10.14639/0392-100X-N2197
Objective. Metalloproteinases (MMPs) are implicated in tissue remodeling in chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to evaluate the expression profiles of MMP-9 and the extracellular matrix metalloproteinase inducer (EMMPRIN) in nasal polyps compared to healthy mucosa. Methods. Tissue samples from 37 CRSwNP patients undergoing functional endoscopic sinus surgery and mucosal specimens from 12 healthy controls were obtained intra-operatively. MMP-9 and EMMPRIN mRNA levels were assessed by reverse transcriptionpolymerase chain reaction and their protein expression by Western blot analysis. Results. MMP-9 mRNA expression levels were significantly elevated in CRSwNP compared to controls (p &lt; 0.05). MMP-9 protein levels presented an increasing trend but with no statistical significance (p &gt; 0.05). No statistically significant difference in EMMPRIN mRNA and protein levels was identified. Conclusions. Upregulation of MMP-9 in nasal polyps is evident and highlights its role in the pathogenesis of CRSwNP. The lack of concordance between MMP-9 mRNA and protein levels may be attributed to post-translational gene expression regulation. Although EMMPRIN expression was not significantly different between the two groups, its role remains to be elucidated. MMP-9 may be a valuable biomarker and treatment target in CRSwNP.
text/xml
1827-675X
2197
en
Vasileios Danielides
http://creativecommons.org/licenses/by-nc-nd/4.0
ACTA Otorhinolaryngologica Italica
sinusitis
Expression profiles of MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps
Text.Serial.Journal
https://www.actaitalica.it/
43
Rhinology
2023-07-28
2022-06-27
2023-12-19
2023-12-19
400-408
1
Vasileios Danielides
2023/07/28
10.14639/0392-100X-N2197
400
Department of Otorhinolaryngology, School of Medicine, University of Patras, Patras, Greece
1827-675X
sinusitis
en
408
Expression profiles of MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps
https://www.actaitalica.it/article/download/2197/1024
43
ACTA OTORHINOLARYNGOLOGICA ITALICA 2023;43:400-408; doi: 10.14639/0392-100X-N2197




Rhinology


Expression profiles of MMP-9 and EMMPRIN
in chronic rhinosinusitis with nasal polyps
Profili dell’espressione di MMP-9 e EMMPRIN nella rinosinusite cronica con poliposi
nasali
Spyridon Lygeros1, Gerasimos Danielides1, George C. Kyriakopoulos2, Foteini Tsapardoni3, Katerina Grafanaki2,4,
Constantinos Stathopoulos2, Vasileios Danielides1
1
  Department of Otorhinolaryngology, School of Medicine, University of Patras, Patras, Greece; 2 Department of Biochemistry, School
of Medicine, University of Patras, Patras, Greece; 3 Department of Ophthalmology, University Hospital of Patras, Patras, Greece;
4
  Department of Dermatology, School of Medicine, University of Patras, Patras, Greece



SUMMARY
Objective. Metalloproteinases (MMPs) are implicated in tissue remodeling in chronic
rhinosinusitis with nasal polyps (CRSwNP). This study aimed to evaluate the expression
profiles of MMP-9 and the extracellular matrix metalloproteinase inducer (EMMPRIN) in
nasal polyps compared to healthy mucosa.
Methods. Tissue samples from 37 CRSwNP patients undergoing functional endoscopic
sinus surgery and mucosal specimens from 12 healthy controls were obtained intra-op-
eratively. MMP-9 and EMMPRIN mRNA levels were assessed by reverse transcription-
polymerase chain reaction and their protein expression by Western blot analysis.
Results. MMP-9 mRNA expression levels were significantly elevated in CRSwNP com-
pared to controls (p < 0.05). MMP-9 protein levels presented an increasing trend but with
no statistical significance (p > 0.05). No statistically significant difference in EMMPRIN
mRNA and protein levels was identified.
Conclusions. Upregulation of MMP-9 in nasal polyps is evident and highlights its role in
                                                                                                 Received: June 28, 2022
the pathogenesis of CRSwNP. The lack of concordance between MMP-9 mRNA and protein               Accepted: March 15, 2023
levels may be attributed to post-translational gene expression regulation. Although EMM-         Published online: July 28, 2023
PRIN expression was not significantly different between the two groups, its role remains
to be elucidated. MMP-9 may be a valuable biomarker and treatment target in CRSwNP.              Correspondence
KEY WORDS: matrix metalloproteinases, nasal mucosa, nasal polyps, sinusitis                      Spyridon Lygeros
                                                                                                 Academic Fellow in Otorhinolaryngology, Uni-
                                                                                                 versity Hospital of Patras, GR 26504, Rion Patras,
RIASSUNTO                                                                                        Greece. Tel. +30 69 47804091, +30 26 13603265
Obiettivo. Le metalloproteinasi (MMPs) sono coinvolte nel rimodellamento tissutale pro-          Fax +30 26 13603265
prio della rinosinusite cronica con poliposi (CRSwNP). Questo studio si prefigge lo scopo        E-mail: spyroslygeros@gmail.com
di confrontare l’espressione di MMP-9 e dell’induttore di metalloproteinasi della matrice
extracellulare (EMMPRIN) nei polipi nasali e nella mucosa sana.                                    How to cite this article: Lygeros S, Daniel-
Metodi. I campioni di tessuto sono stati ottenuti da 37 pazienti con CRSwNP sottoposti a           ides G, Kyriakopoulos GC, et al. Expression
chirurgia endoscopica funzionale dei seni paranasali e da 12 controlli sani. I livelli di mRNA     profiles of MMP-9 and EMMPRIN in chronic
di MMP-9 e EMMPRIN sono stati quantificati tramite reazione a catena della polimerasi              rhinosinusitis with nasal polyps. Acta Otorhi-
inversa e la loro espressione proteica è stata analizzata tramite analisi di Western Blot.         nolaryngol Ital 2023;43:400-408. https://doi.
Risultati. I livelli di espressione di mRNA MMP-9 erano significativamente elevati nei poli-       org/10.14639/0392-100X-N2197
pi nasali rispetto ai controlli (p < 0,05). La proteina MMP-9 era tendenzialmente più eleva-
                                                                                                 © Società Italiana di Otorinolaringoiatria
ta nei polipi ma senza una differenza significativa (p > 0,05). Non è stata riscontrata alcuna
                                                                                                 e Chirurgia Cervico-Facciale
differenza statisticamente significativa nell’espressione di mRNA e proteica di EMMPRIN.
Conclusioni. L’incremento di MMP-9 nei polipi nasali dimostra il suo ruolo nella patoge-
                                                                                                                OPEN ACCESS
nesi di CRSwNP. La discordanza tra l’espressione di mRNA e proteica di MMP-9 potrebbe
essere attribuita ad aspetti di regolazione post-trascrizionali. Sebbene nessuna differenza      This is an open access article distributed in accordance with
                                                                                                 the CC-BY-NC-ND (Creative Commons Attribution-Non-
di espressione di EMMPRIN sia stata dimostrata nei due gruppi, il ruolo di questa proteina       Commercial-NoDerivatives 4.0 International) license. The
deve essere ancora chiarito. MMP-9 potrebbe rappresentare un valido biomarker e target           article can be used by giving appropriate credit and mentio-
di trattamento in CRSwNP.                                                                        ning the license, but only for non-commercial purposes and
                                                                                                 only in the original version. For further information: https://
PAROLE CHIAVE: metalloproteinasi, mucosa nasale, polipi nasali, sinusite                         creativecommons.org/licenses/by-nc-nd/4.0/deed.en




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                                                                        MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps




Introduction                                                     Materials and methods
Chronic rhinosinusitis (CRS) is a chronic multifactorial,        Study population and inclusion criteria
inflammatory disease of the nose and paranasal sinuses af-       A total of 49 subjects (37 CRSwNP patients, aged between
fecting around 10% of the world’s population. As reported        18 and 72 years, average 46.3 years and 12 controls, aver-
by the European Position Paper on Rhinosinusitis and Na-         age 45.2 years) were recruited in the period between Sep-
sal Polyps (EPOS2020), CRS is defined as sinonasal in-           tember 2017 and January 2021.
flammation with a length of a minimum of 12 weeks, with          Inclusion criteria for enrollment in the study group were
two or more symptoms, one of which should be either nasal        age over 18 years, with CRSwNP scheduled to undergo
blockage, congestion, obstruction or anterior/posterior na-      functional endoscopic sinus surgery (FESS). The diagnosis
sal discharge, accompanied by facial pain/pressure or de-        of CRSwNP was based on standard preoperative computed
crease/loss of olfactory sensation 1.                            tomography (CT) and endoscopy according to the diagnos-
CRS, being a highly heterogeneous disease, is classified         tic criteria set by the guidelines of the latest EPOS2020 1.
depending on presence of polyps into CRS without (CRSs-          Exclusion criteria for the patient group were considered
NP) and CRS with nasal polyps (CRSwNP), which in turn            CRSsNP, allergic fungal rhinosinusitis, antrochoanal pol-
also exhibits various endotypes. In the Western world,           yps, secondary forms of CRSwNP, neoplastic disease and
CRSwNP usually presents as Th2-based immune response             cases with orbital, intracranial, and osseous extra-sinus
with abundant tissue eosinophilic infiltrates, mast cells, and   complications of CRSwNP. In 2 cases originally included,
basophils and oedema. In Asian populations, it is charac-        an inverse papilloma, identified intra-operatively, was con-
terised by a mixed T cell immune response and primarily          firmed by pathology and these subjects were also excluded.
neutrophilic infiltration. Eosinophilic CRSwNP presents          Participants were selected randomly solely based on con-
with basement membrane thickening and goblet cell hy-            secutive appointments for scheduled surgery.
                                                                 The control group consisted of 12 participants who under-
perplasia 1.
                                                                 went septoplasty for nasal septum deviation without any
An imbalance in the dynamic process between extracellular
                                                                 other nasal pathology. Controls were selected arbitrarily
matrix (ECM) accumulation and turnover, alongside thick-
                                                                 based on the date of presentation for surgery.
ening of the basement membrane and rupture, is postulated
to be involved in tissue remodeling and the pathogenesis
                                                                 Biological samples acquisition
of nasal polyposis in CRSwNP. Interestingly, in the early
                                                                 Nasal polyp tissue samples were collected during FESS
stages of nasal polyp development, fibroblasts differenti-
                                                                 from 37 patients (30 males and 7 females) with CRSwNP,
ated into myofibroblasts overproduce ECM proteins such
                                                                 aged between 18 and 72 years. A portion of the excised tis-
as collagen type I, IV, VI, and fibronectin 2.                   sue was sent for histopathological examination, while the
Extracellular matrix metalloproteinase inducer (EMM-             remainder was stored at -800C until processed. Nasal mu-
PRIN), also known as basigin or CD147, is a cell-surface-        cosal specimens from healthy controls were received from
glycosylated transmembrane protein, that belongs to the          the inferior turbinate after separation from the underlying
immunoglobulin superfamily. Its glycosylated form po-            bone.
tently induces the production of matrix metalloproteinas-
es (MMPs), especially MMP-9 expression, and has a role           Real-time polymerase chain reaction (qRT-PCR)
in signaling pathways and transcellular communication 3.         for mRNA quantification
MMP-9 (also referred to gelatinase B) is mainly impli-           RNA extraction was performed with the use of NucleoSpin
cated in type IV collagen degradation, and is an essen-          RNA/protein kit (Macherey-Nagel) according to the manu-
tial element of the ECM and basement membrane of the             facturer’s instructions. All samples were cryopreserved at
nasal epithelium. Except from its role in cell migration         -80°C and then lysed by adding 700 μl Lysis solution (RP1)
and proliferation, MMP-9 has also been found to regulate         and 3.5 μl of β-mercaptoethanol. After homogenisation, the
microvascular permeability and resultant oedema in the           homogenisation product was added in the column provided
lower airways 4.                                                 for disposal of particles not homogenised by centrifugation
The aim of the present study was to assess the expression        at 11,000 g for 1 minute. 350 μl of ethanol 70% were added
of MMP-9 and its inducer EMMPRIN in terms of mRNA                to the repeatedly eluted suspension, until a clear suspen-
and protein in patients with CRSwNP and compare them to          sion was received for total RNA extraction. The tissue elute
healthy controls, hypothesising that they may play a role in     was further centrifuged at 11,000 g for 1 min and stored
tissue remodeling encountered in the disease.                    at 4°C until RNA extraction was complete. 350 μl MDB



                                                                                                                                401
S. Lygeros et al.




were added and the elute was recentrifuged at 11,000 g          Statistical analysis
for 1 minute. Total RNA was measured with an Multiskan          Statistical analysis was performed using the GraphPad
Sky High Microplate Spectrophotometer (ThermoFischer).          Prism 8 software package. The unpaired parametric Stu-
Measurements were performed at 230 nm and 280 nm to             dent’s t-test was applied and data for all samples are pre-
ensure RNA purity. The estimated ideal absorbance ratio         sented as mean ± standard deviation (SD). Statistical sig-
at 260 nm versus 280 nm was 1.8. A 1.5% agarose gel in          nificance was set at p < 0.05.
the presence of formaldehyde was used for the assessment
of RNA quality, permitting visualisation of the 28S rRNA        Results
and 18S rRNA bands as markers of RNA integrity. Reverse
transcription to complementary DNA (cDNA) of a total of         Participant characteristics
5 µg mRNA of total RNA per sample was performed by the          The detailed demographic data and clinical characteristics
reverse transcriptase enzyme Superscrip tII (Invitrogen).       of the participants are shown in Table I. No significant dif-
                                                                ferences regarding age and gender were present between
Random nucleotide hexamers, serving as primers, were em-
                                                                the study and control groups (p < 0.05). 14 of the CRS pa-
ployed for cDNA synthesis, to warrant DNA synthesis from
                                                                tients were smokers and 14 had concurrent asthma, while 5
the whole mRNA. qPC reactions were performed with the
                                                                of the controls were smokers and 2 had a history of asthma.
KAPA SYBR FAST qPCR Kit (Kapa Biosystems) using
50 ng cDNA as template. Reactions were set up in 96-well
                                                                mRNA levels of MMP-9 and EMMPRIN in CRSwNP
plates and executed on an MX3000P qPCR system (Agi-
                                                                Expressional profiles of MMP-9 in CRSwNP
lent). The Ct values were analysed using the 2-ΔΔCT method
                                                                Enhanced expression of the MMP-9 gene was detected in nasal
after normalization against ACTB levels 5. The normali-
                                                                polyp tissue samples (Figs. 1A-B). More specifically, MMP-
sation procedure was performed using one of the control
                                                                9 was estimated to be 2.24-fold higher in the patient group
samples (sample C1), which was analysed in each 96-well
                                                                (n = 37) compared to healthy controls (n = 12), a significant
plate to ensure that all samples were equally compared to       increase (p-value < 0.01) with a standard deviation ± 2.29.
the same control. All reactions were performed in tripli-       Smoking status did not correlate to a significant increase in
cates and the primer sequences are as follows: ACTB_F:          MMP-9 expression in the CRSwNP group (Fig. 1C). The rela-
AGCGAGCATCCCCCAAAGTT, ACTB_R: GGGCAC-                           tive MMP-9 expression was estimated to be 2.96 (± 2.59) in
GAAGGCTCATCATT, MMP9_F: CTTTGGACACG-                            smokers compared to 1.05 (± 0.52) in non-smokers (p-val-
CACGACG, MMP9_R: CACCTGGTTCAACTCACTC-                           ue < 0.05). Furthermore, no significant difference in MMP-9
CG, EMMPRIN_F: CTTCATCTACGAGAAGCGCCG,                           expression was observed in patients with a history of asthma
EMMPRIN_R: AATCTACGGGGTGGGTTTTCT.                               in comparison to those without asthma. The relative MMP-9
                                                                expression in asthma patients was 2.02 (± 1.49), while in non-
Western blotting                                                asthmatic patients was 2.31 (± 2.68) (Fig. 1D).
Protein lysates were extracted using the NucleoSpin RNA/
protein kit (Macherey-Nagel) according to the manufac-          mRNA levels of EMMPRIN in CRSwNP
turer’s instructions. 30-50 μg of total protein extracts were   Expression profiles of EMMPRIN in CRSwNP
separated by SDS-PAGE and transferred to Immobilon-             EMMPRIN gene expression was found to be elevated in
P PVDF membranes (Millipore, Massachusetts, USA).               tissue samples from CRSwNP patients (Figs. 2A-B). This
Blocking in 5% (w/v) non-fat dry milk in TBS/0.05%              increase was estimated 0.95-fold higher (SD ± 0.60) for
Tween 20 was followed by incubation with primary anti-          the nasal polyp samples (n = 37) compared with healthy
bodies overnight at 4oC and with goat anti-rabbit secondary     mucosa (n = 12). Nevertheless, this increase was not sig-
HRP-conjugated antibody for 1h at room temperature. The         nificant. EMMPRIN expression did not differ significantly
Image Lab software (Bio-Rad, version 6.1) was employed          between smoking and non-smoking patients with CRSwNP
to measure the band intensity of each experiment. For           (Fig. 2C). The relative EMMPRIN expression was esti-
each set of experiments, normalisation of the protein lev-      mated to be 1.07 (± 0.70) in smokers as opposed to 0.88
els against β-actin levels was performed, using one of the      (± 0.53) in non-smokers (p-value < 0.05). Moreover, no
control samples (sample C1), which was separated in every       significant increase in EMMPRIN expression was noticed
SDS-PAGE confirming equal comparison of each sample             in asthmatic CRSwNP patients compared to patients with-
to the same control. The following antibodies were utilised:    out asthma. The relative EMMPRIN expression in asthma
anti-β-actin (#4967, Cell Signaling), anti-MMP-9 (#13667,       patients was 0.95 (± 0.66), while in non-asthmatic patients
Cell Signaling), anti-EMMPRIN (#13287, Cell Signaling).         was 0.96 (± 0.58) (Fig. 2D).



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                                                                                            MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps




Table I. Demographic and clinical characteristics of the patient and control groups.
 Age (years)                         N                    Percentage (%)             Patients (n = 37)                  Controls (n = 12)
                                                                                          Mean                               Mean
 < 30                                  8                        16.4                      46.3                                45.2
 31-40                                11                        22.4                   (31.1-61.4)                         (29.2-61.1)
 41-50                                11                       22.4
 51-60                                 8                        16.4
 > 61                                 11                        22.4
 Total                                49                       100.0
 Gender                                         Patients                                                                    Controls
                                       N                   Percentage (%)                                           N                  Percentage (%)
 Male                                 30                        81.1                                                8                       66.7
 Female                                7                        18.9                                                4                       33.3
 Smoking                                        Patients                                                                    Controls
                                       N                   Percentage (%)                                           N                  Percentage (%)
 Yes                                  14                        37.8                                                5                       41.7
 No                                   22                        59.5                                                7                       58.3
 Discontinued                          1                         2.7                                                -                        -
 Asthma                                         Patients                                                                    Controls
                                       N                   Percentage (%)                                           N                  Percentage (%)
 Yes                                  14                        37.8                                               2                        16.7
 No                                   23                        62.2                                               10                       83.3



Protein expression of MMP-9 and EMMPRIN in CRSwNP                                 The relative expression of EMMPRIN was 212.70%
The results of Western blot analysis demonstrated a                               (± 92.36) in CRSwNP patients with asthma, while in non-
trend to increased expression of the protein levels of                            asthmatic patients EMMPRIN expression was calculated
MMP-9 and EMMPRIN, but with no statistical signifi-                               to be 234.01% (± 160.60).
cance (Figs. 3A-B). MMP-9 expression was estimated at
328.36% (± 258.07) for the CRSwNP group compared to                               Discussion
204.57% (± 85.17) for the healthy controls (n = 12). Ad-
ditionally, in the study group, EMMPRIN expression was                            In the present study, we aimed to evaluate the expression
calculated at 225.94% (± 137.67) as opposed to 160.76%                            of MMP-9 and EMMPRIN in patients with CRSwNP. Our
(± 51.05) in the control group. Smoking was not asso-                             findings demonstrate that MMP-9 is highly expressed in
ciated with a significant increase in MMP-9 and EMM-                              polyp tissue samples from patients with chronic sinusal
PRIN expression in patients with CRSwNP. It should be                             inflammation in terms of mRNA compared to healthy na-
noted, however, that a trend towards increased expression                         sal mucosa and offer potentially significant insights into
of MMP-9 was observed, in accordance with the mRNA                                the role of MMP-9 in CRSwNP. Our results are overall in
levels (Fig. 3C). The relative MMP-9 expression was es-                           agreement with previous studies and support the involve-
timated to be 414.59% (± 260.58) in smokers as opposed                            ment of MMP-9 in tissue remodeling observed in the dis-
to 269.80% (± 243.65) in non-smokers. The relative ex-                            ease. The protein levels of MMP-9 showed a slight upregu-
pression of EMMPRIN was 238.30% (± 91.26) in smok-                                lation but with no statistical significance, a finding that is
ers with CRSwNP, while in non-smoking patients EMM-                               not in accordance with the mRNA levels of MMP-9 gene,
PRIN expression was estimated at 218.42% (± 161.04).                              where a statistically significant upregulation was observed.
Also, no significant increase in MMP-9 and EMMPRIN                                A possible reason for this finding is the small number of ex-
expression was noticed in asthmatic patients compared to                          perimental samples. Moreover, a post-transcriptional gene
patients without asthma (Fig. 3D). The relative MMP-9                             expression regulation program could take place involving
expression in asthma patients was 344.20% (± 260.87),                             different classes of non-coding RNAs (ncRNAs), such as
while in non-asthmatic patients was 312.64% (± 261.49).                           miRNAs. Their involvement in the regulation of gene ex-



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S. Lygeros et al.




           A                                                                        B




           C                                                                        D




Figure 1. mRNA levels of MMP-9 in CRSwNP. (A-B) relative expression of MMP-9 in controls and patients. MMP-9 expression was significantly increased in the
patient group; (C) comparison of the relative expression of MMP-9 between smoking and non-smoking CRSwNP patients showed upregulation of MMP-9 among
the smoking group; (D) Asthma was not associated with a significant increase in MMP-9 expression in CRSwNP patients.
CRSwNP: chronic rhinosinusitis with nasal polyps.


pression is well studied and involves targeting of specific                    correlated with the expression of MMP-9 in CRSwNPs 6.
mRNAs and repression of translation. Several miRNAs                            Other miRNAs that directly or indirectly affect MMP-9 ex-
have been reported to possibly regulate the expression lev-                    pression include let-7e 7, miR-129, miR-335 8, miR-211 9,
els of MMP-9. It was recently reported that a miR29b3p/                        miR-491-5p 10 and miR-1253 11. Moreover, novel classes
MMP-9 axis is involved in the pathogenesis of CRSwNPs.                         of ncRNAs that can regulate gene expression are tRNA-
miR-29b-3p has been found to directly target MMP-9 mR-                         derived fragments (tRFs) and long ncRNAs 12,13, whose role
NA and that miR29b3p expression is moderately positively                       in the pathogenesis of CRSwNP remains to be elucidated.



404
                                                                                     MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps




           A                                                                     B




           C                                                                     D




Figure 2. mRNA levels of EMMPRIN in CRSwNP. (A-B) relative expression of EMMPRIN in controls and patients. EMMPRIN expression showed no change
between the two groups; (C) comparison of the relative expression of EMMPRIN between smoking and non-smoking CRSwNP patients showed no alterations;
(D) asthma was not associated with differential expression of EMMPRIN in CRSwNP patients.
CRSwNP: chronic rhinosinusitis with nasal polyps.


MMP-9 exhibits an important role in modifying extracel-                    zyme activation and inhibition by specific and nonspecific
lular components and participates in normal embryonic                      inhibitors are the major points implicated 14.
development, as well as healing through tissue remod-                      Due to its potent role in tissue remodeling, MMP-9 has
eling. It is mainly produced by inflammatory cells such as                 been evaluated as a possible biomarker or therapeutic target
macrophages, neutrophils, fibroblasts and airway epithelial                in CRSwNP. However, these studies have identified equal
cells. While the regulation of its expression remains mul-                 distribution of either non-significant or increased MMP-9
tifaceted and not fully clarified, gene transcription, proen-              levels in patients with CRSwNP. Topographic distribution



                                                                                                                                              405
S. Lygeros et al.




          A                                                                        B




                                                                                   C




                                                                                   D




Figure 3. Protein levels of MMP-9 and EMMPRIN in CRSwNP. (A) Western blots of MMP-9 and EMMPRIN in controls (C1-C12) and patients (P1-P37). (B) relative
percentages in protein levels of MMP-9 and EMMPRIN in controls and patients; (C) comparison of relative expression of MMP-9 and EMMPRIN between smok-
ing and non-smoking CRSwNP patients showed no significant difference between the two groups but a trend of the MMP-9 protein to increase on smokers; (D)
asthma was not associated with a significant increase in MMP-9 and EMMPRIN expression in CRSwNP patients. CRSwNP: Chronic rhinosinusitis with nasal polyps.



studies of MMP-9 have identified the protein in different                       trols, without a significant difference in terms of expression
tissues, with increased staining in the epithelial layer 15. In                 between the two patient groups, presumably due to differ-
the majority of those studies, the authors agree that nasal                     ent pathogenetic mechanisms responsible for recurrent
polyp tissue exhibits increased MMP-9 expression in the                         disease, outside of MMPs 13. Moreover, higher amounts of
superficial epithelium, endothelial cells, adenoid cells and                    MMP-9 in nasal secretions and connective tissue have been
extracellular matrix in comparison to healthy controls tis-                     associated with worse healing following sinus surgery 18,
sue 16.                                                                         while it also appears to contribute to the development of
Similar to the findings of our study, increased expression of                   osteitis in CRS 19.
MMP-9 has been shown in nasal polyp tissue samples with                         EMMPRIN constitutes an upstream inducer of MMPs, ca-
immunohistochemistry and RT-PCR 14. An upregulation of                          pable of modulating MMP expression, composed of two
MMP-9 has been demonstrated in both polyp tissue (evalu-                        domains, one transmembrane and a short cytoplasmic one.
ated with RT-PCR) as well as in CRSwNP patient serum                            Although first identified in tumour cells where it stimulated
(assessed with ELISA) and correlated this increase to the                       interstitial collagenase (MMP-1) production by regional
severity of the disease 17.                                                     fibroblasts, overexpression of EMMPRIN has also been
MMP-9 expression has been found elevated in recurrent                           documented in diverse inflammatory lower airway disease
and non-recurrent CRSwNP in comparison to healthy con-                          including chronic obstructive pulmonary disease (COPD) 20



406
                                                                          MMP-9 and EMMPRIN in chronic rhinosinusitis with nasal polyps




and smoking 21 with a subsequent increase in MMP expres-         CRSwNP, the nasal polyp tissue received and assessed may
sion.                                                            not be indicative of the early stages of polypogenesis and
One study has previously investigated EMMPRIN in nasal           may not be indicative of the changes in MMP expression in
polyps and found increased EMMPRIN levels, suggest-              the initial phases of the disease.
ing increased ECM degradation in the disease 22. In a sim-
ilar study in COPD, blockage of EMMPRIN has led to a
                                                                 Conclusions
decrease in MMP-9 20. Moreover, patients with COPD had
higher levels of EMMPRIN expression in bronchoalveolar           Our findings confirm the contribution of MMP-9 in the
lavage compared to healthy smokers and non-smokers.              occurrence and progression of CRSwNP, as is manifested
In the present study, no alterations were observed regarding     by its increased expression and as suggested by previous
the mRNA and protein levels of EMMPRIN in CRSwNP                 research. Larger studies may help assess the proteolytic
patients compared to healthy controls. This finding              spectrum of CRSwNP and the implication and clinical sig-
though does not exclude the involvement of EMMPRIN               nificance of EMMPRIN in the pathophysiological pathway
in CRSwNP. It is known that EMMPRIN induces the pro-             of MMP, as well as the regulatory mechanisms involved in
duction of MMP-9 through a post-translational modifica-          the disease.
tion that involves glycosylation. Moreover, EMMPRIN
has been indicated to act as a key regulatory molecule in        Conflict of interest statement
various signaling routes and cell to cell interactions 3. Tak-   The authors declare no conflict of interest.
ing into consideration the aforementioned, the possible in-
volvement of EMMPRIN in the pathogenesis and progres-            Funding
sion of CRSwNP could involve other mechanisms and not            This research did not receive any specific grant from fund-
upregulation of its protein.                                     ing agencies in the public, commercial, or not-for-profit
Smoking has been correlated with an increase in MMP-9 in         sectors.
CRSwNP patients. In a study including smoking and non-
smoking CRSwNP patients, PCR and Western Blot showed             Author contributions
elevated MMP-9 expression 23. However, in the present            SL: data collection, preparation and final review of the
study, cigarette smoking was not associated with MMP-9           manuscript; GD: data collection, review of the manuscript;
and EMMPRIN upregulation.                                        KG: data processing, preparation of the manuscript; FT:
CRS and asthma are currently being regarded as different         literature review and collection, preparation of the manu-
manifestations of the spectrum of an inflammatory disease
                                                                 script; KG: data processing, review of the manuscript; CS:
of a ‘united airway’ considering the frequent comorbidity
                                                                 design of the research, supervision of data analysis, review
of asthma and CRS, as well as the presence of comparable
                                                                 of the manuscript; VD: design, implementation and super-
pathologic processes such edema and airway remodeling.
                                                                 vision of the research and final review of the manuscript.
The levels of MMP-9 have been correlated to the severity
of asthma and lung function decrease. According to Katain-
                                                                 Ethical consideration
en et al. patients with asthma, CRSwNP, or both tend to
                                                                 This study was approved by the Institutional Ethics Com-
have lower MMP-9 levels in nasal secretions, but higher
MMP-9 serum levels compared to healthy controls 24. Ac-          mittee (Research and Ethics Committee of the University
cording to our results, the expression of MMP-9 in tissue        Hospital of Patras) (approval number/protocol number
samples was found increased in both CRSwNP subgroups,            403/01.08.2017).
independently of the presence of asthma, without a signifi-      The research was conducted ethically, with all study pro-
cant difference between the two. This finding may reflect        cedures being performed in accordance with the require-
enhanced expression of MMP-9 due to the underlying CRS           ments of the World Medical Association’s Declaration of
irrespective of the presence of asthma.                          Helsinki.
Our study has certain limitations, most important of which       Written informed consent was obtained from each partici-
is the relatively small sample size, preventing solid deduc-     pant/patient for study participation and data publication.
tions especially as far as correlation parameters are con-
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