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Fenofibrate Inhibits Subretinal Fibrosis Through Suppressing TGF??—Smad2/3 signaling and Wnt signaling in Neovascular Age?Related Macular Degeneration

ABSTRACT: Subretinal fibrosis is a common pathological change that causes vision loss in neovascular age-related macular degeneration (nAMD). Treatment modalities for subretinal fibrosis are limited. In the present study, the effects of fenofibrate, a specific peroxisome proliferator–activated receptor alpha agonist, on subretinal fibrosis of nAMD were tested, and its molecular mechanisms of action were delineated. Collagen deposition and protein expression of fibrotic markers, such as vimentin, collagen-1, alpha-smooth muscle actin, and fibronectin, were increased in very low–density lipoprotein receptor (VLDLR) knockout mouse, indicating Vldlr?/? mice can be used as a model for subretinal fibrosis. Fenofibrate suppressed subretinal fibrosis of Vldlr?/? mice by reducing collagen deposition and protein expression of fibrotic markers. Two fibrotic pathways, TGF-?—Smad2/3 signaling and Wnt signaling, were significantly up-regulated, while inhibited by fenofibrate in Vldlr?/? retinas. Moreover, fenofibrate significantly reduced the downstream connective tissue growth factor (CTGF) expression of these two pathways. Müller cells were a major source of CTGF in Vldlr?/? retinas. Fenofibrate was capable of suppressing Müller cell activation and thus reducing the release of CTGF in Vldlr?/? retinas. In cultured Müller cells, fenofibrate reversed TGF-?2–induced up-regulation of Wnt signaling and CTGF expression. These findings suggested that fenofibrate inhibits subretinal fibrosis by suppressing TGF-?—Smad2/3 signaling and Wnt signaling and reducing CTGF expression, and thus, fenofibrate could be a potential treatment for nAMD with subretinal fibrosis.

SUBMITTER: Chen Q

PROVIDER: S-EPMC7797782 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundMacular fibrosis causes irreparable vision loss in neovascular age-related macular degeneration (nAMD) even with anti-vascular endothelial growth factor (VEGF) therapy. Inflammation is known to play an important role in macular fibrosis although the underlying mechanism remains poorly defined. The aim of this study was to understand how infiltrating macrophages and complement proteins may contribute to macular fibrosis.MethodsSubretinal fibrosis was induced in C57BL/6J mice using the two-stage laser protocol developed by our group. The eyes were collected at 10, 20, 30 and 40 days after the second laser and processed for immunohistochemistry for infiltrating macrophages (F4/80 and Iba-1), complement components (C3a and C3aR) and fibrovascular lesions (collagen-1, Isolectin B4 and α-SMA). Human retinal sections with macular fibrosis were also used in the study. Bone marrow-derived macrophages (BMDMs) from C57BL/6J mice were treated with recombinant C3a, C5a or TGF-β for 48 and 96 h. qPCR, Western blot and immunohistochemistry were used to examine the expression of myofibroblast markers. The involvement of C3a-C3aR pathway in macrophage to myofibroblast transition (MMT) and subretinal fibrosis was further investigated using a C3aR antagonist (C3aRA) and a C3a blocking antibody in vitro and in vivo.ResultsApproximately 20~30% of F4/80+ (or Iba-1+) infiltrating macrophages co-expressed α-SMA in subretinal fibrotic lesions both in human nAMD eyes and in the mouse model. TGF-β and C3a, but not C5a treatment, significantly upregulated expression of α-SMA, fibronectin and collagen-1 in BMDMs. C3a-induced upregulation of α-SMA, fibronectin and collagen-1 in BMDMs was prevented by C3aRA treatment. In the two-stage laser model of induced subretinal fibrosis, treatment with C3a blocking antibody but not C3aRA significantly reduced vascular leakage and Isolectin B4+ lesions. The treatment did not significantly alter collagen-1+ fibrotic lesions.ConclusionsMMT plays a role in macular fibrosis secondary to nAMD. MMT can be induced by TGF-β and C3a but not C5a. Further research is required to fully understand the role of MMT in macular fibrosis. Macrophage to myofibroblast transition (MMT) contributes to subretinal fibrosis. Subretinal fibrosis lesions contain various cell types, including macrophages and myofibroblasts, and are fibrovascular. Myofibroblasts are key cells driving pathogenic fibrosis, and they do so by producing excessive amount of extracellular matrix proteins. We have found that infiltrating macrophages can transdifferentiate into myofibroblasts, a phenomenon termed macrophage to myofibroblast transition (MMT) in macular fibrosis. In addition to TGF-β1, C3a generated during complement activation in CNV can also induce MMT contributing to macular fibrosis. RPE = retinal pigment epithelium. BM = Bruch's membrane. MMT = macrophage to myofibroblast transition. TGFB = transforming growth factor β. a-SMA = alpha smooth muscle actin. C3a = complement C3a.

Project description:Fenofibrate is a peroxisome proliferator-activated receptor ? (PPAR?) agonist and has been shown to have therapeutic effects on diabetic retinopathy (DR). However, the effects of fenofibrate through systemic administration are not as potent as desired due to inefficient drug delivery to the retina. The present study aimed to explore the sustained therapeutic effects of fenofibrate-loaded biodegradable nanoparticles (NP) on both DR and neovascular age-related macular degeneration (AMD). Fenofibrate was successfully encapsulated into poly(lactic- co-glycolic acid) (PLGA) NP (Feno-NP), and Feno-NP were optimized by varying polymer composition to achieve high drug loading and prolonged drug release. The Feno-NP made of PLGA 34 kDa demonstrated a drug content of 6% w/w and a sustained drug release up to 60 days in vitro. Feno-NP (PLGA 34 kDa) was selected for following in vivo studies, and one single intravitreal (IVT) injection of Feno-NP into rat eyes with a 30G fine needle maintained sustained fenofibric acid drug level in the eye for more than 60 days. The efficacy of Feno-NP in DR and neovascular AMD was investigated using streptozotocin (STZ)-induced diabetic rats, laser-induced choroidal neovascularization (CNV) rats, and very low-density lipoprotein receptor knockout ( Vldlr -/-) mice. Therapeutic effects of Feno-NP were evaluated by measuring electroretinogram (ERG), retinal vascular leakage, leukostasis, CNV size, and retinal levels of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). In diabetic rats, Feno-NP ameliorated retinal dysfunctions, reduced retinal vascular leakage, inhibited retinal leukostasis, and downregulated the overexpression of VEGF and ICAM-1 at 8 weeks after one IVT injection. In addition, Feno-NP reduced retinal vascular leakage and CNV formation in both CNV rats and Vldlr -/- mice. Moreover, no toxicity of Feno-NP or Blank-NP to retinal structure and function was detected. Feno-NP exhibited good physiochemical characteristics and controlled drug release profile, conferring prolonged beneficial effects on DR and neovascular AMD.

Project description:ObjectiveThe study aims to investigate the protective effect of Mingjing granule (MG) in a fibrovascular membrane rat model of neovascular age-related macular degeneration (nAMD) and explore the underlying mechanism.MethodsThe nAMD fibrovascular membrane model was established by two-stage laser photocoagulation. BN rats were randomly divided into four groups: the model group was gavaged with distilled water, the anti-VEGF group was given an intravitreous injection of ranibizumab, the MG + anti-VEGF group was gavaged with MG combined with an intravitreous injection of ranibizumab, and the normal group not modeled only fed conventionally. Lesions were evaluated by color fundus photograph, optical coherence tomography, fundus fluorescein angiography, and retinal pigment epithelial-choroid-sclera flat mount. The changes in the retinal structure were observed by histopathology. The expression of inflammatory cell markers F4/80, Iba-1, and glial fibrillary acidic protein (GFAP); the fibrosis-related factors collagen-1, fibronectin, α-smooth muscle actin (α-SMA), and transforming growth factor-beta (TGF-β); and the complement system-related factors C3a and C3aR in the retina were detected by immunofluorescence or qRT-PCR.ResultsThe current study revealed that MG + anti-VEGF administration more significantly reduced the thickness of fibrovascular lesions, suppressed vascular leakage (exudation area and mean density value), inhibited the area of fibrovascular lesions, and restrained the formation of the fibrovascular membrane than the anti-VEGF agent alone in the two-stage laser-induced rat model. The fluorescence intensities of F4/80, Iba-1, collagen-1, fibronectin, TGF-β, and C3aR showed more significant inhibition in MG + anti-VEGF-treated rats than the anti-VEGF agent alone. The mRNA expression levels of F4/80, Iba-1, GFAP, collagen-1, fibronectin, α-SMA, TGF-β, and C3a showed lower levels in rats treated with MG + anti-VEGF than the anti-VEGF agent alone.ConclusionCombining MG with anti-VEGF treatment inhibits the growth of the fibrovascular membrane more effectively than using anti-VEGF treatment alone. The mechanism underlying this effect may involve limiting inflammatory cell aggregation, controlling complement system activation, and decreasing the expression of the fibrotic protein.

Project description:BackgroundNeovascular age-related macular degeneration (nAMD), accounts for up to 90% of AMD-associated vision loss, ultimately resulting in the formation of fibrotic scar in the macular region. The pathogenesis of subretinal fibrosis in nAMD involves the process of epithelial-mesenchymal transition (EMT) occurring in retinal pigment epithelium (RPE). Here, we aim to investigate the underlying mechanisms involved in the Wnt signaling during the EMT of RPE cells and in the pathological process of subretinal fibrosis secondary to nAMD.MethodsIn vivo, the induction of subretinal fibrosis was performed in male C57BL/6J mice through laser photocoagulation. Either FH535 (a β-catenin inhibitor) or Box5 (a Wnt5a inhibitor) was intravitreally administered on the same day or 14 days following laser induction. The RPE-Bruch's membrane-choriocapillaris complex (RBCC) tissues were collected and subjected to Western blot analysis and immunofluorescence to examine fibrovascular and Wnt-related markers. In vitro, transforming growth factor beta 1 (TGFβ1)-treated ARPE-19 cells were co-incubated with or without FH535, Foxy-5 (a Wnt5a-mimicking peptide), Box5, or Wnt5a shRNA, respectively. The changes in EMT- and Wnt-related signaling molecules, as well as cell functions were assessed using qRT-PCR, nuclear-cytoplasmic fractionation assay, Western blot, immunofluorescence, scratch assay or transwell migration assay. The cell viability of ARPE-19 cells was determined using Cell Counting Kit (CCK)-8.ResultsThe in vivo analysis demonstrated Wnt5a/ROR1, but not Wnt3a, was upregulated in the RBCCs of the laser-induced CNV mice compared to the normal control group. Intravitreal injection of FH535 effectively reduced Wnt5a protein expression. Both FH535 and Box5 effectively attenuated subretinal fibrosis and EMT, as well as the activation of β-catenin in laser-induced CNV mice, as evidenced by the significant reduction in areas positive for fibronectin, alpha-smooth muscle actin (α-SMA), collagen I, and active β-catenin labeling. In vitro, Wnt5a/ROR1, active β-catenin, and some other Wnt signaling molecules were upregulated in the TGFβ1-induced EMT cell model using ARPE-19 cells. Co-treatment with FH535, Box5, or Wnt5a shRNA markedly suppressed the activation of Wnt5a, nuclear translocation of active β-catenin, as well as the EMT in TGFβ1-treated ARPE-19 cells. Conversely, treatment with Foxy-5 independently resulted in the activation of abovementioned molecules and subsequent induction of EMT in ARPE-19 cells.ConclusionsOur study reveals a reciprocal activation between Wnt5a and β-catenin to mediate EMT as a pivotal driver of subretinal fibrosis in nAMD. This positive feedback loop provides valuable insights into potential therapeutic strategies to treat subretinal fibrosis in nAMD patients.

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Fenofibrate Inhibits Subretinal Fibrosis Through Suppressing TGF??—Smad2/3 signaling and Wnt signaling in Neovascular Age?Related Macular Degeneration

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