Possible involvement of the mesenchymal cell marker Meflin in angiogenesis promotion and fibrosis suppression after retinal injury

Possible involvement of the mesenchymal cell marker Meflin in angiogenesis promotion and fibrosis suppression after retinal injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Possible involvement of the mesenchymal cell marker Meflin in angiogenesis promotion and fibrosis suppression after retinal injury Daishi Okuda, Shinji Mii, Yuki Miyai, Katsuhiro Kato, Ryota Ando, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8930515/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract Pathological tissue remodeling, including choroidal neovascularization (CNV) and fibrosis, is central to the development of retinal diseases such as age-related macular degeneration (AMD); however, its underlying molecular mechanisms remain incompletely defined. In this study, we examined the intraocular expression patterns of Meflin, a mesenchymal stromal cell marker with anti-fibrotic properties, and evaluated its role in the pathophysiology of retinal diseases using a mouse model. In wild-type mice, Meflin expression was detected in the ciliary body, lens epithelium, retinal pigment epithelium, optic nerve meningeal cells, and choroidal perivascular fibroblasts. Lineage-tracing analysis using a Meflin reporter mouse line revealed accumulation of Meflin-lineage cells within laser-induced CNV lesions. Interestingly, adeno-associated virus-mediated overexpression of Meflin increased CNV volume in the acute phase of retinal injury but significantly suppressed subretinal fibrosis in the chronic phase. These findings suggest that Meflin promotes angiogenesis to support tissue repair and inhibits fibrosis after retinal injury in a temporally dependent manner, consistent with its previously reported roles in mouse models of cancer and other fibrotic diseases. These results improve our understanding of retinal disease pathology and highlight Meflin as a potential therapeutic target in diseases such as AMD. Biological sciences/Cell biology Health sciences/Diseases Health sciences/Medical research Health sciences/Pathogenesis Meflin immunoglobulin superfamily containing leucine-rich repeat fibrosis angiogenesis choroidal neovascularization age-related macular degeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Retinal diseases such as proliferative vitreoretinopathy (PVR) [ 1 ], proliferative diabetic retinopathy (PDR) [ 2 ], and age-related macular degeneration (AMD) [ 3 ] lead to severe vision loss and substantial reduction in quality of life. These disorders are difficult to treat and share a common pathological feature: retinal dysfunction associated with angiogenesis and excessive fibrosis [ 4 ]. Angiogenesis and fibrosis are essential for tissue repair and maintenance of homeostasis after injury [ 5 , 6 ]. However, the aforementioned retinal diseases are characterized by prolonged intraocular neovascular responses and fibrotic scar formation, leading to irreversible destruction and remodeling of retinal architecture [ 4 ]. Accordingly, elucidating the mechanisms that govern tissue repair and its regulation in the retina remains an important challenge in the development of therapeutic strategies for diverse retinal disorders. AMD is a representative disease in which pathological tissue repair is a critical determinant of visual prognosis [ 3 , 7 ]. AMD is broadly categorized into two major subtypes: dry AMD and neovascular (wet) AMD (nAMD). In nAMD, choroidal neovascularization (CNV) causes subretinal fluid exudation, hemorrhage, and subsequent scar formation, leading to central vision loss [ 3 ]. The introduction of anti-vascular endothelial growth factor (VEGF) therapy has markedly improved visual outcomes for patients with nAMD [ 8 , 9 ]. However, effective management of nAMD is hindered by clinical challenges such as reduced therapeutic responsiveness during long-term treatment [ 10 , 11 ]. Drusen, which are deposits beneath the retinal pigment epithelium (RPE), are believed to cause chronic inflammation in the early stages of nAMD. This promotes upregulation of angiogenic factors, recruitment of immune cells and fibroblasts, and progression of CNV [ 4 , 12 , 13 ]. Although anti-VEGF therapy effectively suppresses neovascular growth, it does not resolve the underlying persistent inflammation. Under conditions of persistent inflammation, ongoing tissue injury and a sustained reparative response can drive progression of fibrosis [ 13 ]. These observations suggest that, in addition to targeting CNV, understanding the molecular mechanisms underlying chronic inflammation and prolonged tissue repair is essential for the developing effective therapeutic strategies for nAMD. However, the molecular basis of retinal tissue repair remains incompletely defined. Although mesenchymal cells, including fibroblasts and perivascular cells, play central roles in fibrosis under pathological conditions [ 4 , 13 , 14 ], the molecular mechanisms that regulate the complex cellular networks formed by these cells remain unclear. Therefore, identifying key molecular regulators that govern the balance between repair and fibrosis in retinal tissue is crucial. To provide novel insights into the mechanisms underlying retinal tissue repair, we focused on Meflin, a mesenchymal stromal cell and fibroblast marker with anti-fibrotic properties [ 15 – 20 ]. Meflin is a glycosylphosphatidylinositol (GPI)-anchored membrane protein encoded by the ISLR/Islr (immunoglobulin superfamily containing leucine-rich repeat) gene. It has been identified as a marker of tumor-restraining cancer-associated fibroblasts (rCAFs) [ 21 – 29 ] and has suppressed fibrotic progression in mouse models of cardiac, pulmonary, and renal fibrosis [ 16 – 18 ]. Mechanistically, Meflin enhances bone morphogenetic protein (BMP) signaling and suppresses transforming growth factor-β (TGF-β) signaling through its interaction with BMP7 [ 16 , 24 ]. Meflin also inhibits collagen crosslinking by binding to lysyl oxidase (LOX), thereby limiting tissue stiffening [ 30 ]. Furthermore, our cancer research demonstrated that Meflin positively regulates tumor vascularization, which improves drug delivery and antitumor immune responses [ 23 , 30 , 31 ]. Moreover, Meflin-positive CAFs produce the inflammatory chemokine Chemerin (Rarres2), which induces polarization of macrophages to the M1-like phenotype [ 32 ]. Although the Meflin gene was originally cloned in 1997 and its expression in the retina was noted [ 33 ], its precise localization within the eye and its functional roles in retinal tissue repair responses remain unexplored. In this study, we characterized the detailed expression patterns of Meflin in the eye and investigated its role in retinal tissue repair using a laser-induced CNV mouse model. Results Expression of Meflin in human fibrovascular membranes and cultured cells To determine the involvement of Meflin in human retinal diseases, we first analyzed its expression patterns in clinical surgical specimens. In situ hybridization (ISH) was performed to detect ISLR mRNA (hereafter referred to as Meflin mRNA) in fibrovascular membranes excised from patients who underwent vitrectomy for PVR or PDR (Fig. 1 A, B). In both types of specimens (PVR and PDR), Meflin mRNA signals were detected in fibroblast-like stromal cells within the fibrovascular membranes, suggesting the involvement of Meflin in the pathogenesis of these retinopathies. To identify the specific cell types that express Meflin in the human retina, we subsequently analyzed its expression in human RPE cells and Müller glial cells, both of which contribute to tissue repair and formation of fibrovascular membranes [ 1 , 2 , 14 , 34 ]. Specifically, we conducted Western blotting and quantitative polymerase chain reaction (qPCR) to evaluate Meflin protein and Meflin mRNA expression, respectively, using the human RPE cell line hTERT-RPE1, primary human RPE cells (hRPEpiC), and the human Müller glial cell line MIO-M1. Western blotting revealed a distinct Meflin protein band only in hRPEpiC, whereas no bands were detected in MIO-M1 or hTERT-RPE1 (Fig. 1 C). Similarly, qPCR analysis revealed Meflin mRNA expression only in hRPEpiC, whereas the results for both MIO-M1 and hTERT-RPE1 remained below the limit of detection (Fig. 1 D). Meflin expression in the murine eye We investigated Meflin expression patterns in the murine eye by conducting ISH for Meflin mRNA using ocular tissue sections obtained from C57BL/6J wild-type mice, with Meflin-deficient (KO) mice [ 15 ] analyzed in parallel as a negative control. The ISH revealed Meflin mRNA signals in meningeal cells surrounding the optic nerve, ciliary epithelium, RPE, lens epithelium, and fibroblast-like mesenchymal cells in the choroid and subjacent sclera (Fig. 2 A). To avoid signal masking by the melanin pigment in C57BL/6J mice, we performed the same analyses using albino BALB/c wild-type mice, with a DapB probe as a negative control. In the BALB/c mice, Meflin mRNA signals were detected in the same regions as those in C57BL/6J mice. Notably, the signals observed in the ciliary epithelium, RPE, and choroidal fibroblast-like mesenchymal cells were visualized more clearly in the BALB/c mice than in the C57BL/6J mice (Fig. 2 B). Between the two layers of the ciliary epithelium, Meflin mRNA expression was detected mainly in the basal layer cells that correspond to the pigmented epithelium in C57BL/6J mice (Fig. 2 Ba). These findings differ considerably from previous reports of Meflin expression being largely restricted to fibroblasts in other organs [ 15 – 18 ], highlighting the unique characteristics of intraocular epithelial cells. In contrast, the Meflin expression patterns detected in optic nerve meningeal cells and choroidal fibroblast-like mesenchymal cells are consistent with the expression patterns reported for other organs [ 35 ], suggesting the potential involvement of Meflin-positive fibroblast-like cells in tissue repair and fibrotic responses in these regions. Meflin deficiency does not affect ocular structure or physiological function We compared the eyes of Meflin KO mice with those of wild-type mice to determine the role of Meflin in the maintenance of ocular architecture and physiological function. Hematoxylin and eosin (H&E) staining of ocular sections obtained from 10-week-old mice revealed no significant histological abnormalities or disruption of the retinal laminar structure in Meflin KO mice compared with wild-type mice (Fig. 3 A). Given that Meflin is expressed in multiple intraocular tissues including the RPE, we evaluated retinal electrophysiological function using electroretinography (ERG). ERGs of the wild-type and Meflin KO mice were recorded under scotopic (rod-dominant) and photopic (cone-dominant) conditions (Fig. 3 B, D). The b/a ratio (b-wave amplitude relative to a-wave amplitude) in the scotopic ERG and the b-wave amplitude in the photopic ERG were used as functional readouts. No significant differences in these parameters were observed between the wild-type and Meflin KO mice (Fig. 3 C, E). These results indicate that Meflin is not critically involved in maintaining the basic structure or physiological function of the eye. Meflin-lineage cells accumulate at sites of retinal injury To investigate the involvement of Meflin-positive cells in angiogenesis and tissue remodeling following retinal injury, we subjected Meflin reporter mice to laser-induced CNV injuries [ 36 ] and performed Meflin lineage-tracing experiments. To visualize Meflin-lineage cells, we utilized Meflin-CreERT2; Rosa26-LSL-tdTomato mice, generated by crossing a line expressing tamoxifen-inducible CreERT2 under the control of the Meflin promoter ( Meflin-CreERT2 ) with a reporter line harboring a loxP-STOP-loxP-tdTomato cassette at the Rosa26 locus ( Rosa26-LSL-tdTomato ) (Fig. 4 A) [ 19 , 21 ]. Following the labeling of Meflin-positive cells with tdTomato via tamoxifen administration, laser photocoagulation was performed and CNV lesions were subsequently evaluated using RPE–choroid flatmounts and frozen ocular sections (Fig. 4 B). Significant accumulation of tdTomato-positive Meflin-lineage cells was identified within the CNV lesions in the Meflin reporter mice, whereas no tdTomato signal was detected in the control ( Meflin-CreERT2 +/− ) mice (Fig. 4 C). Evaluation of the frozen sections revealed tdTomato fluorescence in the internal limiting membrane (ILM), the inner nuclear layer (INL), and the choroid (Fig. 4 D). No tdTomato-positive cells were observed in the intact areas of the retina in the reporter and control mice (Fig. 4 E). These results suggest that Meflin-lineage cells are recruited to sites of tissue repair or expand there in response to retinal injury. Although Meflin expression was not detected in the MIO-M1 cell line in vitro, tdTomato signals were localized along the ILM in vivo (Fig. 4 D). As the ILM represents the basement membrane of Müller cells, this pattern is consistent with the possibility that Müller cells, which proliferate during the injury response, contribute at least in part to the Meflin-lineage cell population. Meflin deficiency does not affect tissue repair outcomes in a laser-induced CNV model To investigate the role of Meflin in angiogenesis and tissue remodeling accompanied by excessive fibrosis, we subjected Meflin KO mice to laser-induced CNV injuries and quantified CNV and subretinal fibrosis volume. CNV volume on RPE–choroid flatmounts was quantified using immunostaining (Fig. 5 A, B). The CNV volume (mean ± SD) was 252,000 ± 164,000 µm³ (n = 19) in wild-type mice and 165,000 ± 117,000 µm³ (n = 24) in Meflin KO mice. Although the Meflin KO mice tended to have lower CNV volumes, Welch’s t-test revealed no significant difference between the two groups (p = 0.060) (Fig. 5 C). Subsequently, we quantified the volume of subretinal fibrosis in the fibrotic phase of the laser-induced CNV (Fig. 5 D, E). The subretinal fibrosis volume (mean ± SD) was 264,000 ± 138,000 µm³ (n = 17) in the Meflin KO mice and 261,000 ± 106,000 µm³ (n = 18) in the wild-type mice, with no significant difference between two groups (Welch’s t-test, p = 0.942) (Fig. 5 F). Collectively, these results indicate that Meflin deficiency does not cause detectable changes in CNV or subretinal fibrosis formation following laser-induced injury. Establishment of an adeno-associated virus-mediated intraocular Meflin overexpression model We previously demonstrated that Meflin expression in fibroblasts confers tumor-suppressive and anti-fibrotic functions [ 21 , 23 , 30 ]. We established an adeno-associated virus (AAV)-mediated overexpression model to examine whether intraocular Meflin overexpression influences CNV and fibrosis formation after retinal injury. We generated an AAV9-CBh-Islr (hereafter referred to as AAV9-Meflin) vector using the AAV9 serotype, which exhibits broad cellular tropism [ 37 ], and the strong ubiquitous CBh promoter [ 38 ]. An AAV9-CBh-EGFP (hereafter AAV9-EGFP) vector was used as a control. Seven days after subretinal injection of AAV9-Meflin or AAV9-EGFP into the wild-type mice, their eyes were harvested to assess Meflin expression (Fig. 6 ). The AAV9-Meflin-injected eyes showed markedly increased Meflin mRNA signals in the retina and RPE, as well as in fibroblast-like cells in the choroid and the subjacent sclera. Within the retina, strong Meflin expression was observed particularly in the INL. In contrast, no appreciable increase in Meflin expression was observed in these regions in AAV9-EGFP-injected eyes (Fig. 6 ). We also evaluated intravitreal delivery of AAV9-Meflin; however, the transduction efficiency in the RPE and choroidal cells was lower than that achieved via subretinal injection (Supplementary Fig. 1). Therefore, subretinal administration was employed for all subsequent experiments. Meflin overexpression regulates angiogenesis and fibrosis after retinal injury We examined the effects of AAV9-mediated Meflin overexpression on CNV growth and subretinal fibrosis in the laser-induced CNV model. To assess the impact of Meflin overexpression on acute-phase angiogenesis, we performed laser photocoagulation 7 days after subretinal injection of AAV9-Meflin or AAV9-EGFP and evaluated CNV volume 14 days after the laser injury (Fig. 7 A, B). The CNV volume (mean ± SD) was 272,000 ± 187,000 µm³ (n = 33) in the AAV9-Meflin group and 174,000 ± 117,000 µm³ (n = 34) in the AAV9-EGFP group, indicating a significant increase in CNV volume in the Meflin-overexpression group (Welch’s t-test, p = 0.013) (Fig. 7 C). Subsequently, we quantified the volume of subretinal fibrosis in the fibrotic phase (Fig. 7 D, E). The subretinal fibrosis volume (mean ± SD) was 291,000 ± 177,000 µm³ (n = 31) in the AAV9-Meflin group and 306,000 ± 247,000 µm³ (n = 33) in the AAV9-EGFP group, with no significant difference between the two groups (p = 0.776) (Fig. 7 F). We performed additional experiments using a protocol designed to selectively overexpress Meflin during the fibrotic phase, which follows the proliferative phase of CNV. Previous studies have shown that CNV volume peaks at approximately 7 days after laser injury and subsequently regresses [ 39 , 40 ], followed by the development of subretinal fibrosis [ 41 ]. Based on these reports, we administered subretinal injections of AAV9-Meflin or AAV9-EGFP on day 7 after laser photocoagulation (day 0) and evaluated subretinal fibrosis volume on day 35 (Fig. 7 G, H). The results indicated that the subretinal fibrosis volume (mean ± SD) in the AAV9-Meflin group (261,000 ± 103,000 µm³, n = 24) was significantly lower than that in the AAV9-EGFP group (384,000 ± 214,000 µm³, n = 24; p = 0.015) (Fig. 7 I). These results indicate that Meflin overexpression during the subretinal fibrotic phase can significantly suppress fibrotic scar formation. Discussion In the present study, we characterized the intraocular expression patterns of Meflin and demonstrated that Meflin-positive and Meflin-lineage cells accumulate at sites of laser-induced CNV lesions. In addition, we found that AAV-mediated Meflin overexpression increased CNV volume during the acute phase of the tissue repair response. Given that angiogenesis is essential for formation of granulation tissue during the repair of damaged tissues [ 5 , 6 ], these findings indicate that Meflin or Meflin-positive cells may positively regulate tissue repair. In contrast, induction of Meflin overexpression after CNV formation in the laser-induced CNV model suppressed subretinal fibrosis. These findings suggest that Meflin may regulate retinal tissue repair in a temporally dependent manner. In normal mice, Meflin expression was observed in the RPE, ciliary epithelium, and lens epithelium, in fibroblast-like cells in the choroid and subjacent sclera, and in the meningeal cells surrounding the optic nerve. Previous reports have suggested that Meflin is specifically expressed in mesenchymal cells, such as bone marrow-derived mesenchymal stromal cells and fibroblasts, and is largely absent in epithelial cells [ 15 , 19 , 20 ]. However, we identified Meflin expression in multiple intraocular epithelial cell types. Interestingly, these intraocular epithelial cells possess embryological and functional characteristics that distinguish them from epithelial cells in other organs [ 42 ]. For example, under pathological stress, RPE cells undergo epithelial–mesenchymal transition (EMT) and differentiate into myofibroblasts, thereby contributing to the formation of fibroproliferative membranes [ 14 , 43 ]. Furthermore, RPE cells have a high capacity for extracellular matrix production [ 44 ] and the ability to phagocytose shed photoreceptor outer segments [ 45 ]. Similarly, in the lens epithelium, TGF-β-regulated EMT has been implicated in the pathogenesis of anterior subcapsular cataract and posterior capsule opacification [ 46 , 47 ]. Moreover, the ciliary epithelium produces microfibrils, the primary component of the ciliary zonules [ 48 ]. These intraocular epithelia exhibit diverse mesenchymal-like traits and can be regarded as hybrid cell populations positioned at the interface between epithelial and mesenchymal phenotypes. Thus, Meflin expression in these intraocular epithelial cells is consistent with their mesenchymal-like properties. The Meflin lineage-tracing experiments conducted in this study revealed the appearance of Meflin-lineage cells at sites of retinal injury. This finding suggests that Meflin-expressing cells, including intraocular epithelial cells, may proliferate at sites of tissue repair or be recruited there through mechanisms that remain to be determined. Interestingly, tdTomato signals were also detected in the INL and along the ILM. Given that the ILM represents the basement membrane of Müller cells, this signal distribution suggests that Meflin may serve as a marker of Müller cells involved in the tissue repair response. However, further studies are warranted to test this possibility and clarify the role of Meflin in these cells. Notably, Meflin overexpression in the laser-induced CNV model led to an increase in CNV volume alongside suppression of subretinal fibrosis—an outcome that may initially seem counterintuitive. Meflin binds to LOX and inhibits its collagen crosslinking activity, thereby preventing fibrosis of the interstitial tissue [ 30 ]. A soft stroma provides a favorable scaffold for cell migration and tube formation, which may result in dilation of vascular lumens and increased vascular volume. In previous studies conducted using pancreatic cancer models, Meflin expression led to the repair of damaged tissue, increased tumor vessel diameter, and improved drug delivery [ 21 , 30 , 31 ]. In the context of oncology, “vascular normalization” is considered crucial for an effective therapeutic response [ 49 – 51 ]; therefore, therapeutic strategies are being increasingly designed to target formation of functional vascular networks rather than complete suppression of pathological angiogenesis. However, whether the increase in CNV volume observed in the present study is beneficial for retinal tissue repair cannot be concluded based on the current data alone. Future evaluations of vascular function, including assessment of structural integrity and blood flow dynamics, are warranted. The suppressive effect of Meflin overexpression on subretinal fibrosis observed in this study is consistent with the previously reported anti-fibrotic properties of Meflin in other organs [ 16 – 18 ]. As described above, Meflin inhibits fibroblast-to-myofibroblast differentiation by enhancing BMP7 signaling and antagonizing TGF-β signaling [ 16 , 24 ]. In addition, it further restrains excessive fibrotic progression by inhibiting collagen crosslinking [ 30 ]. Similar mechanisms may operate intraocularly, thereby suppressing the formation of subretinal fibrosis. However, further studies conducted using in vivo and in vitro approaches are warranted to clarify the detailed molecular mechanisms underlying these anti-fibrotic effects, particularly the mechanisms that involve retina-specific cell populations and the local microenvironment. No overt ophthalmic phenotype was observed in the Meflin KO mice analyzed in the present study. The absence of apparent abnormalities in ocular morphology or ERG findings suggests the presence of compensatory mechanisms that involve other molecules with overlapping functions. In addition, it is possible that the single-laser injury CNV model used in this study did not induce tissue damage severe enough to unmask the functional consequences of Meflin deficiency. Thus, whether the impact of Meflin deficiency becomes more evident in models of more severe tissue injury, chronic inflammation, or age-related changes remains to be determined. The present study suggests a new paradigm in retinal research by elucidating the functions of Meflin in promoting angiogenesis while suppressing pathological fibrosis. The findings of this study may deepen our understanding of the pathogenesis of nAMD as well as a broad spectrum of intractable fibroproliferative retinal diseases, including PVR and PDR. Furthermore, this study may provide a foundation for the development of novel therapeutic strategies aimed at promoting retinal tissue repair. Methods Study Approval All animal protocols were approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine (approval number: M240018-002). All animal experiments were performed in accordance with Nagoya University’s Animal Facility regulations. The study is reported in accordance with the ARRIVE guidelines. The study was conducted in accordance with the Declaration of Helsinki and relevant institutional guidelines and regulations and was approved by the Ethics Committee of Nagoya University Graduate School of Medicine (approval number: 2013-0010). Written informed consent for the use of surgical specimens for research purposes was obtained from all patients. Human surgical specimens Fibrovascular membranes were obtained from patients who underwent pars plana vitrectomy for PVR or PDR at Nagoya University Hospital (Nagoya, Japan). Membrane tissues excised during surgery and that would otherwise be discarded were collected and processed for histological analyses. Cell culture The human Müller glial cell line MIO-M1 was purchased from e-lucid (University College London, London, UK). The immortalized human retinal pigment epithelial cell line hTERT-RPE1 was purchased from ATCC (Manassas, VA, USA). The human osteosarcoma cell line HS-Os-1 was purchased from RIKEN BioResource Research Center (Tsukuba, Japan). Human dermal fibroblasts fHDF/TERT166 were obtained from Evercyte (Vienna, Austria). Primary human retinal pigment epithelial cells (hRPEpiC; Cat. no. 6540) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). All cells were maintained at 37°C in a humidified incubator with 5% CO₂ and cultured according to the manufacturers’ or providers’ instructions using the recommended media. For the experiments, cells were grown to 90–100% confluence, then protein lysates and total RNA were extracted for Western blotting and qPCR analyses. Western blot analysis Confluent cell monolayers were washed twice with phosphate-buffered saline (PBS) and lysed directly in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, and bromophenol blue). Lysates were collected and briefly sonicated on ice to reduce viscosity. Protein concentrations were determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20–30 µg per lane) were prepared for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were reduced using 2-mercaptoethanol, heated at 100°C for 2 min, and separated using SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes (Merck, Darmstadt, Germany), which were pre-wetted in 100% methanol and equilibrated in transfer buffer containing 10% methanol. Blotting was performed at a constant voltage of 30 V (approximately 200 mA) overnight at 4°C. Membranes were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, incubated with primary antibodies diluted in TBST (0.1% Tween 20), and then with horseradish peroxidase-conjugated secondary antibodies. The primary antibodies used were rabbit anti-Meflin ( ISLR ) (HPA050811; Merck) and mouse anti-β-actin (clone AC-74; Merck). After washing in TBST (5 min, three times), immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection system (Cytiva, Marlborough, MA, USA) and imaged using a LAS-4000 system (GE Healthcare, Chicago, IL, USA). For detection of Meflin, primary antibodies were diluted in Can-Get-Signal Solution 1 (TOYOBO, Osaka, Japan) to enhance antibody–antigen binding. Full-length blots are provided in Supplementary Fig. 2. Quantitative PCR Total RNA was purified from cultured cells using the RNeasy Mini Kit (Cat. no. 74104; QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Purified RNA samples were reverse-transcribed using ReverTra Ace (Cat. no. TRT-101; TOYOBO) with oligo dT and random primers. qPCR of the generated cDNA was performed using TaqMan Gene Expression Master Mix (Cat. no. 4369016; Thermo Fisher Scientific, Waltham, MA, USA) on an Mx3005P thermal cycler (Agilent Technologies, Santa Clara, CA, USA). TaqMan probes/primers for human ISLR (Meflin) and ACTB (Thermo Fisher Scientific) were used according to the manufacturer’s instructions. Relative gene expression was calculated using the comparative threshold cycle (Ct) method and normalized to ACTB . Animals All animal experiments were performed at Nagoya University Graduate School of Medicine. The generation and characterization of Meflin knockout mice have been described previously [ 15 ]. Genomic DNA was extracted from mouse tail biopsies and subjected to polymerase chain reaction (PCR) genotyping. The primer sequences were as follows: PCR1 forward, 5′-GCTGCATTTGAGCTGAGCCTCTGG-3′; PCR1 reverse, 5′-AACCCCTTCCTCCTACATAGTTGG-3′; PCR2 forward, 5′-TGAGGTTAGCCTGGGGACTTCAC-3′; and PCR2 reverse, 5′-GGCTAGAACTCTCAAAGTAGGTCAGG-3′. The generation of Meflin-CreERT2 knock-in mice has been described previously [ 21 ]. For lineage-tracing experiments, the Meflin-CreERT2 mice were crossed with Rosa26-LSL-tdTomato mice (JAX stock no. 007909; The Jackson Laboratory, Bar Harbor, ME, USA) to generate Meflin-CreERT2; Rosa26-LSL-tdTomato mice. Rosa26-LSL-tdTomato mice were obtained from The Jackson Laboratory and housed at Nagoya University Graduate School of Medicine. The animals were maintained under controlled temperature, humidity, and light conditions (12h light/dark cycle) with free access to water and standard chow. Unless otherwise indicated, 8–10-week-old male mice on a C57BL/6J background were used. In situ hybridization RNA in situ hybridization was performed using the RNAscope 2.5 HD Reagent Kit-Brown (Cat. No. 322300; Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer’s protocol. The probes used in this study were RNAscope probe-Mm- Islr (450041; Advanced Cell Diagnostics), probe-Hs- ISLR (455481; Advanced Cell Diagnostics), and the negative control probe- DapB (310043; Advanced Cell Diagnostics). Tissue processing and paraffin embedding for in situ hybridization were performed as previously described [ 23 ] or by Genostaff (Tokyo, Japan). Giemsa staining was performed for counterstaining of C57BL/6J tissues, whereas hematoxylin counterstaining was used for BALB/c tissues. Laser-induced choroidal neovascularization model Laser-induced CNV was generated as previously described [ 36 ] with minor modifications. Mice were anesthetized through intraperitoneal injection of ketamine (37.5 mg/kg) and medetomidine (0.625 mg/kg), with supplemental dosing administered as required. The mice’s pupils were dilated through topical instillation of 0.5% tropicamide and 0.5% phenylephrine (Mydrin-P; Santen, Osaka, Japan). Laser photocoagulation was performed using a Novus Verdi laser system (532 nm; 180 mW; 100 ms; 75 µm spot diameter; Coherent Inc., Santa Clara, CA, USA). Four laser burns were applied per eye at approximately 1–2 disc diameters from the optic nerve head. Lesions that exhibited bubble formation at the time of laser application, indicating rupture of Bruch’s membrane, were included for analysis. Eyes were collected 10–14 days after laser induction for evaluation of CNV and 35 days after induction for analysis of subretinal fibrosis. Analysis of CNV and subretinal fibrosis volume CNV and subretinal fibrosis volumes were quantified from confocal z-stack images. Specimens were prepared as eye cups and subjected to immunofluorescence staining as described below. The lesions were imaged using a confocal microscope (AX R; Nikon, Tokyo, Japan). Horizontal optical sections were acquired at 0.5-µm intervals from the lesion surface to the RPE. The lesion area in each section was measured using Fiji (ImageJ) software (National Institutes of Health, Bethesda, MD, USA), and the sum of the lesion areas across all sections was used as an index of CNV or subretinal fibrosis volume. Imaging and quantification were performed by an investigator masked to group assignments. Electroretinography Electroretinograms (ERGs) were recorded using a commercial Ganzfeld ERG recording system. The mice were dark-adapted overnight, and procedures were performed under dim red light. After induction of anesthesia and pupil dilation were performed as described above, the mice were placed on a heated platform to maintain a stable body temperature. A contact lens electrode was positioned on the cornea, and reference and ground electrodes were placed subcutaneously. Scotopic ERGs were elicited by white flash stimuli ranging from − 7.0 to 0.0 log cd·s/m². Photopic ERGs were recorded following light adaptation on a steady background light, with flash intensities ranging from − 0.5 to 1.5 log cd·s/m². Responses obtained from multiple trials were band-pass filtered and averaged. The a-wave amplitude was measured from baseline to the a-wave trough, whereas the b-wave amplitude was measured from the a-wave trough to the b-wave peak. The b/a ratio for scotopic ERGs and the b-wave amplitude for photopic ERGs were used for quantitative analyses. Lineage tracing Tamoxifen (200 mg/kg; Cat. no. T5648; Merck, Darmstadt, Germany) dissolved in corn oil (C8267; Merck) was administered intraperitoneally to eight-week-old male Meflin-CreERT2; Rosa26-LSL-tdTomato mice three times on alternate days. Laser-induced CNV was performed concurrently with the third tamoxifen injection. Tissues were harvested 5 days after the final injection. Specimens were prepared as eye cups and subjected to immunofluorescence staining as described below. Immunofluorescence staining and confocal imaging The eyes were enucleated and immersion-fixed in 4% paraformaldehyde (PFA) for 30 min. After removal of the anterior segment, including the lens and vitreous, the eye cups were further fixed in 4% PFA for 1.5 h at 4°C, rinsed with PBS, cryoprotected in 30% sucrose overnight at 4°C, embedded in optimal cutting temperature (OCT) compound, and cryosectioned at 10 µm using a cryostat (CM3050 S; Leica Microsystems, Wetzlar, Germany). Sections were washed with PBS, blocked with 1% bovine serum albumin (BSA) for 30 min, and incubated with primary antibodies overnight at 4°C. The sections were then incubated with Alexa Fluor-conjugated secondary antibodies (1:400; Thermo Fisher Scientific) for 1 h at room temperature, washed with PBS, and mounted with Immu-Mount (Epredia, Kalamazoo, MI, USA). RPE flatmounts were prepared for immunofluorescence staining for the laser-induced CNV, subretinal fibrosis, and lineage-tracing experiments. After preparation of eye cups, the neural retina was gently removed, and the tissues were fixed in 4% PFA for 90 min at 4°C. Samples were washed with PBS, blocked in PBST (PBS containing 0.5% Triton X-100) with 1% BSA, and incubated with primary antibodies overnight at 4°C. After washing, the samples were incubated with Alexa Fluor-conjugated secondary antibodies (1:400; Thermo Fisher Scientific) for 1 h at room temperature. Fluorescence images were acquired using a confocal microscope (AX R; Nikon). The primary antibodies were rabbit anti-RFP (polyclonal, 1:1,000; 600-401-379; Rockland Immunochemicals, Limerick, PA, USA), rabbit anti-collagen I and III (polyclonal, 1:200; ab34710; Abcam, Cambridge, UK), and rat anti-mouse CD31 (monoclonal, 1:200; 550274; BD Biosciences, Franklin Lakes, NJ, USA). AAV vectors and subretinal injection Recombinant AAV9 vectors encoding Flag-tagged mouse Islr (Flag tag inserted immediately downstream of the endogenous signal peptide; AAV9-CBh-Islr) or Flag-tagged enhanced green fluorescent protein (AAV9-CBh-EGFP), both driven by the CBh promoter, were purchased from VectorBuilder (Chicago, IL, USA). The viral titers were determined using qPCR and adjusted to a final concentration of 1.0 × 10 12 viral genomes (vg) / mL in PBS. For the subretinal injection, the mice were anesthetized and their pupils were dilated as described above. A total volume of 2 µL of the vector solution (total dose: 2.0 × 10 9 vg) was injected into the subretinal space using a trans-scleral approach with a 33-gauge needle (Ito Corporation, Shizuoka, Japan). Successful delivery into the subretinal space was confirmed by visual observation of a subretinal bleb. Statistical analysis Statistical analyses were performed using Python (version 3.12.11). Data are presented as mean ± standard deviation (n = number of samples). Group comparisons were conducted using Welch’s t-test. A two-sided p < 0.05 was considered statistically significant. Investigators were blinded to group assignments during data analysis. Graphs were generated using standard Python libraries. Declarations Acknowledgements We are grateful to Kozo Uchiyama, Kayoko Endo and Yasuko Sobue (Department of Pathology, Nagoya University Graduate School of Medicine) for their assistance. We also appreciate the support of the staff of the Division for Medical Research Engineering for the use of the confocal microscope AX R, and the Division of Experimental Animals (Nagoya University) for their help with the animal experiments. Our thanks also go to our colleagues at the Department of Pathology and the Department of Ophthalmology, Nagoya University Graduate School of Medicine, for their helpful discussions and technical assistance. We would like to thank Editage (Cactus Communications) for English language editing. Funding This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grants 22K07000 to S.M., 22H02848 and 22K18390 to A.E.); the Japan Agency for Medical Research and Development (AMED) (grants JP24gm1210009 and JP25ama221333 to A.E.); the Chukyo Longevity Medical and Promotion Foundation (to S.M.); the Naito Foundation (to A.E.); the Takamatsunomiya Cancer Foundation (to A.E.); the Toyoaki Foundation (to A.E.); and the Japan Science and Technology Agency (JST) (grant number JPMJSP2125 to D.O.). Author contributions D.O. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. S.M. and A.E. supervised the project, provided experimental guidance, interpreted the data, and revised the manuscript. Y.M., R.A., Y.S., and N.E. assisted with the experiments and data analysis. K.K. and K.Y. provided advice on the experimental designs. H.U. and H.S. collected clinical samples. H.K. and K.M.N. contributed to the study design, data interpretation, and manuscript revision. All the authors discussed the results and approved the final version of the manuscript. Correspondence and requests for materials should be addressed to S.M. or A.E. Data availability The data generated in this study are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. References Pastor, J. C. et al. Proliferative vitreoretinopathy: A new concept of disease pathogenesis and treatment. Prog Retin Eye Res. 51 , 125–155. https://doi.org/10.1016/j.preteyeres.2015.07.005 (2016). Cheung, N., Mitchell, P. & Wong, T. Y. Diabetic retinopathy. Lancet 376 , 124–136. https://doi.org/10.1016/S0140-6736(09)62124-3 (2010). Lim, L. S., Mitchell, P., Seddon, J. M., Holz, F. G. & Wong, T. Y. Age-related macular degeneration. 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Drug Discov . 10 , 417–427. https://doi.org/10.1038/nrd3455 (2011). Goel, S. et al. Normalization of the vasculature for treatment of cancer and other diseases. Physiol. Rev. 91 , 1071–1121. https://doi.org/10.1152/physrev.00038.2010 (2011). Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation20260227.pdf Supplementary Figure 1. AAV9-mediated overexpression of Meflin in the retina–choroid complex. Intravitreal injection of AAV9-Meflin led to Meflin overexpression in the ganglion cell layer (pink arrows), the inner nuclear layer (pink arrowheads), and the retinal pigment epithelium (yellow arrows) in the AAV9-Meflin group, but not in the AAV9-EGFP controls. Boxed areas are further magnified in the right panels. Scale bars: 200 μm. GCL, ganglion cell layer; INL, inner nuclear layer; RPE, retinal pigment epithelium; Ch, choroid; Sc, sclera. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8930515","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":614793172,"identity":"7dd2fea0-1077-4988-bd9a-b7d0b601dd87","order_by":0,"name":"Daishi Okuda","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Daishi","middleName":"","lastName":"Okuda","suffix":""},{"id":614793173,"identity":"270f200f-f93c-49aa-b1de-0abaf74e5a0c","order_by":1,"name":"Shinji Mii","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Shinji","middleName":"","lastName":"Mii","suffix":""},{"id":614793174,"identity":"014557c2-7f53-47fd-91f3-2d30994b17c9","order_by":2,"name":"Yuki Miyai","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Miyai","suffix":""},{"id":614793175,"identity":"5c84d441-bb68-4b8a-b1d5-5fbca30feedb","order_by":3,"name":"Katsuhiro Kato","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Katsuhiro","middleName":"","lastName":"Kato","suffix":""},{"id":614793176,"identity":"6af37c18-4629-4582-849e-0a8aad014d03","order_by":4,"name":"Ryota Ando","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Ryota","middleName":"","lastName":"Ando","suffix":""},{"id":614793177,"identity":"12226871-b33b-4d01-93be-37f8a0389ad6","order_by":5,"name":"Yukihiro Shiraki","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Yukihiro","middleName":"","lastName":"Shiraki","suffix":""},{"id":614793178,"identity":"1bd169a1-09f0-4ea5-9a1e-72197b86f7fa","order_by":6,"name":"Nobutoshi Esaki","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Nobutoshi","middleName":"","lastName":"Esaki","suffix":""},{"id":614793179,"identity":"5efb81c2-2d28-4cd9-8589-0c7e9e2e6f66","order_by":7,"name":"Kazuhisa Yamada","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Kazuhisa","middleName":"","lastName":"Yamada","suffix":""},{"id":614793180,"identity":"d050a506-4822-4930-ab59-ebd780652497","order_by":8,"name":"Hideyuki Shimizu","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Hideyuki","middleName":"","lastName":"Shimizu","suffix":""},{"id":614793181,"identity":"3a8632aa-8fc5-4339-80c4-e8e684d2f106","order_by":9,"name":"Hiroaki Ushida","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Hiroaki","middleName":"","lastName":"Ushida","suffix":""},{"id":614793182,"identity":"ac3ef4f3-290a-42ff-a938-3d11f86e2288","order_by":10,"name":"Hiroki Kaneko","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Kaneko","suffix":""},{"id":614793183,"identity":"94f96361-e957-46a0-b60d-a2836a20d5e6","order_by":11,"name":"Koji M. 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Boxed areas \u003cstrong\u003e(a, a', b and b')\u003c/strong\u003e in the left panels are magnified in the right panels. In both cases, Meflin mRNA signals were detected in fibroblast-like stromal cells (arrowheads). Scale bars: 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Western blot analysis of Meflin expression in cultured retinal cells. No clear Meflin band was detected in the Müller glial cell line MIO-M1 or the RPE cell line hTERT-RPE1. However, a distinct band was observed in primary RPE cells (hRPEpiC). HS-Os-1 and human dermal fibroblasts (fHDF) were used as positive controls. Blots were cropped for presentation; full-length blots are provided in Supplementary Fig. 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e qPCR analysis of Meflin mRNA expression in cultured retinal cells. Meflin mRNA expression was not detected in MIO-M1 or hTERT-RPE1 but was detected in hRPEpiC. The expression levels are shown as log2 fold-change calculated using the 2\u003csup\u003e−ΔΔCt\u003c/sup\u003e method relative to the positive-control cell line (fHDF). The Ct values were normalized to \u003cem\u003eACTB\u003c/em\u003e. Bars show mean ± SD; dots show individual replicates (n = 3).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/959789cb49140299254fbbbc.png"},{"id":105909071,"identity":"c15373a8-f68c-4e91-a8f1-b720da05c5db","added_by":"auto","created_at":"2026-04-01 10:41:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2549149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of Meflin mRNA in the murine eye\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e In situ hybridization (ISH) for Meflin mRNA in ocular sections obtained from wild-type (WT) C57BL/6J mice (upper panels). Meflin knockout (KO) mice were used as negative controls (lower panels). The anterior segment \u003cstrong\u003e(a, a′)\u003c/strong\u003e, the optic nerve area \u003cstrong\u003e(b, b′)\u003c/strong\u003e, and the retina \u003cstrong\u003e(c, c′)\u003c/strong\u003e are shown. Boxed areas in the left panels are magnified in the right or lower panels.\u003cstrong\u003e \u003c/strong\u003eA simplified schematic illustrating the retinal layers and adjacent tissues is shown beneath the low-magnification image. Sections were counterstained with Giemsa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e ISH for Meflin mRNA (upper panels) and negative control (\u003cem\u003eDapB, \u003c/em\u003elower panels) probes in ocular sections obtained from WT BALB/c mice. Sections were counterstained with hematoxylin\u003cstrong\u003e.\u003c/strong\u003e In both \u003cstrong\u003e(A)\u003c/strong\u003e and \u003cstrong\u003e(B)\u003c/strong\u003e, Meflin mRNA signals were detected in the ciliary epithelium (yellow arrows), lens epithelium (yellow arrowheads), meningeal cells of the optic nerve (green arrows), retinal pigment epithelium (red arrows), and fibroblast-like mesenchymal cells in the choroid and subjacent sclera (red arrowheads).\u003c/p\u003e\n\u003cp\u003eScale bars: 100 μm.\u003c/p\u003e\n\u003cp\u003eCB, ciliary body; LE, lens epithelium; ON, optic nerve; ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; Ch, choroid; Sc, sclera.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/039ad37af8f36d7f2402e7a1.png"},{"id":105908971,"identity":"c1d71097-ea59-4206-b24f-928085b9be19","added_by":"auto","created_at":"2026-04-01 10:40:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":766067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMeflin deficiency does not affect ocular morphology or physiological function in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eHematoxylin and eosin (H\u0026amp;E)-stained ocular sections from WT and Meflin KO mice. The anterior segment \u003cstrong\u003e(a, a′)\u003c/strong\u003e and posterior pole \u003cstrong\u003e(b, b′)\u003c/strong\u003e in the left panels are magnified in the corresponding right panels. No apparent morphological differences were observed between WT and KO mice. Scale bars: 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eRepresentative electroretinography (ERG) traces recorded under scotopic conditions (rod responses).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003eQuantification of b/a-wave ratios of scotopic ERG responses show no significant differences between WT and KO mice (n = 10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003eRepresentative ERG traces recorded under photopic conditions (cone responses).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003eQuantification of b-wave amplitudes of photopic ERG responses show no significant differences between WT and KO mice (n = 10).\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/54511a092858ba470acaa16d.png"},{"id":105909069,"identity":"33f54cb7-3ef2-4729-846a-5df972ffad5c","added_by":"auto","created_at":"2026-04-01 10:41:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of Meflin-lineage cells in laser-induced choroidal neovascularization (CNV) lesions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic of the \u003cem\u003eMeflin-CreERT2; Rosa26-LSL-tdTomato\u003c/em\u003e reporter system used in the study. Following tamoxifen administration, Meflin-lineage cells are permanently labeled with the red fluorescent protein tdTomato.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eMeflin-CreERT2; Rosa26-LSL-tdTomato\u003c/em\u003e reporter mice were subjected to laser-induced CNV injuries. Tamoxifen was administered via intraperitoneal injection on days 0, 2, and 4, followed by laser photocoagulation on day 4 and harvesting of ocular tissues on day 10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Immunofluorescence of RPE–choroid flatmounts showing the presence of tdTomato\u003csup\u003e+\u003c/sup\u003e Meflin-lineage cells accumulating within CD31-positive CNV lesions in the Meflin reporter mice (upper panels), but not in the control mice (\u003cem\u003eMeflin-CreERT2\u003c/em\u003e\u003csup\u003e+/–\u003c/sup\u003e) (lower panels).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Immunofluorescence staining of retinal sections from CNV regions. tdTomato-positive Meflin-lineage cells are observed in the inner limiting membrane (white arrow), the inner nuclear layer (yellow arrow), and the choroid (white arrowheads) in the Meflin reporter mice (upper panels), but not in control mice. Boxed areas are magnified in right panels. Nuclei were visualized using DAPI staining (blue).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Immunofluorescence staining of the intact areas of the retina. tdTomato-positive Meflin-lineage cells are absent in both the Meflin reporter mice (upper panels) and control mice (lower panels).\u003c/p\u003e\n\u003cp\u003eScale bars: 200 μm.\u003c/p\u003e\n\u003cp\u003eILM, inner limiting membrane; INL, inner nuclear layer; ONL, outer nuclear layer; Ch, choroid.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/f73efb43748ff7f016d9d129.png"},{"id":105909073,"identity":"6ba186c3-fa2b-4315-9e9b-442531e84fc3","added_by":"auto","created_at":"2026-04-01 10:41:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1958548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the effect of Meflin deficiency on CNV and fibrosis in a laser-induced CNV model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A–C)\u003c/strong\u003e WT and Meflin KO mice were subjected to laser-induced CNV injuries. A schematic of the experimental protocol \u003cstrong\u003e(A)\u003c/strong\u003e, representative RPE–choroid flatmount images stained for CD31 \u003cstrong\u003e(B)\u003c/strong\u003e, and quantification of CNV volumes using confocal microscopy \u003cstrong\u003e(C)\u003c/strong\u003e are shown. There was no significant difference between the WT and KO mice (WT, n = 19; Meflin KO, n = 24). Scale bars: 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D–F)\u003c/strong\u003e WT and Meflin KO mice were subjected to laser-induced CNV injuries, followed by analysis of subretinal fibrosis. A schematic of the experimental protocol \u003cstrong\u003e(D)\u003c/strong\u003e, representative RPE–choroid flatmount images stained for type I and III collagen \u003cstrong\u003e(E)\u003c/strong\u003e, and quantification of subretinal fibrosis volumes by confocal microscopy \u003cstrong\u003e(F)\u003c/strong\u003e are shown. There was no significant difference between the WT and KO mice (WT, n = 18; Meflin KO, n = 17). Scale bars: 200 μm.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/bed252a4d8d829f335a99767.png"},{"id":105909041,"identity":"b176ac6f-a03e-419f-9f3c-a98ea00b637a","added_by":"auto","created_at":"2026-04-01 10:40:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":798923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAAV9-mediated overexpression of Meflin in the retina–choroid complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWT mice were injected subretinally with AAV9-Meflin to induce ocular-specific overexpression of Meflin or with AAV9-EGFP as a control. In situ hybridization for Meflin mRNA performed 7 days after the AAV9 injection shows elevated Meflin mRNA signals in the cells of the inner nuclear layer (pink arrows), the retinal pigment epithelium (yellow arrows), and the choroid and subjacent sclera (yellow arrowheads) in AAV9-Meflin-injected eyes, but not in AAV9-EGFP controls. Boxed areas are further magnified in the right panel. Scale bars: 200 μm.\u003c/p\u003e\n\u003cp\u003eINL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; Ch, choroid; Sc, sclera.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/2e1e2c548d4814256cfbb066.png"},{"id":105909150,"identity":"f8f31ba3-1fa5-42ad-aefd-8447e3912894","added_by":"auto","created_at":"2026-04-01 10:41:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":466300,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Meflin overexpression on choroidal neovascularization and subretinal fibrosis in a retinal disease model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e WT mice were administered AAV9-Meflin or AAV9-EGFP (control) via subretinal injection, followed by laser photocoagulation. Eyes were collected 14 days after induction of the laser injuries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003eRepresentative RPE–choroid flatmount images stained for CD31 showing CNV lesions in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003eQuantification of CNV volumes demonstrates a significant increase in Meflin-overexpressing eyes compared with EGFP controls (p = 0.013; control, n = 34; Meflin, n = 33). Scale bars: 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e AAV was injected subretinally before laser photocoagulation, and eyes were collected 35 days after induction of the laser injuries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003eRepresentative RPE–choroid flatmount images immunostained for type I and III collagen showing fibrotic lesions in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003eQuantification of subretinal fibrosis volumes shows no significant difference between Meflin-overexpressing and control eyes (p = 0.776; control, n = 33; Meflin, n = 31). Scale bars: 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Laser photocoagulation was performed before AAV subretinal injection, and eyes were collected 35 days after induction of the laser injuries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003eRepresentative RPE–choroid flatmount images immunostained for type I and III collagen showing fibrotic lesions in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003eQuantification of subretinal fibrosis volumes demonstrates a significant decrease in the Meflin-overexpressing group compared with controls (p = 0.015; n = 24 per group). Scale bars: 200 μm.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/25a15c66cfbda7eb1705a0eb.png"},{"id":105911191,"identity":"9e314ad8-9314-4287-ad85-0e90dcb3aca0","added_by":"auto","created_at":"2026-04-01 10:52:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10149264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/c2644198-0652-417e-8cd9-770372084912.pdf"},{"id":105908952,"identity":"4a31e160-74d3-416e-ac4c-a54fe6f55991","added_by":"auto","created_at":"2026-04-01 10:40:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":369322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. AAV9-mediated overexpression of Meflin in the retina–choroid complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntravitreal injection of AAV9-Meflin led to Meflin overexpression in the ganglion cell layer (pink arrows), the inner nuclear layer (pink arrowheads), and the retinal pigment epithelium (yellow arrows) in the AAV9-Meflin group, but not in the AAV9-EGFP controls. Boxed areas are further magnified in the right panels. Scale bars: 200 μm.\u003c/p\u003e\n\u003cp\u003eGCL, ganglion cell layer; INL, inner nuclear layer; RPE, retinal pigment epithelium; Ch, choroid; Sc, sclera.\u003c/p\u003e","description":"","filename":"SupplementaryInformation20260227.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8930515/v1/65436fef6b63f67a29b2b080.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Possible involvement of the mesenchymal cell marker Meflin in angiogenesis promotion and fibrosis suppression after retinal injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRetinal diseases such as proliferative vitreoretinopathy (PVR) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], proliferative diabetic retinopathy (PDR) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and age-related macular degeneration (AMD) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] lead to severe vision loss and substantial reduction in quality of life. These disorders are difficult to treat and share a common pathological feature: retinal dysfunction associated with angiogenesis and excessive fibrosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Angiogenesis and fibrosis are essential for tissue repair and maintenance of homeostasis after injury [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the aforementioned retinal diseases are characterized by prolonged intraocular neovascular responses and fibrotic scar formation, leading to irreversible destruction and remodeling of retinal architecture [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Accordingly, elucidating the mechanisms that govern tissue repair and its regulation in the retina remains an important challenge in the development of therapeutic strategies for diverse retinal disorders.\u003c/p\u003e \u003cp\u003eAMD is a representative disease in which pathological tissue repair is a critical determinant of visual prognosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. AMD is broadly categorized into two major subtypes: dry AMD and neovascular (wet) AMD (nAMD). In nAMD, choroidal neovascularization (CNV) causes subretinal fluid exudation, hemorrhage, and subsequent scar formation, leading to central vision loss [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The introduction of anti-vascular endothelial growth factor (VEGF) therapy has markedly improved visual outcomes for patients with nAMD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, effective management of nAMD is hindered by clinical challenges such as reduced therapeutic responsiveness during long-term treatment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDrusen, which are deposits beneath the retinal pigment epithelium (RPE), are believed to cause chronic inflammation in the early stages of nAMD. This promotes upregulation of angiogenic factors, recruitment of immune cells and fibroblasts, and progression of CNV [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although anti-VEGF therapy effectively suppresses neovascular growth, it does not resolve the underlying persistent inflammation. Under conditions of persistent inflammation, ongoing tissue injury and a sustained reparative response can drive progression of fibrosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These observations suggest that, in addition to targeting CNV, understanding the molecular mechanisms underlying chronic inflammation and prolonged tissue repair is essential for the developing effective therapeutic strategies for nAMD. However, the molecular basis of retinal tissue repair remains incompletely defined. Although mesenchymal cells, including fibroblasts and perivascular cells, play central roles in fibrosis under pathological conditions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], the molecular mechanisms that regulate the complex cellular networks formed by these cells remain unclear. Therefore, identifying key molecular regulators that govern the balance between repair and fibrosis in retinal tissue is crucial.\u003c/p\u003e \u003cp\u003eTo provide novel insights into the mechanisms underlying retinal tissue repair, we focused on Meflin, a mesenchymal stromal cell and fibroblast marker with anti-fibrotic properties [\u003cspan additionalcitationids=\"CR16 CR17 CR18 CR19\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Meflin is a glycosylphosphatidylinositol (GPI)-anchored membrane protein encoded by the \u003cem\u003eISLR/Islr\u003c/em\u003e (immunoglobulin superfamily containing leucine-rich repeat) gene. It has been identified as a marker of tumor-restraining cancer-associated fibroblasts (rCAFs) [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and has suppressed fibrotic progression in mouse models of cardiac, pulmonary, and renal fibrosis [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Mechanistically, Meflin enhances bone morphogenetic protein (BMP) signaling and suppresses transforming growth factor-β (TGF-β) signaling through its interaction with BMP7 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Meflin also inhibits collagen crosslinking by binding to lysyl oxidase (LOX), thereby limiting tissue stiffening [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, our cancer research demonstrated that Meflin positively regulates tumor vascularization, which improves drug delivery and antitumor immune responses [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Moreover, Meflin-positive CAFs produce the inflammatory chemokine Chemerin (Rarres2), which induces polarization of macrophages to the M1-like phenotype [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Although the Meflin gene was originally cloned in 1997 and its expression in the retina was noted [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], its precise localization within the eye and its functional roles in retinal tissue repair responses remain unexplored. In this study, we characterized the detailed expression patterns of Meflin in the eye and investigated its role in retinal tissue repair using a laser-induced CNV mouse model.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExpression of Meflin in human fibrovascular membranes and cultured cells\u003c/h2\u003e \u003cp\u003eTo determine the involvement of Meflin in human retinal diseases, we first analyzed its expression patterns in clinical surgical specimens. In situ hybridization (ISH) was performed to detect \u003cem\u003eISLR\u003c/em\u003e mRNA (hereafter referred to as Meflin mRNA) in fibrovascular membranes excised from patients who underwent vitrectomy for PVR or PDR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). In both types of specimens (PVR and PDR), Meflin mRNA signals were detected in fibroblast-like stromal cells within the fibrovascular membranes, suggesting the involvement of Meflin in the pathogenesis of these retinopathies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the specific cell types that express Meflin in the human retina, we subsequently analyzed its expression in human RPE cells and M\u0026uuml;ller glial cells, both of which contribute to tissue repair and formation of fibrovascular membranes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Specifically, we conducted Western blotting and quantitative polymerase chain reaction (qPCR) to evaluate Meflin protein and Meflin mRNA expression, respectively, using the human RPE cell line hTERT-RPE1, primary human RPE cells (hRPEpiC), and the human M\u0026uuml;ller glial cell line MIO-M1. Western blotting revealed a distinct Meflin protein band only in hRPEpiC, whereas no bands were detected in MIO-M1 or hTERT-RPE1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Similarly, qPCR analysis revealed Meflin mRNA expression only in hRPEpiC, whereas the results for both MIO-M1 and hTERT-RPE1 remained below the limit of detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeflin expression in the murine eye\u003c/h3\u003e\n\u003cp\u003eWe investigated Meflin expression patterns in the murine eye by conducting ISH for Meflin mRNA using ocular tissue sections obtained from C57BL/6J wild-type mice, with Meflin-deficient (KO) mice [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] analyzed in parallel as a negative control. The ISH revealed Meflin mRNA signals in meningeal cells surrounding the optic nerve, ciliary epithelium, RPE, lens epithelium, and fibroblast-like mesenchymal cells in the choroid and subjacent sclera (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo avoid signal masking by the melanin pigment in C57BL/6J mice, we performed the same analyses using albino BALB/c wild-type mice, with a \u003cem\u003eDapB\u003c/em\u003e probe as a negative control. In the BALB/c mice, Meflin mRNA signals were detected in the same regions as those in C57BL/6J mice. Notably, the signals observed in the ciliary epithelium, RPE, and choroidal fibroblast-like mesenchymal cells were visualized more clearly in the BALB/c mice than in the C57BL/6J mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Between the two layers of the ciliary epithelium, Meflin mRNA expression was detected mainly in the basal layer cells that correspond to the pigmented epithelium in C57BL/6J mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eBa). These findings differ considerably from previous reports of Meflin expression being largely restricted to fibroblasts in other organs [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], highlighting the unique characteristics of intraocular epithelial cells. In contrast, the Meflin expression patterns detected in optic nerve meningeal cells and choroidal fibroblast-like mesenchymal cells are consistent with the expression patterns reported for other organs [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], suggesting the potential involvement of Meflin-positive fibroblast-like cells in tissue repair and fibrotic responses in these regions.\u003c/p\u003e\n\u003ch3\u003eMeflin deficiency does not affect ocular structure or physiological function\u003c/h3\u003e\n\u003cp\u003eWe compared the eyes of Meflin KO mice with those of wild-type mice to determine the role of Meflin in the maintenance of ocular architecture and physiological function. Hematoxylin and eosin (H\u0026amp;E) staining of ocular sections obtained from 10-week-old mice revealed no significant histological abnormalities or disruption of the retinal laminar structure in Meflin KO mice compared with wild-type mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that Meflin is expressed in multiple intraocular tissues including the RPE, we evaluated retinal electrophysiological function using electroretinography (ERG). ERGs of the wild-type and Meflin KO mice were recorded under scotopic (rod-dominant) and photopic (cone-dominant) conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D). The b/a ratio (b-wave amplitude relative to a-wave amplitude) in the scotopic ERG and the b-wave amplitude in the photopic ERG were used as functional readouts. No significant differences in these parameters were observed between the wild-type and Meflin KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, E). These results indicate that Meflin is not critically involved in maintaining the basic structure or physiological function of the eye.\u003c/p\u003e\n\u003ch3\u003eMeflin-lineage cells accumulate at sites of retinal injury\u003c/h3\u003e\n\u003cp\u003eTo investigate the involvement of Meflin-positive cells in angiogenesis and tissue remodeling following retinal injury, we subjected Meflin reporter mice to laser-induced CNV injuries [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and performed Meflin lineage-tracing experiments. To visualize Meflin-lineage cells, we utilized \u003cem\u003eMeflin-CreERT2; Rosa26-LSL-tdTomato\u003c/em\u003e mice, generated by crossing a line expressing tamoxifen-inducible CreERT2 under the control of the Meflin promoter (\u003cem\u003eMeflin-CreERT2\u003c/em\u003e) with a reporter line harboring a \u003cem\u003eloxP-STOP-loxP-tdTomato\u003c/em\u003e cassette at the \u003cem\u003eRosa26\u003c/em\u003e locus (\u003cem\u003eRosa26-LSL-tdTomato\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Following the labeling of Meflin-positive cells with tdTomato via tamoxifen administration, laser photocoagulation was performed and CNV lesions were subsequently evaluated using RPE\u0026ndash;choroid flatmounts and frozen ocular sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSignificant accumulation of tdTomato-positive Meflin-lineage cells was identified within the CNV lesions in the Meflin reporter mice, whereas no tdTomato signal was detected in the control (\u003cem\u003eMeflin-CreERT2\u003c/em\u003e\u003csup\u003e+/\u0026minus;\u003c/sup\u003e) mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Evaluation of the frozen sections revealed tdTomato fluorescence in the internal limiting membrane (ILM), the inner nuclear layer (INL), and the choroid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). No tdTomato-positive cells were observed in the intact areas of the retina in the reporter and control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results suggest that Meflin-lineage cells are recruited to sites of tissue repair or expand there in response to retinal injury. Although Meflin expression was not detected in the MIO-M1 cell line in vitro, tdTomato signals were localized along the ILM in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). As the ILM represents the basement membrane of M\u0026uuml;ller cells, this pattern is consistent with the possibility that M\u0026uuml;ller cells, which proliferate during the injury response, contribute at least in part to the Meflin-lineage cell population.\u003c/p\u003e\n\u003ch3\u003eMeflin deficiency does not affect tissue repair outcomes in a laser-induced CNV model\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of Meflin in angiogenesis and tissue remodeling accompanied by excessive fibrosis, we subjected Meflin KO mice to laser-induced CNV injuries and quantified CNV and subretinal fibrosis volume.\u003c/p\u003e \u003cp\u003eCNV volume on RPE\u0026ndash;choroid flatmounts was quantified using immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). The CNV volume (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) was 252,000\u0026thinsp;\u0026plusmn;\u0026thinsp;164,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;19) in wild-type mice and 165,000\u0026thinsp;\u0026plusmn;\u0026thinsp;117,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;24) in Meflin KO mice. Although the Meflin KO mice tended to have lower CNV volumes, Welch\u0026rsquo;s t-test revealed no significant difference between the two groups (p\u0026thinsp;=\u0026thinsp;0.060) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we quantified the volume of subretinal fibrosis in the fibrotic phase of the laser-induced CNV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). The subretinal fibrosis volume (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) was 264,000\u0026thinsp;\u0026plusmn;\u0026thinsp;138,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;17) in the Meflin KO mice and 261,000\u0026thinsp;\u0026plusmn;\u0026thinsp;106,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;18) in the wild-type mice, with no significant difference between two groups (Welch\u0026rsquo;s t-test, p\u0026thinsp;=\u0026thinsp;0.942) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Collectively, these results indicate that Meflin deficiency does not cause detectable changes in CNV or subretinal fibrosis formation following laser-induced injury.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of an adeno-associated virus-mediated intraocular Meflin overexpression model\u003c/h2\u003e \u003cp\u003eWe previously demonstrated that Meflin expression in fibroblasts confers tumor-suppressive and anti-fibrotic functions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We established an adeno-associated virus (AAV)-mediated overexpression model to examine whether intraocular Meflin overexpression influences CNV and fibrosis formation after retinal injury. We generated an AAV9-CBh-Islr (hereafter referred to as AAV9-Meflin) vector using the AAV9 serotype, which exhibits broad cellular tropism [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and the strong ubiquitous CBh promoter [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. An AAV9-CBh-EGFP (hereafter AAV9-EGFP) vector was used as a control.\u003c/p\u003e \u003cp\u003eSeven days after subretinal injection of AAV9-Meflin or AAV9-EGFP into the wild-type mice, their eyes were harvested to assess Meflin expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The AAV9-Meflin-injected eyes showed markedly increased Meflin mRNA signals in the retina and RPE, as well as in fibroblast-like cells in the choroid and the subjacent sclera. Within the retina, strong Meflin expression was observed particularly in the INL. In contrast, no appreciable increase in Meflin expression was observed in these regions in AAV9-EGFP-injected eyes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also evaluated intravitreal delivery of AAV9-Meflin; however, the transduction efficiency in the RPE and choroidal cells was lower than that achieved via subretinal injection (Supplementary Fig.\u0026nbsp;1). Therefore, subretinal administration was employed for all subsequent experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeflin overexpression regulates angiogenesis and fibrosis after retinal injury\u003c/h3\u003e\n\u003cp\u003eWe examined the effects of AAV9-mediated Meflin overexpression on CNV growth and subretinal fibrosis in the laser-induced CNV model.\u003c/p\u003e \u003cp\u003eTo assess the impact of Meflin overexpression on acute-phase angiogenesis, we performed laser photocoagulation 7 days after subretinal injection of AAV9-Meflin or AAV9-EGFP and evaluated CNV volume 14 days after the laser injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). The CNV volume (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) was 272,000\u0026thinsp;\u0026plusmn;\u0026thinsp;187,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;33) in the AAV9-Meflin group and 174,000\u0026thinsp;\u0026plusmn;\u0026thinsp;117,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;34) in the AAV9-EGFP group, indicating a significant increase in CNV volume in the Meflin-overexpression group (Welch\u0026rsquo;s t-test, p\u0026thinsp;=\u0026thinsp;0.013) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, we quantified the volume of subretinal fibrosis in the fibrotic phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E). The subretinal fibrosis volume (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) was 291,000\u0026thinsp;\u0026plusmn;\u0026thinsp;177,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;31) in the AAV9-Meflin group and 306,000\u0026thinsp;\u0026plusmn;\u0026thinsp;247,000 \u0026micro;m\u0026sup3; (n\u0026thinsp;=\u0026thinsp;33) in the AAV9-EGFP group, with no significant difference between the two groups (p\u0026thinsp;=\u0026thinsp;0.776) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eWe performed additional experiments using a protocol designed to selectively overexpress Meflin during the fibrotic phase, which follows the proliferative phase of CNV. Previous studies have shown that CNV volume peaks at approximately 7 days after laser injury and subsequently regresses [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], followed by the development of subretinal fibrosis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Based on these reports, we administered subretinal injections of AAV9-Meflin or AAV9-EGFP on day 7 after laser photocoagulation (day 0) and evaluated subretinal fibrosis volume on day 35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG, H). The results indicated that the subretinal fibrosis volume (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) in the AAV9-Meflin group (261,000\u0026thinsp;\u0026plusmn;\u0026thinsp;103,000 \u0026micro;m\u0026sup3;, n\u0026thinsp;=\u0026thinsp;24) was significantly lower than that in the AAV9-EGFP group (384,000\u0026thinsp;\u0026plusmn;\u0026thinsp;214,000 \u0026micro;m\u0026sup3;, n\u0026thinsp;=\u0026thinsp;24; p\u0026thinsp;=\u0026thinsp;0.015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). These results indicate that Meflin overexpression during the subretinal fibrotic phase can significantly suppress fibrotic scar formation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we characterized the intraocular expression patterns of Meflin and demonstrated that Meflin-positive and Meflin-lineage cells accumulate at sites of laser-induced CNV lesions. In addition, we found that AAV-mediated Meflin overexpression increased CNV volume during the acute phase of the tissue repair response. Given that angiogenesis is essential for formation of granulation tissue during the repair of damaged tissues [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], these findings indicate that Meflin or Meflin-positive cells may positively regulate tissue repair. In contrast, induction of Meflin overexpression after CNV formation in the laser-induced CNV model suppressed subretinal fibrosis. These findings suggest that Meflin may regulate retinal tissue repair in a temporally dependent manner.\u003c/p\u003e \u003cp\u003eIn normal mice, Meflin expression was observed in the RPE, ciliary epithelium, and lens epithelium, in fibroblast-like cells in the choroid and subjacent sclera, and in the meningeal cells surrounding the optic nerve. Previous reports have suggested that Meflin is specifically expressed in mesenchymal cells, such as bone marrow-derived mesenchymal stromal cells and fibroblasts, and is largely absent in epithelial cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, we identified Meflin expression in multiple intraocular epithelial cell types. Interestingly, these intraocular epithelial cells possess embryological and functional characteristics that distinguish them from epithelial cells in other organs [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. For example, under pathological stress, RPE cells undergo epithelial\u0026ndash;mesenchymal transition (EMT) and differentiate into myofibroblasts, thereby contributing to the formation of fibroproliferative membranes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Furthermore, RPE cells have a high capacity for extracellular matrix production [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and the ability to phagocytose shed photoreceptor outer segments [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Similarly, in the lens epithelium, TGF-β-regulated EMT has been implicated in the pathogenesis of anterior subcapsular cataract and posterior capsule opacification [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, the ciliary epithelium produces microfibrils, the primary component of the ciliary zonules [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These intraocular epithelia exhibit diverse mesenchymal-like traits and can be regarded as hybrid cell populations positioned at the interface between epithelial and mesenchymal phenotypes. Thus, Meflin expression in these intraocular epithelial cells is consistent with their mesenchymal-like properties.\u003c/p\u003e \u003cp\u003eThe Meflin lineage-tracing experiments conducted in this study revealed the appearance of Meflin-lineage cells at sites of retinal injury. This finding suggests that Meflin-expressing cells, including intraocular epithelial cells, may proliferate at sites of tissue repair or be recruited there through mechanisms that remain to be determined. Interestingly, tdTomato signals were also detected in the INL and along the ILM. Given that the ILM represents the basement membrane of M\u0026uuml;ller cells, this signal distribution suggests that Meflin may serve as a marker of M\u0026uuml;ller cells involved in the tissue repair response. However, further studies are warranted to test this possibility and clarify the role of Meflin in these cells.\u003c/p\u003e \u003cp\u003eNotably, Meflin overexpression in the laser-induced CNV model led to an increase in CNV volume alongside suppression of subretinal fibrosis\u0026mdash;an outcome that may initially seem counterintuitive. Meflin binds to LOX and inhibits its collagen crosslinking activity, thereby preventing fibrosis of the interstitial tissue [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A soft stroma provides a favorable scaffold for cell migration and tube formation, which may result in dilation of vascular lumens and increased vascular volume. In previous studies conducted using pancreatic cancer models, Meflin expression led to the repair of damaged tissue, increased tumor vessel diameter, and improved drug delivery [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the context of oncology, \u0026ldquo;vascular normalization\u0026rdquo; is considered crucial for an effective therapeutic response [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]; therefore, therapeutic strategies are being increasingly designed to target formation of functional vascular networks rather than complete suppression of pathological angiogenesis. However, whether the increase in CNV volume observed in the present study is beneficial for retinal tissue repair cannot be concluded based on the current data alone. Future evaluations of vascular function, including assessment of structural integrity and blood flow dynamics, are warranted.\u003c/p\u003e \u003cp\u003eThe suppressive effect of Meflin overexpression on subretinal fibrosis observed in this study is consistent with the previously reported anti-fibrotic properties of Meflin in other organs [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As described above, Meflin inhibits fibroblast-to-myofibroblast differentiation by enhancing BMP7 signaling and antagonizing TGF-β signaling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, it further restrains excessive fibrotic progression by inhibiting collagen crosslinking [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similar mechanisms may operate intraocularly, thereby suppressing the formation of subretinal fibrosis. However, further studies conducted using in vivo and in vitro approaches are warranted to clarify the detailed molecular mechanisms underlying these anti-fibrotic effects, particularly the mechanisms that involve retina-specific cell populations and the local microenvironment.\u003c/p\u003e \u003cp\u003eNo overt ophthalmic phenotype was observed in the Meflin KO mice analyzed in the present study. The absence of apparent abnormalities in ocular morphology or ERG findings suggests the presence of compensatory mechanisms that involve other molecules with overlapping functions. In addition, it is possible that the single-laser injury CNV model used in this study did not induce tissue damage severe enough to unmask the functional consequences of Meflin deficiency. Thus, whether the impact of Meflin deficiency becomes more evident in models of more severe tissue injury, chronic inflammation, or age-related changes remains to be determined.\u003c/p\u003e \u003cp\u003eThe present study suggests a new paradigm in retinal research by elucidating the functions of Meflin in promoting angiogenesis while suppressing pathological fibrosis. The findings of this study may deepen our understanding of the pathogenesis of nAMD as well as a broad spectrum of intractable fibroproliferative retinal diseases, including PVR and PDR. Furthermore, this study may provide a foundation for the development of novel therapeutic strategies aimed at promoting retinal tissue repair.\u003c/p\u003e "},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eStudy Approval\u003c/h2\u003e \u003cp\u003eAll animal protocols were approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine (approval number: M240018-002). All animal experiments were performed in accordance with Nagoya University\u0026rsquo;s Animal Facility regulations. The study is reported in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and relevant institutional guidelines and regulations and was approved by the Ethics Committee of Nagoya University Graduate School of Medicine (approval number: 2013-0010). Written informed consent for the use of surgical specimens for research purposes was obtained from all patients.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHuman surgical specimens\u003c/h2\u003e \u003cp\u003eFibrovascular membranes were obtained from patients who underwent pars plana vitrectomy for PVR or PDR at Nagoya University Hospital (Nagoya, Japan). Membrane tissues excised during surgery and that would otherwise be discarded were collected and processed for histological analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe human M\u0026uuml;ller glial cell line MIO-M1 was purchased from e-lucid (University College London, London, UK). The immortalized human retinal pigment epithelial cell line hTERT-RPE1 was purchased from ATCC (Manassas, VA, USA). The human osteosarcoma cell line HS-Os-1 was purchased from RIKEN BioResource Research Center (Tsukuba, Japan). Human dermal fibroblasts fHDF/TERT166 were obtained from Evercyte (Vienna, Austria). Primary human retinal pigment epithelial cells (hRPEpiC; Cat. no. 6540) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). All cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂ and cultured according to the manufacturers\u0026rsquo; or providers\u0026rsquo; instructions using the recommended media. For the experiments, cells were grown to 90\u0026ndash;100% confluence, then protein lysates and total RNA were extracted for Western blotting and qPCR analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eConfluent cell monolayers were washed twice with phosphate-buffered saline (PBS) and lysed directly in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, and bromophenol blue). Lysates were collected and briefly sonicated on ice to reduce viscosity. Protein concentrations were determined using a DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (20\u0026ndash;30 \u0026micro;g per lane) were prepared for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were reduced using 2-mercaptoethanol, heated at 100\u0026deg;C for 2 min, and separated using SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes (Merck, Darmstadt, Germany), which were pre-wetted in 100% methanol and equilibrated in transfer buffer containing 10% methanol. Blotting was performed at a constant voltage of 30 V (approximately 200 mA) overnight at 4\u0026deg;C. Membranes were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, incubated with primary antibodies diluted in TBST (0.1% Tween 20), and then with horseradish peroxidase-conjugated secondary antibodies. The primary antibodies used were rabbit anti-Meflin (\u003cem\u003eISLR\u003c/em\u003e) (HPA050811; Merck) and mouse anti-β-actin (clone AC-74; Merck). After washing in TBST (5 min, three times), immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection system (Cytiva, Marlborough, MA, USA) and imaged using a LAS-4000 system (GE Healthcare, Chicago, IL, USA). For detection of Meflin, primary antibodies were diluted in Can-Get-Signal Solution 1 (TOYOBO, Osaka, Japan) to enhance antibody\u0026ndash;antigen binding. Full-length blots are provided in Supplementary Fig.\u0026nbsp;2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was purified from cultured cells using the RNeasy Mini Kit (Cat. no. 74104; QIAGEN, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions. Purified RNA samples were reverse-transcribed using ReverTra Ace (Cat. no. TRT-101; TOYOBO) with oligo dT and random primers. qPCR of the generated cDNA was performed using TaqMan Gene Expression Master Mix (Cat. no. 4369016; Thermo Fisher Scientific, Waltham, MA, USA) on an Mx3005P thermal cycler (Agilent Technologies, Santa Clara, CA, USA). TaqMan probes/primers for human \u003cem\u003eISLR\u003c/em\u003e (Meflin) and \u003cem\u003eACTB\u003c/em\u003e (Thermo Fisher Scientific) were used according to the manufacturer\u0026rsquo;s instructions. Relative gene expression was calculated using the comparative threshold cycle (Ct) method and normalized to \u003cem\u003eACTB\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAll animal experiments were performed at Nagoya University Graduate School of Medicine. The generation and characterization of Meflin knockout mice have been described previously [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Genomic DNA was extracted from mouse tail biopsies and subjected to polymerase chain reaction (PCR) genotyping. The primer sequences were as follows: PCR1 forward, 5\u0026prime;-GCTGCATTTGAGCTGAGCCTCTGG-3\u0026prime;; PCR1 reverse, 5\u0026prime;-AACCCCTTCCTCCTACATAGTTGG-3\u0026prime;; PCR2 forward, 5\u0026prime;-TGAGGTTAGCCTGGGGACTTCAC-3\u0026prime;; and PCR2 reverse, 5\u0026prime;-GGCTAGAACTCTCAAAGTAGGTCAGG-3\u0026prime;.\u003c/p\u003e \u003cp\u003eThe generation of \u003cem\u003eMeflin-CreERT2\u003c/em\u003e knock-in mice has been described previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For lineage-tracing experiments, the \u003cem\u003eMeflin-CreERT2\u003c/em\u003e mice were crossed with \u003cem\u003eRosa26-LSL-tdTomato\u003c/em\u003e mice (JAX stock no. 007909; The Jackson Laboratory, Bar Harbor, ME, USA) to generate \u003cem\u003eMeflin-CreERT2; Rosa26-LSL-tdTomato\u003c/em\u003e mice. \u003cem\u003eRosa26-LSL-tdTomato\u003c/em\u003e mice were obtained from The Jackson Laboratory and housed at Nagoya University Graduate School of Medicine. The animals were maintained under controlled temperature, humidity, and light conditions (12h light/dark cycle) with free access to water and standard chow. Unless otherwise indicated, 8\u0026ndash;10-week-old male mice on a C57BL/6J background were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIn situ hybridization\u003c/h2\u003e \u003cp\u003eRNA in situ hybridization was performed using the RNAscope 2.5 HD Reagent Kit-Brown (Cat. No. 322300; Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer\u0026rsquo;s protocol. The probes used in this study were RNAscope probe-Mm-\u003cem\u003eIslr\u003c/em\u003e (450041; Advanced Cell Diagnostics), probe-Hs-\u003cem\u003eISLR\u003c/em\u003e (455481; Advanced Cell Diagnostics), and the negative control probe-\u003cem\u003eDapB\u003c/em\u003e (310043; Advanced Cell Diagnostics). Tissue processing and paraffin embedding for in situ hybridization were performed as previously described [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] or by Genostaff (Tokyo, Japan). Giemsa staining was performed for counterstaining of C57BL/6J tissues, whereas hematoxylin counterstaining was used for BALB/c tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLaser-induced choroidal neovascularization model\u003c/h2\u003e \u003cp\u003eLaser-induced CNV was generated as previously described [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] with minor modifications. Mice were anesthetized through intraperitoneal injection of ketamine (37.5 mg/kg) and medetomidine (0.625 mg/kg), with supplemental dosing administered as required. The mice\u0026rsquo;s pupils were dilated through topical instillation of 0.5% tropicamide and 0.5% phenylephrine (Mydrin-P; Santen, Osaka, Japan). Laser photocoagulation was performed using a Novus Verdi laser system (532 nm; 180 mW; 100 ms; 75 \u0026micro;m spot diameter; Coherent Inc., Santa Clara, CA, USA). Four laser burns were applied per eye at approximately 1\u0026ndash;2 disc diameters from the optic nerve head. Lesions that exhibited bubble formation at the time of laser application, indicating rupture of Bruch\u0026rsquo;s membrane, were included for analysis. Eyes were collected 10\u0026ndash;14 days after laser induction for evaluation of CNV and 35 days after induction for analysis of subretinal fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of CNV and subretinal fibrosis volume\u003c/h2\u003e \u003cp\u003eCNV and subretinal fibrosis volumes were quantified from confocal z-stack images. Specimens were prepared as eye cups and subjected to immunofluorescence staining as described below. The lesions were imaged using a confocal microscope (AX R; Nikon, Tokyo, Japan). Horizontal optical sections were acquired at 0.5-\u0026micro;m intervals from the lesion surface to the RPE. The lesion area in each section was measured using Fiji (ImageJ) software (National Institutes of Health, Bethesda, MD, USA), and the sum of the lesion areas across all sections was used as an index of CNV or subretinal fibrosis volume. Imaging and quantification were performed by an investigator masked to group assignments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eElectroretinography\u003c/h2\u003e \u003cp\u003eElectroretinograms (ERGs) were recorded using a commercial Ganzfeld ERG recording system. The mice were dark-adapted overnight, and procedures were performed under dim red light. After induction of anesthesia and pupil dilation were performed as described above, the mice were placed on a heated platform to maintain a stable body temperature. A contact lens electrode was positioned on the cornea, and reference and ground electrodes were placed subcutaneously.\u003c/p\u003e \u003cp\u003eScotopic ERGs were elicited by white flash stimuli ranging from \u0026minus;\u0026thinsp;7.0 to 0.0 log cd\u0026middot;s/m\u0026sup2;. Photopic ERGs were recorded following light adaptation on a steady background light, with flash intensities ranging from \u0026minus;\u0026thinsp;0.5 to 1.5 log cd\u0026middot;s/m\u0026sup2;. Responses obtained from multiple trials were band-pass filtered and averaged. The a-wave amplitude was measured from baseline to the a-wave trough, whereas the b-wave amplitude was measured from the a-wave trough to the b-wave peak. The b/a ratio for scotopic ERGs and the b-wave amplitude for photopic ERGs were used for quantitative analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLineage tracing\u003c/h2\u003e \u003cp\u003eTamoxifen (200 mg/kg; Cat. no. T5648; Merck, Darmstadt, Germany) dissolved in corn oil (C8267; Merck) was administered intraperitoneally to eight-week-old male \u003cem\u003eMeflin-CreERT2; Rosa26-LSL-tdTomato\u003c/em\u003e mice three times on alternate days. Laser-induced CNV was performed concurrently with the third tamoxifen injection. Tissues were harvested 5 days after the final injection. Specimens were prepared as eye cups and subjected to immunofluorescence staining as described below.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eImmunofluorescence staining and confocal imaging\u003c/h2\u003e \u003cp\u003eThe eyes were enucleated and immersion-fixed in 4% paraformaldehyde (PFA) for 30 min. After removal of the anterior segment, including the lens and vitreous, the eye cups were further fixed in 4% PFA for 1.5 h at 4\u0026deg;C, rinsed with PBS, cryoprotected in 30% sucrose overnight at 4\u0026deg;C, embedded in optimal cutting temperature (OCT) compound, and cryosectioned at 10 \u0026micro;m using a cryostat (CM3050 S; Leica Microsystems, Wetzlar, Germany). Sections were washed with PBS, blocked with 1% bovine serum albumin (BSA) for 30 min, and incubated with primary antibodies overnight at 4\u0026deg;C. The sections were then incubated with Alexa Fluor-conjugated secondary antibodies (1:400; Thermo Fisher Scientific) for 1 h at room temperature, washed with PBS, and mounted with Immu-Mount (Epredia, Kalamazoo, MI, USA).\u003c/p\u003e \u003cp\u003eRPE flatmounts were prepared for immunofluorescence staining for the laser-induced CNV, subretinal fibrosis, and lineage-tracing experiments. After preparation of eye cups, the neural retina was gently removed, and the tissues were fixed in 4% PFA for 90 min at 4\u0026deg;C. Samples were washed with PBS, blocked in PBST (PBS containing 0.5% Triton X-100) with 1% BSA, and incubated with primary antibodies overnight at 4\u0026deg;C. After washing, the samples were incubated with Alexa Fluor-conjugated secondary antibodies (1:400; Thermo Fisher Scientific) for 1 h at room temperature. Fluorescence images were acquired using a confocal microscope (AX R; Nikon).\u003c/p\u003e \u003cp\u003eThe primary antibodies were rabbit anti-RFP (polyclonal, 1:1,000; 600-401-379; Rockland Immunochemicals, Limerick, PA, USA), rabbit anti-collagen I and III (polyclonal, 1:200; ab34710; Abcam, Cambridge, UK), and rat anti-mouse CD31 (monoclonal, 1:200; 550274; BD Biosciences, Franklin Lakes, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAAV vectors and subretinal injection\u003c/h2\u003e \u003cp\u003eRecombinant AAV9 vectors encoding Flag-tagged mouse Islr (Flag tag inserted immediately downstream of the endogenous signal peptide; AAV9-CBh-Islr) or Flag-tagged enhanced green fluorescent protein (AAV9-CBh-EGFP), both driven by the CBh promoter, were purchased from VectorBuilder (Chicago, IL, USA). The viral titers were determined using qPCR and adjusted to a final concentration of 1.0 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e viral genomes (vg) / mL in PBS. For the subretinal injection, the mice were anesthetized and their pupils were dilated as described above. A total volume of 2 \u0026micro;L of the vector solution (total dose: 2.0 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e vg) was injected into the subretinal space using a trans-scleral approach with a 33-gauge needle (Ito Corporation, Shizuoka, Japan). Successful delivery into the subretinal space was confirmed by visual observation of a subretinal bleb.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Python (version 3.12.11). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;number of samples). Group comparisons were conducted using Welch\u0026rsquo;s t-test. A two-sided p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Investigators were blinded to group assignments during data analysis. Graphs were generated using standard Python libraries.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Kozo Uchiyama, Kayoko Endo and Yasuko Sobue (Department of Pathology, Nagoya University Graduate School of Medicine) for their assistance. We also appreciate the support of the staff of the Division for Medical Research Engineering for the use of the confocal microscope AX R, and the Division of Experimental Animals (Nagoya University) for their help with the animal experiments. Our thanks also go to our colleagues at the Department of Pathology and the Department of Ophthalmology, Nagoya University Graduate School of Medicine, for their helpful discussions and technical assistance. We would like to thank Editage (Cactus Communications) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grants 22K07000 to S.M., 22H02848 and 22K18390 to A.E.); the Japan Agency for Medical Research and Development (AMED) (grants JP24gm1210009 and JP25ama221333 to A.E.); the Chukyo Longevity Medical and Promotion Foundation (to S.M.); the Naito Foundation (to A.E.); the Takamatsunomiya Cancer Foundation (to A.E.); the Toyoaki Foundation (to A.E.); and the Japan Science and Technology Agency (JST) (grant number JPMJSP2125 to D.O.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.O. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. S.M. and A.E. supervised the project, provided experimental guidance, interpreted the data, and revised the manuscript. Y.M., R.A., Y.S., and N.E. assisted with the experiments and data analysis. K.K. and K.Y. provided advice on the experimental designs. H.U. and H.S. collected clinical samples. H.K. and K.M.N. contributed to the study design, data interpretation, and manuscript revision. All the authors discussed the results and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to S.M. or A.E.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003cstrong\u003e\u003cbr clear=\"all\"\u003e\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePastor, J. C. et al. 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In this study, we examined the intraocular expression patterns of Meflin, a mesenchymal stromal cell marker with anti-fibrotic properties, and evaluated its role in the pathophysiology of retinal diseases using a mouse model. In wild-type mice, Meflin expression was detected in the ciliary body, lens epithelium, retinal pigment epithelium, optic nerve meningeal cells, and choroidal perivascular fibroblasts. Lineage-tracing analysis using a Meflin reporter mouse line revealed accumulation of Meflin-lineage cells within laser-induced CNV lesions. Interestingly, adeno-associated virus-mediated overexpression of Meflin increased CNV volume in the acute phase of retinal injury but significantly suppressed subretinal fibrosis in the chronic phase. These findings suggest that Meflin promotes angiogenesis to support tissue repair and inhibits fibrosis after retinal injury in a temporally dependent manner, consistent with its previously reported roles in mouse models of cancer and other fibrotic diseases. These results improve our understanding of retinal disease pathology and highlight Meflin as a potential therapeutic target in diseases such as AMD.\u003c/p\u003e","manuscriptTitle":"Possible involvement of the mesenchymal cell marker Meflin in angiogenesis promotion and fibrosis suppression after retinal injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 10:14:32","doi":"10.21203/rs.3.rs-8930515/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"54329738768433460532388732884798758941","date":"2026-03-30T18:02:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268594944319769980398550127911583640442","date":"2026-03-30T16:01:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22401645231129277751108258145253064648","date":"2026-03-30T14:04:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T12:46:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-09T13:51:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-27T19:51:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-27T12:57:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f1528643-f670-4011-b3dd-1b51c49f6ba0","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":65418326,"name":"Biological sciences/Cell biology"},{"id":65418327,"name":"Health sciences/Diseases"},{"id":65418328,"name":"Health sciences/Medical research"},{"id":65418329,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-05-11T08:40:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 10:14:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8930515","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8930515","identity":"rs-8930515","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}