Macrophage to myofibroblast transition contributes to subretinal fibrosis secondary to neovascular 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.

Subretinal fibrosis in neovascular age-related macular degeneration: current concepts, therapeutic avenues, and future perspectives

“…Luo et al identified choroidal pericytes as a source of myofibroblasts in a model of laser-induced CNV and showed that choroidal pericytes play a role in the formation of subretinal fibrosis by expressing ECM components (Luo et al 2018). Finally, myeloid cells such as macrophages transdifferentiate into myofibroblasts in experimental fibrotic kidney disease (Meng et al 2016) and express α-SMA under TGFβ induction in vitro (Little et al In line with these findings, approximately 20% of infiltrating Iba-1 + myeloid cells in human MNV were reported to be immunoreactive for α-SMA, suggesting that MMT is present in macular fibrosis secondary to nAMD (Little et al 2020b) (Fig. 5).…”

Macrophage elastase (MMP12) critically contributes to the development of subretinal fibrosis

“…The study complies with the Declaration of Helsinki and the protocol was approved by the Ethical Review Board of Queen’s University Belfast. The samples were then wax embedded and processed for immunohistochemistry detailed previously by us [ 22 ].…”

The interplay of fibroblasts, the extracellular matrix, and inflammation in scar formation

“…In both human and murine fibrotic tissues, this activation state is maintained via direct contact enabled by macrophages' cadherin-11 ( 138 ). In addition to their ability to directly stimulate myofibroblasts, recent work has shown that macrophages are capable of transitioning into smooth muscle cells or myofibroblasts in the context of atherosclerosis, subretinal, or renal fibrosis, respectively ( 139 , 140 , 141 ). In conclusion, macrophages are complex and jack-of-all-trades components of the innate response, but their direct interaction with fibroblasts should serve as motivation to manipulate and further understand their role in scarless wound healing and fibrosis.…”