Research directions
Deciphering the role of MAPK in Age-Related Macular Degeneration (ARMD)
ARMD is one of the most common irreversible causes of loss of vision occurring in aged patients (over 50 years). Despite intensive research, the mechanisms leading to ARMD remain vague and no really effective treatment is available yet. Features of ARMD have been well characterized and the role of RPE cell death seems to be central at the onset of the disease. Moreover, there are strong evidence for an important role of reactive oxygen species (ROS) and macrophages in ARMD, but how these compounds are induced by external stress and how they act on cells remains unclear.
The role of mitogen-activated protein kinase (MAPK) in degenerative processes has been described in a large number of studies, from in vitro systems to animal models. The involvement of these kinases has been highlighted in human pathologies, including neurodegenerative diseases (Parkinson, Alzheimer and Stroke), metabolism diseases (diabetes, obesity) and cancer. To date no specific analyses or studies on the role of MAPK in ARMD disease have been performed, while several lines of evidence suggest the implication of these kinases in this pathology.
We here propose to analyze in detail the role of MAPK in the mechanisms of RPE cell death and in ARMD mouse models. Two main projects are led to this aim *:
1) Analyze the effect of bis-retinoids, A2E, on MAPK(s) activities in polarized ARPE19 and mouse isolated RPE cells.
2) Decipher the role of ERK2 in ARMD using a mouse model with a disruption of the kinase specifically in RPE.
We have recently shown the key role of kinases in various models, including glucose- and leptin-induced pancreatic β-cell apoptosis (Meadler et al.2008) and UV-induced ARPE19 cell death (Roduit et al.2008).
* Studies supported by Gelbert Foundation and Fritz-Tobler Foundation.
Acute Hypoglycemia Induces Retinal Cell Death in Mouse
Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. Very little is known about the deleterious effect of hypoglycemia. Therefore, we decided to study the role of acute hypoglycemia in mouse retina **.
We showed that hypoglycemia (5h) induces retinal cell death. Many genes are affected by hypoglycemia, among them few implicated in glutathion (GSH) metabolism, a scavenger protein that protect cells against oxidative stress. Decrease of GHS induces cell death while restauration of normal GSH level during low glucose, protect these cells.
We showed that low glucose induces also autophagy process and affect autophagosomes/lysosomes fusion. Inhibition of autophagy, either chemically or genetically, induced a more pronounce effect when cells are cultured at low glucose.
We confirmed that hypoglycemia induces retinal cell death in mouse, with a key role of GSH in the process. In addition, a defect in the protective autophagy pathway is implicated and may play a role in diabetic retinopathy. Actually, our studies are focus on the protective role of GSH in hypoglycemia-induced retinal cell death.
** project supported by «la fondation romande pour la recherche sur le diabète », by the « Art & Vie » foundation and by « Rouffy&Cardi » foundation.
Proteomic study in refractory ARMD
The standard treatment for neovascular age-related macular degeneration (nAMD) consists of intravitreal anti-vascular endothelial growth factors (VEGF). However, for some patients, even maximal anti-VEGF treatment does not entirely suppress exudative activity. The goal of this study was to identify molecular biomarkers in nAMD with incomplete response to anti-VEGF treatment.
Aqueous humor (AH) samples were collected from three groups of patients: 17 patients with nAMD responding incompletely to anti-VEGF (18 eyes), 17 patients affected by nAMD with normal treatment response (21 eyes), and 16 control patients without any retinopathy (16 eyes). Proteomic and multiplex analyses were performed on these samples.
Proteomic analyses showed that nAMD patients with incomplete anti-VEGF response displayed an increased inflammatory response, complement activation, cytolysis, protein-lipid complex, and vasculature development pathways. Multiplex analyses revealed a significant increase of soluble vascular cell adhesion molecule-1 (sVCAM-1), interleukin-6 (IL-6), bioactive interleukin-12 (IL-12p40), plasminogen activator inhibitor type 1 (PAI-1), and hepatocyte growth factor (HGF) levels in incomplete responders in comparison to normal responders. In addition, we confirmed by AlphaLISA the increase of sVCAM-1 and IL-6 in the incomplete responder group.
Inflammatory response after anti-VEGFA injection
Age-related macular degeneration (AMD) is a multifactorial disease, involving a large number of molecular factors, such as growth factors, inflammation, complement cascade, fluid regulation, radicals and anti-oxidative factors, interstitial factors, visual cycle products, and others. Currently, the established treatment for the neovascular form of AMD consists of intravitreal injections of anti-VEGF, mainly aflibercept (Eylea), ranibizumab (Lucentis), and recently brolucizumab (Beovu). Efficiencies are comparable, with a potential superiority for the Beovu, despite some side effects involving inflammatory response (uveitis, endophtalmitis).
The aim of the project is to analyze the levels of inflammatory response in the aqueous humor (AH) of AMD patients (60 AMD patients and 20 control participants) treated with the three different anti-VEGF-A treatments, including aflibercept, ranibizumab and brolucizumab (20 patients per group of drug). The main goal is to monitor cytokines levels during the entire treatment period (before any injection and after each injection planned at 1, 2, 4, 6, and 12 months). AH will be taken before any injection and analyzed by the multiplex system able to detect simultaneously around 23 diverse cytokines in a very low volume of AH.
In parallel, we planned to test the remaining levels of the anti-VEGF-A drugs using magnetic beads covalently linked to VEGF-A followed by a proteomic approach that enable us to quantify anti-VEGF-A drugs linked to VEGF-A or free.
Correlation between remained active drug and the inflammatory response will give us good indications and could partly explain higher inflammation observed in brolucizumab treated patient.
Omics analysis from mild to severe Diabetic Retinopathy (DR) vs no DR
Background.
Diabetes mellitus (DM) is among the leading causes of irreversible blindness worldwide in the working age population. Estimates point out that about 360 mio people will be affected by DM by 2030. Among them, a large number will develop a diabetic retinopathy (DR) as secondary effect. Therefore, it is crucial to classify DR into categories ranging from mild to severe non-proliferative DR, ending with proliferative DR. In addition to diabetes-established rules for this classification, it will be essential to precisely define each stage using biomarkers.
Project.
We propose to perform “omics” analyses on aqueous humor (AH) of diabetic patients affected by different stages of non-proliferative DR, from mild to severe, including proliferative DR patients. We will compare results with diabetic patients who are not affected by DR and non-diabetic patients, as controls. We will first perform epigenomics, proteomics and metabolomics analyses on AH to generate a kind of identification card for each group of DR patients. Then we will evaluate the presence of identified biomarkers in both tears and blood plasma.
Perspective.
The stratification and classification of all patients affected by DR into subgroups will be possible with this approach and will enable a more personalized treatment in order to give the right treatment to the right person at the right time. Moreover, identified biomarkers detectable in tears and/or in plasma serum would allow a non-invasive testing method of these biomarkers in patients.
