Strong expression of HSP47 in metaplastic nasal mucosa may predict a poor outcome after primary endoscopic dacryocystorhinostomy: a prospective study

Purpose: Dacryocystorhinostomy (DCR) is an effective and safe procedure for patients with post-saccal obstruction of the nasolacrimal pathway. The aim of DCR is to relieve symptoms by creating a bypass between the lacrimal sac and the nasal cavity. The most common reason for failure is stenosis caused by a fibrotic process at the rhinostomy site. In this prospective study we assessed the expression of heat shock protein 47 (HSP47), a regulator of fibrosis, in the biopsies of nasal mucosa isolated from patients undergoing primary endoscopic DCR (EN-DCR). Methods: Thirty consecutive primary EN-DCR procedures in 30 patients were performed using the powered instrumentation technique. The nasal mucosa specimens over the rhinostomy site were collected for histological analysis at the beginning of the operation and the expression of HSP47 was evaluated by immunohistochemistry. The outcome of EN-DCR was estimated in follow-up visits at 1 week, 2 months and 6 months after surgery. Results: At the 6-month follow-up, the overall success rate after primary EN-DCR was 83%. A metaplastic change and strong expression of HSP47 in nasal mucosa were associated with EN-DCR failure (p = 0.009). Conclusions: HSP47 may be regarded as a novel marker to predict impaired EN-DCR outcome. Obstruction of the lacrimal pathway is manifested by infection, epiphora, blurred vision and facial pain. In recent years, endoscopic dacryocystorhinostomy (EN-DCR) has become accepted as a suitable treatment for patients with post-saccal obstruction. The procedure involves the creation of a bypass between the lacrimal sac and the nasal cavity. If surgery is successful, the rhinostoma remains open. Adhesions of the rhinostomy site, predisposing to the failure of EN-DCR, may be linked to the biology of nasal mucosa wound healing (Goldberg 2004). One of the most important factors affecting wound healing may be related to heat shock protein (HSP) upregulation. The ubiquitously expressed HSPs are classified into different families (HSP90, HSP70, HSP60, HSP40 and small HSPs) according to their function or molecular size (Georgopoulos & Welch 1993). A wide variety of stress stimuli such as heat shock, hydrostatic pressure, oxidative stress, ultraviolet radiation, viral or bacterial infections and different chemical exposures evoke the expression of highly conserved HSPs (Georgopoulos & Welch 1993;Kaarniranta et al. 1998, 2005). Heat shock proteins have been associated with many autoimmune and inflammatory processes (Salminen et al. 2008). Moreover, HSP47 is closely implicated in fibrotic processes, functioning as a specific chaperone for procollagen molecules as they transit through the endoplasmic reticulum (Nagata 1996). We hypothesized that HSP47 expression in nasal mucosa might influence the outcome of operations in the nasal cavity. The aim of our study was to evaluate the HSP47 expression of the nasal mucosa in patients who had undergone endoscopic surgery of the nasolacrimal pathway. A prospective study design was used. Recruitment started in autumn 2004, and study subjects were consecutively recruited from patients referred to the outpatient clinic of the Department of Otorhinolaryngology at Kuopio University Hospital, Kuopio, Finland. Patients were eligible for participation if they were adults (aged > 18 years), had an American Society of Anesthesiologists (ASA) physical status of I–III, and were scheduled for primary lacrimal pathway surgery for recurrent or chronic watering eyes or conjunctival discharge. The study was approved by the Research Ethics Committee of the Hospital District of Northern Savo, Kuopio, Finland. Patients were given oral and written information on the trial protocol and were asked to provide written consent. Preoperatively, the objective assessment was carried out by an otorhinolaryngologist. Irrigation of the lacrimal system was performed to check the patency of the nasolacrimal pathway and to rule out pre-saccal obstructions. Thirty consecutive primary EN-DCRs in 30 patients suffering from dacryostenosis or recurrent or chronic dacryocystitis were included in the study. Patient demographics are shown in Table 1. There were no drop-outs during the follow-up. Standardized general anaesthesia was used. The detailed endoscopic powered instrumentation technique used has been described previously (Wormald 2002; Smirnov et al. 2006, 2008). The operations were performed by the same three otorhinolaryngologists (JS, HT, GS) Additional nasal surgery in order to make space for DCR, such as septoplasty, hypertrophic middle turbinate resection or infundibulotomy, was performed in three cases. The specimen of nasal mucosa over the rhinostomy site was taken at the beginning of the operation for histological analysis. The postoperative care, follow-up and successful EN-DCR outcome criteria have been described in our previous paper (Smirnov et al. 2008). Histological samples from the nasal mucosa were fixed in formalin and processed according to routine protocols. The adequacy of the samples was confirmed by having the haematoxylin and eosin-stained sections analysed by an experienced histopathologist (RP). The samples were cut into 5-μm thick sections, which were used in immunohistochemical analyses. The sections were deparaffinized in xylene and rehydrated in graded ethanols according to standard procedures. Endogenous peroxidase was blocked by 3% hydrogen peroxide. Histostain™ Plus Mouse Primary Bulk Kit (Zymed Laboratories Inc., San Francisco, California, USA) was used for immunostaining HSP47 (1 : 100 dilution; StressGen Biotechnologies, Inc., Ann Arbor, Michigan, USA) as described earlier (Rönkkö et al. 2007). The appropriate dilution and the functionality of the antibody had been tested previously. The negative controls were processed without the primary antibody and showed no positivity. All stainings were analysed by the same observer (RP), who was unaware of the clinical data of the patients. The staining signal for HSP47 was located in the cell cytoplasm. Positive staining was found in both epithelial and stromal cells. Staining intensities in the epithelium and stromal tissue were analysed separately and were graded on a scale of 0–3, where 0 = negative, 1 = weak, 2 = moderate and 3 = strong. Strong intensity was comparable with that seen in controls. Because of the heterogeneous staining intensity found in many slides, a 50% cut-off level was used (i.e. the case was considered moderately or strongly stained if > 50% of the epithelial or stromal cells showed that level of intensity). Patient characteristics and variables were analysed using spss for Windows, Version 14 (SPSS Inc., Chicago, Illinois, USA). Differences according to treatment assignment for categorical variables were assessed with Pearson’s chi-square and Fisher’s exact tests. The correlations between variables were assessed with Pearson’s correlation coefficient. A 95% confidence interval was calculated for the main results. All analyses were performed on an intent-to-treat basis. Differences were regarded as statistically significant if a two-sided p-value was < 0.05. Data are expressed as the number of cases or as means with standard deviation (SD). The overall success rate after primary EN-DCR was 83% (25/30) at the 6-month follow-up. Eight patients (8/30) had a history of previous nasolacrimal duct intubation, but there was no association between previous nasolacrimal sac intubation and poor outcome after DCR (p > 0.05, Fisher’s exact test). All patients were discharged from hospital on the first postoperative day and no intraoperative or immediate postoperative complications occurred during the study. The samples represented the histology of normal nasal mucosa and included some inflammatory and reactive changes, such as fibrosis and calcification (Fig. 1A). The pseudostratified surface epithelium had variable thickness, being partly exfoliated. In 10 of 30 cases, squamous metaplasia was noted in the surface epithelium, associated with the mild chronic lymphoplasmacytic inflammation in the stroma surrounding the mucus-secreting glands (Fig. 1B). Haematoxylin and eosin stain. (A) Normal pseudostratified epithelium of the nasal mucosa (*).Chronic inflammation in the stromal tissue surrounding the seromucinous glands (arrow). (B) Nasal mucosa showing squamous metaplasia in surface epithelium (○). (C) Weak expression of heat shock protein 47 (HSP47) in the normal pseudostratified epithelium (*). (D) Strong expression of HSP47 in the metaplastic epithelium (○). HSP47 expression also noted in stromal cells (arrows). A positive expression of HSP47 was noted in the cells of the surface epithelium and in the cells of the columnar epithelium of the stromal glands. Usually, the normal pseudostratified epithelium showed only weak staining for HSP47. By contrast, a metaplastic change in the surface epithelium was associated more often with moderate or strong expression of HSP47 (Fig. 1D). The stromal tissue was always positive for HSP47. The positive staining signal was located in the fibroblasts and some inflammatory cells. In the samples without metaplasia, moderate or strong stromal expression for HSP47 was found in only seven of 20 cases, whereas in the samples showing metaplastic change, moderate or strong expression was noted in eight of 10 cases (p = 0.009) (Table 2). Squamous metaplasia in the surface epithelium was associated with unsuccessful EN-DCR (p = 0.031) (Table 3). Moreover, all cases with failed outcome showed either strong or moderate expression of HSP47 (p = 0.002) (Table 4). Normal histology (without metaplasia) was found in 20 cases, among which only one EN-DCR failed. Chronic inflammation in the mucosa was found in most cases, but was not correlated with expression of HSP47 (Fig. 1C). Further, it was observed in follow-up visits that nine of the 10 patients with metaplasia in the surface epithelium also had either scar or granulation tissue over the rhinostomy site (Fig. 2). The association between these postoperative findings and metaplasia was statistically significant (p = 0.049) (Table 5). Scar formation over the lacrimal sac rhinostoma. LNW = lateral nasal wall; MT = middle turbinate; SF = scar formation; LSR = lacrimal sac rhinostoma (arrow). Endoscopic DCR has become increasingly common as a treatment for patients with post-saccal obstruction of the nasolacrimal pathway. The overall success rate of primary EN-DCR varies from 63% to 99% (Sprekelsen & Barberan 1996; Hartikainen et al. 1998). In this study, the overall success rate was 83%, which is comparable with rates in previous studies. A history of chronic or recurrent sinusitis and additional surgery in the nasal cavity have been shown to increase the risk of EN-DCR failure (Allen et al. 1988; Nussbaumer et al. 2004). In this study a history of recurrent rhinosinusitis and additional surgery in three patients did not affect the outcome. The main reason for failure in our study was scar formation at the rhinostomy site. In all failures, the watering eye symptom recurred between the second and third follow-up visits, at 2–6 months post-surgery (Fig. 2). In the present study, we analysed the structure of the nasal mucosa over the rhinostomy site in order to evaluate preoperative histopathological changes which may affect the final outcome of EN-DCR. The specimen was taken for histological analysis at the beginning of primary EN-DCR. Interestingly, squamous metaplasia in the surface epithelium was found in 10 of the 30 cases and was associated with strong expression of HSP47. Of the five cases in which surgery failed, metaplasia was found in four. Heat shock proteins are known to prevent the denaturation or aggregation of proteins. As molecular chaperones, they assist in the refolding of damaged proteins and facilitate their translocation to the correct intracellular localization (Hartl 1996). Many HSPs are stress-inducible, whereas others are expressed constitutively or related to developmental processes (Hartl 1996). Heat shock stress response is usually transcriptionally regulated via transcription factor heat shock factor-1 (HSF1), which binds to the promoter of heat shock genes and initiates transcription, although in certain forms of stress post-transcriptional gene regulation may be implicated (Kaarniranta et al. 1998). HSP47 is a 47-kDa glycoprotein that is localized in the endoplasmic reticulum of collagen-producing cells and is implicated in the molecular maturation of various types of collagens in that it assists the correct folding of the procollagens. Unlike most other HSPs, which have various client proteins, collagens are only substrates for HSP47, which has been shown to be highly expressed in all the studied human and experimental fibrotic diseases (Nagata 1996; Razzaque & Taguchi 1999). Previous studies have demonstrated a close relationship between strong expression of HSP47 and excessive collagen accumulation in scar formation and have shown that the expression of HSP47 in various organs is closely associated with increased accumulation of various types of collagens (Razzaque et al. 1998; Chen et al. 2007). In our study, it was not possible to analyse the exact amount of fibrosis in the stromal tissue. However, strong or moderate stromal expression of HSP47 was found in the nasal mucosa of all failure cases, which supports the role of HSP47 in scar formation (Fig. 3). Proposed role of heat shock protein (HSP) 47 in scar formation of nasal mucosa. To date, there are no effective anti-fibrotic drugs that target fibrogenic factors or block their receptors (Taguchi & Razzaque 2006). A novel therapeutic target to control the progression of fibrosis is currently sought. The present study demonstrates that, in patients undergoing EN-DCR, squamous metaplasia and strong expression of HSP47 of the nasal mucosa may predict poor surgical results. Because of its profibrotic effects, HSP47 is a promising focus for future studies because it represents a potential target in the development of anti-fibrotic therapy for application in different surgical problems or diseases in order to prevent scar formation. This work was supported by the Finnish Eye Foundation, the Finnish Eye and Tissue Bank Foundation, the Finnish Funding Agency for Technology and Innovation, and the EVO Fund of Kuopio University Hospital. The study sponsors had no role in the study design, data collection, analysis or interpretation, or the writing of the report. The corresponding author had full access to all data pertaining to the study and is the study guarantor.

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