Mutant Best1 Expression and Impaired Phagocytosis in an iPSC Model of Autosomal Recessive Bestrophinopathy.

Autosomal recessive bestrophinopathy (ARB) is caused by mutations in the gene BEST1 which encodes bestrophin 1 (Best1), an anion channel expressed in retinal pigment epithelial (RPE) cells. It has been hypothesized that ARB represents the human null phenotype for BEST1 and that this occurs due to nonsense mediated decay (NMD). To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with ARB and her parents. After differentiation to retinal pigment epithelial (iPSC-RPE) cells, both BEST1 mRNA and Best1 protein expression were compared to controls. BEST1 mRNA expression levels, determined by quantitative PCR, were similar in ARB iPSC-RPE, parental cells, and genetically unrelated controls. Western blotting revealed that CRALBP and RPE65 were expressed within the range delineated by unrelated controls in iPSC-RPE from the ARB donor and her parents. Best1 protein was detected in different clones of ARB iPSC-RPE, but at reduced levels compared to all controls. When tested for the ability to phagocytose photoreceptor outer segments, ARB iPSC-RPE exhibited impaired internalization. These data suggest that impaired phagocytosis is a trait common to the bestrophinopathies. Furthermore, ARB is not universally the result of NMD and ARB, in this patient, is not due to the absence of Best1.

Mutant Fibulin-3 Causes Proteoglycan Accumulation and Impaired Diffusion Across Bruch's Membrane.

Purpose: The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1-/- mice. Methods: Proteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. Results: HSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1-/- Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1-/- Bruch's membrane was enhanced. Conclusions: Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.

Early AMD-like defects in the RPE and retinal degeneration in aged mice with RPE-specific deletion of Atg5 or Atg7.

PURPOSE: To examine the effects of autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 in mice as a function of age. METHODS: Conditional knockout mice with a floxed allele of Atg5 or Atg7 were crossed with inducible VMD2-rtTA/Cre transgenic mice. VMD2-directed RPE-specific Cre recombinase expression was induced with doxycycline feeding in the resulting mice. Cre-mediated deletion of floxed Atg5 or Atg7 resulted in RPE-specific inactivation of the Atg5 or Atg7 gene. Plastic and thin retinal sections were analyzed with light and electron microscopy for histological changes. Photoreceptor outer segment (POS) thickness in plastic sections was measured using the Adobe Photoshop CS4 extended ruler tool. Autophagic adaptor p62/SQSTM1 and markers for oxidatively damaged lipids, proteins, and DNA were examined with immunofluorescence staining of cryosections. Fluorescence signals were quantified using Image J software. RESULTS: Accumulation of p62/SQSTM1 reflecting autophagy deficiency was observed in the RPE of the Atg5ΔRPE and Atg7ΔRPE mice. 3-nitrotyrosine, advanced glycation end products (AGEs), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), markers for oxidatively damaged proteins and DNA, were also found to accumulate in the RPE of these mice. We observed retinal degeneration in 35% of the Atg5ΔRPE mice and 45% of the Atg7ΔRPE mice at 8 to 24 months old. Degeneration severity and the number of mice with degeneration increased with age. The mean POS thickness of these mice was 25 µm at 8-12 months, 15 µm at 13-18 months, and 3 µm at 19-24 months, compared to 35 µm, 30 µm, and 24 µm in the wild-type mice, respectively. Early age-related macular degeneration (AMD)-like RPE defects were found in all the Atg5ΔRPE and Atg7ΔRPE mice 13 months old or older, including vacuoles, uneven RPE thickness, diminished basal infoldings, RPE hypertrophy/hypotrophy, pigmentary irregularities, and necrosis. The severity of the RPE defects increased with age and in the mice with retinal degeneration. RPE atrophy and choroidal neovascularization (CNV) were occasionally observed in the Atg5ΔRPE and Atg7ΔRPE mice with advanced age. CONCLUSIONS: Autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 predisposes but does not necessarily drive the development of AMD-like phenotypes or retinal degeneration.

Deletion of Efemp1 Is Protective Against the Development of Sub-RPE Deposits in Mouse Eyes.

Purpose: EFEMP1 (fibulin-3) is mutated in Malattia Leventinese/Doyne's honeycomb retinal dystrophy (ML/DHRD), an inherited macular dystrophy similar to AMD. Both ML/DHRD and AMD are characterized by the presence of sub-RPE deposits. Efemp1 knockout mice do not develop sub-RPE deposits. This study was to test whether sub-RPE deposits can be induced in Efemp1 knockout mice by experimentally applied stress conditions that cause wild-type mice to develop sub-RPE deposits. Methods: Efemp1 knockout and control mice at 6, 18, or 24 months old were fed with a synthetic high-fat diet (HFD). Beginning 1 month after starting the HFD, one group of mice was exposed to cigarette smoke daily for 1 month, and another group of mice was subjected to photochemical injury every other day for 2 weeks from a 488-nm argon laser. After the treatments, histologic analysis was performed to assess whether sub-RPE deposits were induced. Results: Basal laminar deposits (BLamDs), a form of sub-RPE deposits, were observed in the 18- and 24-month-old wild-type mice but not in Efemp1 knockout mice in any age groups after exposure to HFD and cigarette smoke or laser injury. Conclusions: Mice lacking fibulin-3 do not develop sub-RPE deposits. Environmental oxidative stressors (HFD/cigarette smoke or HFD/laser) known to cause BLamD formation in wild-type mice failed to induce BLamD formation in Efemp1 knockout mice. These results suggest that fibulin-3 is a central player in the development of BLamD, and deletion of fibulin-3 is protective against the development of BLamD.

Bestrophin 1 and retinal disease.

Mutations in the gene BEST1 are causally associated with as many as five clinically distinct retinal degenerative diseases, which are collectively referred to as the "bestrophinopathies". These five associated diseases are: Best vitelliform macular dystrophy, autosomal recessive bestrophinopathy, adult-onset vitelliform macular dystrophy, autosomal dominant vitreoretinochoroidopathy, and retinitis pigmentosa. The most common of these is Best vitelliform macular dystrophy. Bestrophin 1 (Best1), the protein encoded by the gene BEST1, has been the subject of a great deal of research since it was first identified nearly two decades ago. Today we know that Best1 functions as both a pentameric anion channel and a regulator of intracellular Ca2+ signaling. Best1 is an integral membrane protein which, within the eye, is uniquely expressed in the retinal pigment epithelium where it predominantly localizes to the basolateral plasma membrane. Within the brain, Best1 expression has been documented in both glial cells and astrocytes where it functions in both tonic GABA release and glutamate transport. The crystal structure of Best1 has revealed critical information about how Best1 functions as an ion channel and how Ca2+ regulates that function. Studies using animal models have led to critical insights into the physiological roles of Best1 and advances in stem cell technology have allowed for the development of patient-derived, "disease in a dish" models. In this article we review our knowledge of Best1 and discuss prospects for near-term clinical trials to test therapies for the bestrophinopathies, a currently incurable and untreatable set of diseases.

Role of Fibulin 3 in Aging-Related Joint Changes and Osteoarthritis Pathogenesis in Human and Mouse Knee Cartilage.

OBJECTIVE: The EFEMP1 gene encoding fibulin 3 is specifically expressed in the superficial zone (SZ) of articular cartilage. The aims of this study were to examine the expression patterns of fibulin 3 in the knee joints during aging and during osteoarthritis (OA) and to determine the role of fibulin 3 in the pathogenesis of OA. METHODS: Immunohistochemical analysis was performed on normal and OA knee cartilage samples from humans and mice. Experimental OA was induced in wild-type and fibulin 3-/- mice, and the severity of OA was evaluated by histologic scoring. To examine fibulin 3 function, human chondrocyte monolayer cultures were transfected with small interfering RNA (siRNA), followed by quantitative polymerase chain reaction and Western blot analyses. Human bone marrow-derived mesenchymal stem cells (BM-MSCs) were transduced with an EFEMP1 lentivirus and analyzed for markers of chondrogenesis. RESULTS: Fibulin 3 was specifically expressed in the SZ of normal knee joint cartilage from humans and mice, and the expression levels declined with aging. Both aging-related OA and experimental OA were significantly more severe in fibulin 3-/- mice compared with wild-type mice. Fibulin 3 expression was high in undifferentiated human BM-MSCs and decreased during chondrogenesis. Suppression of fibulin 3 by siRNA significantly increased the expression of SOX9, type II collagen, and aggrecan in human articular chondrocytes, while overexpression of fibulin 3 inhibited chondrogenesis in BM-MSCs. CONCLUSION: Fibulin 3 is specifically expressed in the SZ of articular cartilage and its expression is reduced in aging and OA. Fibulin 3 regulates differentiation of adult progenitor cells, and its aging-related decline is an early event in the pathogenesis of OA. Preventing aging-associated loss of fibulin 3 or restoring it to normal levels in SZ chondrocytes has the potential to delay or prevent the onset of OA.

Control of Maintenance and Regeneration of Planarian Eyes by ovo.

PURPOSE: Following decapitation, the planarian Schmidtea mediterranea regenerates its head and eyes. The gene ovo is required for eye maintenance and regeneration in response to wounding. In this study, we investigated whether eye regeneration in S. mediterranea could occur absent a wound healing response. METHODS: One hundred twenty S. mediterranea were treated with ovo RNA interference (RNAi) or control (unc-22) RNAi by feeding double-stranded RNA (dsRNA). Following eye loss, ovo RNAi treatment was halted and replaced with control RNAi treatment. Quantitative real-time PCR (qPCR) was used to monitor ovo expression. Eye functionality was monitored via a phototaxis assay. Photoreceptor neurons were visualized via immunofluorescence staining of arrestin. RESULTS: Treatment with ovo RNAi caused eyes to gradually shrink until they were completely absent. One hundred percent of ovo RNAi-treated planarians lost both eyes within 137 days of treatment onset. ovo RNAi-treated planarians were unable to regenerate eyes in response to decapitation. Upon removal of ovo RNAi, eyes became visible as small pigmented spots in the head within 28 days. The eyes slowly developed, appearing to gain pigmented cells first and then nonpigmented photoreceptors. Phototaxis assays demonstrated functional eye loss and eye restoration. ovo mRNA was significantly decreased following treatment with ovo RNAi and significantly increased following removal of ovo RNAi. Arrestin staining was present in the eyes, optic nerves, and optic chiasm of worms with regenerated eyes but not in eyeless worms. CONCLUSIONS: S. mediterranea have the ability to generate functional eyes in the absence of a wound healing response. This ability requires the expression of ovo.

Autosomal Recessive Bestrophinopathy Is Not Associated With the Loss of Bestrophin-1 Anion Channel Function in a Patient With a Novel BEST1 Mutation.

PURPOSE: Mutations in BEST1, encoding bestrophin-1 (Best1), cause autosomal recessive bestrophinopathy (ARB). Encoding bestrophin-1 is a pentameric anion channel localized to the basolateral plasma membrane of the RPE. Here, we characterize the effects of the mutations R141H (CGC > CAC) and I366fsX18 (c.1098_1100+7del), identified in a patient in our practice, on Best1 trafficking, oligomerization, and channel activity. METHODS: Currents of Cl- were assessed in transfected HEK293 cells using whole-cell patch clamp. Best1 localization was assessed by confocal microscopy in differentiated, human-induced pluripotent stem cell-derived RPE (iPSC-RPE) cells following expression of mutants via adenovirus-mediated gene transfer. Oligomerization was evaluated by coimmunoprecipitation in iPSC-RPE and MDCK cells. RESULTS: Compared to Best1, Best1 I366fsX18 currents were increased while Best1 R141H Cl- currents were diminished. Coexpression of Best1 R141H with Best1 or Best1 I366fsX18 resulted in rescued channel activity. Overexpressed Best1, Best1 R141H, and Best1 I366fsX18 were all properly localized in iPSC-RPE cells; Best1 R141H and Best1 I366fsX18 coimmunoprecipitated with endogenous Best1 in iPSC-RPE cells and with each other in MDCK cells. CONCLUSIONS: The first 366 amino acids of Best1 are sufficient to mediate channel activity and homo-oligomerization. The combination of Best1 and Best1 R141H does not cause disease, while Best1 R141H together with Best1 I366fsX18 causes ARB. Since both combinations generate comparable Cl- currents, this indicates that ARB in this patient is not caused by a loss of channel activity. Moreover, Best1 I366fsX18 differs from Best1 in that it lacks most of the cytosolic C-terminal domain, suggesting that the loss of this region contributes significantly to the pathogenesis of ARB in this patient.

Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium.

PURPOSE: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. METHODS: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. RESULTS: Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling. CONCLUSIONS: These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.

Soluble adenylyl cyclase in the eye.

Adenylyl cyclases (ACs) are a family of enzymes which convert ATP to cAMP, an essential intermediate in many signal transduction pathways. Of the 10 AC genes in man, 9 fall into the category of transmembrane ACs (tmACs), which associate with G-protein coupled receptors (GPCRs) and are activated by forskolin. The 10th AC, termed soluble AC (sAC) is neither activated by forskolin nor does it interact with GPCRs. Rather, sAC can be found in many compartments within the cell and is activated by bicarbonate. As such, sAC is considered a major sensor of bicarbonate in many tissues. The pathways involving sAC vary in different tissues and organ systems, and are as diverse as facilitating sperm capacitation and regulating pressure in the eye. The role of sAC in the eye has only recently begun to receive significant attention. Here we summarize what is known about the roles of sAC in the eye. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

Disease-causing mutations associated with four bestrophinopathies exhibit disparate effects on the localization, but not the oligomerization, of Bestrophin-1.

BEST1 encodes Bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium. Mutations in BEST1 cause five distinct retinal degenerative diseases, including adult vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). The mechanisms underlying these diseases and why mutations cause one disease over another are, for the most part, unknown. To gain insights into these four diseases, we expressed 28 Best1 mutants fused to YFP in polarized MDCK monolayers and, via confocal microscopy and immunofluorescence, live-cell FRET, and reciprocal co-immunoprecipitation experiments, screened these mutants for defects in localization and oligomerization. All 28 mutants exhibited comparable FRET efficiencies to and co-immunoprecipitated with WT Best1, indicating unimpaired oligomerization. RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When co-expressed, all mislocalized mutants caused mislocalization of WT Best1 to intracellular compartments. Our current and past results indicate that mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, occurring in AVMD, BVMD, and ARB. Furthermore, some ARB mutants that do not also cause dominant disease cause mislocalization of Best1, indicating that mislocalization is not a cause of disease, and that absence of Best1 activity from the plasma membrane is tolerated. Lastly, we find that the ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first ∼174 amino acids of Best1 are sufficient for oligomerization to occur.

Differential effects of Best disease causing missense mutations on bestrophin-1 trafficking.

Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently in MDCK and RPE cells, and MDCK cells do not express endogenous Best1. Thus, effects of Best1 mutations on localization in MDCK cells may not translate to RPE cells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M, W93C, and R218C. In MDCK cells, Best1 and Best1(R218C) were basolaterally localized. Best1(W93C) and Best1(V9M) accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1(R218C) and Best1(W93C) were basolateral. Best1(V9M) was intracellular. All three mutants exhibited similar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1(V9M) accumulated in intracellular compartments in mouse RPE. Co-expression of Best1 and Best1(W93C) in MDCK cells resulted in basolateral localization of both Best1 and Best1(W93C), but co-expression of Best1 with Best1(V9M) resulted in mislocalization of both proteins. We conclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.

Regulation of anterior chamber drainage by bicarbonate-sensitive soluble adenylyl cyclase in the ciliary body.

Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.

Relationship of stokes radius to the rate of diffusion across Bruch's membrane.

PURPOSE: To determine the effect of Stokes radius (R(S)) on the diffusion of molecules through Bruch's membrane (BM), and to establish a system suitable for the analysis of diffusion through small (<2 mm(2)) samples of BM. METHODS: Porcine BM/choroid (BM/Ch) was mounted in a modified Ussing chamber. A concentration gradient was simultaneously established for four tracers with R(S) values ranging from <1.0 to 6.15 nm. Samples were collected from both chambers at various time points up to 36 hours and the amount of each tracer was determined using quantitative gel exclusion chromatography. The integrity of samples was determined using scanning electron microscopy. RESULTS: BM/Ch mounted in the chamber exhibited no obvious damage even after 36 hours in the chamber. Flux was significantly (P < 0.05) greater in the BM to Ch direction than that in the Ch to BM direction for only two of the tracers: cytosine and RNase A. Flux also was dependent on R(S); cytosine, the smallest tracer (R(S) < 1 nm), exhibited the greatest flux and ferritin (R(S) = 6.15 nm) the least. Permeability coefficients for each tracer were determined and exhibited a power relationship with R(S). CONCLUSIONS: Flux was dependent on the direction of the concentration gradient and the R(S) of the individual tracers. We have successfully demonstrated that quantitative gel exclusion chromatography can be used to follow diffusion of a mixture of tracers across BM/Ch, and that we can measure flux across BM/Ch preparations with an exposed surface area as small as 1.8 mm(2).

Suppression of Ca2+ signaling in a mouse model of Best disease.

Mutations in BEST1, encoding bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited macular degeneration characterized by a diminished electrooculogram light peak (LP), lipofuscin in retinal pigment epithelial cells (RPE), and fluid- and debris-filled retinal detachments. To understand the pathogenesis of BVMD we generated knock-in mice carrying the BVMD-causing mutation W93C in Best1. Both Best1(+/W93C)and Best1(W93C/W93C) mice had normal ERG a- and b-waves, but exhibited an altered LP luminance response reminiscent of that observed in BVMD patients. Morphological analysis identified fluid- and debris-filled retinal detachments in mice as young as 6 months of age. By 18-24 months of age Best1(+/W93C)and Best1(W93C/W93C) mice exhibited enhanced accumulation of lipofuscin in the RPE, and a significant deposition of debris composed of unphagocytosed photoreceptor outer segments and lipofuscin granules in the subretinal space. Although Best1 is thought to function as a Ca(2+)-activated Cl(-) channel, RPE cells from Best1(W93C) mice exhibited normal Cl(-) conductances. We have previously shown that Best1(-/-) mice exhibit increased [Ca(2+)](i) in response to ATP stimulation. However, ATP-stimulated changes in [Ca(2+)](i) in RPE cells from Best1(+/W93C) and Best1(W93C/W93C) mice were suppressed relative to Best1(+/+) littermates. Based on these data we conclude that mice carrying the Best1(W93C) mutation are a valid model for BVMD. Furthermore, these data suggest that BVMD is not because of Best1 deficiency, as the phenotypes of Best1(+/W93C) and Best1(W93C/W93C) mice are distinct from that of Best1(-/-) mice with regard to lipofuscin accumulation, and changes in the LP and ATP Ca(2+) responses.

Fibulin-4 deficiency results in ascending aortic aneurysms: a potential link between abnormal smooth muscle cell phenotype and aneurysm progression.

RATIONALE: Loss of fibulin-4 during embryogenesis results in perinatal lethality because of aneurysm rupture, and defective elastic fiber assembly has been proposed as an underlying cause for the aneurysm phenotype. However, aneurysms are never seen in mice deficient for elastin, or for fibulin-5, which absence also leads to compromised elastic fibers. OBJECTIVE: We sought to determine the mechanism of aneurysm development in the absence of fibulin-4 and establish the role of fibulin-4 in aortic development. METHODS AND RESULTS: We generated germline and smooth muscle cell (SMC)-specific deletion of the fibulin-4 gene in mice (Fbln4(GKO) and Fbln4(SMKO), respectively). Fbln4(GKO) and Fbln4(SMKO) aortic walls fail to fully differentiate, exhibiting reduced expression of SM-specific contractile genes and focal proliferation of SMCs accompanied by degenerative changes of the medial wall. Marked upregulation of extracellular signal-regulated kinase 1/2 signaling pathway was observed in the aneurysmal wall of Fbln4(GKO) and Fbln4(SMKO) mice and both mutants developed aneurysm predominantly in the ascending thoracic aorta. In vitro, Fbln4(GKO) SMCs exhibit an immature SMC phenotype with a marked reduction of SM-myosin heavy chain and increased proliferative capacity. CONCLUSIONS: The vascular phenotype in Fbln4 mutant mice is remarkably similar to a subset of human thoracic aortic aneurysms caused by mutations in SMC contractile genes. Our study provides a potential link between the intrinsic properties of SMCs and aneurysm progression in vivo and supports the dual role of fibulin-4 in the formation of elastic fibers as well as terminal differentiation and maturation of SMCs in the aortic wall.

Fibulin-4 conducts proper elastogenesis via interaction with cross-linking enzyme lysyl oxidase.

Great arteries, as well as lungs and skin, contain elastic fibers as important components to maintain their physiological functions. Although recent studies have revealed that a glycoprotein fibulin-4 (FBLN4) is indispensable for the assembly of mature elastic fibers, it remains to be elucidated how FBLN4 takes part in elastogenesis. Here, we report a dose-dependent requirement for FBLN4 in the development of the elastic fibers in arteries, and a specific role of FBLN4 in recruiting the elastin-cross-linking enzyme, lysyl oxidase (LOX). Reduced expression of Fbln4, which was achieved with a smooth muscle-specific Cre-mediated gene deletion, caused arterial stiffness. Electron-microscopic examination revealed disorganized thick elastic laminae with aberrant deposition of elastin. Aneurysmal dilation of the ascending aorta was found when the Fbln4 expression level was reduced to an even lower level, whereas systemic Fbln4 null mice died perinatally from rupture of the diaphragm. We also found a specific interaction between FBLN4 and the propeptide of LOX, which efficiently promotes assembly of LOX onto tropoelastin. These data suggest a mechanism of elastogenesis, in which a sufficient amount of FBLN4 is essential for tethering LOX to tropoelastin to facilitate cross-linking.

Lack of fibulin-3 alters regenerative tissue responses in the primary olfactory pathway.

The adult olfactory epithelium has maintained the ability to reconstitute its olfactory sensory neurons (OSNs) from a basal progenitor cell compartment. This allows for life-long turnover and replacement of receptor components as well as repair of the primary olfactory pathway in response to injury and environmental insults. The present study investigated whether fibulin-3, a glycoprotein in the extracellular matrix and binding partner of tissue inhibitor of metalloproteinases-3 (TIMP-3), plays a role in ongoing plasticity and regenerative events in the adult primary olfactory pathway. In wild-type control mice, fibulin-3 protein was detected on IB4(+)CD31(+) blood vessels, nerve fascicles and the basement membrane underneath the olfactory epithelium. After target ablation (olfactory bulbectomy), fibulin-3 was also abundantly present in the central nervous system (CNS) scar tissue that occupied the bulbar cavity. Using two different lesion models, i.e. intranasal Triton X-100 lesion and olfactory bulbectomy, we show that fibulin-3 deficient (Efemp1(-/-)) mice have impaired recovery of the olfactory epithelium after injury. Ten days post-injury, Efemp1(-/-) mice showed altered basal stem/progenitor cell proliferation and increased overall numbers of mature (olfactory marker protein (OMP) -positive) versus immature OSNs. However, compromised regenerative capacity of the primary olfactory pathway in Efemp1(-/-) mice was evidenced by reduced numbers of mature OSNs at the later time point of 42 days post-injury. In addition to these neural differences there were consistent changes in blood vessel structure in the olfactory lamina propria of Efemp1(-/-) mice. Overall, these data suggest a role for fibulin-3 in tissue maintenance and regeneration in the adult olfactory pathway.

Failure of pelvic organ support in mice deficient in fibulin-3.

Fibulin-5 is crucial for normal elastic fiber synthesis in the vaginal wall; more than 90% of fibulin-5-knockout mice develop pelvic organ prolapse by 20 weeks of age. In contrast, fibulin-1 and -2 deficiencies do not result in similar pathologies, and fibulin-4-knockout mice die shortly after birth. EFEMP1 encodes fibulin-3, an extracellular matrix protein important in the maintenance of abdominal fascia. Herein, we evaluated the role of fibulin-3 in pelvic organ support. Pelvic organ support was impaired significantly in female Efemp1 knockout mice (Fbln3(-[supi]/-)), and overt vaginal, perineal, and rectal prolapse occurred in 26.9% of animals. Prolapse severity increased with age but not parity. Fibulin-5 was up-regulated in vaginal tissues from Fbln3(-[supi]/-) mice regardless of prolapse. Despite increased expression of fibulin-5 in the vaginal wall, pelvic organ support failure occurred in Fbln3(-[supi]/-) animals, suggesting that factors related to aging led to prolapse. Elastic fiber abnormalities in vaginal tissues from young Fbln3(-[supi]/-) mice progressed to severe elastic fiber disruption with age, and vaginal matrix metalloprotease activity was increased significantly in Fbln3(-[supi]/-) animals with prolapse compared with Fbln3(-[supi]/-) mice without prolapse. Overall, these results indicate that both fibulin-3 and -5 are important in maintaining pelvic organ support in mice. We suggest that increased vaginal protease activity and abnormal elastic fibers in the vaginal wall are important components in the pathogenesis of pelvic organ prolapse.