PEDF-Mediated Mitophagy Triggers the Visual Cycle by Enhancing Mitochondrial Functions in a H2O2-Injured Rat Model

Retinal degenerative diseases result from oxidative stress and mitochondrial dysfunction, leading to the loss of visual acuity. Damaged retinal pigment epithelial (RPE) and photoreceptor cells undergo mitophagy. Pigment epithelium-derived factor (PEDF) protects from oxidative stress in RPE and improves mitochondrial functions. Overexpression of PEDF in placenta-derived mesenchymal stem cells (PD-MSCs; PD-MSCsPEDF) provides therapeutic effects in retinal degenerative diseases. Here, we investigated whether PD-MSCsPEDF restored the visual cycle through a mitophagic mechanism in RPE cells in hydrogen peroxide (H2O2)-injured rat retinas. Compared with naïve PD-MSCs, PD-MSCsPEDF augmented mitochondrial biogenesis and translation markers as well as mitochondrial respiratory states. In the H2O2-injured rat model, intravitreal administration of PD-MSCsPEDF restored total retinal layer thickness compared to that of naïve PD-MSCs. In particular, PTEN-induced kinase 1 (PINK1), which is the major mitophagy marker, exhibited increased expression in retinal layers and RPE cells after PD-MSCPEDF transplantation. Similarly, expression of the visual cycle enzyme retinol dehydrogenase 11 (RDH11) showed the same patterns as PINK1 levels, resulting in improved visual activity. Taken together, these findings suggest that PD-MSCsPEDF facilitate mitophagy and restore the loss of visual cycles in H2O2-injured rat retinas and RPE cells. These data indicate a new strategy for next-generation MSC-based treatment of retinal degenerative diseases.


Introduction
Retinal degenerative diseases are heterogeneous conditions and include diabetic retinopathy and age-related macular degeneration (AMD) [1]. Loss of the visual cycle and photoreceptor damage in AMD are the part of causes of blindness due to mitochondrial dysfunction [2,3]. The stages of AMD include hard drusen, which is associated with localized malfunctions of the retinal pigment epithelium, soft drusen associated with diffuse dysfunction of the retinal pigment epithelium, and damage to photoreceptors [4]. In retinal degeneration, pigment epithelium-derived factor (PEDF), which is produced in retinal pigment epithelial (RPE) cells and maintains retinal homeostasis, is decreased, while the expression of vascular endothelial growth factor (VEGF) is increased [5,6].
PEDF is a 50-kDa secreted glycoprotein known as serine proteinase inhibitor (serpin) F1 and is synthesized by cultured RPE cells [7]. In the human retina, PEDF localizes to photoreceptor cells, the inner nuclear layer (INL), the ganglion cell (GC) layer, the inner plexiform layer (IPL), and pigment epithelial cells (PECs). PEDF plays important roles in protecting retinal functions [8]. Deficiencies or defects in PEDF are intimately associated with the progression of angiogenic diseases such as diabetic retinopathy [9]. In a diabetic mouse model, PEDF has antioxidant effects that protect against oxidative stress by promoting cell survival and reducing pathological angiogenesis [10]. Moreover, PEDF treatment prevents oxidative stress by improving mitochondrial structure and function and activating the PI3K/AKT and MAPK pathways in H 2 O 2 -injured RPE cells [11].
The classical cycle is involved in the cycling of retinoids from the rod outer segment and the retinal pigment epithelium [12][13][14]. All-trans retinal is released from opsin and decreased by all-trans retinol dehydrogenase (at-RDH). In retinal degeneration, a lack of the PEDF receptor induces the regressive survival of photoreceptor cells [15]. In cone photoreceptor cell damage, PEDF signaling through the PEDF receptor induces marked increases in S cone numbers and survival [16]. Downregulated PEDF levels in RPE cells decrease the expression of LRAT and RDH11. However, the effect of PEDF on the visual cycle in H 2 O 2 -damaged RPE cells is still unknown.
Autophagy is a programmed and evolutionarily conserved catabolic pathway that degrades excessive or damaged organelles and cellular proteins through the formation of autophagosomes [17]. Additionally, specific autophagy processes have been reported, including mitophagy, that are mediated by PINK1/PARKIN signaling and selectively remove damaged or excessive mitochondria [18]. In AMD-like pathology, oxidative stressinduced RPE cells activate mitochondrial clearance by a mitophagic mechanism [19]. Knockdown of PINK1 expression downregulated phosphorylated PARKIN levels in RPE cells treated with H 2 O 2 [20]. In addition, the effect of PEDF expression on mitophagy in RPE cells is still unclear.
Previously, a mitochondrial oxidative stress model was established using H 2 O 2 in rats based on this study [21]. We found that overexpressed PEDF in PD-MSCs (PD-MSCs PEDF ) improved antioxidant effects and mitochondrial biogenesis associated with retinal regeneration in a H 2 O 2 -injured rat model and RPE cells [22]. In this study, we focused on recovery of the visual cycle through mitophagy and mitochondrial functions by PD-MSC PEDF in a H 2 O 2 -injured rat model and RPE cells.

Animals and MSC Transplantation
Seven-week-old male Sprague-Dawley rats (Orient Bio Inc., Seongnam, Korea) were maintained in an air-conditioned facility. All rats were anesthetized by 75 mg/mL of tribromoethanol (avertin; Sigma-Aldrich) by intraperitoneal injection. Acute eye disease was induced by a intravitreally single injection of H 2 O 2 (10 µg/µL; Sigma-Aldrich) for 2 weeks, and rats in the sham group were injected with balanced salt solution (BSS). The rats were divided into the following groups: the sham (sham; n = 6), which received H 2 O 2 injection for 2 weeks (H 2 O 2 ; n = 6); naïve PD-MSCs (PD-MSCs; 2 × 10 5 ; n = 6); and PD-MSCs PEDF (PD-MSCs PEDF ; 2 × 10 5 ; n = 6). A week after MSC transplantation, the rats were euthanized using a CO 2 chamber, body and tissue weights were measured, and eyeballs and serum were collected. The experimental protocol was approved by the Institutional Animal Care and Use Committee of CHA University, Seongnam, Korea (IACUC-200033).

Histopathological and Immunofluorescence Analysis
Enucleated eyeballs were examined and fixed after immersion with neutral buffered formalin (NBF) from each group (n = 6). Each sample was embedded in paraffin and cut into 5-µm-thick sections in the central region on the optic disc of the eyeball for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). To analyze the genes in retinal layers of the eyeball, each eyeball section was stained with anti-VEGF  (Table S3) and nuclei was stained with 4 6-diamidino-2-phenylindole (DAPI; Invitrogen). The images were observed by confocal microscopy (LSM880) and analyzed using ZEN blue software (ZEISS, Oberkochen, Germany).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from rat retinas or cells using TRIzol LS (Invitrogen) based on the manufacturer's method. cDNA was synthesized using the SuperScript III reverse transcriptase (Invitrogen). qRT-PCR was assessed on a CFX Connect™ Real-Time System (Bio-Rad, Hercules, CA, USA), with primers (Tables S1 and S2) and SYBR Green PCR master mix (Roche, Basel, Switzerland). Gene expression was quantified by the 2-∆∆CT method, and all data were analyzed in triplicate.

XF Mito Stress Assay
To analyze mitochondrial metabolic conditions in live naïve PD-MSCs and PD-MSCs PEDF , the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed using an XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Naïve PD-MSCs and PD-MSCs PEDF (7 × 10 3 cells/well) were cultured in XF 24-well microplates and equilibrated in XF buffer for 60 min by repeated cycles of mixing for 3 min, incubation for 2 min, and measurement for 30 min in non-CO 2 conditions. Each cell type was sequentially treated with 0.5 µM oligomycin, 0.5 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and a 1 µM rotenone/antimycin A (AA) mixture according to the manufacturer's recommendation by the Seahorse XF24 Analyzer. The OCR/ECAR was normalized to the total cell number.

Enzyme-Linked Immunosorbent Assay (ELISA)
To confirm PEDF overexpression in PD-MSCs, human PEDF in cell culture supernatant was analyzed according to the instructions using the human PEDF ELISA kit (Abcam). IL-6 and IL-10 levels in rat serum (n = 6/group) were analyzed by rat ELISA kit (Abcam) to determine proinflammatory and anti-inflammatory factor levels. Samples and standards were added to appropriate wells. Then, each antibody was incubated at room temperature. These were aspirated and each well was washed four times. TMB solution was incubated and stop solution was added. The OD at 450 nm was measured by an Epoch microplate reader (BioTek, Winooski, VT, USA). Each concentration was assessed by the trend line equation. All reactions were performed in triplicate.

Statistical Analysis
All data were analyzed using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA), and statistically significant differences were assessed using two-tailed unpaired Student's t-tests or one-way analysis of variance (ANOVA), followed by an appropriate post hoc test. P values less than 0.05 were considered statistically significant.

PD-MSC PEDF Transplantation Restores Retinal Function in a H 2 O 2 -Injured Rat Model
To induce an acute retinal degeneration model, H 2 O 2 (10 µg/µL) injection was conducted for 1 and 2 weeks, while the sham group was injected with the same volume of BSS. Naïve PD-MSCs (H 2 O 2 + Naïve) and PD-MSCs PEDF (H 2 O 2 + PEDF+) were intravitreally injected into rats. After 1 and 2 weeks, eyeball weight was significantly decreased in the H 2 O 2 -injured groups ( Figure S2). However, naïve PD-MSC transplantation induced a slight increase. Interestingly, the PD-MSCs PEDF group showed a significant difference (Figure 2A; p < 0.05). We analyzed inflammatory cytokines in rat serum and confirmed proinflammatory IL-6 and anti-inflammatory cytokine IL-10 levels. After H 2 O 2 treatment, increased IL-6 and decreased IL-10 were observed compared with those in the sham group. After transplantation of naïve PD-MSCs or PD-MSCs PEDF , decreases in IL-6 and increases in IL-10 levels were assessed. In particular, IL-10 in the PD-MSCs PEDF group was markedly greater than that in the naïve PD-MSCs group (Figure 2B,C; p < 0.05). We further evaluated histological structural changes in retinal layers in the eyeball using H&E staining. Following H 2 O 2 injection, total retinal layers showed abnormal conditions and neutrophil infiltration.
After transplantation of naïve PD-MSCs or PD-MSCs PEDF , decreases in IL-6 and increases in IL-10 levels were assessed. In particular, IL-10 in the PD-MSCs PEDF group was markedly greater than that in the naïve PD-MSCs group (Figure 2B, C; p < 0.05). We further evaluated histological structural changes in retinal layers in the eyeball using H&E staining. Following H2O2 injection, total retinal layers showed abnormal conditions and neutrophil infiltration.  Interestingly, the administration of PD-MSCs PEDF preserved the integrity of the retinal layers ( Figure 2D). Additionally, the thickness of the total retina was reduced in the H 2 O 2 group compared to the sham group. Other layers, including the ganglion cell layer (GCL) and inner plexiform layer (IPL), were damaged. The effects in the naïve PD-MSCs and PD-MSCs PEDF groups were restored. In particular, PD-MSC PEDF transplantation increased the length of the total retina layer ( Figure 2E). In detail, the thicknesses of the outer nuclear layer

PD-MSCs PEDF Balanced VEGF and PEDF Levels in a H 2 O 2 -Induced Rat Model
Retinal degenerative diseases, including AMD, are characterized by the growth of abnormal blood vessels, resulting in increased levels of the angiogenic factor VEGF and decreased levels of the antiangiogenic factor PEDF [26]. We confirmed the mRNA expression of VEGF and PEDF in the rat retina. After H 2 O 2 treatment, the expression of VEGF was highly increased, while the expression of PEDF was significantly decreased. Compared to naïve PD-MSC transplantation, PD-MSCs PEDF reduced VEGF levels and enhanced PEDF levels ( Figure 3A; p < 0.05). The mRNA expression of these two genes showed a negative correlation value of R 2 = −0.7575 ( Figure 3B). Moreover, endogenous VEGF and PEDF expression was analyzed by immunohistochemistry. Representative images showed the same trend as that of the mRNA expression ( Figure 3C). The VEGF-and PEDF-positive areas were measured in each group. H 2 O 2 treatment significantly increased the intensity of VEGF, whereas it decreased that of PEDF. After naïve PD-MSC and PD-MSC PEDF transplantation, decreased VEGF-positive areas and increased PEDF-positive areas were observed. In particular, PD-MSCs PEDF significantly improved PEDF expression in retinal layers (Figure 3D,E). Endogenous gene expression levels of VEGF and PEDF in the retina exhibited a negative correlation value of R 2 = −0.8349 ( Figure 3F). These results indicated that VEGF and PEDF expression were modulated by PD-MSC PEDF transplantation in H 2 O 2 -injured rat retinas.

PD-MSCs PEDF Improved Visual Cycles in Rat Retinal Layers and RPE Cells
The structural relationship between the retinal pigment epithelium and photoreceptors is critical for the visual cycle [12]. In RPE cells, all-trans retinol is converted into 11-cis retinal and is transferred to cellular retinoid-binding protein (RBP). All-trans-retinol is delivered by lecithin retinol acyl transferase (LRAT). Then, retinol dehydrogenase (RDH) 5 and 11 are involved in the oxidation of 11-cis retinol.
The generated 11-cis retinal crosses the interphotoreceptor matrix and enters photoreceptor cells. All-trans retinal is converted into all-trans-retinol by RDH12, 13, and 14 ( Figure 4A). We analyzed the expression of RDH11, which is a key enzyme in the visual cycle, in the rat retina by IHC. Interestingly, the H 2 O 2 injection group exhibited decreased gene expression. On the other hand, RDH11 levels in the naïve PD-MSC and PD-MSC PEDF groups were significantly restored. In particular, the administration of PD-MSCs PEDF significantly increased the expression levels of visual cycle enzymes (e.g., RDH11, 12, 13, and 14) compared to that of naïve PD-MSCs ( Figure 4C; p < 0.05).
To further analyze visual enzymes in RPE cells, H 2 O 2 stimulation was conducted for 2 h to induce oxidative stress, and naïve PD-MSCs or PD-MSCs PEDF were indirectly cocultured with ARPE-19 cells for 24 h. Previously, we confirmed the gene expression of retinal function markers in ARPE-19 cells in response to PD-MSCs PEDF [22]. We further analyzed the major enzymes involved in the visual cycle in RPE cells. The mRNA expression of RDH5 and RDH11 in H 2 O 2 -treated cells was reduced, as shown by qRT-PCR and immunofluorescence analysis ( Figure 4D,E). Compared to coculture with naïve PD-MSCs, coculture with PD-MSCs PEDF induced significant levels of RDH5 and RDH11. In addition, LRAT and RBP1 expression were also restored after coculture with PD-MSCs PEDF compared to naïve PD-MSCs ( Figure 4F,G; p < 0.05). These results indicate that the administration of PD-MSCs PEDF improves visual cycles in H 2 O 2 -injured rat retinas and RPE cells.

The Administration of PD-MSCs PEDF Induces Mitophagy in H 2 O 2 -Injured Rat Retinas
In retinal degenerative diseases, PEDF results in elevated autophagy in RPE cells [6]. Previously, our reports showed that PD-MSCs PEDF enhanced antioxidant effects and mitochondrial functions in RPE cells [22]. We further analyzed mitochondrial autophagy, which is known as mitophagy, in H 2 O 2 -injured rat retinas. The mRNA expression of autophagy markers (e.g., microtubule-associated proteins 1A/1B light chain 3B (LC3), autophagy-related 7 (ATG7), and Beclin1 (BECN1)) was increased in the H 2 O 2 group compared with the sham group. Additionally, the expression of autophagy-related markers after PD-MSC PEDF transplantation was significantly different from that after naïve PD-MSC transplantation ( Figure 5A; p < 0.05). In the rat retina, PD-MSCs PEDF induced predominant LC3 expression, as shown by IHC ( Figure 5B). Furthermore, mitophagy markers (e.g., mitofusin 2 (MFN2), PTEN-induced kinase 1 (PINK1), and Parkin RBR E3 ubiquitin protein kinase (PARKIN)) were analyzed by qRT-PCR. The expression of these factors was also increased in the H 2 O 2 group. Compared to the naïve PD-MSCs group, the PD-MSCs PEDF group showed significant enhancement ( Figure 5C; p < 0.05). Endogenous PINK1 expression in rat retinal sections was confirmed by IHC. Compared to naïve PD-MSC transplantation, PD-MSC PEDF transplantation also increased expression patterns ( Figure 5D). These findings indicate that PD-MSCs PEDF promote mitophagy in a PINK1-dependent manner in the rat retina. To further analyze visual enzymes in RPE cells, H2O2 stimulation was conducted for 2 h to induce oxidative stress, and naïve PD-MSCs or PD-MSCs PEDF were indirectly cocultured with ARPE-19 cells for 24 h. Previously, we confirmed the gene expression of retinal function markers in ARPE-19 cells in response to PD-MSCs PEDF [22]. We further analyzed the major enzymes involved in the visual cycle in RPE cells. The mRNA expression of

Coculturing RPE Cells with PD-MSCs PEDF Enhances Mitophagy
In H2O2-injured rat retinas, improvements in PINK1-dependent mitophagy occurred. Additionally, autophagy and mitophagy in H2O2-damaged RPE cells were analyzed after the cells were cocultured with naïve PD-MSCs or PD-MSCs PEDF . The mRNA expression of LC3 and ATG7 was decreased in H2O2-induced ARPE-19 cells. After coculture with naïve PD-MSCs or PD-MSCs PEDF , the expression of these genes was elevated. In particular, PD-MSCs PEDF induced higher levels than naïve cells ( Figure 6A; p < 0.05). In addition, the mitophagy-related genes PINK1 and PARKIN tended to have the same expression patterns

Coculturing RPE Cells with PD-MSCs PEDF Enhances Mitophagy
In H 2 O 2 -injured rat retinas, improvements in PINK1-dependent mitophagy occurred. Additionally, autophagy and mitophagy in H 2 O 2 -damaged RPE cells were analyzed after the cells were cocultured with naïve PD-MSCs or PD-MSCs PEDF . The mRNA expression of LC3 and ATG7 was decreased in H 2 O 2 -induced ARPE-19 cells. After coculture with naïve PD-MSCs or PD-MSCs PEDF , the expression of these genes was elevated. In particular, PD-MSCs PEDF induced higher levels than naïve cells ( Figure 6A; p < 0.05). In addition, the mitophagy-related genes PINK1 and PARKIN tended to have the same expression patterns ( Figure 6B). To further analyze PINK1 levels in the mitochondria of RPE cells, immunostaining with PINK1 and mtTracker was performed in ARPE-19 cells.

Discussion
The treatment of retinal degenerative diseases is ongoing in the developed world. Approximately 42 phase 1-3 clinical trials for retinal degenerative disease treatment have been conducted (www.clinicaltrials.gov). As standard treatments, ranibizumab and bevacizumab, which are monoclonal antibodies that inhibit angiogenesis by targeting H 2 O 2 treatment induced PINK1 expression, whereas mitochondrial signals were decreased. However, coculture with naïve PD-MSCs slightly decreased PINK1 expression and increased mtTracker signals. In particular, PD-MSCs PEDF significantly improved both signals by increasing PINK1 expression ( Figure 6C). In addition, the protein expression of LC3 type 2 and Beclin1 tended to be increased in PD-MSCs PEDF . After H 2 O 2 treatment, PINK1 and ATG7 expression was induced in RPE cells compared with normal control cells; however, naïve PD-MSCs did not mediate this expression. In particular, coculture with PD-MSCs PEDF significantly improved PINK1 and ATG7 levels ( Figures 6D,E and S3). These data suggest that PD-MSC PEDF coculture enhances mitophagy signaling in H 2 O 2 -damaged RPE cells.

Discussion
The treatment of retinal degenerative diseases is ongoing in the developed world. Approximately 42 phase 1-3 clinical trials for retinal degenerative disease treatment have been conducted (www.clinicaltrials.gov (accessed on 22 March 2021)). As standard treatments, ranibizumab and bevacizumab, which are monoclonal antibodies that inhibit angiogenesis by targeting VEGF, are usually used for drug therapy. The outcomes indicated changes in the visual acuity scores of patients with AMD or diabetes. However, these therapies have several side effects, including conjunctival hemorrhage, intraocular inflammation, and increased intraocular pressure [27]. Additionally, several trials are necessary to assess long-term observations in an ongoing follow-up trial phase and continuous improvements in the relief of symptoms.
Currently, stem cell therapy has been applied for acute and chronic retinal degenerative disorders [28]. Embryonic stem cell (ESC)-and induced pluripotent stem cell (iPSC)derived RPE cells are mainly used for visual restoration [29]. The methods have procedural difficulties and risk factors such as graft rejection and tumor formation [30]. In particular, differentiated RPE transplantation has stringent controls associated with retinal detachment [31]. To overcome these limitations, mesenchymal stem cell (MSC)-based therapy has become a promising strategy to restore retinal functions through various secreted factors, resulting in antiapoptotic and anti-inflammatory effects [32]. In patients with retinitis pigmentosa, implantation of umbilical cord-derived MSCs (UC-MSCs) into the suprachoroidal area resulted in the best corrected visual acuity and visual field after 6 months of follow-up in a phase 3 trial [33]. However, the mechanism of MSC therapy is still unknown. Consequently, the next generation of MSCs expressing PEDF was generated to examine the efficacy of MSC-based therapy based on our previous reports [23]. Compared with naïve PD-MSCs, PD-MSCs PEDF highly secreted PEDF and augmented mitochondrial biogenesis and translation, as well as improved respiratory metabolism (Figure 1).
Since the main function of the retinal pigment epithelium is to structurally maintain photoreceptors, RPE degeneration results in the dysfunction of photoreceptors, leading to the loss of visual cycles. Photoreceptors depend on the retinal pigment epithelium for numerous metabolic functions, including the visual cycle. Typically, visual cycles regenerate 11-cis retinal through multiple steps involving specialized enzymes, such as retinoid binding protein (IRBP), and begin in the outer segment with all-trans retinal release from the opsin [12]. Therefore, the photoisomerization of 11-cis retinal to all-trans retinal in rod and cone photoreceptors is the initial step in vision induced by light, but the continued function of photoreceptors demands that all-trans retinal be converted into 11-cis retinal through the visual cycle.
In damaged RPE and photoreceptor cells, the autophagic mechanism converges for the recovery of vision loss. Zhou et al. reported that ATG5 knockdown in photoreceptors resulted in retinal degeneration and the deterioration of phototransduction protein levels [34]. During light-exposed photoreceptor degeneration, the deletion of Beclin1 or ATG7 in rod photoreceptor cells induced retinal degeneration via Parkin. Moreover, ABCA4and RDH8-knockdown mice exhibited increased LC3B conversion, leading to the loss of photoreceptor cells [35]. Based on previous evidence, RDH11 expression in H 2 O 2 -injured rat retinas showed similar patterns to those of LC3B and PINK1 in our study (Figure 4). In RPE cells stimulated with glucose and diabetic retinas, decreased PEDF levels reduced the expression of RPE65, LRAT, and RDH11, which are visual cycle enzymes [36], and this effect occurred in AMD patients [37]. Similarly, our results indicate that H 2 O 2 -injured RPE cells cocultured with PD-MSCs PEDF exhibited markedly increased RDH5, RDH11, LRAT, and RBP1 expression compared with those of cells cocultured with naïve PD-MSCs. Interestingly, the synthesis and secretion of PEDF by RPE cells were apically distributed to the retinal pigment epithelium around the outer segments of photoreceptors [38].
The pathogenesis of retinal degenerative diseases, including AMD and diabetic retinopathy, is highly associated with mitochondrial dysfunction, oxidative stress, and mitochondrial DNA damage [2]. Our previous study reported that PD-MSCs PEDF promoted antioxidant defenses against mitochondrial ROS levels and improved mitochondrial biogenesis in oxidative stress-injured RPE cells as well as mitochondrial translation ( Figure S4). Moreover, in an acute retinal degenerative rat model injured by H 2 O 2 , PD-MSC PEDF transplantation had a prominent effect on mitochondrial dynamics compared to that of naïve PD-MSC transplantation, resulting in the recovery of retinal functions [22]. The coordination of mitochondrial biogenesis and mitophagy regulates mitochondrial contents and metabolism, preserving homeostasis [39]. Mitochondria impairment in RPE degeneration and the pathology of AMD induce a cellular defense mechanism known as mitophagy [19].
In the present study, we focused on facilitating mitophagy (mitochondrial autophagy) through PD-MSC PEDF transplantation in H 2 O 2 -injured retinas. In cardiomyocytes, PEDF expression improved mitophagy to protect against hypoxic conditions by modulating the PEDF receptor (PEDF-R)/protein kinase C alpha (PKC-α) signaling pathway [40]. Although increased PKC-α expression by PEDF activates AMPK, which contributes to mitophagy in cardiovascular diseases [41], the effect of PEDF on mitophagy in the context of RPE degeneration is still unknown. We determined that the administration of PD-MSCs PEDF augmented the expression of both the autophagosome marker LC3 and the mitophagy marker PINK1 in H 2 O 2 -induced rat retinas ( Figure 5). Of the 10 retinal layers, LC3B expression was shown in ganglion cells (GCs), the inner plexiform layer (IPL), the inner nuclear layer (INL), the outer plexiform layer (OPL), photoreceptor inner segments (ISs), and pigment epithelial cells (PECs) [42]. In H 2 O 2 -injured rat retinas, these expression levels were observed in more than six layers ( Figure 5). Interestingly, RPE cells cocultured with PD-MSC PEDF exhibited significantly enhanced mitophagy, as indicated by the colocalization of PINK1 and the mitochondrial marker mtTracker compared to that of cells cocultured with naïve PD-MSCs. In addition, naïve PD-MSC coculture facilitated autophagy, but it did not induce mitophagy ( Figure 6). These data suggest that PEDF could be highly relevant to mitophagy in H 2 O 2 -injured rat retinas.
Oxidative stress-induced experimental animal models have been reported in various methods [43][44][45][46][47]. Using transgenic mice, exon 3 of the antioxidant enzyme superoxide dismutase 2 (SOD2) was flanked, and confirmed to induce mitochondrial oxidative stress levels and the death of photoreceptor cells. However, transgenic methods have a longer time of 9 months, and do not induce drusen-like deposits [48]. In addition, radiation exposure can cause retinal damage and impaired vision. This method has several disadvantages, including cataracts and permanent blindness [49]. Especially, intravitreal injection using H 2 O 2 can induce acute retinal degeneration against oxidative stress [21]. Additionally, H 2 O 2 -induced animal models have many benefits, such as taking a short time and having consistency, resulting in elevated oxidative stress and cellular apoptosis.
Previously, we confirmed therapeutic effects by PD-MSCs PEDF in RPE cells and an acute retinal degenerative model. However, H 2 O 2 -injured rats acquired cataracts, and albino rats were known to have weak vision and be less resistant to stress levels [50]. For further study, we need to further establish chronic degenerative models, including streptozotocin (STZ)-induced diabetic or transgenic models, and investigate their molecular mechanisms.

Conclusions
In conclusion, the present study investigated whether functionally enhanced PD-MSCs PEDF stimulated mitochondrial biogenesis and mitochondrial translation, as well as mitophagy for cellular clearance, resulting in the recovery of the visual cycle. Therefore, functionally enhanced PD-MSCs may be a new strategy for next-generation stem cell therapy for retinal degenerative diseases.