Sorsby Fundus Dystrophy Research Articles

Tissue Inhibitor of Metalloproteinase 3 (TIMP3) mutations increase glycolytic activity and dysregulate glutamine metabolism in RPE cells

ObjectivesMutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsby's Fundus Dystrophy (SFD), a dominantly inherited, rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells, which constitute the outer blood-retinal barrier. One major function of RPE is the synthesis and transport of vital nutrients, such as glucose, to the retina. Recently, metabolic dysfunction in RPE cells has emerged as an important contributing factor in retinal degenerations. We set out to determine if RPE metabolic dysfunction was contributing to SFD pathogenesis. MethodsQuantitative proteomics was conducted on RPE of mice expressing the S179C variant of TIMP3, known to be causative of SFD in humans. Proteins found to be differentially expressed (P < 0.05) were analyzed using statistical overrepresentation analysis to determine enriched pathways, processes, and protein classes using g:profiler and PANTHER Gene Ontology. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. RPE metabolism was directly probed using isotopic tracing coupled with GC/MS analysis. Steady state [U–13C6] glucose isotopic tracing was preliminarily conducted on S179C ARPE-19 followed by [U–13C6] glucose and [U–13C5] glutamine isotopic tracing in SFD iRPE cells. ResultsQuantitative proteomics and enrichment analysis conducted on RPE of mice expressing mutant S179C TIMP3 identified differentially expressed proteins that were enriched for metabolism-related pathways and processes. Notably these results highlighted dysregulated glycolysis and glucose metabolism. Stable isotope tracing experiments with [U–13C6] glucose demonstrated enhanced glucose utilization and glycolytic activity in S179C TIMP3 APRE-19 cells. Similarly, [U–13C6] glucose tracing in SFD iRPE revealed increased glucose contribution to glycolysis and the TCA cycle. Additionally, [U–13C5] glutamine tracing found evidence of altered malic enzyme activity. ConclusionsThis study provides important information on the dysregulation of RPE glucose metabolism in SFD and implicates a potential commonality with other retinal degenerative diseases, emphasizing RPE cellular metabolism as a therapeutic target.

Metabolic phenotyping of healthy and diseased human RPE cells.

Metabolic defects in the retinal pigment epithelium (RPE) underlie many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells. We profiled nutrient utilization of various human RPE cells, including differentiated and dedifferentiated fetal RPE (fRPE), induced pluripotent stem cell derived-RPE (iPSC RPE), Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE, CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and ARPE-19 cell lines using Biolog Phenotype MicroArray Assays. Differentiated fRPE cells and healthy iPSC RPE cells can utilize 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose their epithelial phenotype through dedifferentiation, nutrient utilization becomes restricted to 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can utilize 37 nutrients; however, compared to cSFD RPE and healthy iPSC RPE, they are unable to utilize lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased utilization of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. Dedifferentiated ARPE-19 cells grown in traditional culture media cannot utilize lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation towards an epithelial phenotype, restoring the ability to use these nutrients. Epithelial phenotype confers metabolic flexibility to healthy RPE for utilizing various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the potentially important roles of nutrient availability and utilization in RPE differentiation and diseases.

Generation of an induced pluripotent stem cell line (SJTUGHi003-A) from a patient with Sorsby fundus dystrophy carrying c.484G>A mutation in TIMP3 gene

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disorder with macular dystrophy and severe visual loss. Mutations in TIMP3 gene has been related to SFD with mechanisms unclear. We have successfully reprogrammed the peripheral blood mononuclear cells (PBMCs) from an SFD patient carrying c.484G>A mutation in TIMP3 gene to induced pluripotent stem cells (iPSCs) and characterized their pluripotency and genetic stability. This line may serve as a useful tool to explore the role of TIMP3 in SFD pathogenesis.

Disease-Causing TIMP3 Variants and Deep Phenotyping of Two Czech Families with Sorsby Fundus Dystrophy Associated with Novel p.(Tyr152Cys) Mutation.

We aim to report the ocular phenotype and molecular genetic findings in two Czech families with Sorsby fundus dystrophy and to review all the reported TIMP3 pathogenic variants. Two probands with Sorsby fundus dystrophy and three first-degree relatives underwent ocular examination and retinal imaging, including optical coherence tomography angiography. The DNA of the first proband was screened using a targeted ocular gene panel, while, in the second proband, direct sequencing of the TIMP3 coding region was performed. Sanger sequencing was also used for segregation analysis within the families. All the previously reported TIMP3 variants were reviewed using the American College of Medical Genetics and the Association for Molecular Pathology interpretation framework. A novel heterozygous variant, c.455A>G p.(Tyr152Cys), in TIMP3 was identified in both families and potentially de novo in one. Optical coherence tomography angiography documented in one patient the development of a choroidal neovascular membrane at 54 years. Including this study, 23 heterozygous variants in TIMP3 have been reported as disease-causing. Application of gene-specific criteria denoted eleven variants as pathogenic, eleven as likely pathogenic, and one as a variant of unknown significance. Our study expands the spectrum of TIMP3 pathogenic variants and highlights the importance of optical coherence tomography angiography for early detection of choroidal neovascular membranes.

The extracellular microenvironment in immune dysregulation and inflammation in retinal disorders.

Inherited retinal dystrophies (IRDs) as well as genetically complex retinal phenotypes represent a heterogenous group of ocular diseases, both on account of their phenotypic and genotypic characteristics. Therefore, overlaps in clinical features often complicate or even impede their correct clinical diagnosis. Deciphering the molecular basis of retinal diseases has not only aided in their disease classification but also helped in our understanding of how different molecular pathologies may share common pathomechanisms. In particular, these relate to dysregulation of two key processes that contribute to cellular integrity, namely extracellular matrix (ECM) homeostasis and inflammation. Pathological changes in the ECM of Bruch's membrane have been described in both monogenic IRDs, such as Sorsby fundus dystrophy (SFD) and Doyne honeycomb retinal dystrophy (DHRD), as well as in the genetically complex age-related macular degeneration (AMD) or diabetic retinopathy (DR). Additionally, complement system dysfunction and distorted immune regulation may also represent a common connection between some IRDs and complex retinal degenerations. Through highlighting such overlaps in molecular pathology, this review aims to illuminate how inflammatory processes and ECM homeostasis are linked in the healthy retina and how their interplay may be disturbed in aging as well as in disease.

Electrophysiological Evaluation of Macular Dystrophies

Macular dystrophies are a heterogeneous group of genetic disorders that often severely threatens the bilateral central vision of the affected patient. While advances in molecular genetics have been instrumental in the understanding and diagnosis of these disorders, there remains significant phenotypical variation among patients within any particular subset of macular dystrophies. Electrophysiological testing remains a vital tool not only to characterize vision loss for differential diagnosis but also to understand the pathophysiology of these disorders and to monitor the treatment effect, potentially leading to therapeutic advances. This review summarizes the application of electrophysiological testing in macular dystrophies, including Stargardt disease, bestrophinopathies, X-linked retinoschisis, Sorsby fundus dystrophy, Doyne honeycomb retina dystrophy, autosomal dominant drusen, occult macular dystrophy, North Carolina macular dystrophy, pattern dystrophy, and central areolar choroidal dystrophy.

Generation of a TIMP3 knockout stem cell line via CRISPR/Cas9 system

The tissue inhibitors of metalloproteinases 3 (TIMP3) play an essential role in the tumorigenesis of human pancreatic endocrine tumors and Sorsby fundus dystrophy. To further investigate the significance of TIMP3 in disease, we used CRISPR/Cas9 to create a TIMP3 knock out human embryonic stem cell line (WAe009-A-89) that can differentiate into any desired cell type. Our results show that the WAe009-A-89 cell line retains the typical colony form and normal karyotype of stem cells. The cells strongly expressed pluripotency markers and could differentiate into tissues of all three germ layers in vivo. This cell line allowed exploring the role of the TIMP3 gene in related diseases.

Deglycosylation Increases the Aggregation and Angiogenic Properties of Mutant Tissue Inhibitor of Metalloproteinase 3 Protein: Implications for Sorsby Fundus Dystrophy.

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular disorder caused by mutations in tissue Inhibitor of the metalloproteinase-3 (TIMP3) gene with the onset of symptoms including choroidal neovascularization as early as the second decade of life. We have previously reported that wild-type TIMP3 is an endogenous angiogenesis inhibitor that inhibits Vascular Endothelial Growth Factor (VEGF)-mediated signaling in endothelial cells. In contrast, SFD-related S179C-TIMP3 when expressed in endothelial cells, does not have angiogenesis-inhibitory properties. To evaluate if this is a common feature of TIMP3 mutants associated with SFD, we examined and compared endothelial cells expressing S179C, Y191C and S204C TIMP3 mutants for their angiogenesis-inhibitory function. Western blot analysis, zymography and reverse zymography and migration assays were utilized to evaluate TIMP3 protein, Matrix Metalloproteinase (MMP) and MMP inhibitory activity, VEGF signaling and in vitro migration in endothelial cells expressing (VEGF receptor-2 (VEGFR-2) and wild-type TIMP3 or mutant-TIMP3. We demonstrate that mutant S179C, Y191C- and S204C-TIMP3 all show increased glycosylation and multimerization/aggregation of the TIMP3 protein. In addition, endothelial cells expressing TIMP3 mutations show increased angiogenic activities and elevated VEGFR-2. Removal of N-glycosylation by mutation of Asn184, the only potential N-glycosylation site in mutant TIMP3, resulted in increased aggregation of TIMP3, further upregulation of VEGFR-2, VEGF-induced phosphorylation of VEGFR2 and VEGF-mediated migration concomitant with reduced MMP inhibitory activity. These results suggest that even though mutant TIMP3 proteins are more glycosylated, post-translational deglycosylation may play a critical role in the aggregation of mutant TIMP3 and contribute to the pathogenesis of SFD. The identification of factors that might contribute to changes in the glycome of patients with SFD will be useful. Future studies will evaluate whether variations in the glycosylation of mutant TIMP3 proteins are contributing to the severity of the disease.

Sorsby fundus dystrophy (SFD): A narrative review.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disorder with complete penetrance affecting the macula. This is caused by a mutation in the TIMP-3. This objective narrative review aims to provide an overview of the pathophysiology, current treatment modalities, and future perspectives. A literature search was performed using “PubMed,” “Web of Science,” “Scopus,” “ScienceDirect,” “Google Scholar,” “medRxiv,” and “bioRxiv.” The molecular mechanisms underlying SFD are not completely understood. Novel advancements in cell culture techniques, including induced pluripotent stem cells, may enable more reliable modeling of SFD. These cell culture techniques aim to shed more light on the pathophysiology of SFD, and hopefully, this may lead to the future development of treatment strategies for SFD. Currently, no gene therapy is available. The main treatment is the use of anti-vascular endothelial growth factors (anti-VEGF) to treat secondary choroidal neovascular membrane (CNV), which is a major complication observed in this condition. If CNV is detected and treated promptly, patients with SFD have a good chance of maintaining a functional central vision. Other treatment modalities have been tried but have shown limited benefit, and therefore, have not managed to be more widely accepted. In summary, although there is no definitive cure yet, the use of anti-VEGF treatment for secondary CNV has provided the opportunity to maintain functional vision in individuals with SFD, provided CNV is detected and treated early.

Localized Choroidal Dystrophies

Localized choroidal dystrophies are a group of progressive hereditary diseases characterized by loss of retinal pigment epithelium, photoreceptors, and choriocapillaris. They are extremely rare. The first symptom is generally a decrease in visual acuity. In addition to imaging methods and electrophysiological tests, genetic tests are also used in diagnosis. In this article, the genetic features, clinic and course of central areolar choroidal dystrophy, Sorsby fundus dystrophy, North Carolina macular dystrophy, peripapillary choroidal dystrophy, and progressive bifocal chorioretinal atrophy will be reviewed under the title of localized choroidal dystrophies.

Evidence that all Sorsby's fundus dystrophy mutations cause TIMP3 dimerization resulting in impaired inhibition of VEGFR2

Evidence that all Sorsby's fundus dystrophy mutations cause TIMP3 dimerization resulting in impaired inhibition of VEGFR2

Extracellular matrix dysfunction in Sorsby patient-derived retinal pigment epithelium

Sorsby Fundus Dystrophy (SFD) is a rare form of macular degeneration that is clinically similar to age-related macular degeneration (AMD), and a histologic hallmark of SFD is a thick layer of extracellular deposits beneath the retinal pigment epithelium (RPE). Previous studies of SFD patient-induced pluripotent stem cell (iPSC) derived RPE differ as to whether these cultures recapitulate this key clinical feature by forming increased drusenoid deposits. The primary purpose of this study is to examine whether SFD patient-derived iPSC-RPE form basal deposits similar to what is found in affected family member SFD globes and to determine whether SFD iPSC RPE may be more oxidatively stressed. We performed a careful comparison of iPSC RPE from three control individuals, multiple iPSC clones from two SFD patients’ iPSC RPE, and post-mortem eyes of affected SFD family members. We also examined the effect of CRISPR-Cas9 gene correction of the S204C TIMP3 mutation on RPE phenotype. Finally, targeted metabolomics with liquid chromatography and mass spectrometry analysis and stable isotope-labeled metabolite analysis were performed to determine whether SFD RPE are more oxidatively stressed. We found that SFD iPSC-RPE formed significantly more sub-RPE deposits (∼6–90 μm in height) compared to control RPE at 8 weeks. These deposits were similar in composition to the thick layer of sub-RPE deposits found in SFD family member globes by immunofluorescence staining and TEM imaging. S204C TIMP3 correction by CRISPR-Cas9 gene editing in SFD iPSC RPE cells resulted in significantly reduced basal laminar and sub-RPE calcium deposits. We detected a ∼18-fold increase in TIMP3 accumulation in the extracellular matrix (ECM) of SFD RPE, and targeted metabolomics showed that intracellular 4-hydroxyproline, a major breakdown product of collagen, is significantly elevated in SFD RPE, suggesting increased ECM turnover. Finally, SFD RPE cells have decreased intracellular reduced glutathione and were found to be more vulnerable to oxidative stress. Our findings suggest that elements of SFD pathology can be demonstrated in culture which may lead to insights into disease mechanisms.

Choriocapillaris Flow Signal Impairment in Sorsby Fundus Dystrophy

Purpose: The aim of the study was to quantify choriocapillaris (CC) flow alterations in early Sorsby fundus dystrophy (SFD) and to investigate the relationship of the CC flow deficits with the choroidal and outer retinal microstructure. Methods: In this prospective case-control study, 18 eyes of 11 patients with early SFD and 31 eyes of 31 controls without ocular pathology underwent multimodal imaging, including spectral-domain optical coherence tomography (OCT), followed by deep-learning-based layer segmentation. OCT angiography (OCTA) was performed to quantify CC flow signal deficits (FDs). Differences in CC FD density between SFD patients and controls were determined, and the relationships with choroidal thickness, retinal pigment epithelium-drusen complex (RPEDC) thickness and outer retinal layer thicknesses were analyzed using mixed-model analysis. Results: SFD patients exhibited a significantly greater CC FD density than controls (estimate [95% CI]: +20.0%FD [13.3; 26.7], p < 0.001 for SFD patients), even when adjusted for age. Square-root transformed choroidal thickness was a structural OCT surrogate of the CC FD density (−2.1%FD per √µm, p < 0.001), whereas RPEDC thickness was not informative regarding CC FD (p = 0.061). The CC FD density was associated with an altered microstructure of the overlying photoreceptors (outer segments, inner segments, and outer nuclear layer thinning of −0.19 μm, −0.08 μm and −0.30 μm per %FD, respectively, all p < 0.001). Conclusions: Patients with early SFD exhibit pronounced abnormalities of CC flow signal on OCTA, which are not limited to areas of sub-RPE deposits seen on OCT imaging. Thus, analysis of the CC flow may enable clinical trials at earlier disease stages in SFD.

Visual Dysfunction and Structural Correlates in Sorsby Fundus Dystrophy

To elucidate morphological determinants of rod and cone dysfunction in Sorsby fundus dystrophy (SFD), and to systematically compare visual function tests for interventional trials. Prospective cross-sectional study. Patients with SFD (n=16) and controls (n=20) underwent visual function testing (best-corrected visual acuity [BCVA] and low luminance visual acuity [LLVA], contrast sensitivity, mesopic and dark-adapted (DA) fundus-controlled perimetry [FCP], rod-mediated dark adaptation [RMDA]), and multimodal imaging. Vision-related quality of life was evaluated. FCP and RMDA thresholds were analyzed using mixed models and structure-function correlation using machine learning (ML). Longitudinal data of 1 patient with high-dose vitamin A supplementation were available. Although photopic BCVA was normative in SFD, LLVA was impaired (0.30 LogMAR [0.20; 0.45] vs 0.20 LogMAR [0.03; 0.28], P < .05). Scotopic visual function exhibited a delayed rod-intercept time (21 minutes [12.15; 21] vs 4.05 minutes [3.22; 5.36], P < .001), and marked DA cyan mean sensitivity loss (-11.80 dB [-3.47; -19.85]), paralleled by a reduced vision-related quality of life. ML-based structure-function correlation allowed prediction of mesopic, DA cyan, and red sensitivity with high accuracy (cross-validated mean absolute error: 4.36, 7.77, and 5.31 dB, respectively), whereas RMDA could be slowed even in the absence of fundus alterations on multimodal imaging. After high-dose vitamin A supplementation, RMDA and DA thresholds improved markedly. Patients with SFD exhibit severely impaired scotopic visual function even in the absence of funduscopic alterations on multimodal imaging. In contrast to BCVA, scotopic visual function tests are suitable to quantify dysfunction in the early stages. Improvement of scotopic dysfunction after (off-label) high-dose vitamin A intake, as observed in one patient in our study, is compatible with the hypothesized local deficiency of vitamin A secondary to Bruch's membrane alterations.

Sorsby Pseudoinflammatory Fundus Dystrophy, North Carolina Macular Dystrophy, and Occult Macular Dystrophy; Pathophysiology, Findings, Diagnosis, and Treatment

In this review, Sorsby's pseudoinflammatory fundus dystrophy, North Carolina macular dystrophy, and occult macular dystrophy, which can lead to a decrease in visual acuity in both eyes, are discussed. The current pathophysiology, findings, diagnosis, and treatment of these diseases, which are rarely seen in our country, are discussed.

The retinal pigment epithelium in Sorsby Fundus Dystrophy shows increased sensitivity to oxidative stress-induced degeneration

Sorsby Fundus Dystrophy (SFD) is a rare inherited autosomal dominant macular degeneration caused by specific mutations in TIMP3. Patients with SFD present with pathophysiology similar to the more common Age-related Macular Degeneration (AMD) and loss of vision due to both choroidal neovascularization and geographic atrophy. Previously, it has been shown that RPE degeneration in AMD is due in part to oxidative stress. We hypothesized that similar mechanisms may be at play in SFD. The objective of this study was to evaluate whether mice carrying the S179C-Timp3 mutation, a variant commonly observed in SFD, showed increased sensitivity to oxidative stress. Antioxidant genes are increased at baseline in the RPE in SFD mouse models, but not in the retina. This suggests the presence of a pro-oxidant environment in the RPE in the presence of Timp3 mutations. To determine if the RPE of Timp3 mutant mice is more susceptible to degeneration when exposed to low levels of oxidative stress, mice were injected with low doses of sodium iodate. The RPE and photoreceptors in Timp3 mutant mice degenerated at low doses of sodium iodate, which had no effect in wildtype control mice. These studies suggest that TIMP3 mutations may result in a dysregulation of pro-oxidant—antioxidant homeostasis in the RPE, leading to RPE degeneration in SFD.

In vitro stem cell modelling demonstrates a proof-of-concept for excess functional mutant TIMP3 as the cause of Sorsby fundus dystrophy.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease-related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-hiPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis-related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Quantifying the Separation Between the Retinal Pigment Epithelium and Bruch's Membrane using Optical Coherence Tomography in Patients with Inherited Macular Degeneration.

PurposeTo describe and quantify Bruch's membrane (BM) and retinal pigment epithelium (RPE) separation using spectral-domain (SD) optical coherence tomography (OCT) in patients affected by inherited macular degenerations associated with BM thickening.MethodsPatients with molecularly confirmed Sorsby fundus dystrophy (SFD), dominant drusen (DD), and late-onset retinal degeneration (L-ORD) were included in this retrospective study. Each disease was classed as early stage if subjects were asymptomatic, intermediate stage if they had nyctalopia alone, and late stage if they described loss of central vision. The main outcome was measurement of BM-RPE separation on SD-OCT. The BM-RPE separation measurements were compared against those in normal age-matched controls.ResultsSeventeen patients with SFD, 22 with DD, and eight with L-ORD were included. BM-RPE separation on SD-OCT demonstrated a high test-retest and interobserver reproducibility (intraclass correlation coefficients >0.9). BM-RPE separation was not identified in normal subjects. In SFD, there was greater BM-RPE separation in late-stage disease compared with intermediate-stage patients both at subfoveal (P < 0.05) and juxtafoveal (P < 0.01) locations. In DD, there was increased BM-RPE separation in late-stage disease compared with early stage at subfoveal (P < 0.001) and juxtafoveal (P < 0.05) topographies. There was no significant difference in BM-RPE separation between disease stages in L-ORD.ConclusionsBM-RPE separation is a novel, quantifiable phenotype in the three monogenic macular dystrophies studied, and may be an optical correlate of the histopathological thickening in BM that is known to occur. BM-RPE separation, as measured by OCT, varies with stage of disease in SFD and DD, but not in L-ORD.Translational Relevance SFD, DD, and L-ORD are associated with BM thickening. In this group of patients, OCT assessment of macular structure identifies a separation of the usually single, hyperreflective line thought to represent BM and the overlying RPE. This separation is a novel and quantifiable feature of disease staging in SFD and DD.

Role of FGF and Hyaluronan in Choroidal Neovascularization in Sorsby Fundus Dystrophy

Sorsby’s fundus dystrophy (SFD) is an inherited blinding disorder caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene. The SFD pathology of macular degeneration with subretinal deposits and choroidal neovascularization (CNV) closely resembles that of the more common age-related macular degeneration (AMD). The objective of this study was to gain further insight into the molecular mechanism(s) by which mutant TIMP3 induces CNV. In this study we demonstrate that hyaluronan (HA), a large glycosaminoglycan, is elevated in the plasma and retinal pigment epithelium (RPE)/choroid of patients with AMD. Mice carrying the S179C-TIMP3 mutation also showed increased plasma levels of HA as well as accumulation of HA around the RPE in the retina. Human RPE cells expressing the S179C-TIMP3 mutation accumulated HA apically, intracellularly and basally when cultured long-term compared with cells expressing wildtype TIMP3. We recently reported that RPE cells carrying the S179C-TIMP3 mutation have the propensity to induce angiogenesis via basic fibroblast growth factor (FGF-2). We now demonstrate that FGF-2 induces accumulation of HA in RPE cells. These results suggest that the TIMP3-MMP-FGF-2-HA axis may have an important role in the pathogenesis of CNV in SFD and possibly AMD.

The Diverse Roles of TIMP-3: Insights into Degenerative Diseases of the Senescent Retina and Brain.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a component of the extracellular environment, where it mediates diverse processes including matrix regulation/turnover, inflammation and angiogenesis. Rare TIMP-3 risk alleles and mutations are directly linked with retinopathies such as age-related macular degeneration (AMD) and Sorsby fundus dystrophy, and potentially, through indirect mechanisms, with Alzheimer’s disease. Insights into TIMP-3 activities may be gleaned from studying Sorsby-linked mutations. However, recent findings do not fully support the prevailing hypothesis that a gain of function through the dimerisation of mutated TIMP-3 is responsible for retinopathy. Findings from Alzheimer’s patients suggest a hitherto poorly studied relationship between TIMP-3 and the Alzheimer’s-linked amyloid-beta (Aβ) proteins that warrant further scrutiny. This may also have implications for understanding AMD as aged/diseased retinae contain high levels of Aβ. Findings from TIMP-3 knockout and mutant knock-in mice have not led to new treatments, particularly as the latter does not satisfactorily recapitulate the Sorsby phenotype. However, recent advances in stem cell and in vitro approaches offer novel insights into understanding TIMP-3 pathology in the retina-brain axis, which has so far not been collectively examined. We propose that TIMP-3 activities could extend beyond its hitherto supposed functions to cause age-related changes and disease in these organs.