Effects of the Calix[4]arene Derivative Compound OTX008 on High Glucose-Stimulated ARPE-19 Cells: Focus on Galectin-1/TGF-β/EMT Pathway

Diabetic retinopathy (DR) is a neurovascular disease characterized by the reduction of retina integrity and functionality, as a consequence of retinal pigment epithelial cell fibrosis. Although galectin-1 (a glycan-binding protein) has been associated with dysregulated retinal angiogenesis, no evidence has been reported about galectin-1 roles in DR-induced fibrosis. ARPE-19 cells were cultured in normal (5 mM) or high glucose (35 mM) for 3 days, then exposed to the selective galectin-1 inhibitor OTX008 (2.5–5–10 μM) for 6 days. The determination of cell viability and ROS content along with the analysis of specific proteins (by immunocytochemistry, Western blotting, and ELISA) or mRNAs (by real time-PCR) were performed. OTX008 5 μM and 10 μM improved cell viability and markedly reduced galectin-1 protein expression in cells exposed to high glucose. This was paralleled by a down-regulation of the TGF-β/, NF-kB p65 levels, and ROS content. Moreover, epithelial–mesenchymal transition markers were reduced by OTX008 5 μM and 10 μM. The inhibition of galectin-1 by OTX008 in DR may preserve retinal pigment epithelial cell integrity and functionality by reducing their pro-fibrotic phenotype and epithelial–mesenchymal transition phenomenon induced by diabetes.


Introduction
Recent results suggest galectin-1 (Gal-1) as a new possible therapeutic target for fibrosis in diabetes [1]. Gal-1 belongs to a group of proteins binding β-galactoside sugars by N-linked or O-linked glycosylation and has been associated to cell adhesion/proliferation, immune responses, apoptosis, and inflammation [2]. Gal-1 was found upregulated in the intraocular microenvironment of diabetic retinopathic patients along with the progression of clinical stages of retinopathy but not in the intraocular microenvironment of non-diabetic retinopathic patients [3], indicating a role for Gal-1 in diabetic retinopathy (DR) and diabetes or related hyperglycemia, a primum movens in its production.
Similarly, there is increasing evidence that transforming growth factor beta (TGF-β) signaling plays a critical role in DR. In fact, TGF-β1 expression in DR patients, DR rats, and high glucose-incubated human retinal endothelial cells (HRECs) and human adult

Discussion
Diabetic retinopathy is generally considered a debilitating microvascular complication, and the effects on the RPE have received much less attention. RPE forms the outer blood-retinal barrier between the choroid and neurosensory retina to protect the retina from damaging stimuli [9], and in diabetic retinopathy's later stages, it begins to settle on the retina, transform into fibroblast-like cells, and disassemble in a proliferative fibrotic phenotype. Some controversies still exist on the pattern of mediators and pathways involved in this, and in-vivo-like RPE culture models may identify new targets and tools for preventing how diabetes affects RPE-specific functions.
Here, it is reported for the first time that OTX008 (or PTX-008), a calixarene-based compound and galectin-1 (Gal-1) inhibitor, is able to block the deleterious effects exerted

Discussion
Diabetic retinopathy is generally considered a debilitating microvascular complication, and the effects on the RPE have received much less attention. RPE forms the outer bloodretinal barrier between the choroid and neurosensory retina to protect the retina from damaging stimuli [9], and in diabetic retinopathy's later stages, it begins to settle on the retina, transform into fibroblast-like cells, and disassemble in a proliferative fibrotic phenotype. Some controversies still exist on the pattern of mediators and pathways involved in this, and in-vivo-like RPE culture models may identify new targets and tools for preventing how diabetes affects RPE-specific functions.
Here, it is reported for the first time that OTX008 (or PTX-008), a calixarene-based compound and galectin-1 (Gal-1) inhibitor, is able to block the deleterious effects exerted by high concentrations of glucose on the cell viability of ARPE-19 cells. Interestingly, the compound reduced the burst of free radicals released into the medium by hyper glucose. Effects paralleled by the reduction of an important marker in these cells, the Gal-1, were in line with the reduction of Gal-1 expression previously reported in hypoxic human retinal Müller glial cells (HRMECs) exposed to OTX008 [17]. Interestingly, the pro-fibrotic cytokine TGF-β, hyper-expressed by high glucose, was also reduced by the treatment of ARPE-19 cells with OTX008.
Calix [n]arenes are synthetic skeletons that support functionalization of several molecules and derivatives applied in the biomedical field as drugs [18]. They function as nanocarriers for drugs delivery and optimize their activities [19][20][21]. Particularly, OTX008, N-[2-dimethylamino)ethyl] acetamidyl calix [4]arene, previously showed to bind Gal-1 and lead to its oxidation and proteosomal degradation through a mechanism that is not yet elucidated, displayed anti-angiogenic and antineoplastic activities by Gal-1 and Erk1/2 and protein kinase B (AKT)-dependent survival pathways inhibition, and induced G2/M cell cycle arrest through cyclin-dependent kinase 1 (CDK1) [13,22].
Worthy of note is that Gal-1 is considered a new fibrotic promoter protein in type 1 and type 2 diabetes [1], whose production is activated by the AP4 transcription factor through AKT pathway and is translated in excess deposition of the extracellular matrix (ECM). Fibroblasts are the main cells responsible for regulating ECM homeostasis along with other mesenchymal cell types, such as mesangial cells in the kidney and stellate cells in the liver [23]. The activation, proliferation, and persistence of those cells are mediated by cytokines, chemokines, and growth factors, such as TGF-β, platelet-derived growth factor (PDGF) and matrix metalloproteinases (MMPs) [24]. Among them, TGF-β signaling is considered the key profibrogenic pathway, which promotes the secretion of ECM components and deposition and induces terminal differentiation of fibroblasts into myofibroblasts and tissue contraction [25].
Pioneer studies done in a clinical setting by Connor et al. [26] and later confirmed by ref. [27] evidenced the strong and pushing role of TGF-β in ocular fibrosis, with levels of the cytokines as high as three times that in the non-fibrotic eye. Recently, Gal-1-knockout mice and RPE cells have linked the TGF-β to Gal-1 as two pathways talking each other to control a series of fibrosis transducers such as, for example, type 1 collagen and Fn1 [28]. OTX008, which is a well-established inhibitor of Gal-1, was able to counteract the putative actions derived from these agents, as seen in the various settings tested on ARPE-19 cells, through its proteasomal degradation of Gal-1 protein and not through its ubiquitination [1].
Signaling downstream of TGF-β includes canonical (through SMAD) or non-canonical pathways through Erk, MAPK, NF-kB, and AKT, which crosstalk with SMAD [23]. OTX008 was able to counteract the TGF-β and the putative actions derived from it, as seen in the various settings tested on ARPE-19 cells.
The role played by inflammation in the genesis of fibrosis is widely known, with a major role driven by interleukins (IL)-1β, IL-6, IL-8, tumor necrosis factor-alpha (TNFα), and NF-kB [29].
In line with this, OTX008 restored the content of IkB-β, the NF-kB protein blocker, while decreasing the levels of the transcription factor. These evidences are in line with the attenuation of NF-kB in hypoxic HRMECs exposed to OTX008 [17].
Another aspect of the OTX008 that, however, has to be considered for future perspective studies is that by blocking the Gal-1/TGF-β pathway, the compound may candidate itself as a drug conditioning the EMT process of ARPE-19 cells under high glucose. Indeed, here, it is shown that the canonical markers of EMT such as, for instance, ACTA2 and FnI [10,30] are reduced by the treatment of the cells with OTX008. Worthy of note is that the EMT of polarized RPE cells is a process activated under pathological circumstances, such as inflammation, wound healing, and carcinogenesis, enabling epithelial cells to obtain an enhanced migration ability and increase their production of extracellular matrix components, leading to RPE dysfunction [31]. Loss of RPE functional integrity is the base for development of numerous retinal diseases, including inherited cone-rod degenerations, inherited macular degeneration, age-related macular degeneration, and proliferative vitreoretinopathy [28]. OTX008, by an activity triggered by Gal-1 inhibition, may orchestrate and direct various actors on the complex scenario of high-glucose-induced damage of ARPE-19 cells towards a stabilization of their physiological functions (e.g., prevention of vision loss and photoreceptor integrity preservation) instead of pathological ones.
The authors do not exclude that OTX may have additional effects to the inhibition of Gal-1, but it is not possible to know this completely due to the few studies done on the compound and given its recent introduction into experimentation. Similarly, it is not clear whether Gal-1 is expressed by other cells of the retina involved in fibrosis, and thus, in vivo studies or in vitro studies on different cell lines may corroborate the concept expressed in the present study.
In conclusion, OTX008, by preventing the pro-fibrotic processes activated by Gal-1 in a cell line strictly involved in the pathogenic mechanisms of diabetes-or hyperglycemiarelated eye diseases, candidates itself as good therapeutic tool to treat them.
For the daily observation of ARPE-19 morphology with optic microscope (Leica DMi1, Mannheim, Germany), cells were plated in 6-well plates at a density of 2 × 10 5 cells/well as well as for protein and RNA isolation [34]. After the stimulation period, cells and supernatants were collected and preserved for down-stream analysis. For each assay, three independent experiments were performed, each done in triplicate.

ROS Assessment
ROS levels were detected by the conversion of the fluorescent probe 2 ,7 -dichlorodihydr ofluorescein diacetate (DCFH-DA) to highly fluorescent dichlorofluorescein (DFC) diacetate within cells by ROS. ARPE-19 were seeded in 96-well plates (5 × 10 3 cells/well) [36] and exposed to normal or high glucose, with or without OTX. At the end of the stimulation period, cells were loaded with 20 µM DCFH-DA in medium with 5% FBS at 37 • C for 30 min and then were trypsinized. Total intracellular ROS production was measured with a fluorometric plate reader at an excitation of 485 nm and an emission of 530 nm. Both cell types were exposed to medium 5% FBS without DCFH-DA as negative control (CTR−) or incubated with H 2 O 2 (100 µM) 30 min before trypsinization as a positive control (CTR+) [32].
Fluorescent-labeled anti-rabbit (Invitrogen 11,008; dilution 1:1000) and anti-mouse (Life Technologies A21202; dilution 1:1000) secondary antibodies were used to locate the specific antigens in each slide. Cells were counterstained and mounted with VECTASHIELD Antifade Mounting Medium with 4 ,6-diamidino-2-phenylindole (DAPI) (Novus Biologicals H-1200-NB). Fluorescently labeled slides were viewed with a fluorescence microscope (Leica, Wetzlar, Germany) and with a fluorescence confocal microscope (LSM 710, Zeiss, Oberkochen, Germany). Immunofluorescence images were analyzed with Leica FW4000 software (Leica, Wetzlar, Germany) and with Zen Zeiss software (Zeiss, Oberkochen, Germany). The percentage of positive cells in each microscope field was calculated by the number of positive cells of 350 cells in four different microscope fields for each treatment by considering only DAPI counterstained cells as positive profiles. Data were reported as mean percentage of positive cells/total cells counted ± standard deviation (SD).

Statistical Analysis
The results are reported as mean ± SD of three independent experiments, with each performed in triplicate. Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Tukey's comparison test by using GraphPad Prism 6.0. A p-value less than 0.05 was considered significant to reject the null hypothesis.

Conflicts of Interest:
The authors declare no conflict of interest.