Expression of p97/VCP and ubiquitin during postnatal development of the degenerating rat retina

In this study, we aimed to investigate the distribution pattern of ubiquitin and p97/VCP in the rat retina during postnatal development. Eyeballs from 1-, 4-, 10-, 36- and 72-week-old rats were examined by immunohistochemistry, and protein colocalization was determined by immunofluorescence microscopy. In the 1-week-old rat retina, p97/VCP was strongly expressed in the neuroblast layer, however no ubiquitin immunoreactivity was observed. p97/VCP immunoreactivity was present in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS) of the photoreceptor layer, and retinal pigment epithelium in the 4- and 10-week-old rat retinas. p97/VCP immunoreactivity increased significantly in the 10-week-old rat retinas. Ubiquitin was barely seen in the 4-week-old rat retinas, and ubiquitin expression was weak in the GCL and the IPL of the 10-week-old rat retinas. In the 36- and 72-week-old rats, the presence of ubiquitin was remarkable in the IS, INL, IPL and GCL, however, p97/VCP immunoreactivity was significantly decreased. Colocalization of ubiquitin and p97/VCP was also observed in the INL, IS, GCL and ONL of 36- and 72-week-old rat retinas. Our results indicate that p97/VCP immunoreactivity in the retina significantly decreases after rats reach 10 weeks of age, whereas ubiquitin immunoreactivity increases with aging. These results suggest that an altered expression pattern of p97/VCP and ubiquitin in the developing rat retina may associate with age-related retinal degeneration.     

表皮生长因子及其受体和erb-B在鼠视网膜的表达

用免疫组织化学方法,对鼠视网膜的表皮生长因子(EGF)及其受体(EGF-R),和erb-B的表达,作了研究.结果表明:①EGF,EGF-R和erb-B分别在视网膜各层有不同表达;②EGF免疫反应着色不强,但有特异性地均匀分布在节细胞层(GCL)、内核层(INL)、外核层(ONL)和视网膜色素上皮层(RPE);③EGF-R免疫反应着色以GCL和INL的细胞明显,分布于细胞浆和细胞核,以细胞核更明显;④erb-B的分布类似EGF.但在ONL内出现一条较明显的着色带.对以上结果的可能意义进行了讨论.     

Impaired retinal vascular development in anencephalic human fetus

This study was to investigate the effect of the absence of ganglion cells on the development of human retinal vasculature. Anencephaly (AnC) and age-matched control eyes derived from each three spontaneously aborted fetus (ranging from 15 to 20 weeks gestation) were subjected to immunofluorescence staining for HIF-1α, Thy-1, glial fibrillary acidic protein (GFAP) and platelet/endothelial cell adhesion molecule (PECAM) and apoptosis assay. In developing mouse retina, Western blotting for hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) was performed. Under hypoxic condition (O₂ < 1%), cellular proliferation and VEGF mRNA expression in astrocytes were measured. Apoptotic cells in AnC retina were primarily localized in the ganglion cell layer (GCL), whereas apoptotic cells in normal retina were distributed in the retinoblastic layer. With increase of apoptotic cells in GCL of AnC retina, HIF-1α expression were severely distinguished in avascular retina and GFAP expression in junctional area between avascular and vascular retina was much reduced, accompanied by decrease of PECAM expression compared to normal retina. In developing mouse retina, HIF-1α and VEGF expression were high in hypoxic retina of early stage with incomplete vascular development and then progressively decreased with regression to arborous pattern of matured vascular networks. In hypoxic condition, a significant increase in cellular proliferation and VEGF mRNA expression was observed in astrocytes. Therefore, our results suggest that vascular attenuation in AnC retina could be closely related to the absence of ganglion cells as the metabolic demander to induce retinal vascular development.     

Increased expression of VEGF in retinal pigmented epithelial cells is not sufficient to cause choroidal neovascularization

Increased expression of vascular endothelial cell growth factor (VEGF) in the retina is sufficient to stimulate sprouting of neovascularization from the deep capillary bed of the retina, but not the superficial retinal capillaries or the choriocapillaris. Coexpression of VEGF and angiopoietin 2 (Ang2) results in sprouting of neovascularization from superficial and deep retinal capillaries, but not the choriocapillaris. However, retina-derived VEGF and Ang2 may not reach the choriocapillaris, because of tight junctions between retinal pigmented epithelial (RPE) cells. To eliminate this possible confounding factor, we used the human vitelliform macular dystrophy 2 (VMD2) promoter, an RPE-specific promoter, combined with the tetracycline-inducible promoter system, to generate double transgenic mice with inducible expression of VEGF in RPE cells. Adult mice with increased expression of VEGF in RPE cells had normal retinas and choroids with no choroidal neovascularization (CNV), but when increased expression of VEGF in RPE cells was combined with subretinal injection of a gutless adenoviral vector containing an expression construct for Ang2 (AGVAng2), CNV consistently occurred. In contrast, triple transgenic mice with induced expression of Ang2 and VEGF in RPE cells, did not develop CNV. These data suggest that increased expression of VEGF and/or Ang2 in RPE cells is not sufficient to cause CNV unless it is combined with a subretinal injection of a gutless adenoviral vector, which is likely to perturb RPE cells. These data also suggest that the effects of angiogenic proteins may vary among vascular beds, even those that are closely related, and, therefore, generalizations should be avoided.     

Retinal development and the expression profiles of opsin genes during larval development in Takifugu rubripes

The light-sensitive capacity of fish larvae is determined by the structure of the retina and the opsins expressed in the retinal and nonretinal photoreceptors. In this study, the retinal structure and expression of opsin genes during the early developmental stage of Takifugu rubripes larvae were investigated. Histological examination showed that at 1 days after hatching (dah), seven layers were observed in the retina of T. rubripes larva, including the pigment epithelial layer [retinal pigment epithelium layer (RPE)], photoreceptor layer (PRos/is), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL). At 2 dah, optic fibre layer (OFL) can be observed, and all eight layers were visible in the retina. By measuring the thickness of each layer, opposing developmental trends were found in the thickness of ONL, OPL, INL, IPL, GCL and OFL. The nuclear density of ONL, INL and GCL and the ratios of ONL/INL, ONL/GCL and INL/GCL were also measured and the ratio of ONL/GCL ranged from 1.9 at 2 dah to 3.4 at 8 dah and no significant difference was observed between the different developmental stages (P > 0.05). No significant difference was observed for the INL/GCL ratio between the different developmental stages, which ranged from 1.2 at 2 dah to 2.0 at 18 dah (P > 0.05). The results of quantitative real-time polymerase chain reaction (PCR) showed that the expression of RH1, LWS, RH2-1, RH2-2, SWS2, rod opsin, opsin3 and opsin5 could be detected from 1 dah. These results suggest that the well-developed retina and early expression of the opsins of T. rubripes during the period of transition from endogenous to mixed feeding might be critical for vision-based survival skills during the early life stages after hatching.     

Hyperoxia causes decreased expression of vascular endothelial growth factor and endothelial cell apoptosis in adult retina

Mice or humans with photoreceptor degenerations experience permeability and dropout of retinal capillaries. Loss of photoreceptors results in decreased oxygen usage and thinning of the retina with increased oxygen delivery to the inner retina. To investigate the possibility that increased tissue oxygen plays a role in the vascular damage, we exposed adult mice to hyperoxia, which also increases oxygen in the retina. After 1, 2, or 3 weeks of hyperoxia, there was a statistically significant decrease in retinal vascular density that was not reversible, and endothelial cell apoptosis was demonstrated by TUNEL staining. Mice exposed to hyperoxia and mice with photoreceptor degeneration both showed decreased expression of VEGF in the retina. After complete or near-complete degeneration of photoreceptors, there was increased expression of VEGF in RPE cells, which may explain the association of photoreceptor degeneration and neovascularization in or around the RPE. Increased expression of VEGF in photoreceptors of transgenic mice failed to prevent hyperoxia-induced retinal capillary dropout. These data suggest that increased oxygen in the retina, either by increased inspired oxygen or by photoreceptor degeneration, results in endothelial cell death and dropout of capillaries. Decreased expression of VEGF may be a contributing factor, but the situation may be more complicated for mature retinal vessels than it is for immature vessels, because VEGF replacement does not rescue mature retinal vessels, suggesting that other factors may also be involved.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.     

Prolonged blockade of VEGF receptors does not damage retinal photoreceptors or ganglion cells

It has recently been reported that relatively short‐term inhibition of vascular endothelial growth factor (VEGF) signaling can cause photoreceptor cell death, a potentially clinically important finding since VEGF blockade has become an important modality of treatment of ocular neovascularization and macular edema. However, in a set of studies in which we achieved extended and complete blockage of VEGF‐induced vascular leakage through retinal expression of a VEGF binding protein, we did not observe any toxicity to retinal neurons. To follow‐up on these apparently discrepant findings, we designed a set of experiments with the kinase inhibitor SU4312, which blocks phosphorylation of VEGF receptors, to look directly for evidence of VEGF inhibition‐related retinal toxicity. Using transgenic mice with sustained expression of VEGF in photoreceptors, we determined that periocular injection of 3 µg of SU4312 every 5 days markedly suppressed subretinal neovascularization, indicating effective blockade of VEGF signaling. Wild‐type mice given periocular injections of 5 µg of SU4312 every 5 days for up to 12 weeks showed normal scotopic and photopic electroretinograms (ERGs), no TUNEL stained cells in the retina, and no reduction in outer nuclear layer thickness. Incubation of cultured ganglion cells or retinal cultures containing photoreceptors with high doses of SU4312 did not reduce cell viability. These data suggest that blocking VEGF signaling in the retina for up to 12 weeks does not damage photoreceptors nor alter ERG function and should reassure patients who are receiving frequent injections of VEGF antagonists for choroidal and retinal vascular diseases. J. Cell. Physiol. 224:262–272, 2010 © 2010 Wiley‐Liss, Inc.     

Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.     

Expression of the cyclin-dependent kinase inhibitor p27Kip1 by developing retinal pigment epithelium

The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 contributes to the timing of cell cycle withdrawal during development and, consequently, in organogenesis. Within the retina, this effector protein is up-regulated during the birth of neuronal and glial cells [Dev. Biol. (2000) 299]. However, its expression within the retinal pigment epithelium (RPE), a supporting cell layer that is essential for neural retina development and function, has not previously been reported. We show that p27Kip1 protein expression in the RPE occurs in two phases: an up-regulation during mid-to late embryonic stages and a down-regulation during the subsequent postnatal period. In the early phase of up-regulation, an inverse relationship is seen between expression of p27Kip1 and PCNA, an indicator of cycling cells. During both up-and down-regulation, the change in spatial pattern of expression proceeds in a central to peripheral manner, with p27Kip1 up-regulation paralleling retinal maturation. These data suggest that this cell cycle regulator may be an important factor controlling the timing of RPE cell cycle withdrawal.     

The retinal pigment epithelium of Cr-deficient rats

The purpose of this study is to investigate the effect of Cr deficiency on the rat retina. Three-week-old Wistar Kyoto rats were divided into 2 groups. Cr-deficient rats were fed AIN-93G diet without Cr and deionized distilled water. Control rats were fed AIN-93G diet and deionized distilled water. The Cr and sugar concentrations in the whole blood and cholesterol concentration in the serum were measured. We observed the retina with an electron microscope, and counted phagocytized lamellar structures in the retinal pigment epithelium (RPE) before and after the start of light exposure on negative electron microscopic films. The whole blood Cr level of Cr-deficient rats was less than 0.2 microg/l. The blood sugar level of Cr-deficient rats was significantly higher than that of normal rats (p < 0.05). There were significantly more phagocytized lamellar structures in the RPE of Cr-deficient rats 1, 2, 7, 11 and 12 h after the start of light exposure than in that of normal rats (p < 0.05). However, no morphological abnormalities were found in the photoreceptor cells of Cr-deficient rats. Phagocytosis in the photoreceptor outer segment discs in the RPE was accelerated, but the pattern of the retinal circadian rhythm with maximum phagocytosis 2 h after exposure to light was unchanged. The Cr-deficient state may cause the membrane to degenerate, and phagocytosis of the photoreceptor outer segment discs in the RPE may be accelerated. This study provided an evidence of the nutritional importance of Cr in rat retina.     

Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells

We previously demonstrated that differentiated retinal pigment epithelial (RPE) cells express high levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), and a critical balance between VEGF and PEDF is important to prevent the development of choroidal neovascularization. We report here that VEGF secreted by RPE cells upregulates PEDF expression via VEGFR-1 in an autocrine manner. PEDF mRNA and protein expression was downregulated by neutralizing antibody against VEGF in differentiated human RPE cells. VEGFR-1 neutralization decreased PEDF mRNA and protein expression whereas anti-VEGFR-2 antibody had no effect. Addition of placenta growth factor (PlGF) restored PEDF expression in the presence of anti-VEGF antibody. These results demonstrate a regulatory interaction between angiogenesis stimulators and inhibitors to maintain homeostasis in normal human retina.     

Conditional deletion of activating protein 2alpha (AP-2alpha) in the developing retina demonstrates non-cell-autonomous roles for AP-2alpha in optic cup development

Activating protein 2alpha (AP-2alpha) is known to be expressed in the retina, and AP-2alpha-null mice exhibit defects in the developing optic cup, including patterning of the neural retina (NR) and a replacement of the dorsal retinal pigmented epithelium (RPE) with NR. In this study, we analyzed the temporal and spatial retinal expression patterns of AP-2alpha and created a conditional deletion of AP-2alpha in the developing retina. AP-2alpha exhibited a distinct expression pattern in the developing inner nuclear layer of the retina, and colocalization studies indicated that AP-2alpha was exclusively expressed in postmitotic amacrine cell populations. Targeted deletion of AP-2alpha in the developing retina did not result in observable retinal defects. Further examination of AP-2alpha-null mutants revealed that the severity of the RPE defect was variable and, although defects in retinal lamination occur at later embryonic stages, earlier stages showed normal lamination and expression of markers for amacrine and ganglion cells. Together, these data demonstrate that, whereas AP-2alpha alone does not play an intrinsic role in retinogenesis, it has non-cell-autonomous effects on optic cup development. Additional expression analyses showed that multiple AP-2 proteins are present in the developing retina, which will be important to future studies.     

Morphological and functional changes in the retina after chronic oxygen-induced retinopathy

The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment.     

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors

The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.     

Angiopoietin-2 enhances retinal vessel sensitivity to vascular endothelial growth factor

Increased expression of vascular endothelial growth factor (VEGF) in the retina starting after postnatal day (P)7 results in neovascularization originating from deep retinal capillaries, but not those in the superficial capillary bed. Doxycycline was administered starting P0 to double transgenic mice with inducible expression of VEGF in the retina. These mice showed proliferation and dilation of superficial retinal capillaries, indicating that at this stage of development, the superficial capillaries are sensitive to the effects of VEGF. Angiopoietin-2 (Ang2) is expressed along the surface of the retina for several days after birth, but by P7 and later, Ang2 is only expressed in the region of the deep capillary bed. In mice with ubiquitous doxycycline-inducible expression of Ang2, in the absence of doxycycline, intravitreous injection of a gutless adenoviral vector expressing VEGF (AGV.VEGF) resulted in neovascularization of the cornea and iris, but no retinal neovascularization. After treatment with doxycycline to induce Ang2 expression, intravitreous injection of AGV.VEGF caused retinal neovascularization in addition to corneal and iris neovascularization. The retinal neovascularization originated from both the superficial and deep capillary beds. These data suggest that Ang2 promotes sensitivity to the angiogenic effects of VEGF in retinal vessels.     

Bipolar assembly of caveolae in retinal pigment epithelium

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function. caveolin; raft microdomains; membrane traffic; normal rat kidney     

Variegated expression and delayed retinal pigmentation during development in transgenic mice with a deletion in the locus control region of the tyrosinase gene

Deletion of the tyrosinase locus control region (LCR) in transgenic mice results in variegated expression in the skin. Here we investigate the pigmentation pattern of other tissues that express tyrosinase: iris, choroid, and retina in the same animals. A mosaic distribution of pigmentation appears in the iris and choroid. Interestingly, a markedly different mosaic pattern is found in the retina, where central areas contain little or no melanin while pigmentation rises to normal levels towards periphery. Further, there is a temporal delay in the initiation and accumulation of pigment in retinal pigmented epithelium (RPE) cells during development, and patterns of adult retinal melanisation in these mice appear arrested at a stage found in early embryogenesis in wild-type mice. These results demonstrate that the tyrosinase LCR is needed for the correct establishment and maintenance of this expression domain throughout development, but particularly during the later stages of retinal melanisation.