The epiretinal membrane is a common pathological condition characterized by the formation of fibrocontractile tissue at the vitreoretinal interface. Various cell types, including Müller glial cells, astrocytes, hyalocytes, macrophages, retinal pigment epithelium cells, and especially myofibroblasts have been identified in this membrane (Da Silva et al., 2022). Additionally, a substantial amount of extracellular matrix is present in epiretinal membranes (Altera et al., 2021).

Müller glial cells play critical roles in the retina, as they perform several functions related to structural and metabolic support, in addition to contributing to local signaling (Reichenbach and Bringmann, 2020). In response to changes in the retinal microenvironment, these cells undergo reactive gliosis, which may indicate a neuroprotective response. However, if prolonged, it may have detrimental effects (Bringmann and Wiedemann, 2012).

Furthermore, it has been shown that Müller cells can undergo a process of glial-mesenchymal transdifferentiation, in which they acquire a myofibroblast phenotype, characterized by an increase in the expression of smooth muscle α -actin (α-SMA) and even a contractile capacity (Guidry, 1996, 1997; Kanda et al., 2019). Myofibroblasts have different origins and play an important role in fibrosis by synthesizing extracellular matrix and generating traction (Shu and Lovicu, 2017). Therefore, in pathological conditions, Müller cells can contribute to the formation of fibrocontractile membranes and constitute a possible source of myofibroblasts (Guidry, 2005; Bu et al., 2014a). Besides their structural role in fibrotic tissue, extracellular matrix proteins could modulate cell behavior, so their decrease could be beneficial in pathological conditions.

Fibronectin and laminin were diffusely expressed amid collagen fibers in fibrocontractile membranes as shown by immunohistochemistry (Okada et al., 1995; Ioachim et al., 2005; Altera et al. 2021). Collagen V was detected by western blotting and immunofluorescence techniques (Scheiffarth et al., 1988; Bu et al., 2014b). Higher levels of fibronectin expression were detected in idiopathic epiretinal membrane (George et al., 2009; Christakopoulos et al., 2019) and proliferative diabetic retinopathy compared to the vitreous of non-diabetic patients (George et al., 2009).

In particular, the deposition of structural proteins in the epiretinal membrane that are also present in the internal limiting membrane, such as laminin and collagen IV, suggests a disorderly process of basement membrane formation (Altera et al., 2021). Furthermore, there is a peculiar pattern of these proteins deposition as an event that is related to disease progression (Regoli et al., 2020), and the deposition of these basal lamina proteins probably aid in cell adhesion. Collagen IV and laminin stimulate the migration of glial Muller cells (Wu et al., 2021).

Membranes containing higher amounts of fibrillar collagen types I and II are usually whitish, opaque and more difficult to peel (Kritzenberger et al., 2011). In proliferative vitreoretinopathy (PVR) membranes, single-cell and transcriptomic analyses have shown that genes such as COL1A1, FN1, and SPARC are differentially expressed, suggesting an important role in disease progression (Laich et al., 2022). In fact, the deposition of collagen I may increase matrix stiffness, and cells respond by increasing their contractile apparatus (stress fibers, i.e.) and forming focal adhesions to the substrate. In fibrocontractile membranes, activated GFAP- expressing Muller cells contribute to the collagen deposition (Bu et al, 2014b, 2015a, 2015b). Certain fibronectin isoforms such as ED-A may induce transdifferentiation into myofibroblasts (Serini et al., 1998; Bochaton-Piallat et al., 2000).

There is a search for novel therapeutic approaches for epiretinal membranes since there is currently no available pharmacological treatment. Interesting results, however, were obtained in retinal fibrosis induced with the use of a Rho kinase inhibitor, which attenuated the fibrotic process (Kita et al., 2008).

Rho kinase (ROCK) is a serine/threonine kinase downstream of the RhoA GTPase, a widely expressed member of the Rho Family of small GTPases. Among other functions, ROCK regulates the actin cytoskeleton, cellular contractility, and the maturation of focal adhesions to the extracellular matrix (Guan et al., 2023). There are two ROCK isoforms (ROCK1 and ROCK2), and several specific ROCK inhibitors are commercially available. The ROCK inhibitors Fasudil and its fluorinated analog Ripasudil (K-115) were approved for clinical glaucoma treatment in Japan and China, whereas Netarsudil (AR-13, 324) was approved for the same purpose in USA (Al-Humimat et al., 2021). These inhibitors lower intraocular pressure by promoting the relaxation of the trabecular meshwork cells, but more studies are still necessary regarding safety, as well as other potential effects (Defert and Boland, 2017). There is, however, a growing interest in the potential use of ROCK inhibitors as therapeutic tools in epiretinal membranes (Julian and Olson, 2014).

Considering the participation of Muller cells in the progression of epiretinal membranes, as well as the role of RhoA-ROCK pathway in regulating cellular contractility and migration, our aim was to evaluate the effects of the ROCK1 and ROCK2 inhibitor, Y27632, on Muller cells viability, proliferation, migration, contractility, and extracellular matrix expression.