Investigating The Effect Of Age In Subretinal Fibrosis And The Underlying Mechanisms

Aims:This study aimed to understand the impact of age on subretinal fibrosis secondary to neovascular age-related macular degeneration（n AMD）and the underlying mechanisms,including the systemic factors such as bone marrow-derived macrophages（BMDMs）and circulating fibrocytes,and local RPE/choroidal microenvironment.Part 1Purpose:To investigate the effect of age on subretinal fibrosis and the mechanism of action,particularly the role of age-related alterations in BMDMs and circulating fibrocytes.Methods:（1）Young（2-2.5 months）and aged（15-16 months）mice were subjected to two-stage laser-induced subretinal fibrosis.Eyes,blood and bone marrows were collected 5 and 30 days after the second laser for further investigation.Eyes were processed for immunostaining of collagen-1 and CD45 in RPE/choroid flatmounts or cryosections.Samples were imaged by confocal microscopy for the measurement of subretinal fibrotic lesions and CD45⁺collagen-1⁺cells.Bone marrows and blood were collected for the analysis of circulating fibrocytes（CD45⁺collagen-1⁺）using flow cytometry.（2）Bone marrow cells from 22-month old Ly5.2 donor mice were transplanted into the 2-month old young x-ray irradiated recipient mice.Six weeks later,the recipient mice were subjected to subretinal fibrosis induction and collagen-1⁺fibrotic lesions were evaluated 30 days after the second laser.（3）BMDMs from young and aged mice were cultured with/without10ng/ml of recombinant transforming growth factor beta 1（TGF-β1）for 96h.The cells were collected for immunocytochemistry of collagen-1⁺α-SMA⁺cells and q RT-PCR analysis of fibrosis-related genes（Col1a1,Acta2,Fn1）and Tgfb1.The Luminex multiplex cytokine assay was used to measure 15 cytokines/chemokines in BMDM supernatants.Results:（1）Confocal microscopy of RPE/choroid flatmounts and cryosections showed that aged mice had a significantly larger size of subretinal collagen-1⁺fibrotic lesion and an increased number of CD45⁺collagen-1⁺cells at lesion site than young mice.Under normal conditions,the population of blood CD45⁺collagen-1⁺fibrocytes was significantly higher in aged mice than that in young mice.Induction of subretinal fibrosis increased the population of CD45⁺collagen-1⁺cells in bone marrows in both young and aged mice.The population of blood and bone marrow CD45⁺collagen-1⁺fibrocytes was significantly higher in aged subretinal fibrosis mice compared to that in young subretinal fibrosis mice.Young mice transplanted with old bone marrows had significantly larger size of subretinal fibrosis compared with age-matched mice without old bone marrow transplantation.（2）BMDMs from aged mice expressed higher levels of collagen-1,α-SMA and Tgfb1,had higher potentials to undergo TGF-β1-induce macrophage-to-myofibroblast transition（MMT）,produced higher levels of profibrotic cytokines EGF and OPN,but less anti-fibrotic chemokine CXCL10.Upon induction of subretinal fibrosis,BMDMs from both young and old mice expressed higher levels of myofibroblast signature genes（Col1a1,Acta2,Fn1）and produced more pro-fibrotic factor u PAR,but less anti-fibrotic factor CXCL10.Furthermore,the induction of subretinal fibrosis in aged mice also increased the production of VEGF and Ang-2 in BMDMs.Conclusion:Old age aggregates subretinal fibrosis through increased circulating fibrocytes and pro-fibrotic bone marrow-derived macrophages.Part 2Purpose:To investigate the molecular mechanism involved in the development of subretinal fibrosis using RNA sequencing（RNA-seq）technology.Methods:The normal and subretinal fibrotic RPE/choroid from young mice were collected and processed for RNA sequencing（RNA-seq）analysis.Differentially expressed genes（DEGs）were identified by DESeq2.Gene Ontology（GO）and KEGG were used to analyze the enriched pathways.The expression of the selected DEGs including matrix metalloproteinase 12（Mmp12）was verified by q PCR.The expression of MMP12 in subretinal fibrosis of mouse and n AMD donor eyes was examined by confocal microscopy.The expression of collagen-1,α-SMA,fibronectin and cytokines in BMDMs from control and subretinal fibrosis mice were examined by q PCR,immunocytochemistry and Luminex multiplex cytokine assay.A MMP12-specific inhibitor MMP408 was used to evaluate the effect of MMP12 on TGFβ1-induced macrophage-to-myofibroblast transition（MMT）in vitro and its role in subretinal fibrosis in vivo.Results:RNA-seq analysis of RPE/choroid from control and subretinal fibrosis eyes uncovered 139 DEGs（log₂FC≥0.5,FDR<0.05）,including 104 up-regulated and 35 down-regulated genes.The top 25enrichment 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 25GO terms.Higher levels of MMP12 were detected in subretinal fibrotic lesions in n AMD patients and the mouse model.Many F4/80⁺or Iba1⁺macrophages express MMP12.BMDMs from subretinal fibrosis mice expressed higher levels of MMP12,collagen-1,αSMA and fibronectin.MMP408 dose-dependently suppressed TGFβ-induced MMT in vitro,reduced the size of subretinal fibrosis and the infiltration of F4/80⁺macrophage in vivo.Conclusion:Macrophages may contribute to the development of subretinal fibrosis through secreting MMP12.Part 3Purpose:To investigate the effect of the local microenvironment of aged RPE/choroid on subretinal fibrosis using RNA-seq technology.Methods:The control and subretinal fibrotic RPE/choroid from young and aged mice were collected and processed for RNA-seq analysis.m MCP-counter were used to analyse all genes,DEGs of young and aged normal RPE/choroid,and DEGs of young and aged fibrosis RPE/choroid.GO and KEGG enrichment analysis was carried out using DEGs between young and aged normal RPE/choroid or DEGs between young and aged subretinal fibrosis RPE/choroid.Results:m MCP-counter analysis showed that aged RPE/choroid had lower vessel and lymphatic cell scores,higher fibroblast scores compared to young RPE/choroid.The GO enrichment analysis of DEGs between normal young and old RPE/choroid showed the top 25 terms were related to immune regulation,protein metabolism and ribosome function.Similar pathways were observed in the KEGG analysis.After induction of subretinal fibrosis,2066 DEGs were detected in age RPE/choroid and 139DRGs were detected in young mice.m MCP-counter analysis revealed significant higher scores of monocytes/macrophages and fibroblasts in the aged RPE/choroid compared with young RPE/choroid.GO and KEGG enrichment analysis of shared DEGs between young and aged subretinal fibrosis RPE/choroid showed that the enriched pathways were relate to immune response（e.g.complement-coagulation cascade pathway）and extracellular matrix-receptor interactions.GO and KEGG enrichment analysis of DEGs that are specific to subretinal fibrosis of aged RPE/choroid showed that the immune response,angiogenesis,extracellular matrix-receptor interactions,cellular chemotaxis and activation,chemokine and TGFβsignaling pathways were enriched.Mitochondrial respiratory chain complex synthesis and oxidative phosphorylation related genes were down-regulated in aged subretinal fibrosis RPE/choroid.Conclusion:Old age promotes subretinal fibrosis through the pro-inflammatory and pro-fibrotic microenvironment of RPE/choroid.