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The subretinal fibrosis model:
The new norm for modelling wet AMD

April 2024

Introduction

Age-related macular degeneration (AMD) is a growing concern for vision health, affecting over a million people in the UK alone and with projections indicating a staggering 288 million diagnoses globally by 20401. Amidst the intricacies of its pathology, fibrosis is emerging as a pivotal factor, especially in the advanced stages of neovascular AMD (nAMD). Fibrosis in nAMD is associated with particularly poor long-term vision outcomes despite the use of anti-VEGFs, a key mainstay of current treatment strategies.

In this blog, we provide an overview of fibrosis in AMD, potential therapeutic avenues and a new model to meet the challenge, the subretinal fibrosis model. By unravelling the molecular pathways involved in fibrosis and considering this knowledge in the preclinical models we use, we can pave the way for more potent treatments to combat vision loss in AMD patients.

The impact of fibrosis on vision loss, just how common is it?

AMD is a complex disease with diverse pathways interacting with environmental factors to drive pathology. Several of these pathways, including inflammatory and complement related pathways, are heavily implicated in fibrosis.   As for how common subretinal fibrosis is in AMD, studies have revealed fibrosis in as many as 36% of patients after year one, 45% by year two and 71% by year 10, regardless of whether or not they have received the regular anti-VEGF treatment 2-7
These statistics raise alarms, especially considering the terrible impact that fibrosis has on visual outcomes. Even with anti-VEGF treatment, half of nAMD patients still battle subretinal fibrosis
6 and half of these again will suffer an ETDRS loss of more than 15 letters over 10 years as a result8. Subretinal fibrosis can result in as much as a 30-letter drop in BCVA9.

Pathophysiology of fibrosis in AMD

AMD belfast street.png
Fibrosis AMD stats.png

AMD patient perspective - City centre street, Belfast, UK

While we’re still unravelling the complexities of subretinal fibrosis in nAMD, fibrosis formation initially follows a familiar pattern to that seen in other organs like the lungs, liver, and skin. When the retina suffers injury, epithelial cells release signals that attract inflammatory cells, endothelial cells, and fibroblasts. These signals cause local cells to undergo various mesenchymal transitions, depending on the original cell type, transforming into myofibroblasts. The new myofibroblasts then produce excessive extracellular matrix (ECM) to cover the damaged tissue in a normal repair response. In the case of the nAMD eye, this deposition surrounds new diseased vessels, and this lends to the resistance to anti-VEGF therapy that is apparent in patients with fibrosis. More, because the original injury or inflammation persists, the fibrotic scar builds and persists. This happens in a number of ways.

One way is the dysfunction of the RPE cells themselves, driving their transition to myofibroblasts. This transition can be observed in several ways, including less pigmentation and more α-SMA expression the closer the cell is to the site of the choroidal neovascularisation (CNV) , but the loss of this contact can occur as new abnormal blood vessels form around and through the RPE layer12.

 

Neovascularization also fuels fibrosis in what can be thought of as a vicious cycle. Namely, the new blood vessels, created because of high levels of growth factors, are leaky and hemorrhagic, allowing even more inflammatory cells and fibroblasts to be recruited and so feeding fibrosis. In turn, the ECM that builds as a result plus the inflammation and complement dysfunction then further stabilises the abnormal vessels.

 

Eventually, as these and other pathways persist, CNV is replaced by a grey-yellow fibrovascular membrane that later becomes an area of atrophy on both the retina and choroid, with the devastating consequences for vision mentioned above.

What can we do about it?

The impressive success of anti-VEGF therapies has somewhat hindered the exploration of alternative treatments for nAMD and other similar diseases; they are difficult to compete with in clinical trial. However, as we better understand AMD pathology and longer follow-up studies become available, it has become increasingly apparent that not everyone responds to anti-VEGF treatment. Simply put, anti-VEGF medications do not address fibrosis.The involvement of multiple cell types and their complex interactions make developing effective strategies for fibrosis challenging. Therapeutic targets for fibrosis range from TGF-β inhibitors to integrin antagonists to myofibroblast depletion strategies, each with their own challenges. The prospect of an altered immune response has also captured wide research interest, with patients with fibrosis in the macula also possessing an altered systemic immune profile including elevated complement fragments like C3a and C5a13
and reduced complement regulators like CD4614. The complement system is central to chronic subretinal inflammation, directly driving fibrosis in a myriad of ways15. With a better understanding of these diseases and their progression, coupled with the recognition of this significant population, the pursuit of new treatment avenues has become more imperative than ever. This call is being answered by a wave of exciting developments in the field. From innovative drug therapies, like EOM Pharmaceutical’s asset EOM147, which targets multiple growth factors including bFGF in addition to VEGF, to cutting-edge gene therapies and regenerative medicine approaches, researchers are exploring a diverse array of options to address the shortcomings of current treatments.

Fibrosis therapeutic targets.png

Fig 1. Therapies beyond anti-VEGFs. Pathways that are altered in AMD related retinal fibrosis include complement pathways, integrins and inflammatory responses among others. Investigation of each of these presents us with additional therapeutic targets and strategies. These have been explored to different extends, with some already leading to new drugs at various trial levels.

A better model for subretinal fibrosis

Improving preclinical models is key to accelerating the development of new treatments to addresses fibrosis in addition to angiogenesis. One of the most commonly used models in nAMD, the laser-induced choroidal neovascularisation (CNV) model, has been used in the past. This has several limitations, however, that limit its applicability to fibrosis and wider human disease. One limitation is that the lesions produced by this model resolve within as little as one month. As such, a fibrovascular membrane doesn’t form, and while microglia can be observed in the periphery of the lesion, these rarely if ever actually infiltrate the site of injury. More recently, an improvement on this model has been proposed, the subretinal fibrosis model introduced and characterised by the Chen & Xu laboratories in Queen’s University Belfast16.

CNV SRF Figure.png

Fig. 2 Schematic differences between one-laser CNV and SRF model. The differences between the CNV and SRF model include a larger lesion that lasts for longer and a larger immune response. While traditional CNV lesions show blood vessel regression by day 30, the SRF lesion continues to expand, with concurrent fibrosis and vasculature formation forming a fibrovascular membrane—a key feature of subretinal fibrosis in nAMD.

Unlike the traditional CNV model, the subretinal fibrosis model shows no sign of abating even 40 days after injury, and boasts lesions with COL-1+ fibres and IBA1+/CD31+ blood vessels as many as 40 days after laser injury, higher proinflammatory and VEGF expression and, crucially, profibrotic gene expression in the form of TGF-β and FGF2. Ultimately, the subretinal fibrosis model produces a lesion that much better represents the chronic fibrosis in nAMD, complete with features like a fibrovascular membrane and microglial infiltration.  

The future of fibrosis treatment

To sum up, fibrosis plays a critical role in the progression of AMD, particularly in nAMD, leading to irreversible vision loss despite current treatment strategies. Confronting fibrosis poses a significant challenge, but with innovative approaches and improved preclinical models, there is hope for developing more effective therapies. Collaborative efforts across the scientific community are essential to advance our understanding and treatment of fibrosis in AMD, ultimately improving outcomes for patients worldwide.

 

The complex mechanisms of fibrosis in the retina have been reviewed in depth elsewhere, including the following:

Xu et al., 2024

Ishikawa et al., 2015;
Higashijima et al., 2023;
Sorenson et al., 2024;
Liu et al., 2023

We want your help. Is there analysis we are lacking? Tell us.....

Or reach out to us - we have tissue/samples available to identify whether your therapeutic targets are upregulated in this model.

References:

1 Wong, W. L. et al. Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis. The Lancet. Global Health 2 (2014). https://doi.org/10.1016/S2214-109X(13)70145-1
2 Bhisitkul, R. B. et al. Macular atrophy progression and 7-year vision outcomes in subjects from the ANCHOR, MARINA, and HORIZON studies: the SEVEN-UP study. American Journal of Ophthalmology 159 (2015). https://doi.org/10.1016/j.ajo.2015.01.032
3 Rofagha, S., Bhisitkul, R. B., Boyer, D. S., Sadda, S. R. & Zhang, K. Seven-year outcomes in ranibizumab-treated patients in ANCHOR, MARINA, and HORIZON: a multicenter cohort study (SEVEN-UP). Ophthalmology 120 (2013). https://doi.org/10.1016/j.ophtha.2013.03.046
4 Bloch, S. B., Lund-Andersen, H., Sander, B. & Larsen, M. Subfoveal fibrosis in eyes with neovascular age-related macular degeneration treated with intravitreal ranibizumab. American Journal of Ophthalmology 156 (2013). https://doi.org/10.1016/j.ajo.2013.02.012
5 Willoughby, A. S. et al. Subretinal Hyperreflective Material in the Comparison of Age-Related Macular Degeneration Treatments Trials. Ophthalmology 122 (2015). https://doi.org/10.1016/j.ophtha.2015.05.042
6 Daniel, E. et al. Risk of scar in the comparison of age-related macular degeneration treatments trials. Ophthalmology 121 (2014). https://doi.org/10.1016/j.ophtha.2013.10.019
7 Wolff, B. et al. Ten-year outcomes of anti-vascular endothelial growth factor treatment for neovascular age-related macular disease: A single-centre French study. Clinical & Experimental Ophthalmology 48 (2020). https://doi.org/10.1111/ceo.13742
8 Chandra, S. et al. Ten-year outcomes of antivascular endothelial growth factor therapy in neovascular age-related macular degeneration. Eye 34 (2020).
https://doi.org/10.1038/s41433-020-0764-9
9 Brynskov, T., Munch, I. C., Larsen, T. M., Erngaard, L. & Sørensen, T. L. Real-world 10-year experiences with intravitreal treatment with ranibizumab and aflibercept for neovascular age-related macular degeneration. Acta Ophthalmologica 98 (2020). https://doi.org/10.1111/aos.14183
10 Lopez, P. F., Sippy, B. D., Lambert, H. M., Thach, A. B. & Hinton, D. R. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Investigative Ophthalmology & Visual Science 37 (1996).
11 Tamiya, S., Liu, L. & Kaplan, H. J. Epithelial-mesenchymal transition and proliferation of retinal pigment epithelial cells initiated upon loss of cell-cell contact. Investigative Ophthalmology & Visual Science 51 (2010). https://doi.org/10.1167/iovs.09-4725
12 Ambati, J. & Fowler, B. J. Mechanisms of age-related macular degeneration. Neuron 75 (2012). https://doi.org/10.1016/j.neuron.2012.06.018

13 Lechner, J. et al. Higher plasma levels of complement C3a, C4a and C5a increase the risk of subretinal fibrosis in neovascular age-related macular degeneration: Complement activation in AMD. Immunity & Ageing 13 (2016). https://doi.org/10.1186/s12979-016-0060-5
14 Singh, A. et al. Altered expression of CD46 and CD59 on leukocytes in neovascular age-related macular degeneration. American Journal of Ophthalmology 154 (2012). https://doi.org/10.1016/j.ajo.2012.01.036
15 Xu, H., Yi, C. & Chen, M. (Current Opinion in Pharmacology, 2024).
16 Little, K. et al. A Two-Stage Laser-Induced Mouse Model of Subretinal Fibrosis Secondary to Choroidal Neovascularization. Translational Vision Science & Technology 9, 3-3 (2023).https://doi.org/10.1167/tvst.9.4.3

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