both in VFP and CVF groups. However, there was no significant difference in epithelial expression of MMP-2 and MMP-9 between VFP and CVF samples. It can be explained by the fact that in most cases VFP is covered by normal epithelium. Andrian et al. (20) showed that normal human oral epithelial cells had a higher expression of MMP-9 mRNR than MMP-2 mRNR.

Conclusions
Expression of MMP-2 in stroma was significant higher in polyps than in normal tissue of the 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 benign, premalignant, and malignant lesions.