The complement pathway as a therapeutic target for neovascular age-related macular degeneration-mediated subretinal fibrosis.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the elderly in developed countries. Intravitreal injection of VEGF inhibitors is the mainstream therapy for nAMD, although nearly 50% of the patients do not respond or respond poorly to the therapy. One of the main reasons for the poor outcome of the therapy is the development of subretinal macular fibrosis, a process of excessive deposition of extracellular matrix proteins around the diseased blood vessels. Currently, there is no medication to prevent or treat the condition. Here, we discussed recent advances in the pathogenesis of nAMD-mediated macular fibrosis, with a focus on the role of the complement system. We further proposed approaches to target the complement system for the management of macular fibrosis and highlighted the area of further research for future clinical applications of complement-based therapy.

Vascular Cell Adhesion Molecule-1 (VCAM-1) contributes to macular fibrosis in neovascular age-related macular degeneration through modulating macrophage functions.

BACKGROUND: Neovascular age-related macular degeneration (nAMD) is a major cause of blindness in the elderly. The disease is due to the growth of abnormal blood vessels into the macula, leading to the loss of central vision. Intravitreal injection of vascular endothelial growth factor (VEGF) inhibitors (e.g., anti-VEGF) is the standard of care for nAMD. However, nearly 50% of patients do not respond or respond poorly to the therapy. More importantly, up to 70% of nAMD patients develop macular fibrosis after 10 years of anti-VEGF therapy. The underlying mechanism of nAMD-mediated macular fibrosis is unknown although inflammation is known to play an important role in the development of abnormal macular blood vessels and its progression to fibro-vascular membrane. In this study, we measured the intraocular levels of adhesion molecule VCAM-1, ICAM-1, CD44, CD62L, and CD62P in nAMD patients with and without macular fibrosis and investigated the link between the levels of adhesion molecule and clinical features (e.g., visual improvement, retinal thickness, etc.). We further investigated the effect of VCAM-1 in macrophage function in vitro and the development of subretinal fibrosis in vivo using a two-stage laser-induced protocol. RESULTS: The aqueous levels of ICAM-1, VCAM-1, CD44, and CD62L were significantly higher in nAMD patients compared to cataract controls. The aqueous level of VCAM-1 (but not other adhesion molecules) was significantly higher in patients with macular fibrosis than those without and the level correlated positively with the retinal thickness. VCAM-1 was highly expressed at the lesion site in the mouse model of subretinal fibrosis. Blocking VCAM-1 or its receptor VLA-4 significantly prevented macrophage infiltration and reduced subretinal fibrosis in vivo. VCAM-1 induced macrophage migration and upregulated the expression of Arg-1, Mmp12 and Il6 but down-regulated the expression of iNOS and Il1b in macrophages. CONCLUSIONS: VCAM-1 may contribute to the development of macular fibrosis in nAMD patients by modulating macrophage functions, including migration and profibrotic polarization.

Old age promotes retinal fibrosis in choroidal neovascularization through circulating fibrocytes and profibrotic macrophages.

BACKGROUND: Retinal fibrosis affects 40-70% of neovascular age-related macular degeneration patients. This study investigated the effect of ageing on subretinal fibrosis secondary to choroidal neovascularization and the mechanism of action. METHODS: Subretinal fibrosis was induced in young (2.5-month) and aged (15-16-month) C57BL/6J mice using the two-stage laser protocol. Five and 30 days later, eyes were collected and stained for CD45 and collagen-1 and observed by confocal microscopy. Fibrocytes (CD45+collagen-1+) were detected in the bone marrow (BM), blood and fibrotic lesions by flow cytometry and confocal microscopy, respectively. BM-derived macrophages (BMDMs) were cultured from young and aged mice with or without TGF-ß1 (10 ng/mL) treatment. The expression of mesenchymal marker αSMA (Acta2), fibronectin (Fn1) and collagen-1 (Col1a1) was examined by qPCR and immunocytochemistry, whereas cytokine/chemokine production was measured using the Luminex multiplex cytokine assay. BM were transplanted from 22-month (Ly5.2) aged mice into 2.5-month (Ly5.1) young mice and vice versa. Six weeks later, subretinal fibrosis was induced in recipient mice and eyes were collected for evaluation of fibrotic lesion size. RESULTS: Under normal conditions, the number of circulating fibrocytes (CD45+collagen-1+) and the expression levels of Tgfb1, Col1a1, Acta2 and Fn1 in BMDMs were significantly higher in aged mice compared to young mice. Induction of subretinal fibrosis significantly increased the number of circulating fibrocytes, enhanced the expression of Col1a1, Acta2 and Fn1 and the production of soluble urokinase plasminogen activator surface receptor (suPAR) but decreased the production of CXCL10 in BMDMs. BMDMs from aged subretinal fibrosis mice produced significantly higher levels of VEGF, angiopoietin-2 and osteopontin than cells from young subretinal fibrosis mice. The subretinal fibrotic lesion in 15-16-month aged mice was 62% larger than that in 2.5-month young mice. The lesion in aged mice contained a significantly higher number of fibrocytes compared to that in young mice. The number of circulating fibrocytes positively correlated with the size of subretinal fibrotic lesion. Transplantation of BM from aged mice significantly increased subretinal fibrosis in young mice. CONCLUSIONS: A retina-BM-blood-retina pathway of fibrocyte/macrophage recruitment exists during retinal injury. Ageing promotes subretinal fibrosis through higher numbers of circulating fibrocytes and profibrotic potential of BM-derived macrophages.

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

BACKGROUND: Macular subretinal fibrosis is the end-stage complication of neovascular age-related macular degeneration (nAMD). We previously developed a mouse model of two-stage laser-induced subretinal fibrosis that mimics closely the dynamic course of macular fibrosis in nAMD patients. This study was aimed to understand the molecular mechanism of subretinal fibrosis. METHODS: Subretinal fibrosis was induced in C57BL/6J mice using the two-stage laser-induced protocol. Twenty days later, eyes were collected and processed for RNA sequencing (RNA-seq) analysis. DESeq2 was used to determine the differentially expressed genes (DEGs). Gene Ontology (GO) and KEGG were used to analyze the enriched pathways. The expression of the selected DEGs including Mmp12 was verified by qPCR. The expression of MMP12 in subretinal fibrosis of mouse and nAMD donor eyes was examined by immunofluorescence and confocal microscopy. The expression of collagen 1, αSMA and fibronectin and cytokines in bone marrow-derived macrophages from control and subretinal fibrosis mice were examined by qPCR, immunocytochemistry and Luminex multiplex cytokine assay. The MMP12 specific inhibitor MMP408 was used to evaluate the effect of MMP12 on TGFß-induced macrophage-to-myofibroblast transition (MMT) in vitro and its role in subretinal fibrosis in vivo. RESULTS: RNA-seq analysis of RPE-choroid from subretinal fibrosis eyes uncovered 139 DEGs (fold change log2(fc) ≥ 0.5, FDR < 0.05), including 104 up-regulated and 35 were down-regulated genes. The top 25 enrichment GO terms were related to inflammation, blood vessels/cardiovascular development and angiogenesis. One of the most significantly upregulated genes, Mmp12, contributed to 12 of the top 25 GO terms. Higher levels of MMP12 were detected in subretinal fibrotic lesions in nAMD patients and the mouse model, including in F4/80+ or Iba1+ macrophages. BMDMs from subretinal fibrosis mice expressed higher levels of MMP12, collagen-1, αSMA and fibronectin. MMP408 dose-dependently suppressed TGFß-induced MMT in BMDMs. In vivo treatment with MMP408 (5 mg/kg) significantly reduced subretinal fibrosis accompanied by reduced F4/80+ macrophage infiltration. CONCLUSIONS: MMP12 critically contributes to the development of subretinal fibrosis, partially through promoting MMT.

Retinal Pigment Epithelial Cells Express Antimicrobial Peptide Lysozyme - A Novel Mechanism of Innate Immune Defense of the Blood-Retina Barrier.

Purpose: For this study we aimed to understand if retinal pigment epithelial (RPE) cells express antimicrobial peptide lysozyme as a mechanism to protect the neuroretina from blood-borne pathogens. Methods: The expression of lysozyme in human and mouse RPE cells was examined by RT-PCR or immune (cyto)histochemistry in cell cultures or retinal sections. RPE cultures were treated with different concentrations of Pam3CSK4, lipopolysaccharides (LPS), staphylococcus aureus-derived peptidoglycan (PGN-SA), Poly(I:C), and Poly(dA:dT). The mRNA expression of lysozyme was examined by qPCR and protein expression by ELISA. Poly(I:C) was injected into the subretinal space of C57BL/6J mice and eyes were collected 24 hours later and processed for the evaluation of lysozyme expression by confocal microscopy. Bactericidal activity was measured in ARPE19 cells following LYZ gene deletion using Crispr/Cas9 technology. Results: The mRNA and protein of lysozyme were detected in mouse and human RPE cells under normal conditions, although the expression levels were lower than mouse microglia BV2 or human monocytes THP-1 cells, respectively. Immunohistochemistry showed punctate lysozyme expression inside RPE cells. Lysozyme was detected by ELISA in normal RPE lysates, and in live bacteria-treated RPE supernatants. Treatment of RPE cells with Pam3CSK4, LPS, PGN-SA, and Poly(I:C) enhanced lysozyme expression. CRISPR/Cas9 deletion of lysozyme impaired bactericidal activity of ARPE19 cells and reduced their response to LPS and Poly(I:C) stimulation. Conclusions: RPE cells constitutively express antimicrobial peptide lysozyme and the expression is modulated by pathogenic challenges. RPE cells may protect the neuroretina from blood-borne pathogens by producing antimicrobial peptides, such as lysozyme.

Retrobulbarly injecting nerve growth factor attenuates visual impairment in streptozotocin-induced diabetes rats.

PURPOSE: To explore whether retrobulbar administration of nerve growth factor (NGF) can restore visual function of streptozotocin-induced diabetes rats. METHODS: A high-sucrose/high-fat diet and single injection of streptozotocin (STZ) were used in modeling diabetes. During week 13-15 after STZ injection, diabetic rats were received retrobulbar ßNGF injection. On week 17 after STZ injection, the rats were tested with flash visual evoked potential (FVEP) to reflect visual function and with both optical coherence tomography (OCT) and hematoxylin and eosin (H&E) staining to show retinal morphological changes. Furthermore, periodic acid-Schiff (PAS) staining for retinal vascular digest preparations was performed to investigate retinal microvascular alterations, and immunofluorescences for slides of the optic nerve or retina were checked to assess astrocyte activation, autophagy level, and the unfolded protein response (UPR). RESULTS: Retrobulbar ßNGF injection significantly improved FVEP of diabetic rats. It also significantly alleviated retinal ganglion cell (RGC) loss and scarcely elicited other retinal/microvascular morphological changes, in OCT, H&E staining, and microvascular preparation. Moreover when diabetes rats treated with NGF, immunostaining of the optic nerve showed downregulation of complement 3d (C3d) and upregulations of glial fibrillary acidic protein (GFAP), S100-A10, microtubule-associated proteins 1A/1B light chain 3b (LC3b), and activating transcription factor 4 (ATF-4), while immunostaining of the retina showed upregulation of LC3b and no expression of ATF-4. CONCLUSION: Our findings demonstrate that retrobulbar administration of ßNGF reduces visual impairment with RGC-loss attenuation and without retinal-microvascular morphological alteration in diabetic rats. Furthermore, enhancements of A2 astrocyte activation, autophagy-protein expression, and ATF-4-mediated UPR may play crucial roles in the protective mechanism of NGF in diabetic visual-pathway neurodegeneration.