Epiretinal membranes: cell receptors involved in cell migration and their interaction with the extracellular matrix

Epiretinal membranes (ERMs) are fibro-cellular membranes that form at the vitreoretinal interface, potentially causing vision impairment, particularly when they exhibit a contractile behavior, leading to macular puckers. They are classified in idiopathic (iERM) and secondary membranes and are known to be composed by different cell types and extracellular matrix (ECM). Numerous studies have characterized ERM cells identifying Müller cells, astrocytes, retinal pigmented epithelium (RPE) cells, hyalocytes and myofibroblasts. In the ECM, the presence of collagen protein family and of some matrix associated proteins such as fibrillin and tenascin has been reported. Nevertheless, there is still a lack of information on ERM membrane composition and formation, i.e. ECM components such as proteoglycans (PGs) and glycosaminoglycans (GAGs) and on how the cells migrate to reach the vitreoretinal interface. These are the two aspects on which this thesis work is focused on. In a first bioinformatics analysis on previously reported iERM-associated proteins, we identified cluster of differentiation 44 (CD44) as a central molecule in ERM development. Since CD44 is reported to interact with the glycoprotein podoplanin (PDPN), we added this protein to our network; PDPN entered the net mainly thanks to transcription factors (TFs), among which we selected the ones involved in fibrotic, inflammatory or trans-differentiation processes. These TFs were SRY-box transcription factor 2 (SOX2), yes associated protein 1 (YAP1) and signal transducer and activator of transcription 3 (STAT3), that are also all linked to high mobility group box 1 (HMGB1), a protein itself involved in inflammatory response. To test if these bioinformatic-highlighted proteins were present in ERMs, we collected 45 ERM samples from patients undergoing surgery. We performed both immunofluorescence and real time PCR (RT-qPCR) experiments and determined that PDPN and CD44 are expressed in all the samples, regardless of their idiopathic or secondary nature. Moreover, regardless of their glial, epithelial or mesenchymal origin, all cell types showed PDPN and CD44 expression in the plasma membrane. We also detected SOX2, YAP1 and STAT3 mainly located in cells cytoplasm and faintly in the nuclei. Since CD44 is a hyaluronic acid (HA) receptor we investigated HA localization and distribution in ERMs samples, together with some PGs that were previously reported and detected in proteomic analysis, namely decorin (DCN), perlecan (PLC), collagen XVIII/endostatin and agrin (AGR). We found PLC and collagen XVIII in all the sample tested with a reticular or lamellar distribution, often co-localizing with collagen IV and laminin. DCN and PRELP were detected in almost all the samples preferentially co-localizing each other and avoiding PLC+/collagen XVIII+ regions. AGR was the least represented PG (50%) and formed basement membrane-like structures, preferentially co-localizing with collagen XVIII. We also investigated these PGs in ERM associated internal limiting membranes (ILMs), comparing them with a “reference ILM sample” of a 23 years old patient without ERM. ILMs stained positively for PLC and collagen XVIII extensively on the retinal side, and a thin rim of fluorescence was also detectable on the vitreal side. AGR displayed a diffuse immunofluorescence extending also in ILM bulk. HA was detected in all the samples and preferentially did not co-localize with PGs, showing an immunostaining also in the cytosol of ERM cells. Since we detected CD44 and HA and HA is one of the constituents of the eye vitreous, we decided to investigate HA effects on Müller cells, which represent one cell type found in ERMs. For this reason, we stimulated a human immortalized Müller cell line, MIO-M1, with 10ng/ml of HA, alone or in combination with 10ng/ml of TGFβ2, which is a cytokine involved in ERM formation. We then investigated by RT-qPCR experiments the expression of genes that could uncover a trans-differentiation process of MIO-M1 cells toward the mesenchymal phenotype associated with ECM production. Results showed that TGFβ2 and TGFβ2/HA treatments cause a significant increase in mRNA levels of mesenchymal markers (ACTA2, TAGLN), along with a significant decrease in nervous/glial markers (GFAP, NES), indicating mesenchymal transition. In agreement, we found an increase in mRNA levels of different ECM components, namely collagen I e IV, XVIII and fibronectin. MIO-M1 stimulation caused also PDPN up-regulation and CD44 down-regulation. Given their localization on the plasma membrane and their already known role in cell migration in other contexts, we investigated if their expression could be related to migration necessary for cell reaching the vitreoretinal interface and for ERM formation. To this end we performed a scratch test on MIO-M1 cell line in presence of HA and TGFβ2 and we found that HA accelerated MIO-M1 migration rate. In conclusion, we demonstrated the presence of PDPN and CD44 in ERM samples, regardless of their idiopathic or secondary nature. We observed that the ECM of ERM is composed of different types of PGs, which tend to form two co-localizing groups: PLC and collagen XVIII on one side, and PRELP and DCN on the other; AGR instead showed a basement-membrane localization. Furthermore, we demonstrated the widespread presence of HA in ERM, which appeared also able to enhance the migration rate of the MIO-M1 cell line. PDPN and CD44 were localized in the cytoplasmic protrusions of MIO-M1 cells, suggesting that these cells may utilize these two molecules in the migration process. However, under the influence of TGFβ2, we observed a reduction of the MIO-M1 migration rate, the acquisition of mesenchymal markers, an increase in PDPN and a decrease in CD44 mRNA levels, leading to the hypothesis that under TGFβ2 influence the cells, already migrated, begin to produce ECM proteins resulting in ERM formation. Although some questions remain unresolved, this work shed light on the composition of the ERMs ECM and on potential molecules involved in the complex mechanisms underlying the formation of these membranes.