Approches thérapeutiques des opacités cornéennes par modulation de l'activité de protéinases de la matrice extracellulaire

Pour l'être vivant, la vision constitue un élément important pour la perception de l'environnement. Cette fonction peut être altérée par une perte de transparence de la cornée pouvant conduire à une cécité. Les cécités causées par une perte de transparence cornéenne constituent selon l'Organisation Mondiale de la Santé la 3ème cause de cécité dans le monde après la cataracte et le glaucome. Le seul traitement curatif qui existe à l'heure actuelle pour ce type de cécité est la greffe de cornée. Ce traitement bien que satisfaisant demeure imparfait car il se heurte à de nombreuses complications telles que le rejet de greffe. De ce fait, ce travail vise à étudier des solutions alternatives aux greffes de cornée dans le cadre du traitement des opacités cornéennes et comporte deux axes : Un axe thérapeutique qui évalue l'efficacité de protéinases dans la résorption de l'opacité cornéenne. Les études menées au cours de cette thèse ont montré que la surexpression de la MMP-14 par les kératocytes via un vecteur parvoviral induisait une diminution de la sévérité de l'opacité cornéenne installée après traumatisme. Une autre partie de ses travaux a également montré que les protéinases Tolloïdes et leurs activateurs PCPEs (Procollagen C-proteinase enhancer), protéines qui interviennent dans la maturation du collagène joueraient un rôle important dans la cicatrisation cornéenne notamment dans la régénération de la lame basale et pourraient ainsi constituer une cible thérapeutique pertinente. Un axe plus fondamental qui porte sur l'étude de l'implication des collagènes de type XII et de type XIV dans la mise en place et le maintien de l'opacité cornéenne après blessure. Ces molécules font partie des collagènes FACIT (Fibril Associated Collagens with Interrupted Triple helices). Les investigations entreprises lors de cette étude ont montré que l'expression du collagène de type XII augmente au niveau de la cornée après un traumatisme. De manière intéressante, cet accroissement ne s'observe que dans les zones de la cornée où de l'opacité s'est installée. Cette remarque, d'abord effectuée chez l'homme s'avère également valable chez la souris. De plus, des analyses plus poussées de cette protéine réalisées chez la souris ont montré une expression qui varie en fonction du degré d'opacification de la cornée. En d'autres termes, plus l'opacité de la cornée est sévère, plus l'expression du collagène de type XII est importante. Ceci laisse donc supposer que cette molécule jouerait un rôle important dans le maintien de l'opacité au niveau de la cornée. En conclusion, ce travail de thèse a permis de démontrer d'une part, l'intérêt de la modulation de métalloprotéinases dans la résorption de l'opacité cornéenne et d'autre part, l'implication du collagène FACIT de type XII dans le maintien de l'opacité mise en place après un traumatisme cornéen.Vision constitutes an important element for our perception of the environment. Visual quality can be altered by loss of corneal transparency that can lead to corneal blindness. Loss of corneal transparency represents the 3rd cause of blindness worldwide according to World Health Organization. The only current curative treatment for this type of blindness is corneal transplantation. However, this treatment although satisfactory, faces many complications such as graft rejection. Thus, the purpose of this work was to study alternatives to corneal transplantation in the treatment of corneal opacities. The work consists of two majors goals: A therapeutic axis: This first aim concerns the evaluation of the effectiveness of the modulation of metalloproteinase activity in the resorption of corneal opacity. This project included the examination of the overexpression of the matrix metalloproteinase (MMP) 14 in in vivo mouse corneal scarring. The results showed a decrease of corneal opacity following over expression of MMP14 in the corneal stroma. This project also assessed the variation in the expression of Tolloid proteinases and their enhancers PCPEs (Procollagen C-Proteinase Enhancers) during in vivo corneal wound healing. We observed a significant increase in these proteinases following corneal incision. These observations suggest that these proteinases could play an important role in corneal matrix remodeling observed during wound healing. A more fundamental axis: The purpose of this project was to investigate the implication of FACIT (Fibril Associated Collagens with Interrupted Triple helices) type XII and type XIV collagens in the establishment and maintenance of corneal opacity after injury. We observed an increase in expression of type XII collagen in the cornea after injury, more precisely where opacities persisted. This was demonstrated not only in human corneas, but also in a mouse corneal scarring model. Furthermore, the expression of type XII collagen changed according to the degree of corneal opacity. These results suggest that the type XII collagen molecule could be important in the development and the maintenance of corneal opacity after injury. In conclusion, these projects have demonstrated a novel significance in the modulation of metalloproteinase activity in the resorption of corneal opacity and the implication of the FACIT type XII collagen in the maintenance of opacity after corneal trauma

Mucosal Administration of Collagen V Ameliorates the Atherosclerotic Plaque Burden by Inducing Interleukin 35-dependent Tolerance

We have shown previously that collagen V (col(V)) autoimmunity is a consistent feature of atherosclerosis in human coronary artery disease and in the Apoe(-/-) mouse model. We have also shown sensitization of Apoe(-/-) mice with col(V) to markedly increase the atherosclerotic burden, providing evidence of a causative role for col(V) autoimmunity in atherosclerotic pathogenesis. Here we sought to determine whether induction of immune tolerance to col(V) might ameliorate atherosclerosis, providing further evidence for a causal role for col(V) autoimmunity in atherogenesis and providing insights into the potential for immunomodulatory therapeutic interventions. Mucosal inoculation successfully induced immune tolerance to col(V) with an accompanying reduction in plaque burden in Ldlr(-/-) mice on a high-cholesterol diet. The results therefore demonstrate that inoculation with col(V) can successfully ameliorate the atherosclerotic burden, suggesting novel approaches for therapeutic interventions. Surprisingly, tolerance and reduced atherosclerotic burden were both dependent on the recently described IL-35 and not on IL-10, the immunosuppressive cytokine usually studied in the context of induced tolerance and amelioration of atherosclerotic symptoms. In addition to the above, using recombinant protein fragments, we were able to localize two epitopes of the α1(V) chain involved in col(V) autoimmunity in atherosclerotic Ldlr(-/-) mice, suggesting future courses of experimentation for the characterization of such epitopes

Approches thérapeutiques des opacités cornéennes par modulation de l'activité de protéinases de la matrice extracellulaire

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Deletion of the Unfolded Protein Response Transducer IRE1α Is Detrimental to Aging Photoreceptors and to ER Stress-Mediated Retinal Degeneration

PurposeThe unfolded protein response (UPR) is triggered when the protein folding capacity of the endoplasmic reticulum (ER) is overwhelmed and misfolded proteins accumulate in the ER, a condition referred to as ER stress. IRE1α is an ER-resident protein that plays major roles in orchestrating the UPR. Several lines of evidence implicate the UPR and its transducers in neurodegenerative diseases, including retinitis pigmentosa (RP), a group of inherited diseases that cause progressive dysfunction and loss of rod and cone photoreceptors. This study evaluated the contribution of IRE1α to photoreceptor development, homeostasis, and degeneration.MethodsWe used a conditional gene targeting strategy to selectively inactivate Ire1α in mouse rod photoreceptors. We used a combination of optical coherence tomography (OCT) imaging, histology, and electroretinography (ERG) to assess longitudinally the effect of IRE1α deficiency in retinal development and function. Furthermore, we evaluated the IRE1α-deficient retina responses to tunicamycin-induced ER stress and in the context of RP caused by the rhodopsin mutation RhoP23H.ResultsOCT imaging, histology, and ERG analyses did not reveal abnormalities in IRE1α-deficient retinas up to 3 months old. However, by 6 months of age, the Ire1α mutant animals showed reduced outer nuclear layer thickness and deficits in retinal function. Furthermore, conditional inactivation of Ire1α in rod photoreceptors accelerated retinal degeneration caused by the RhoP23H mutation.ConclusionsThese data suggest that IRE1α is dispensable for photoreceptor development but important for photoreceptor homeostasis in aging retinas and for protecting against ER stress-mediated photoreceptor degeneration

WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM) GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF). Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies

NC1 Long and NC3 Short Splice Variants of Type XII Collagen Are Overexpressed during Corneal Scarring

PURPOSE. To investigate type XII collagen expression in corneal scars in vivo. METHODS. Type XII collagen protein expression was evaluated by immunohistochemistry in human corneal scars and in a mouse model of corneal scarring at several time points (from day 7 to day 210) after full-thickness excision. Alternative splice variants of the NC3 and NC1 domains of type XII collagen were investigated in the mouse wound-healing model using RT-PCR. RESULTS. Type XII collagen was overexpressed in human corneal scars in areas that were also positive for alpha-smooth muscle actin staining. In a mouse model of corneal wound injury we found that at 14 and 21 days postexcision, type XII collagen was largely concentrated in the subepithelial region of the cornea, especially in and near the wound bed. By 28 days postexcision, expression of type XII collagen decreased but remained higher than that in controls. NC3 short form is the main form expressed in the cornea during the wound-healing process. After injury, the NC1 long splice variant mRNA was the most highly overexpressed variant in the cornea, especially in the epithelium (X2.7, 3.72, and 5.57 at days 7, 14, and 21, respectively, P < 0.01 to 0.001 compared with uninjured samples). Corneal scars from a 7-month-old mouse revealed an overexpression of type XII collagen in the wound area similar to what we observed in human corneal scars. CONCLUSIONS. Type XII collagen is overexpressed in permanent human and mouse corneal scars and could represent a new target to treat corneal scarring. (Invest Ophthalmol Vis Sci. 2012; 53: 7246-7256) DOI: 10.1167/iovs.11-859

Upregulation of Bone Morphogenetic Protein-1/Mammalian Tolloid and Procollagen C-Proteinase Enhancer-1 in Corneal Scarring

International audiencePurpose: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. Methods: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries. Results: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. Conclusions: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring

Cardiovascular Function and Structure are Preserved Despite Induced Ablation of BMP1-Related Proteinases

IntroductionBone morphogenetic protein 1 (BMP1) is part of an extracellular metalloproteinase family that biosynthetically processes procollagen molecules. BMP1- and tolloid-like (TLL1) proteinases mediate the cleavage of carboxyl peptides from procollagen molecules, which is a crucial step in fibrillar collagen synthesis. Ablating the genes that encode BMP1-related proteinases (Bmp1 and Tll1) post-natally results in brittle bones, periodontal defects, and thin skin in conditional knockout (BTKO) mice. Despite the importance of collagen to cardiovascular tissues and the adverse effects of Bmp1 and Tll1 ablation in other tissues, the impact of Bmp1 and Tll1 ablation on cardiovascular performance is unknown. Here, we investigated the role of Bmp1- and Tll1-ablation in cardiovascular tissues by examining ventricular and vascular structure and function in BTKO mice.MethodsVentricular and vascular structure and function were comprehensively quantified in BTKO mice (n=9) and in age- and sex-matched controls (n=9). Echocardiography, cardiac catheterization, and biaxial ex vivo arterial mechanical testing were performed to assess tissue function, and histological staining was used to measure collagen protein content.ResultsBmp1- and Tll1-ablation resulted in maintained hemodynamics and cardiovascular function, preserved biaxial arterial compliance, and comparable ventricular and vascular collagen protein content.ConclusionsMaintained ventricular and vascular structure and function despite post-natal ablation of Bmp1 and Tll1 suggests that there is an as-yet unidentified compensatory mechanism in cardiovascular tissues. In addition, these findings suggest that proteinases derived from Bmp1 and Tll1 post-natally have less of an impact on cardiovascular tissues compared to skeletal, periodontal, and dermal tissues