Adrenomedullin-RAMP2 system ameliorates subretinal fibrosis by suppressing epithelial-mesenchymal transition in age-related macular degeneration

Abstract Background Macular 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. Methods Subretinal 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. Results Approximately 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. Conclusions MMT 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. Graphical abstract 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.

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Transcriptome-Wide Analysis of CXCR5 Deficient Retinal Pigment Epithelial (RPE) Cells Reveals Molecular Signatures of RPE Homeostasis

Biomedicines ◽

10.3390/biomedicines8060147 ◽

2020 ◽

Vol 8 (6) ◽

pp. 147 ◽

Cited By ~ 3

Author(s):

Madhu Sudhana Saddala ◽

Anton Lennikov ◽

Anthony Mukwaya ◽

Hu Huang

Keyword(s):

Epithelial Mesenchymal Transition ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Enrichment Analysis ◽

Age Related Macular Degeneration ◽

Pathway Enrichment Analysis ◽

Molecular Signatures ◽

Mesenchymal Transition ◽

Rpe Cells ◽

Age Related

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the elderly population. In our previous studies, we found that deficiency of CXCR5 causes AMD-like pathological phenotypes in mice, characterized by abnormalities and dysfunction of the retinal pigment epithelium (RPE) cells. The abnormalities included abnormal cellular shape and impaired barrier function. In the present study, primary RPE cells were derived separately from CXCR5 knockout (KO) mice and from C57BL6 wild type (WT). The isolated primary cells were cultured for several days, and then total RNA was isolated and used for library preparation, sequencing, and the resultant raw data analyzed. Relative to the WT, a total of 1392 differentially expressed genes (DEG) were identified. Gene ontology analysis showed various biological processes, cellular components, and molecular functions were enriched. Pathway enrichment analysis revealed several pathways, including the PI3K-Akt signaling, mTOR signaling, FoxO, focal adhesion, endocytosis, ubiquitin-mediated proteolysis, TNFα-NF-kB Signaling, adipogenesis genes, p53 signaling, Ras, autophagy, epithelial–mesenchymal transition (EMT), and mitochondrial pathway. This study explores molecular signatures associated with deficiency of CXCR5 in RPE cells. Many of these signatures are important for homeostasis of this tissue. The identified pathways and genes require further evaluation to better understand the pathophysiology of AMD.

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PREVALENCE AND RISK FACTORS FOR THE DEVELOPMENT OF PHYSICIAN-GRADED SUBRETINAL FIBROSIS IN EYES TREATED FOR NEOVASCULAR AGE-RELATED MACULAR DEGENERATION

Retina ◽

10.1097/iae.0000000000002779 ◽

2020 ◽

Vol 40 (12) ◽

pp. 2285-2295 ◽

Cited By ~ 1

Author(s):

Kelvin Yi Chong Teo ◽

Aaron W. Joe ◽

Vuong Nguyen ◽

Alessandro Invernizzi ◽

Jennifer J. Arnold ◽

...

Keyword(s):

Risk Factors ◽

Macular Degeneration ◽

Age Related Macular Degeneration ◽

Age Related ◽

Subretinal Fibrosis

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Caveolin-1 Promotes Cellular Senescence in Exchange for Blocking Subretinal Fibrosis in Age-Related Macular Degeneration

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.61.11.21 ◽

2020 ◽

Vol 61 (11) ◽

pp. 21

Author(s):

Hideyuki Shimizu ◽

Kazuhisa Yamada ◽

Ayana Suzumura ◽

Keiko Kataoka ◽

Kei Takayama ◽

...

Keyword(s):

Macular Degeneration ◽

Cellular Senescence ◽

Age Related Macular Degeneration ◽

Caveolin 1 ◽

Age Related ◽

Subretinal Fibrosis

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Establishment of a New Animal Model of Focal Subretinal Fibrosis That Resembles Disciform Lesion in Advanced Age-Related Macular Degeneration

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.10-5189 ◽

2011 ◽

Vol 52 (9) ◽

pp. 6089 ◽

Cited By ~ 27

Author(s):

Young-Joon Jo ◽

Koh-Hei Sonoda ◽

Yuji Oshima ◽

Atsunobu Takeda ◽

Ri-ichiro Kohno ◽

...

Keyword(s):

Animal Model ◽

Macular Degeneration ◽

Age Related Macular Degeneration ◽

Advanced Age ◽

Age Related ◽

Subretinal Fibrosis

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EMT and EndMT: Emerging Roles in Age-Related Macular Degeneration

International Journal of Molecular Sciences ◽

10.3390/ijms21124271 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4271 ◽

Cited By ~ 4

Author(s):

Daisy Y. Shu ◽

Erik Butcher ◽

Magali Saint-Geniez

Keyword(s):

Macular Degeneration ◽

Transforming Growth Factor Beta ◽

Molecular Mechanisms ◽

Cancer Metastasis ◽

Transforming Growth Factor ◽

Retinal Pigment ◽

Mesenchymal Cells ◽

Age Related Macular Degeneration ◽

Mesenchymal Transition ◽

Age Related

Epithelial–mesenchymal transition (EMT) and endothelial–mesenchymal transition (EndMT) are physiological processes required for normal embryogenesis. However, these processes can be hijacked in pathological conditions to facilitate tissue fibrosis and cancer metastasis. In the eye, EMT and EndMT play key roles in the pathogenesis of subretinal fibrosis, the end-stage of age-related macular degeneration (AMD) that leads to profound and permanent vision loss. Predominant in subretinal fibrotic lesions are matrix-producing mesenchymal cells believed to originate from the retinal pigment epithelium (RPE) and/or choroidal endothelial cells (CECs) through EMT and EndMT, respectively. Recent evidence suggests that EMT of RPE may also be implicated during the early stages of AMD. Transforming growth factor-beta (TGFβ) is a key cytokine orchestrating both EMT and EndMT. Investigations in the molecular mechanisms underpinning EMT and EndMT in AMD have implicated a myriad of contributing factors including signaling pathways, extracellular matrix remodelling, oxidative stress, inflammation, autophagy, metabolism and mitochondrial dysfunction. Questions arise as to differences in the mesenchymal cells derived from these two processes and their distinct mechanistic contributions to the pathogenesis of AMD. Detailed discussion on the AMD microenvironment highlights the synergistic interactions between RPE and CECs that may augment the EMT and EndMT processes in vivo. Understanding the differential regulatory networks of EMT and EndMT and their contributions to both the dry and wet forms of AMD can aid the development of therapeutic strategies targeting both RPE and CECs to potentially reverse the aberrant cellular transdifferentiation processes, regenerate the retina and thus restore vision.

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

Frontiers in Pharmacology ◽

10.3389/fphar.2020.580884 ◽

2020 ◽

Vol 11 ◽

Author(s):

Qian Chen ◽

Nan Jiang ◽

Yuhan Zhang ◽

Sihao Ye ◽

Xu Liang ◽

...

Keyword(s):

Protein Expression ◽

Wnt Signaling ◽

Macular Degeneration ◽

Müller Cells ◽

Tissue Growth ◽

Age Related Macular Degeneration ◽

Collagen Deposition ◽

Muller Cells ◽

Age Related ◽

Subretinal Fibrosis

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-β—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-β2–induced up-regulation of Wnt signaling and CTGF expression. These findings suggested that fenofibrate inhibits subretinal fibrosis by suppressing TGF-β—Smad2/3 signaling and Wnt signaling and reducing CTGF expression, and thus, fenofibrate could be a potential treatment for nAMD with subretinal fibrosis.

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Autotranslocation of the pigment epithelium-choroid complex in treatment of age-related macular degeneration scarring stage

Ophthalmology journal ◽

10.17816/ov26056 ◽

2021 ◽

Vol 13 (3) ◽

pp. 97-104

Author(s):

Sergei V. Sosnovskii ◽

Ernest V. Boiko ◽

Dzhambulat Kh. Oskanov

Keyword(s):

Macular Degeneration ◽

Pigment Epithelium ◽

Antiangiogenic Therapy ◽

Angiogenesis Inhibitors ◽

Original Method ◽

Age Related Macular Degeneration ◽

Age Related ◽

Subretinal Fibrosis ◽

Subretinal Surgery

The gold standard of the neovascular age-related macular degeneration treatment is the intravitreal administration of angiogenesis inhibitors. In subretinal macular fibrosis, antiangiogenic therapy is not effective. In such cases, subretinal surgery is used, in particular, autotranslocation of pigment epithelium-choroid complex. This paper presents a case of successful use of this method in a 77 y.o. female patient with subretinal fibrosis in the macular area as an outcome of neovascular age-related macular degeneration. An original method of translocation of pedicled pigment epithelium-choroid complex from the paramacular area to the macula was used. In 24 months, the visual acuity increased from 0.01 to 0.07; the central fixation was restored; the absolute positive central scotoma disappeared. During all the post-operative follow-up period, the full-rate pigment epithelium-choroid perfusion in the choroid of the translocated flap, the loss of choroidal neovascularization activity signs and of indications for intravitreal administration of angiogenesis inhibitors were proved.

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