In their niches, epithelial stem cells usually neighbour the basement membranes.¹ They are considered anatomically as specialized niches, while non-specialized niches are composed of various types of cells which are distributed in mesenchymal tissues.² Heterologous cell types inhabit the stem niches, including stem cells as well as their committed stem cell progeny which provide versatile feedback signals to their stem cell parents.³

CD146 is a well-known inducer of epithelial-mesenchymal transition (EMT)^4-8 leading to mesenchymal stem-like cells.⁹

It was previously demonstrated that transforming growth factor-beta 1 (TGF-β1) induces alveolar epithelial cells to undergo EMT.^10,11

In the nasal mucosa it was found that in chronic rhinosinusitis the epithelial expression of E-cadherin and cytokeratins decrease while the expression of vimentin, a mesenchymal marker, is increased, this being suggestive for fibrosis, most likely through processes of EMT.¹²Concomitant expression of vimentin and E-cadherin in epithelial cells of the nasal mucosa was found suggestive for ongoing processes of EMT involved in the pathogeny of chronic rhinosinusitis.¹³

The proprotein convertase 1/3, which is expressed in human nasal mucosa, can induce local EMT changes that may contribute to the pathogenesis of nasal polyps.¹⁴

Occurrence of EMT processes was not previously assessed in the sphenoidal sinus mucosa; therefore the role of these processes in the pathogeny of sphenoidal sinusitis might only be speculated by extrapolating the findings in the nasal mucosa. Noteworthy, the sphenoidal sinus mucosa regenerative potential and stem niches were, in our opinion, overlooked.

The purpose of this study was to evaluate the presence of an epithelial stem niche in the sphenoidal sinus mucosa, and we aimed to test this hypothesis through immunohistochemistry by using antibodies against vimentin and CD146.

Tissue samples Post autopsy samples of human sphenoidal sinus mucosa were obtained from six adult donor cadavers. The study was conducted according to the general principles from the Declaration of Helsinki, Cairo revision. Tissue samples were fixed immediately after removal, kept for 24 hours in buffered formalin (8%), and then processed with an automatic histoprocessor (Diapath, Martinengo, BG, Italy) with paraffin embedding. Sections were cut manually at 3 μm, and were mounted on SuperFrost® electrostatic slides for immunohistochemistry (Thermo Scientific, Menzel-Gläser, Braunschweig, Germany). Some of these sections were stained with hematoxylin and eosin for general histology. Antibodies For the purpose of this study we used the following primary antibodies: (1) CD146 (clone N1238, Novocastra-Leica, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK; 1:50); (2) cytokeratin 5 (CK5, clone XM26, Novocastra-Leica, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK; 1:100); (3) cytokeratin 19 (CK19, clone b170, Novocastra-Leica, Leica Biosystems Newcastle Ltd, Newcastle Upon Tyne, UK; 1:100) and (4) vimentin (clone V9, Dako, Glostrup Denmark, 1:50). Immunohistochemistry Tissue samples were fixed for 24 hours in buffered formalin (8%) and processed using an automatic histoprocessor (Diapath, Martinengo, BG, Italy) with paraffin embedding. Sections were cut manually at 3 μm and mounted on SuperFrost® electrostatic slides for immunohistochemistry (Thermo Scientific, Menzel-Gläser, Braunschweig, Germany). Sections were deparaffinized, rehydrated and rinsed in phosphate-buffered solution (PBS) at pH 7.4. Retrieval was completed by cooking in 0.01 M citrate retrieval solution (pH 6.0), for 20 minutes. Appropriate endogenous blocking peroxidase was completed before immune labelling (0.1% BSA in PBS). The next steps consisted of incubation for 5 minutes at room temperature with 3,3'-Diaminobenzidine (DAB, Biocare Medical, Concord, CA) and counterstaining with hematoxylin. Internal negative controls resulted when the primary antibodies were omitted.
The microscopic slides were analysed and results were documented on a Zeiss working station which was previously described.
15

Our results supports the hypothesis that the epithelium of the sphenoidal sinus mucosa is a site for EMT processes, such as was previously found in the epithelium of the maxillary sinus. 16 These could be initiated by CD146 (MCAM, MCAM, MUC18) in a similar manner as in other tissues, by activation of RhoA and up-regulation of Slug. 5 Even though a positive reaction for vimentin in epithelial tissues suggests processes of EMT, the reverse process, of mesenchymal to epithelial transition, should not be ruled out, as multipotent bone marrow-derived mesenchymal stem cells expressing CD146 were found taking up epithelial cell-like properties. 17 Figure 3. Human adult sphenoidal sinus mucosa. The arrow indicates an epithelial cell expressing vimentin. According to Millara et al, mesenchymal markers like vimentin are up-regulated in the epithelial cells of smokers, while epithelial markers, such as CK5, are down-regulated.18 Cigarette smoke was proven to activate EMT processes in vitro through the activation of intracellular reactive oxygen species, the release and action of TGF-β1, the phosphorylation of ERK1/2 and Smad3 and by the down-regulation of cyclic monophosphate. 18 Histological similarities of sinonasal mucosa with airway mucosa could allow extending knowledge from lower airways to the sinonasal tract. Our study found basal cells being involved in EMT processes. Similarly, airway basal epithelial cells were indicated being involved in airway remodelling through TGF-β1-induced EMT. 19 EMT associated with tissue regeneration and organ fibrosis was classified as type II EMT, type I occurring during developmental stages and EMT in the sphenoidal sinus mucosa – Craiu et al.• Original article www.jccp.ro • Journal of Contemporary Clinical Practice 1(1) • September 2015 • page 18 type III in cancers. 20 EMT processes in the sphenoidal sinus mucosa could therefore be caused not only by a normal regenerative status of that mucosa but also by chronic sphenoidal  sinusitis, although the sphenoidal sinus is the most uncommon location of such a pathology. 21

It appears that the epithelium of the sphenoidal sinus has the potential of mesenchymal transformation. Therefore the regenerative potential of the sphenoidal sinus mucosa can be supported by epitheliumderiving stem-like cells. Nevertheless, experimental studies should further test this hypothesis. Authors’ contributions statement: All authors have equally contributed to the present study. All authors read and approved the final version of the manuscript. Conflicts of interest All authors – none to declare

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