Dataset Information

Transcriptomic analysis of choroidal neovascularization reveals dysregulation of immune and fibrosis pathways that are attenuated by a novel anti-fibrotic treatment.

ABSTRACT: Neovascular AMD (nAMD) leads to vision loss and is a leading cause of visual impairment in the industrialised world. Current treatments that target blood vessel growth have not been able to treat subretinal fibrosis and nAMD patients continue to lose vision. The molecular mechanisms involved in the development of fibrotic lesions in nAMD are not well understood. The aim of this study was to further understand subretinal fibrosis in the laser photocoagulation model of choroidal neovascularization (CNV) by studying the whole transcriptome of the RPE/choroid following CNV and the application of an anti-fibrotic following CNV. Seven days after laser induced CNV, RPE and choroid tissue was separated and underwent RNAseq. Differential expression analysis and pathway analysis revealed an over representation of immune signalling and fibrotic associated pathways in CNV compared to control RPE/choroid tissue. Comparisons between the mouse CNV model to human CNV revealed an overlap in upregulated expression for immune genes (Ccl2, Ccl8 and Cxcl9) and extracellular matrix remodeling genes (Comp, Lrcc15, Fndc1 and Thbs2). Comparisons between the CNV model and other fibrosis models showed an overlap of over 60% of genes upregulated in either lung or kidney mouse models of fibrosis. Treatment of CNV using a novel cinnamoyl anthranilate anti-fibrotic (OCX063) in the laser induced CNV model was selected as this class of drugs have previously been shown to target fibrosis. CNV lesion leakage and fibrosis was found to be reduced using OCX063 and gene expression of genes within the TGF-beta signalling pathway. Our findings show the presence of fibrosis gene expression pathways present in the laser induced CNV mouse model and that anti-fibrotic treatments offer the potential to reduce subretinal fibrosis in AMD.

SUBMITTER: Brandli A

PROVIDER: S-EPMC8764037 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Abnormal migration and proliferation of endothelial cells (EC) drive neovascular retinopathies. While anti-VEGF treatment slows progression, pathology is often supported by decrease in intraocular pigment epithelium-derived factor (PEDF), an endogenous inhibitor of angiogenesis. A surface helical 34-mer peptide of PEDF, comprising this activity, is efficacious in animal models of neovascular retina disease but remains impractically large for therapeutic use. We sought smaller fragments within this sequence that mitigate choroidal neovascularization (CNV). Expecting rapid intravitreal (IVT) clearance, we also developed a method to reversibly attach peptides to nano-carriers for extended delivery. Synthetic fragments of 34-mer yielded smaller anti-angiogenic peptides, and N-terminal capping with dicarboxylic acids did not diminish activity. Charge restoration via substitution of an internal aspartate by asparagine improved potency, achieving low nM apoptotic response in VEGF-activated EC. Two optimized peptides (PEDF 335, 8-mer and PEDF 336, 9-mer) were tested in a mouse model of laser-induced CNV. IVT injection of either peptide, 2-5 days before laser treatment, gave significant CNV decrease at day +14 post laser treatment. The 8-mer also decreased CNV, when administered as eye drops. Also examined was a nanoparticle-conjugate (NPC) prodrug of the 9-mer, having positive zeta potential, expected to display longer intraocular residence. This NPC showed extended efficacy, even when injected 14 days before laser treatment. Neither inflammatory cells nor other histopathologic abnormalities were seen in rabbit eyes harvested 14 days following IVT injection of PEDF 336 (>200 ?g). No rabbit or mouse eye irritation was observed over 12-17 days of PEDF 335 eye drops (10 mM). Viability was unaffected in 3 retinal and 2 choroidal cell types by PEDF 335 up to 100 ?M, PEDF 336 (100 ?M) gave slight growth inhibition only in choroidal EC. A small anti-angiogenic PEDF epitope (G-Y-D-L-Y-R-V) was identified, variants (adipic-Sar-Y-N-L-Y-R-V) mitigate CNV, with clinical potential in treating neovascular retinopathy. Their shared active motif, Y - - - R, is found in laminin (Ln) peptide YIGSR, which binds Ln receptor 67LR, a known high-affinity ligand of PEDF 34-mer.

Project description:The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR(-/-) and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR(-/-) mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1(+) ) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C-C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-?1 (TGF?1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that are consistent with a possible role of AhR in wet AMD. The data discussed in this paper have been deposited in NCBI's Gene Expression Omnibus; GEO Submission No. GSE56983, NCBI Tracking System No. 17021116.

Project description:BackgroundRetinal 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.MethodsSubretinal 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-β1 (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.ResultsUnder 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.ConclusionsA 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.

Dataset Information

Transcriptomic analysis of choroidal neovascularization reveals dysregulation of immune and fibrosis pathways that are attenuated by a novel anti-fibrotic treatment.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets