J.B. Stanton

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 Bruchs membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1−/− mice. Methods Proteoglycans in mouse Bruchs 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 Bruchs 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 Bruchs membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruchs membrane was impaired. In contrast, the proteoglycan amount in Efemp1−/− Bruchs 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 Bruchs membrane. Diffusion across Efemp1−/− Bruchs 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 Bruchs membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruchs membrane.

Deletion of Efemp1 is protective against the development of sub-rpe deposits in mouse eyes

Purpose EFEMP1 (fibulin-3) is mutated in Malattia Leventinese/Doynes 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.

In vitro characterization of neonatal, juvenile, and adult porcine islet oxygen demand, β-cell function, and transcriptomes

There is currently a shortage of human donor pancreata which limits the broad application of islet transplantation as a treatment for type 1 diabetes. Porcine islets have demonstrated potential as an alternative source, but a study evaluating islets from different donor ages under unified protocols has yet to be conducted.