No abstract

No abstract

Hypercholesterolemia is a major causative factor for atherosclerotic cardiovascular disease. The molecular mechanisms by which cholesterol initiates and facilitates the process of atherosclerosis are not well understood. Here, we demonstrate that cholesterol treatment suppresses or attenuates TGF-β responsiveness in all cell types studied as determined by measuring TGF-β-induced Smad2 phosphorylation and nuclear translocation, TGF-β-induced PAI-1 expression, TGF-β-induced luciferase reporter gene expression and TGF-β-induced growth inhibition. Cholesterol, alone or complexed in lipoproteins (LDL, VLDL), suppresses TGF-β responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-β receptors and facilitating rapid degradation of TGF-β and thus suppressing TGF-β-induced signaling. Conversely, cholesterol-lowering agents (fluvastatin and lovastatin) and cholesterol-depleting agents (β-cyclodextrin and nystatin) enhance TGF-β responsiveness by increasing non-lipid raft microdomain accumulation of TGF-β receptors and facilitating TGF-β-induced signaling. Furthermore, the effects of cholesterol on the cultured cells are also found in the aortic endothelium of ApoE-null mice fed a high-cholesterol diet. These results suggest that high cholesterol contributes to atherogenesis, at least in part, by suppressing TGF-β responsiveness in vascular cells.

It is commonly assumed that the ultrastructural organization of the rim region of outer segment (OS) discs in rods and lamellae in cones requires functional retinal degeneration slow/rod outer segment membrane protein 1 (Rds/Rom-1) complexes. Cysteine-150 (C150) in Rds has been implicated in intermolecular disulfide bonding essential for functional Rds complexes. Transgenic mice containing the Rds C150S mutation (C150S-Rds) failed to form higher-order Rds oligomers, although interactions between C150S-Rds and Rom-1 occurred in rods, but not in cones. C150S-Rds mice exhibited marked early-onset reductions in cone function and abnormal OS structure. In contrast, C150S-Rds expression in rods partly rescued the rds(+/-) phenotype. Although C150S-Rds was detected in the OSs in rods and cones, a substantial percentage of C150S-Rds and cone opsins were mislocalized to different cellular compartments in cones. The results of this study provide novel insights into the importance of C150 in Rds oligomerization and the differences in Rds requirements in rods versus cones. The apparent OS structural differences between rods and cones may cause cones to be more susceptible to the elimination of higher-order Rds/Rom-1 oligomers (e.g. as mediated by mutation of the Rds C150 residue).

It is commonly assumed that photoreceptor (PR) outer segment (OS) morphogenesis is reliant upon the presence of peripherin/rds, hereafter termed Rds. In this study, we demonstrate a differential requirement of Rds during rod and cone OS morphogenesis. In the absence of this PR-specific protein, rods do not form OSs and enter apoptosis, whereas cone PRs develop atypical OSs and are viable. Such OSs consist of dysmorphic membranous structures devoid of lamellae. These tubular OSs lack any stacked lamellae and have reduced phototransduction efficiency. The loss of Rds only appears to affect the shape of the OS, as the inner segment and connecting cilium remain intact. Furthermore, these structures fail to associate with the specialized extracellular matrix that surrounds cones, suggesting that Rds itself or normal OS formation is required for this interaction. This study provides novel insight into the distinct role of Rds in the OS development of rods and cones.

Peripherin/rds (P/rds) is a membrane glycoprotein essential for the photoreceptor outer segment disc morphogenesis and maintenance. More than half of the disease-causing mutations in P/rds have been linked to different forms of macular dystrophy; the most common one is substitution of tryptophan for arginine at position 172 (R172W). Here we confirm the patient phenotype associated with the expression of R172W mutation in transgenic mice. Functional, structural and biochemical analyses showed that, while R172W P/rds is appropriately localized, a direct correlation exists between transgene expression levels and the onset/severity of the phenotype. In the wild-type background, both cone and rod photoreceptors' structure and function were significantly diminished, which indicates a dominant-negative, cone-rod defect. Whereas rds(+/-) mice maintained the normal cone function at early ages, cone responses in R172W/rds(+/-) mice were diminished to 41% of the wild-type level signifying a preferential damaging effect of the mutation on cones. Conversely, R172W/rds(+/-) mice showed a significant rescue of rod function and improvement of rod outer segment structure. Although rds(-/-) mice have no detectable rod or cone responses, R172W/rds(-/-) animals retained 30% of wild-type structure and rod function, but no significant rescue of cone function was detected at 1 month of age. No biochemical abnormalities were observed in complex formation and association with Rom-1; however, R172W protein was more sensitive to tryptic digestion, indicative of a change in protein conformation, possibly contributing to the cone-dominated phenotype. As the first animal model for P/rds-associated cone-rod dystrophy, R172W mice provide a valuable tool for studying the pathophysiology of P/rds-associated human retinal dystrophies and the development of therapeutic strategies to intervene in these diseases.

Journal of Lipid Research Volume 51, 2010 3399 plus glial cells (Müller cells, astrocytes, and microglia).[For an excellent overview of retinal structure and cytoarchitecture, see ( 1 ) and http://webvision.med.utah. edu/]. The neuronal cells are distributed into discrete histological layers in the fully differentiated retina ( Fig. 1 ), with the ganglion cell layer arranged proximal to the vitreous and the photoreceptor cells (rods and cones) residing at the opposite side of the neural retina, just underneath the retinal pigment epithelium (RPE) layer. The RPE is a monolayer of polarized epithelial cells that serves as the interface between the neural retina and the choroid, the blood supply to the outer cell layers of the retina. The apical tips of the photoreceptor outer segments (OS) are adjacent to and invaginate into the apical face of the RPE (one of the few examples of apical-apical cellular contact in the body) ( Fig. 1C ). Bruch's membrane (BrM; Fig. 1B, C ) is not a membrane, per se, but rather a pentalaminar structure that consists of the basement membranes of the RPE cells and the endothelial cells of the choriocapillaris ( Fig. 1C ), plus the extracellular matrix that fi lls the space between these two membranes [reviewed in ( 2, 3 )].The relatively modest compendium of cell types mentioned above, however, belies the amazing, higher order complexity of this sensory tissue. It has been estimated that there are actually about 55 anatomically distinct neuronal cell types in the adult mammalian retina, of which about 22 have been thoroughly characterized with regard to their fi ne structure and functional repertoire [reviewed in ( 4 )]. For example, photoreceptors consist of both rods and cones, with cones being subdivided into two (three in Abstract The vertebrate retina has multiple demands for utilization of cholesterol and must meet those demands either by synthesizing its own supply of cholesterol or by importing cholesterol from extraretinal sources, or both. Unlike the blood-brain barrier, the blood-retina barrier allows uptake of cholesterol from the circulation via a lipoprotein-based/receptor-mediated mechanism. Under normal conditions, cholesterol homeostasis is tightly regulated; also, cholesterol exists in the neural retina overwhelmingly in unesterifi ed form, and sterol intermediates are present in minimal to negligible quantities. However, under certain pathological conditions, either due to an inborn error in cholesterol biosynthesis or as a consequence of exposure to selective inhibitors of enzymes in the cholesterol pathway, the ratio of sterol intermediates to cholesterol in the retina can rise dramatically and persist, in some cases resulting in progressive degeneration that signifi cantly compromises the structure and function of the retina. The mature vertebrate retina is a highly stratifi ed, multicellular tissue classically described as consisting of six fundamental neuronal cell types (photoreceptor, bipolar, ganglion, horizontal, amacrine, and interplexiform cells) the normal...

Previously, we have shown that Onecut1 (Oc1) and Onecut2 (Oc2) are expressed in retinal progenitor cells, developing retinal ganglion cells (RGCs), and horizontal cells (HCs). However, in Oc1-null mice, we only observed an 80% reduction in HCs, but no defects in other cell types. We postulated that the lack of defects in other cell types in Oc1-null retinas was a result of redundancy with Oc2. To test this theory, we have generated Oc2-null mice and now show that their retinas also only have defects in HCs, with a 50% reduction in their numbers. However, when both Oc1 and Oc2 are knocked out, the retinas exhibit more profound defects in the development of all early retinal cell types, including completely failed genesis of HCs, compromised generation of cones, reduced production (by 30%) of RGCs, and absence of starburst amacrine cells. Cone subtype diversification and RGC subtype composition also were affected in the double-null retina. Using RNA-Seq expression profiling, we have identified downstream genes of Oc1 and Oc2, which not only confirms the redundancy between the two factors and renders a molecular explanation for the defects in the double-null retinas, but also shows that the onecut factors suppress the production of the late cell type, rods, indicating that the two factors contribute to the competence of retinal progenitor cells for the early retinal cell fates. Our results provide insight into how onecut factors regulate the creation of cellular diversity in the retina and, by extension, in the central nervous system in general.transcription factors | neural development | retinal development | cell fate determination | gene regulation