Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

GCL

Figure 26.1. Localization and functional analysis of Cre expression (adopted from Le et al., 2004). A: b- Galactosidase staining of retinal section of double transgenic HRGP-cre/R26R mice. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. B: Fluorescent microscopy of retinal section stained for Cre (cone nuclei) and M-Opsin (cone outer segment). C: Confocal microscopy of retinal section stained for Cre, M- opsin and peanut agglutinin. The scale bar equals to 50 mm in A and B, and 20 mm in C.

3.2. Analysis of Cre Expression

To localize Cre expression and perform Cre functional assays, we decided to use a Creactivatable lacZ reporter mouse line R26R (Soriano, 1999). This mouse strain carries a loxP flanked STOP (un-functional) sequence that prevents the expression of lacZ reporter gene (Soriano, 1999). Upon a Cre-mediated recombination that removes the STOP sequence, the lacZ reporter gene is expressed under the control of a generalized promoter ROSA26; therefore, the cells expressing Cre recombinase are blue after b-galactosidase staining. b- Galactosidase assays using 6 to 8 week-old double transgenic HRGP-cre/R26R mice (F1 of FVB/N and C57B6 genetic background) showed that only two Cre-expressing candidate lines had efficient Cre expression in the retina, judging by X-gal staining in retinal flatmount (Le et al., 2004) and sections (Figure 26.1A). X-gal staining was strong near the center of the retina (Le et al., 2004). This expression pattern is consistent with mouse cone distribution (Jeon et al., 1998). X-gal staining of retinal sections showed that the b- galactosidase activity was localized to the presumptive cone photoreceptor cells (Figure 26.1A). A small number of presumptive ganglion cells were positive with X-gal staining (Figure 26.1A). The remaining candidate transgenic mouse lines had only limited amount of Cre activities and the b-galactosidase staining was punctuated. These strains were not characterized further.

To further confirm that Cre was expressed in cone photoreceptors, retinal sections were stained with anti-Cre antibody and Cre was exclusively localized to the nucleus of cone photoreceptors (data not shown). This result was consistent with our earlier observation that Cre carried a nuclear localization signal (Gagneten et al., 1997; Le et al., 1999). Double labeling of retinal sections with anti-Cre and anti-M-opsin antibodies showed that cells expressing M-opsin (cone outer segment) also had Cre expression (nuclei) (Figure 26.1B). This result suggested that almost all M-opsin-expressing cells expressed Cre recombinase. Since most mouse cone cells express both M- and S-opsins and only a small percentage of cone cells express a single type of pigment (Applebury et al., 2000), retinal sections were

Figure 26.2. Cone distribution in double transgenic HRGP-cre/R26R mice (adopted from Le et al., 2004). Lectin stained cone photoreceptors in 6 month old transgenic (A) and wild-type littermate (B). The scale bars equal to 16 mm. Reprinted with permission from Molecular Vision.

stained for Cre and peanut agglutinin (PNA). The results suggested that almost all cone photoreceptors were Cre-positive in our transgenic mice (Figure 26.1C).

3.3. Distribution and Function of Cone Photoreceptors

Cre is a DNA recombinase and there is evidence suggesting that over-expression of Cre may cause rod photoreceptor degeneration (Le and Anderson, unpublished observation; Fenier L et al., IVOS: 2004 ARVO E-Abstract 3596; Chen, C et al., IVOS: 2004 ARVO E- Abstract 3597), presumably due to undesired recombination that causes chromosomal rearrangement (Loonstra et al., 2001; Schmidt et al., 2000). Since the goal of generating cone-specific Cre transgenic mice is for gene function studies, it is necessary to determine if the cones are normal in the mice. Therefore, the distribution of cones in six-month-old HRGP-cre/R26R mice was analyzed. Fluorescent microscopic analysis of lectin-labeled retinas indicated that there were no significant differences in cone density and distribution between the transgenic mice and wild-type littermates (Figure 26.2). This result suggested that there was no cone photoreceptor degeneration.

To further confirm that cone photoreceptors were normal, six-month-old F1 HRGP- cre/R26R mice were used in ERG analysis. The photopic ERG suggested that there were no significant differences between the transgenic mice and the wild-type littermates, as shown in Figure 26.3. In addition, the scotopic ERG data showed that rod function was normal in HRGP-cre/R26R mice (data not shown). This result indicated that the insertion of the transgene did not cause any changes in retinal function.

4. SUMMARY

To study function of widely expressed essential genes, we established a conditional knockout system for cone photoreceptor cells. Our goal is to generate a useful genetic system that can be utilized to disrupt gene function efficiently in cone photoreceptor cells. Functional assay using a Cre-activatable lacZ reporter gene suggested that HRGP-cre mice

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vival ganglion neurons for up to 6 days in culture (Margiotta and Howard, 1994). Retinal hypoxia has been reported to increase production of vascular endothelial growth factor (VEGF), a known stimulant of retinal neovascularization (Okamoto et al., 1997). The aim of this study was to investigate the regulation of tight junction (TJ) gene expression in cultured retinal pigment epithelial (RPE) cells and in the retina of normal and vascular endothelial growth factor (VEGF) transgenic mice.

2. MATERIALS AND METHODS

2.1. Generation of Transgenic Mice

The human VEGF165 isoform (Chavand et al., 2001) and the truncated murine opsin promoter containing 1.4 kb of a 5¢ upstream regulatory sequence of the opsin gene (May et al., 2003) were used to generate the pcDNA.CMV.VEGF construct for production of transgenic mouse (Lai et al., 2004 BJO-in press). All mice experimentation was performed in accord with the guidelines of the Association for Research in Vision and Ophthalmology on the use of animals in research and following the guidelines of the Animal Ethics Committee at The University of Western Australia, Australia.

2.2. Effects of Eye Extract and Hypoxia on RPE Cell Culture

Human RPE cells were isolated from the retina of a 51-year-old donor as previously described (Kennedy et al., 1996) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. For eye-extract experiments, immediately following enucleation of the eyes of four wild-type female C57/B1 mice, the anterior chamber and lenses were removed and the remaining eyecups were homogenized in culture medium using sterile pestle. The homogenate was centrifuged at 100 g for one minute to remove debris. The supernatant was diluted to final volume of 14 mL with DMEM (Sigma) containing 10% fetal bovine serum. RPE cells were seeded onto 6-well culture plates with or without 13 mm coverslips at 5 ¥ 105 cells/well and cultured in a humidified incubator. Twenty-four hours later plates with or without coverslips received 1 and 2.5 mL of diluted eye extract respectively, and control plates received the same volume of culture medium only. All plates were incubated for an additional 24 hours before being analysed. For hypoxia experiments, RPE cells were seeded onto 6-well culture plates (Australian Biosearch) with or without 13 mm coverslips (Biolab) at 5 ¥ 105 cells/well and cultured under normal (37°C, 5% CO2 and 20% O2) condition. Twenty-four hours later cells were either incubated under normoxic (20% O2) or hypoxic (2% O2) conditions for additional 24 hours prior to being analysed.

2.3. Antibodies and Immunoconfocal Microscopy

For immunoconfocal the eyes of mice were enucleated and one eye per animal was embedded in optimum cutting temperature compound (OCT) (Tissue Tek) and snap frozen using liquid nitrogen. Immunostaining was performed as described previously (Ghassemifar et al., 2002) with minor modifications. Briefly, fixed in ice-cold methanol, cells on coverslips and or cryosections on slides were rehydrated in PBS/0.5% BSA for 15

minutes prior to a 1 hour incubation at room temperature (rt) with either polyclonal anti- ZO-1 (Zymed) (2 mg/mL in PBS/0.5% BSA) or monoclonal anti-occludin (Zymed) (3 mg/mL in PBS/0.5%BSA). A further 3 washes were followed by a 1 hour incubation at rt with FITCconjugated goat anti-mouse antibodies (1 : 500 in PBS/0.5% BSA) and/or TRITC-conjugated goat anti-rabbit antibody (1 : 500 in PBS/0.5% BSA). Specimens were viewed on a Bio-Rad MRC 1024 UV Laser Scanning Confocal Microscope system.

2.4. RT-PCR

Total RNA was extracted from the collected cells and remaining eyes using QIAzol lysis reagent (QIAGEN), according to manufacturer’s instructions. Two-hundred nanograms of total RNAs were reverse transcribed in 50 ml RT reactions into cDNA. Directly from this reaction, 2.5 ml (5%) of each cDNA were used as template for PCRs containing 0.5 mM of each gene-specific primers for detection of OCLN-TM4+/TM4- 966F (5¢- tagtgagtgctatcctgggcat-3¢) and 1585R (5¢-tgcaggtgctctttttgaaggt-3¢), ZO-1a+/a- 3120F (5¢-gagaggactcctctggaatg-3¢) and 3818R (5¢-caaggtcttgagagtgctga-3¢), VEGF 1147F (5¢-catcacgaagtggtgaagtt-3¢) and 1533R (5¢-aacgctccaggacttatacc-3¢), and b-actin 188F (5¢- aggcaccagggcgtgat-3¢) and 711R (5¢-ttaatgtcacgcacgatttc-3¢) (Proligo).

3. RESULTS

3.1. Expression and Immunolocalization of TJs in RPE Cells

The ZO-1a+ transcripts were differentially upregulated in a dose dependant manner (Figure 27.1A). However, the transcription levels of ZO-1a- isoform remained unchanged in all three groups. The transcription levels of OCLN-TM4- isoform was also differentially upregulated in RPE cells incubated with eye extract compared to controls. Moreover, the results showed a downregulation of VEGF transcripts at the highest eye extract concentration compared to the increased transcription levels with the lower eye extract concentration and the control. Moreover, the results showed that within hypoxic group and/or when compared to normoxic controls, there were a noticeable upregulation of ZO-1a- compared to a+ transcript (Figure 27.1B). Moreover, RPE cells under hypoxia upregulated OCLN-TM4+ more than TM4- transcripts both within and/or when compared to normoxic controls. The

A B

Figure 27.1. Composite images of RT-PCR analysis of ZO- 1a+/a-, OCLN-TM4+/TM4-, VEGF and b-actin transcripts during (A) addition of 0 mL, 1 mL and 2.5 mL of eye extract and (B) normoxia and hypoxia, in cultured RPE cells.

Figure 27.2. (A) Occludin and (D) ZO-1 labelling of control RPE cells showing a few number of junctional staining at cell borders (arrows). (B) Occludin and (E) ZO-1 labelling of RPE cells incubated with 1 mL/dish of mouse eye extract; note significant increase in occludin and ZO-1 junctional staining at cell borders (arrows). (C) Occludin and (F) ZO-1 labelling of confluent HEK-293 cells used as a positive control. Bars, 20 mm.

Figure 27.3. Composite images of RT-PCR analysis of ZO-1a+/a-, OCLN-TM4+ and b-actin transcript expression in retinal tissue from transgenic (TG1-TG4) and wild type mice (WT) control.

expression of VEGF transcripts were also significantly higher during hypoxia compared to normoxic controls. RPE cells incubated with eye extract increased the assembly of occludin (Figure 27.2C) and ZO-1 (Figure 27.2D) proteins at the TJ compared to control RPE cells that showed very little if none occludin (Figure 27.2A) and ZO-1 (Figure 27.2B) junctional localization. Panels 2E and 2F are human embryonic kidney cell line (HEK-293) used as positive staining controls for occludin and ZO-1, respectively.

3.2. Expression and Immunolocalization of TJs in Transgenic Mouse Eyes

VEGF over-expressing retinal tissue differentially upregulated the expression of ZO- 1a+ transcripts compared to wild type control retinal tissue (Figure 27.3). However, the expression of ZO-1a- and OCLN-TM4+ transcripts remained uniformly unchanged in all groups. Hematoxylin-eosin stained cryosections showed severe morphological changes in transgenic mice retinas (Figures 27.4F and 27.4K) compared to the retina of wild type control mouse (Figure 27.4A). Immunofluorescence staining revealed an increased TJ localization of occludin (Figures 27.4G-27.4H and 27.4L-27.4N) and ZO-1 (Figures 27.4I-27.4J and 27.4O-27.4P) in the RPE layer of transgenic mice compared to occludin (Figures 27.4B and 27.4C) and ZO-1 (Figures 27.4D and 27.4E) in RPE layer of wild type mouse.

Figure 27.4. Light microscopy and Hematoxylin-eosin stained cryosections show abnormal morphology of transgenic mouse retina (F and K) compared to normal morphology of wild type mouse retina (A). Immunofluorescence staining show occludin [(G with inset H: arrows showing increased junctional staining) and (L with insets M: arrows showing increased junctional and N: arrows showing massive cytoplasmic staining)] and ZO-1 [(I with inset J: arrows showing abnormal punctuated junctional staining) and (O with inset P: arrows showing massive junctional and cytoplasmic staining)] in RPE layer of transgenic mouse retina compared to occludin [(B with inset C: arrows showing normal junctional staining) and ZO-1 (D with inset E: arrows showing normal junctional staining) in the RPE of wild type mouse retina.

4. DISCUSSION

This study demonstrated that human RPE cells cultured in the presence of eye extract differentially upregulated the expression of ZO-1a+ transcripts in a dose dependant manner while, the transcription levels of the ZO-1a- isoform remained unchanged in all groups. Previous studies have demonstrated that factors present in embryonic eye-extract can influence the neurons of chick sympathetic ganglia to express choline acetyl-transferase and also whole-eye extract support full survival of E13 dorsal root ganglion neurons for up to 6 days in culture (Fischer et al., 2002). Although the function of the a-domain is still under investigations, the ZO-1a+ isoform is found in conventional epithelial TJs while ZO-1a- is present in endothelial junctions and highly specialised epithelial TJs characteristic of Sertoli cells and renal podocytes coinciding with our findings showing high expression of ZO-1a- in RPE cells suggesting the highly differentiated and specialized character of RPE cells. Moreover, in RPE cells, eye extract caused the upregulation of the OCLN-TM4- isoform. Nonetheless, it has been suggested that the low level expression of OCLN-TM4- may contribute to the regulation of occludin function (Ghassemifar et al., 2003). These results were supported by immunoconfocal data showing RPE cells incubated with eye extract increased the assembly of ZO-1 and occludin proteins at the TJ. Moreover, RPE cells downregulated VEGF transcripts at the higher eye extract concentration compared to the controls, which could explain the existence of inbuilt mechanisms within the eye to control the overexpression of VEGF in order to minimise the potential for post developmental neovascularization. During hypoxia, ZO-1a- transcripts were upregulated compared to the a+ in RPE cells. Likewise, the OCLN-TM4+ transcripts were upregulated more than TM4- and when compared to normoxic controls, both OCLN-TM4+/TM4- transcripts were notably higher during hypoxia. It has been suggested that hypoxia increases the paracellular flux across the cell monolayer via the release of VEGF, which in turn leads to the relocalization, decreased

expression, and enhanced phosphorylation of ZO-1 (Fischer et al., 2002). Overexpression of occludin has been shown to increase trans-epithelial electrical resistance (TER) in MDCK cells (Mccarthy et al., 1996) and confers adhesiveness in fibroblasts (Van Itallie and Anderson, 1997). It has been shown that RPE cells in the presence of dimethyl sulfoxide increases assembly of occludin and ZO-1 at cell borders parallel with the increases in TER that occurred with decreases in inulin and dextran permeability (Konari et al., 1995). However, anti-occludin antisense oligonucleotides decrease barrier permeability to solutes in arterial endothelial cells (Kevil et al., 1998). The expression of VEGF transcripts was also significantly higher during hypoxia compared to normoxic controls, coinciding with previous findings that have shown hypoxia upregulates the transcription of VEGF (Carmeliet et al., 1998). In the RPE cells, the upregulation of VEGF during hypoxia is directly correlated to the upregulation of TJ proteins in order to create a less permeable RPE layer that could stand against the ingrowth of blood vessels. In summary, we have shown that the eye extract contains constituents, which are to our knowledge directly or indirectly control the regulation of TJ proteins expression and translation. This is the first report to show that in contrast to endothelial cells, hypoxia upregulates and increase the expression and membrane assembly of TJ proteins in RPE cells both in vitro and in vivo. These results suggest that RPE cells, in the presence of excessive amount of VEGF, probably caused by hypoxia maintain their transcriptional and post translational processing of TJs to create a less permeable RPE layer to withstand CNV.

5. ACKNOWLEDGMENT

The authors acknowledge the Lions Eye Institute, Lions Save-Sight Foundation and Juvenile Diabetes Research Foundation, USA for the financial support.

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