A novel tissue inhibitor of metalloproteinases-3 mutation reveals a common molecular phenotype in Sorsby's fundus dystrophy

Sorsby's fundus dystrophy (SFD) is a dominantly inherited degenerative disease of the retina that leads to loss of vision in middle age. It has been shown to be caused by mutations in the gene for tissue inhibitor of metalloproteinases-3 (TIMP-3). Five different mutations have previously been identified, all introducing an extra cysteine residue into exon 5 (which forms part of the C-terminal domain) of the TIMP-3 molecule; however, the significance of these mutations to the disease phenotype was unknown. In this report, we describe the expression of several of these mutated genes, together with a previously unreported novel TIMP-3 mutation from a family with SFD that results in truncation of most of the C-terminal domain of the molecule. Despite these differences, all of these molecules are expressed and exhibit characteristics of the normal protein, including inhibition of metalloproteinases and binding to the extracellular matrix. However, unlike wild-type TIMP-3, they all form dimers. These observations, together with the recent finding that expression of TIMP-3 is increased, rather than decreased, in eyes from patients with SFD, provides compelling evidence that dimerized TIMP-3 plays an active role in the disease process by accumulating in the eye. Increased expression of TIMP-3 is also observed in other degenerative retinal diseases, including the more severe forms of age-related macular degeneration, the most common cause of blindness in the elderly in developed countries. We hypothesize that overexpression of TIMP-3 may prove to be a critical step in the progression of a variety of degenerative retinopathies.     

TIMP3 mutation in Sorsby's fundus dystrophy: molecular insights

Sorsby's fundus dystrophy (SFD) is a rare autosomal dominant disorder that results in degeneration of the macular region of the retina, with onset usually in the fourth to fifth decade of life. It leads to the rapid loss of central vision, often followed by further loss of peripheral vision. SFD shares several pathological features commonly found in the 'wet' or exudative form of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in developed countries. These phenotypic similarities have led to SFD being proposed as an acceptable genetic model for AMD. Whereas AMD appears to have a complex aetiology, with both genetic and environmental factors playing a role, SFD has been shown to be a single-gene disorder, linked to mutations in exon 5 of the tissue inhibitor of metalloproteinases 3 (TIMP3) gene on chromosome 22q12-q13. This review confines itself to a discussion of the known biochemical properties of the wild-type and SFD TIMP3 proteins and attempts to relate these to the pathology encountered in SFD patients. We also discuss briefly how, despite the lack of inherited mutations in the structural gene, the TIMP3 protein might play a role in the onset and progression of AMD.     

Expression of Sorsby's fundus dystrophy mutations in human retinal pigment epithelial cells reduces matrix metalloproteinase inhibition and may promote angiogenesis

Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the macula caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Choroidal neovascularization is a hallmark of this disease, which closely resembles the exudative form of age-related macular degeneration. However, the mechanism by which TIMP-3 mutations induce the disease phenotype in SFD remains unknown. To address this question we established human retinal pigment epithelial cell lines expressing wild type or S156C (Ser(156) changed to cysteine) mutant TIMP-3. S156C TIMP-3 had reduced matrix metalloproteinase (MMP) inhibitory activity in retinal pigment epithelial cells and resulted in increased secretion and activation of gelatinase A and B. The conditioned medium from these cells induced angiogenesis in "in vivo" chick chorioallantoic membrane assays that could be reversed with recombinant wild type TIMP-3. Our data indicate that the choroidal neovascularization in SFD may be a result of increased MMP activity, which could lead to the stimulation of angiogenesis. These results also suggest the potential therapeutic use of TIMP-3 or synthetic MMP inhibitors in this disease.     

Analysis of the collagen VI assemblies associated with Sorsby's fundus dystrophy

Age-related macular degeneration is the leading cause of blindness in the Western world, and the pathophysiology of the condition is largely unknown. However, it shares many clinical and pathological features with Sorsby's fundus dystrophy (SFD), an autosomal dominant disease, known to be associated with mutations in the TIMP-3 gene. In Bruch's membrane of both conditions, there are molecular assemblies with distinct transverse bands occurring with a periodicity of about 100 nm. Similar assemblies were also found in the vitreous of a patient with full-thickness macular holes and were identified as being made of collagen VI. The assemblies found in the eye with SFD can be classified into two types, both with a 105-nm axial repeat, but one showing pairs of narrow bands about 30 nm apart and the other showing a single broad band in every repeat. By comparison with the assemblies in the vitreous, collagen VI is considered to be the most likely protein in these assemblies. Furthermore, both of the assemblies associated with SFD can be explained in terms of collagen VI tetramers, one in which the tetramers bind to the mutant tissue inhibitor of metalloproteinases-3 (the gene product of TIMP-3) and the other in which little or no binding occurs. TIMP-3 bound to collagen VI may be more resistant to degradation and create an imbalance between the normal amount of TIMP-3 and matrix metalloproteinases (the substrate of TIMPs) in Bruch's membrane with consequent disruption of the normal metabolic processes. Understanding the structure of these collagen VI/TIMP assemblies in Bruch's membrane may prove to be important for understanding the pathophysiology of age-related macular degeneration.     

HTRA1, an age‐related macular degeneration protease,processes extracellular matrix proteins EFEMP1 and TSP1

High‐temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age‐related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high‐risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high‐risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP‐1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.     

Compromised mutant EFEMP1 secretion associated with macular dystrophy remedied by proteostasis network alteration

An Arg345Trp (R345W) mutation in epidermal growth factor-containing, fibulin-like extracellular matrix protein 1 (EFEMP1) causes its inefficient secretion and the macular dystrophy malattia leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD). To understand the influence of the protein homeostasis (or proteostasis) network in rescuing mutant EFEMP1 misfolding and inefficient secretion linked to ML/DHRD, we developed a convenient and sensitive cell-based luminescence assay to monitor secretion versus intracellular accumulation. Fusing EFEMP1 to Gaussia luciferase faithfully recapitulates mutant EFEMP1 secretion defects observed previously using more cumbersome methodology. To understand what governs mutant intracellular retention, we generated a series of R345 mutants. These mutants revealed that aromatic residue substitutions (i.e., Trp, Tyr, and Phe) at position 345 cause significant EFEMP1 secretion deficiencies. These secretion defects appear to be caused, in part, by reduced native disulfide bonding in domain 6 harboring the 345 position. Finally, we demonstrate that mutant EFEMP1 secretion and proper disulfide formation are enhanced by adaptation of the cellular environment by a reduced growth temperature and/or translational attenuation. This study highlights the mechanisms underlying the inefficient secretion of R345W EFEMP1 and demonstrates that alteration of the proteostasis network may provide a strategy to alleviate or delay the onset of this macular dystrophy.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Sorsby's fundus dystrophy tissue inhibitor of metalloproteinases-3 (TIMP-3) mutants have unimpaired matrix metalloproteinase inhibitory activities, but affect cell adhesion to the extracellular matrix.

The TIMP family of matrix metalloproteinase inhibitors consists of four members, of which TIMP-1, -2 and -4 are secreted, freely diffusible proteins, whereas TIMP-3 is ECM-associated. Mutations in the TIMP3 gene have been linked to Sorsby's fundus dystrophy (SFD), an autosomal dominant inherited retinal degenerative disease that leads to blindness. The SFD mutations characterized result in introduction of an unpaired cysteine residue in the C-terminal domain of TIMP-3. We have expressed four SFD mutant TIMP-3 proteins in baby hamster kidney (BHK) cells and evaluated their characteristics alongside wild-type TIMP-3. Analysis of the mutant proteins (Ser156Cys, Gly167Cys, Tyr168Cys and Ser181Cys) by SDS-PAGE and reverse zymography revealed that each of the mutants retained gelatinase A and gelatinase B inhibitory activity, and were localized to the ECM. Association rate constants for Ser156Cys TIMP-3 with gelatinase-A, gelatinase-B, stromelysin-1 and collagenase-3 were only moderately reduced compared to wild-type TIMP-3. However, all of the mutants displayed aberrant protein-protein interactions, resulting in the presence of additional proteins or complexes in ECM preparations. Two of the mutants (Ser156Cys and Ser181Cys) showed a marked propensity to form multiple higher molecular-weight complexes that retained TIMP activity on reverse zymography. Expression of the SFD mutant TIMP-3 (and to a lesser extent, wild-type TIMP-3) proteins in BHK cells conferred increased cell adhesiveness to the ECM. Our findings indicate that the pathogenesis of Sorsby's fundus dystrophy cannot be attributed to a failure to localize SFD TIMP-3 proteins to the ECM or defects in MMP inhibition, but may involve the formation of aberrant TIMP-3-containing protein complexes and altered cell adhesion.     

Sorsby's fundus dystrophy mutant tissue inhibitors of metalloproteinase-3 induce apoptosis of retinal pigment epithelial and MCF-7 cells

C-terminal domain tissue inhibitor of metalloproteinases-3 (TIMP-3) mutations cause the rare hereditary blindness Sorsby's fundus dystrophy (SFD), which involves loss of retinal pigment epithelial (RPE) cells. Since wild-type TIMP-3 causes apoptosis, we investigated whether SFD TIMP-3 might kill RPE and other cells. Plasmid-mediated overexpression of Ser-156, Gly-167, Tyr-168 and Ser-181 SFD mutant TIMP-3 decreased RPE viability to 22+/-8, 20+/-6, 32+/-5, 30+/-12% (SFD mutants all P<0.01 versus wild-type 50+/-8%) and similarly increased propidium iodide staining and in situ end labelling. Adenovirus-mediated overexpression of the Gly-167 mutant also caused RPE apoptosis dose-dependently. Apoptosis of RPE cells might therefore contribute to the pathology of SFD.     

Genome-wide analysis of copy number variants in age-related macular degeneration

Age-related macular degeneration (AMD) is a complex genetic disease, with many loci demonstrating appreciable attributable disease risk. Despite significant progress toward understanding the genetic and environmental etiology of AMD, identification of additional risk factors is necessary to fully appreciate and treat AMD pathology. In this study, we investigated copy number variants (CNVs) as potential AMD risk variants in a cohort of 400 AMD patients and 500 AMD-free controls ascertained at the University of Iowa. We used three publicly available copy number programs to analyze signal intensity data from Affymetrix GeneChip SNP Microarrays. CNVs were ranked based on prevalence in the disease cohort and absence from the control group; high interest CNVs were subsequently confirmed by qPCR. While we did not observe a single-locus "risk CNV" that could account for a major fraction of AMD, we identified several rare and overlapping CNVs containing or flanking compelling candidate genes such as NPHP1 and EFEMP1. These and other candidate genes highlighted by this study deserve further scrutiny as sources of genetic risk for AMD.     

Alterations in biochemical components of extracellular matrix in intervertebral disc herniation: role of MMP-2 and TIMP-2 in type II collagen loss

Alterations in the composition of intervertebral disc extracellular matrix, mainly collagen and proteoglycans, may cause changes in mechanical properties of the disc, leading to dysfunction, nerve root compression, and herniation with severe clinical manifestations. Matrix metalloproteinases may be involved in degradation by hydrolysing extracellular matrix components. Inhibitors of matrix metalloproteinases, in contrast, function in the maintenance of degradation control. In this study, we investigated: (i) whether the level of matrix degradation correlated with the duration of the symptomatic disease, (ii) roles of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) in intervertebral disc degeneration. Nucleus pulposus of intervertebral discs were obtained from 22 patients and analysed for collagen and proteoglycan contents, and pro-MMP-2, TIMP-2 levels. Collagen content was determined as hydroxyproline and proteoglycan content was measured as glycosaminoglycans. The loss in matrix components did not correlate with the duration of the degenerative disc disease. Pro-MMP-2 levels were higher at early stages of the degenerative disc disease (r = -0.495, P < 0.05). TIMP-2 levels were similar in all samples. Pro-MMP-2 and TIMP-2 levels negatively correlated in herniated discs samples (r = -0.855, P < 0.01). Pro- MMP-2 levels negatively correlated with the collagen content in herniated disc material. Our findings may suggest a silent period of active disease prior to symptomatic outcome during which irreversible matrix loss occurs. Involvement of other proteolytic enzymes at different stages of the disease should also be investigated to help to control the degradation cascade at relatively early stages of disc degeneration before the clinical onset of disease.     

Definición diagnóstica en una familia con malattia leventinese en Colombia

The malattia leventinese is an autosomal dominant inherited disease whose symptoms appear between the second and fourth decades of life. It is characterized by the appearance of drusen located between the retinal pigment epithelium and the Bruch membrane. It is usually associated with low vision and may progress to blindness. The pathogenic variant p.Arg345Trp in the EFEMP1 gene has been associated with this disease. We characterized clinically and molecularly a family with malattia leventinese using a comprehensive approach that involved ophthalmologists, pediatricians, and geneticists. This approach is of great importance since the phenotype of this disease is often confused with macular degeneration. All family members underwent ophthalmological evaluation and DNA extraction from a peripheral blood sample. All exons of the EFEMP1 gene were amplified and sequenced. The pathogenic variant p.Arg345Trp was identified in affected individuals in this family.This is the first report of malattia leventinese in a family with the p.Arg345Trp pathogenic variant in Colombia. The molecular diagnosis of retinal dystrophies is essential to differentiate this type of pathology.     

Whole Exome Sequencing in Patients with the Cuticular Drusen Subtype of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly people worldwide. Cuticular drusen (CD) is a clinical subtype of AMD, which typically displays an earlier age at onset, and has a strong genetic component. Genetic studies support a role for rare sequence variants in CD susceptibility, and rare sequence variants in the CFH gene have been identified in 8.8% of CD cases. To further explore the role of rare variants in CD, we performed whole exome sequencing (WES) in 14 affected members of six families and 12 sporadic cases with CD. We detected rare sequence variants in CFH and FBLN5, which previously were shown to harbor rare variants in patients with CD. In addition, we detected heterozygous rare sequence variants in several genes encoding components of the extracellular matrix (ECM), including FBLN1, FBLN3/EFEMP1, FBLN5, FBLN6/HMCN1, FBN2, and COL15A1. Two rare pathogenic variants were identified in the COL15A1 gene: one in a sporadic case and another was found to segregate in a family with six affected individuals with CD. In addition, two rare pathogenic variants were identified in the FGL1 gene in three unrelated CD cases. These findings suggest that alterations in the ECM and in the coagulation pathway may play a role in the pathogenesis of CD. The identified candidate genes require further analyses in larger cohorts to confirm their role in the CD subtype of AMD. No evidence was found of rare sequence variants in a single gene that segregate with CD in the six families, suggesting that the disease is genetically heterogeneous.     

Expression level and glycan dynamics determine the net effects of TIMP-1 on cancer progression

Tissue inhibitor of metalloproteinases (TIMPs; TIMP-1, -2, -3 and -4) are endogenous inhibitor for matrix metalloproteinases (MMPs) that are responsible for remodeling the extracellular matrix (ECM) and involved in migration, invasion and metastasis of tumor cells. Unlike under normal conditions, the imbalance between MMPs and TIMPs is associated with various diseased states. Among TIMPs, TIMP-1, a 184-residue protein, is the only N-linked glycoprotein with glycosylation sites at N30 and N78. The structural analysis of the catalytic domain of human stromelysin-1 (MMP-3) and human TIMP-1 suggests new possibilities of the role of TIMP-1 glycan moieties as a tuner for the proteolytic activities by MMPs. Because the TIMP-1 glycosylation participate in the interaction, aberrant glycosylation of TIMP-1 presumably affects the interaction, thereby leading to pathogenic dysfunction in cancer cells. TIMP-1 has not only the cell proliferation activities but also anti-oncogenic properties. Cancer cells appear to utilize these bilateral aspects of TIMP-1 for cancer progression; an elevated TIMP-1 level exerts to cancer development via MMP-independent pathway during the early phase of tumor formation, whereas it is the aberrant glycosylation of TIMP-1 that overcome the high anti-proteolytic burden. The aberrant glycosylation of TIMP-1 can thus be used as staging and/or prognostic biomarker in colon cancer. [BMB Reports 2012; 45(11): 623-628]     

NLRP3 has a protective role in age-related macular degeneration through the induction of IL-18 by drusen components

Age-related macular degeneration (AMD) is the leading cause of central vision loss worldwide. Drusen accumulation is the major pathological hallmark common to both dry and wet AMD. Although activation of the immune system has been implicated in disease progression, the pathways involved are unclear. Here we show that drusen isolated from donor AMD eyes activates the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, causing secretion of interleukin-1b (IL-1b) and IL-18. Drusen component C1Q also activates the NLRP3 inflammasome. Moreover, the oxidative-stress-related protein-modification carboxyethylpyrrole (CEP), a biomarker of AMD, primes the inflammasome. We found cleaved caspase-1 and NLRP3 in activated macrophages in the retinas of mice immunized with CEP-adducted mouse serum albumin, modeling a dry-AMD–like pathology. We show that laser-induced choroidal neovascularization (CNV), a mouse model of wet AMD, is exacerbated in Nlrp3(-/-) but not Il1r1(-/-) mice, directly implicating IL-18 in the regulation of CNV development. These findings indicate a protective role for NLRP3 and IL-18 in the progression of AMD.     

Phosphorylation/inactivation of PTEN by Akt-independent PI3K signaling in retinal pigment epithelium

Retinal pigment epithelium (RPE) plays a critical role in vertebrate vision by providing functional and structural support to the retina. Degeneration of RPE by cumulative oxidative stresses or acute injury frequently results in retinal degenerative diseases, notably age-related macular degeneration (AMD). Moreover, it has been shown that phosphorylation-mediated inactivation of PTEN (phosphatase and tensin homolog) in RPE is closely linked to AMD-like retinal degeneration in mice [1]. In this study, we used AMD mouse models, in which chemokine (C–C motif) ligand 2 (Ccl2) or chemokine (C–C motif) receptor 2 (Ccr2) were genetically ablated, to examine mechanisms linking reactive oxygen species (ROS) to phosphorylation/inactivation of PTEN in RPE. We found that ROS levels were increased in these RPE cells in association with phosphorylation/inactivation of PTEN. Both PTEN phosphorylation/inactivation and consequent Akt activation in the RPE of AMD model mice were inhibited by antioxidant treatment, indicating a functional role for elevated intracellular ROS. We further discovered that PTEN phosphorylation in oxidatively stressed RPE was repressed by a phosphoinositide 3-kinase (PI3K) inhibitor, but not by an Akt inhibitor. Taken together, these results suggest that ROS-activated PI3K potentiates AMD-related RPE pathogenesis through phosphorylation/inactivation of PTEN.     

Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization.

High levels of the plasminogen activators, but also their inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been documented in neovascularization of severe ocular pathologies such as diabetic retinopathy or age-related macular degeneration (AMD). AMD is the primary cause of irreversible photoreceptors loss, and current therapies are limited. PAI-1 has recently been shown to be essential for tumoral angiogenesis. We report here that deficient PAI-1 expression in mice prevented the development of subretinal choroidal angiogenesis induced by laser photocoagulation. When systemic and local PAI-1 expression was achieved by intravenous injection of a replication-defective adenoviral vector expressing human PAI-1 cDNA, the wild-type pattern of choroidal angiogenesis was restored. These observations demonstrate the proangiogenic activity of PAI-1 not only in tumoral models, but also in choroidal experimental neovascularization sharing similarities with human AMD. They identify therefore PAI-1 as a potential target for therapeutic ocular anti-angiogenic strategies.     

Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.