Expression and cellular localization of the Mas receptor in the adult and developing mouse retina

Animals

Adult C57BL/6J and CD1 wild-type mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Different ages of mice used for this study were bred at Animal Care Service at University of Florida. Animals were maintained under standardized conditions with an artificial 12 h:12 h light-dark cycle, with free access to food and water. All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of animals in Ophthalmic and Vision Research and the guidelines of the Institutional Animal Care and Use committee at the University of Florida.

Immunofluorescence detection of the Mas receptor in developing and adult mouse retinas

For the immunofluorescence studies, eyes were collected from P1, P5, P7, P15, P21, and P60 mice. Eyes from P1–P7 mice were enucleated and immersion fixed in 4% paraformaldehyde overnight at 4 °C. The P15–P60 mice were first anaesthetized with a ketamine/xylazine mix (ketamine 60 mg/kg; xylazine 10 mg/kg) and then perfused transcardially with 4% paraformaldehyde freshly made in PBS (1X; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4). Following perfusion, the eyes were enucleated and post-fixed in 4% paraformaldehyde overnight at 4 °C. The eyes were then either embedded in paraffin and 4-µm-thick sections were cut or cryoprotected in 30% sucrose in PBS at 4 °C and frozen in optimum cutting temperature (OCT) compound (Tissue-Tek; Sakura-Finetek, Torrance, CA). Cryostat sections 12 µm thick were cut and mounted on SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA). Paraffin sections were first deparaffinized, which was followed by an antigen retrieval step. The antigen retrieval was performed by boiling the sections in antigen retrieval buffer (10 mM sodium citrate; 0.05% Tween-20, pH 6.0) for 15 min. The paraffin and cryosections were then incubated in blocking solution (5% bovine serum albumin [BSA] + 0.3% Triton X-100 in PBS) for 1 h. This was followed by incubation with different primary antibodies—rabbit anti-Mas (1:500, AAR-013, Alomone Laboratories, Jerusalem, Israel), mouse anti-arrestin (1:200), mouse anti-rhodopsin (1:200), mouse anti-glial fibrillary acidic protein (GFAP; 1:500), mouse anti-Brn3a (1:200, MAB1585, Millipore, Billerica, MA), mouse anti-CRALBP (1:500, ab15051, Abcam, Cambridge, MA), mouse anti-RPE65 (1:100, NB100–355, Novus Biologicals, Littleton, CO), biotinylated peanut agglutinin (PNA-biotin 1:200, B-1075, Vector Laboratories, Burlingame, CA), mouse anti-p27^kip1 (1:200, BD Transduction laboratories, Lexington KY)—diluted in the same blocking solution (overnight at 4 °C) and then with the appropriate secondary antibodies conjugated to Alexa 488 or 594 (Molecular Probes/Invitrogen, Grand Island, NY) for 1 h at room temperature (RT). Sections were washed in PBS containing the nuclear counterstain 4’,6 diamidino-2- phenylindole (DAPI), and mounted in Dako mounting media (Dako North America, Carpinteria, CA). The images were captured on a Keyence confocal microscope (Itasca, IL) or on a spinning disc confocal (UltraVIEW Vox, PerkinElmer, Waltham, MA) using a 20× and/or a 40× objective lens and were prepared for presentation using Adobe Photoshop (San Jose, CA).

Western blot analysis

To quantify the level of Mas receptor protein expression during development, retinal tissue was collected from P1, P15, P21, and P60 wild-type mice in cold RIPA buffer (Sigma, St Louis, MO) supplemented with protease inhibitors and were homogenized by sonication. Homogenized tissues were centrifuged at 12,000 ×g for 15 min at 4 °C, and the supernatant was collected. The protein concentration was detected with the Bio-Rad Protein Assay kit (Hercules, CA). Forty microns of protein samples were loaded and separated on 4–12% gradient gels for 1 h at 120 V in Tris-glycine buffer and electrophoretically transferred onto polyvinylidene difluoride membrane. Immunodetection was performed on blots blocked in fluorescence blocking buffer for 1 h (Rockland Immunochemicals, Gilbertsville, PA) and then incubated with primary (rabbit anti-Mas; 1:2,000; Alomone Laboratories), and secondary (goat anti-rabbit IR Dye 800; 1:5,000; Rockland Immunochemicals) antibodies. β-Actin immunodetection was used as a loading control. Immunoblots were visualized by using a Li-Cor odyssey infrared imager (Odyssey; Lincoln, NE). We used Image J software to quantify the relative level of the Mas protein after normalizing it to β-actin.

Mas receptor mRNA detection with in situ hybridization

Eyes were collected from P1, P5, P7, P15, P21, and P60 mice and immersion fixed in 4% paraformaldehyde, freshly made in phosphate-buffered saline, overnight at RT. The fixed eyes were embedded in paraffin, and 4-μm-thick sections were cut and mounted on SuperFrost Plus slides (Thermo Fisher Scientific). In situ hybridization to look at the Mas receptor gene expression was performed using the RNAscope 2.0 (Red) FFPE Assay kit (Advanced Cell Diagnostics, Hayward, CA) according to the protocol provided by the manufacturer. Briefly, the method involved deparaffinization of the sections by immersion in fresh Xylene (2×5 min) followed by immersion in 100% ethanol (2×3 min) with frequent agitation. The endogenous peroxidase was blocked with the provided pretreatment 1 solution for 10 min at RT, followed by epitope retrieval by boiling in the provided buffer for 10 min, protease digestion for 30 min at 40 °C in a hybridization oven (HybEZ Oven, Advanced Cell Diagnostics) and target hybridization for 2 h at 40 °C followed by six sequential amplification steps each interspersed with washing in the given wash buffer. This was followed by a colorimetric reaction, which involved the addition of fast red substrate on sections for 10 min at RT followed by washing in distilled water (dH₂O). The slides are then baked at 60 °C for 15 min and then mounted in EcoMount mounting media (Biocare Medical, Concord, CA). Control probes against the bacterial gene DapB (negative control) and for the housekeeping gene peptidylprolyl isomerase B (PIPB, positive control) and arrestin-S, which is expressed exclusively in the photoreceptors in the retina were also included to validate the RNAscope technique. Positive staining was identified as red punctate dots in the section.

Trypsin digest preparations of retinal vasculature

Eyes were enucleated from adult mice and immersion fixed in 4% PFA overnight at 4 °C. The retinas were dissected from the eye cups, washed in water for 1 h, and digested in 3% trypsin for 2–3 h at RT. The tissue was then transferred into water, and the network of vessels was freed from adherent retinal tissue by gentle shaking and manipulation under a dissection microscope. The vessels were then mounted on clean slides and allowed to dry. They were then immunostained for CD31 (rat anti-mouse CD31; 1:200; BD PharMingen, San Jose, CA), an endothelial marker, and Mas according to the protocol described.

Mas receptor expression in cultured retinal cells

Four different cell lines were used in this study: a human Müller cell line-MIO-M1 (generously provided by Dr. G. Astrid Limb, University College of Medicine, Institute of Ophthalmology, London, UK) [33], mouse N9-microglial cell line (purchased from American Type Culture Collection, ATCC, Manassas, VA), 661W mouse cone photoreceptor cell line (generously provided by Dr. Muayyad R. Al-Ubaidi, University of Oklahoma Health Science Center, Oklahoma City, OK) [34], and ARPE-19, human RPE cell line (purchased from ATCC). For immunostaining of cultured cells, confluent cells grown in an eight-chambered glass slide were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min followed by incubation with blocking solution (3% BSA+ 0.3% Triton X-100) for 1 h at RT, followed by incubation with primary antibodies in blocking solution (overnight, 4 °C) and secondary antibodies (1 h, RT). Coverslips were washed in PBS containing the nuclear counterstain DAPI and mounted in Dako mounting media.

Real-time RT–PCR analysis of Mas and Ang II receptor expression

Total RNA was isolated from freshly dissected adult mouse retinal samples using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Reverse transcription was performed using the Enhanced Avian HS RT–PCR kit (Sigma) following the manufacturer’s instructions. Real-time PCR was performed on a real-time thermal cycler (iCycler, Bio-Rad Life Sciences) using iQ SYBR Green Supermix (Bio-Rad Life Sciences). The threshold cycle number (Ct) for real-time PCR was set by the cycler software. Optimal primer concentration for PCR (95°C, 3 min, 1 cycle; 95 °C, 10 s , 56 °C, 30 s, 50 cycles; 72 °C, 10 s, 1 cycle) was determined separately for each primer pair. Each reaction was run in duplicate or triplicate, and reaction tubes with target primers and those with actin primers were always included in the same PCR run. The expression levels of the different genes relative to AT₁Ra were established based on the Ct compared with the control housekeeping gene β-actin in each sample (amount of target=2^−∆∆Ct) and presented as fold change. The mouse-specific primers used in this experiment are listed in Table 1.

Developmental expression of the Mas receptor protein in the mouse retina

Immunofluorescence was used to study the pattern of Mas protein expression in developing and adult C57BL/6J mouse retinas as shown in Figure 1. The specificity of the antibody was confirmed by using the control peptide antigen supplied by the manufacturer with the antibody (Alomone Laboratories). At P1, the expression of the Mas protein was mainly confined to the RGC layer. No or low levels of the Mas receptor protein was observed in the neuroblast layer (Figure 1A). At P5, a stronger level of the Mas receptor protein was expressed by the RGCs. In addition, a low level of the Mas receptor protein was expressed in the inner portion of the neuroblast layer (Figure 1B). At P7, expression of the Mas receptor protein increased in the developing inner nuclear layer (INL). Significant levels of the Mas receptor protein continued to be expressed in the retinal ganglion cells; in addition, the Mas receptor protein also seemed to be expressed in the inner segments of the photoreceptors. No protein expression was observed in the outer nuclear layer (ONL; Figure 1C). At P15, strong expression of the Mas receptor protein was evident in the inner segment of the photoreceptor, in the inner nuclear layer where they were expressed by a subset of amacrine cells, bipolar cells, horizontal cells, and in the ganglion cells in the RGC layer (Figure 1D). At P21, the expression pattern remained similar to that seen at P15; however, the Mas receptor expression in the RGCs seemed higher compared to the P15 protein level expression (Figure 1E). At P60, the pattern of the Mas receptor expression remained similar to that observed at P15 and P21 (Figure 1D).

Specific retinal cell markers were used to investigate the cellular colocalization of the Mas receptor protein as shown in Figure 2 and Figure 3. Double immunostaining with PNA, a cone photoreceptor-specific marker, showed that Mas expression was localized to the cone inner segments and was absent from the cone outer segments (Figure 2A-C). Double immunostaining with rod arrestin (Figure 2D-F) and rhodopsin (image not shown), photoreceptor markers, also showed that the Mas receptor protein was localized to the inner segments of the photoreceptors. The level and pattern of expression in photoreceptors suggested that Mas receptor proteins are expressed at higher levels in cone photoreceptors compared to rod photoreceptors. Double immunostaining with Brn3a, a ganglion cell marker, showed that the Mas receptor protein was strongly expressed in the RGCs (Figure 2G-I). Costaining with GFAP, a marker for glial cells and astrocytes, showed that the Mas receptor protein was also expressed in glial cells in addition to neurons in the retina (Figure 3A-C). Coimmunostaining with p27^KIP1 (Figures 3D-F), which stains only the cell bodies of Müller cells, and CRALBP (Figures 3G-I), which labels the cell bodies and the processes of Müller cells, suggested that these cells express the Mas receptor protein at low levels in and around their cell bodies compared to the level of expression in retinal neurons. The observed punctate pattern of Mas receptor protein expression in the INL suggests that this protein may be strongly expressed by a subset of INL neurons or microglial cells (Figure 3D-I). Coimmunostaining with the RPE65 antibody in CD1 (non-pigmented) mice eyes allowed us to study Mas receptor protein expression in the RPE and ciliary body, which was difficult to detect in the C57BL/6J mice due to pigmentation and autofluorescence. Figure 4 shows that the Mas receptor protein is expressed in RPE cells, with the protein expressed uniformly in RPE cells, whereas RPE65 is more abundant on the apical side of the RPE cells. The Mas expression pattern also suggested that the protein is expressed in the choroidal capillaries (Figure 4A-D). Strong Mas receptor protein expression was also observed in the ciliary body of the CD1 mouse eye (Figure 4E-G). Western blot data showed that the level of Mas receptor protein expression in the mouse retina increased with age with the lowest expression seen at P1 and the adult steady level of expression reached by P15 (Figure 5A,B).

Expression of Mas receptor mRNA in developing and adult mouse retinas

Mas receptor mRNA was detected with in situ hybridization in paraffin-embedded sections using the RNAscope kit (Advanced Cell Diagnostics). Figure 6 shows the pattern and level of Mas receptor mRNA expression in the developing mouse retina. Expression of Mas receptor mRNA was observed in the mouse retina as early as P1 (Figure 6A) with a significant level of expression seen in the neuroblast layer and the cell bodies of the retinal ganglion cells. A similar level and pattern of expression were seen at P5 (Figure 6B). At P7 and P15 (Figure 6C,D), with the differentiation of the outer and inner nuclear layer in the developing mouse retina, the in situ hybridization expression pattern suggested that Mas receptor mRNA is expressed in photoreceptors, neurons, and/or glial cells in the inner nuclear layer as well as in a subset of RGCs. The adult pattern and level of Mas receptor mRNA expression seemed to be achieved by P15 (Figure 6D). PIPB (Figure 6G) and arrestin (Figure 6H) were used as two positive controls with PIPB expressed moderately throughout the retinal tissue and arrestin confined to the photoreceptor layer. The specificity of the probe binding and the efficacy of the RNAscope assay were also shown by the complete absence of DapB (used as a negative control) signal in the mouse retina (Figure 6I). This technique also provided quantitative information about the level of expression of a particular mRNA. Thus, the strong expression level of arrestin in the photoreceptor layer was evident as expected while PIPB was expressed at a much lower level. Comparatively, a moderate level of Mas mRNA seemed to be expressed in the developing and adult mouse retinas.

Differential levels of Mas expression in retinal blood vessels and cultured retinal cells

Ang (1-7) has a known vasodilatory function that is mediated by the binding to the specific receptor Mas. Previous Mas receptor expression studies in the brain and other organs have shown that Mas receptors are expressed in blood vessels. Here we show for the first time that the Mas receptor is also expressed strongly in retinal blood vessels. Trypsin digestion was used to isolate retinal blood vessels, and coimmunostaining with CD31, an endothelial cell marker, showed that the Mas receptor protein is strongly expressed in the endothelial cells and pericytes of the retinal vessels (Figure 7A). Four cell lines derived from specific retinal cell types were used to examine the presence or absence of Mas receptor expression in different cell types. The immunofluorescence study showed that the Mas receptor protein is strongly expressed in Müller cells and microglial cells, and a lower level of expression was observed in RPE cells and cone cells (Figure 7B–E).

Quantification of RAS receptor mRNA expression in the mouse retina

Total mRNA was extracted from the adult mouse retina, and a real-time RT–PCR assay was used to determine the relative expression levels of various angiotensin receptor mRNAs in the retina. Real-time RT–PCR data showed that AT₁Ra, AT₁Rb, AT₂R, and Mas receptor mRNA was expressed in the retina. Mas gene expression was about fivefold higher compared to other angiotensin receptor genes tested in the retina (Figure 8).

Conclusion

Immunocytochemistry, immunofluorescence, and in situ hybridization showed that Mas receptor protein and mRNA are expressed locally in the retina. The Mas receptor protein is specifically expressed in retinal ganglion cells, nerve fibers, Müller cells, and endothelial cells. The Mas receptor protein is expressed at a high level in the mouse inner retina from early development to adulthood suggesting that they may play an important role in neurovascular function, not only in adult retinas but also in developing retinas. Immunofluorescence in the cell lines showed that the Mas receptor and AT₁R are expressed in the same cells in the retina, consistent with the counteracting effects mediated by the two receptors. The Mas receptor expression level was about five times higher than AT₁Ra and AT₁Rb in the adult mouse retina, thus suggesting that it plays a central role in the pathophysiology of the retinal neurovascular system.