Mutations in VMD2, encoding bestrophin (best-1), cause Best vitelliform macular dystrophy (BMD), adult-onset vitelliform macular dystrophy (AVMD), and autosomal dominant vitreoretinochoroidopathy (ADVIRC). BMD is distinguished from AVMD by a diminished electrooculogram light peak (LP) in the absence of changes in the flash electroretinogram. Although the LP is thought to be generated by best-1, we find enhanced LP luminance responsiveness with normal amplitude in Vmd2 −/− mice and no differences in cellular Cl− currents in comparison to Vmd2 +/+ littermates. The putative Ca2+ sensitivity of best-1, and our recent observation that best-1 alters the kinetics of voltage-dependent Ca2+ channels (VDCC), led us to examine the role of VDCCs in the LP. Nimodipine diminished the LP, leading us to survey VDCC β-subunit mutant mice. Lethargic mice, which harbor a loss of function mutation in the β4 subunit of VDCCs, exhibited a significant shift in LP luminance response, establishing a role for Ca2+ in LP generation. When stimulated with ATP, which increases [Ca++]I, retinal pigment epithelial cells derived from Vmd2 −/− mice exhibited a fivefold greater response than Vmd2 +/+ littermates, indicating that best-1 can suppress the rise in [Ca2+]I associated with the LP. We conclude that VDCCs regulated by a β4 subunit are required to generate the LP and that best-1 antagonizes the LP luminance response potentially via its ability to modulate VDCC function. Furthermore, we suggest that the loss of vision associated with BMD is not caused by the same pathologic process as the diminished LP, but rather is caused by as yet unidentified effects of best-1 on other cellular processes.

Malattia Leventinese (ML), an inherited macular degenerative disease, is closely reminiscent of age-related macular degeneration (AMD), the most common cause of incurable blindness. Both ML and AMD are characterized by extracellular deposits known as drusen between the retinal pigment epithelium (RPE) and Bruch's membrane. The mechanism underlying drusen formation is unknown. An Arg to Trp mutation in a gene of unknown function, EFEMP1, is responsible for ML, indicating EFEMP1 may be important in drusen formation. Here, we show that wild-type EFEMP1 is a secreted protein whereas mutant EFEMP1 is misfolded, secreted inefficiently, and retained within cells. In normal eyes, EFEMP1 is not present at the site of drusen formation. However, in ML eyes, EFEMP1 accumulates within the RPE cells and between the RPE and drusen, but does not appear to be a major component of drusen. Furthermore, in AMD eyes, EFEMP1 is found to accumulate beneath the RPE immediately overlaying drusen, but not in the region where there is no apparent retinal pathology observed. These data present evidence that misfolding and aberrant accumulation of EFEMP1 may cause drusen formation and cellular degeneration and play an important role in the etiology of both ML and AMD.T he macula, a central circular area of the retina 5 to 6 mm in diameter with the fovea at its center, facilitates central vision and high-resolution visual acuity. Various diseases causing macular degeneration result in severe and irreversible loss of vision. Malattia Leventinese (ML), also known as Doyne honeycomb retinal dystrophy, is a rare autosomal dominant macular degenerative disease with high penetrance (1-3). Onset of ML is generally in midlife but can vary from childhood until old age (4). An early characteristic feature of ML is the presence of amorphous sub-retinal pigment epithelium (RPE) deposits known as drusen between the RPE and Bruch's membrane (1, 5). At a later stage of the disease, ML exhibits a variety of clinical and histopathological features, including decreased visual acuity, geographic atrophy, pigmentary changes, and choroidal neovascularization (6). Drusen are also an early hallmark of age-related macular degeneration (AMD), a heterogeneous late onset macular degenerative condition (7). ML exhibits features more consistent with AMD than any other heritable macular disorder. Except for a late age of onset, AMD shares the typical clinical features of ML (8). AMD accounts for approximately 50% of registered blindness in the developed world (9, 10). More than 20% of the population over 65 years of age is affected with AMD. The molecular mechanism responsible for drusen formation and other retinal pathology observed in ML or AMD is currently unknown.A single mutation, Arg-345 to Trp (R345W) in the gene EFEMP1 (for epidermal growth factor-containing fibrillin-like extracellular matrix protein 1), was found to be responsible for ML (11). To date, no mutation in EFEMP1 has been found to be associated with AMD (11). Initially described as S1-5 (12), also kn...

Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4 ؊/؊ mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4 ؊/؊ embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4 ؊/؊ mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4 ؊/؊ tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4 ؊/؊ mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.Elastic fibers with morphologically distinct architectures are present in the extracellular matrix (ECM) to accommodate elastic requirements and mechanical stresses imposed on different tissues. They are particularly abundant in elastic tissues such as large blood vessels, lung, and skin. Loss of elasticity is a major contributing factor in aging and a myriad of pathological conditions including emphysema, artery diseases, and cutis laxa (39,41,44). Elastic fibers undergo irreversible structural and compositional changes with age and in some pathological conditions (41). Regardless of morphology, all elastic fibers consist of cross-linked elastin, fibrillin-rich microfibrils, and several associated molecules (23,37,38,46). Elastin endows the fiber with the characteristic property of elastic recoil. It is chemically inert, extremely hydrophobic, and insoluble under most conditions. Monomeric elastin, called tropoelastin, is secreted from the cell as a soluble protein. Isolated and purified tropoelastin has been shown to exhibit a great tendency to aggregate (coacervation) in physiological solution and at temperatures in the physiological range, giving rise to supramolecular structures very similar to those found in natural elastic fibers (4,5,11). This self-aggregation property of tropoelastin is thought to contribute to elastic fiber assembly in vivo. However, self-aggregation alone is insufficient to explain the efficiency of the assembly process and the variable form of elastic fibers ...

A mutation in the EFEMP1 gene causes Malattia Leventinese, an inherited macular degenerative disease with strong similarities to age-related macular degeneration. EFEMP1 encodes fibulin-3, an extracellular matrix protein of unknown function. To investigate its biological role, the murine Efemp1 gene was inactivated through targeted disruption. Efemp1(-/-) mice exhibited reduced reproductivity, and displayed an early onset of aging-associated phenotypes including reduced lifespan, decreased body mass, lordokyphosis, reduced hair growth, and generalized fat, muscle and organ atrophy. However, these mice appeared to have normal wound healing ability. Efemp1(-/-) mice on a C57BL/6 genetic background developed multiple large hernias including inguinal hernias, pelvic prolapse and protrusions of the xiphoid process. In contrast, Efemp1(-/-) mice on a BALB/c background rarely had any forms of hernias, indicating the presence of modifiers for fibulin-3's function in different mouse strains. Histological analysis revealed a marked reduction of elastic fibers in fascia, a thin layer of connective tissue maintaining and protecting structures throughout the body. No apparent macular degeneration associated defects were found in Efemp1(-/-) mice, suggesting that loss of fibulin-3 function is not the mechanism by which the mutation in EFEMP1 causes macular degeneration. These data demonstrate that fibulin-3 plays an important role in maintaining the integrity of fascia connective tissues and regulates aging.