Proteomic analysis of rat retinal Hb expression. (A) A 2D-gel obtained from a control rat retinal protein sample. Mass spectrometric analysis of the circled gel spot identified the Hb side chains, α and β. Consistent with the results of the 2D-PAGE-based proteomic analysis, a gel-free proteomic technique involving LC-MS/MS analysis of protein mixtures also identified Hb in the rat retinal proteome. (B) Summary of the mass spectrometric data. To further validate the proteomic data, Western blot analysis was performed with a specific Hb antibody. The 2D-Western blot shown in (A) demonstrates specific immunolabeling in a similar location as the indicated cluster of Hb side chains on the 2D-gel. Rat retinal protein samples were obtained after transcardiac perfusion to wash the blood out of the blood circulation. Data represent four independent experiments using different samples.

Hb expression in the human retina. (A) Summary of the findings of quantitative mass spectrometric analysis, which identified Hb α and β side chains in the human retinal proteome. Based on PAF values representing the protein abundance, retinal Hb expression exhibited a less than two-fold (1.72 ± 0.9) increase in the glaucomatous human donor eyes relative to nonglaucomatous control eyes (Mann-Whitney rank sum test, P < 0.001). In addition to Hb side chains, PAF values for additional proteins are presented as the control. There was prominent increase in the expression of GFAP (a glial activity marker) and αB-crystallin (a stress response protein), although the expression of β-tubulin (a microtubule protein) and rhodopsin (an outer neuronal marker) was constant in glaucomatous samples. (B) Western blot analysis of human blood or retinal protein samples using antibodies to different erythrocyte-specific proteins. Other than Hb, no retinal immunoreactivity was detectable for erythrocyte markers, including glycophorin A and B, and blood group AB and H antigens. Immunoblots were repeated four times with similar results.

In vitro experiments determining Hb expression and hypoxic regulation in primary cultures of rat retinal cells. (A) Hb antibody immunoprecipitates obtained after methionine incorporation into cellular proteins using the click reaction. Eluted proteins were separated by SDS/PAGE. Lanes 2 and 4: 15-kDa bands corresponding to newly synthesized Hb by retinal macroglial cells and ONH astrocytes, respectively, and thereby supporting local synthesis of Hb by these cells. Lanes 1 and 3: control immunoprecipitates of same samples obtained after replacing the Hb antibody with IgG. Experiments were repeated four times with similar results. The role of EPO signaling in Hb induction and cellular survival was determined by treating the cultured cells with rEPO (10 ng/mL) or EPO-R (60 ng/mL) based on the dose-effect curves shown in (B). (C) Western blot analysis of Hb expression in retinal cell cultures. Retinal macroglial cells exposed to hypoxia exhibited a prominent increase in Hb expression (3.64 ± 0.7-fold; Mann-Whitney rank sum test, P < 0.001). This hypoxia-induced upregulation of Hb in glial cells was found parallel to increased expression of HIF-1α and EPO in these cells. RGCs cultured alone did not express a significant amount of HIF-1α or EPO under normoxic condition or hypoxia. However, Hb expression of RGCs exhibited an upregulation after exposure to hypoxia in the presence of hypoxia-conditioned glial medium as well as after rEPO treatment (2.42 ± 0.6-fold; Mann-Whitney rank sum test, P < 0.029). Treatment with EPO-R resulted in a neutralization of these effects. Data are representative of four experiments with new protein samples. (D) Phase-contrast images of RGCs incubated under normal conditions or incubated under hypoxia in the absence or presence of rEPO treatment. Hypoxic RGCs exhibited characteristics of cell death, shrinkage of the cell body and dendrites, and cell debris. (E) Used level of hypoxia or any of the treatments did not prominently affect the glial cell survival. However, incubation of RGCs under hypoxia resulted in a prominent decrease (over 30%) in the number of surviving cells. Addition of the conditioned medium obtained from hypoxic glial cultures or treatment with rEPO provided an approximate 25% protection to these hypoxic RGCs (Mann-Whitney rank sum test: *P = 0.04; **P = 0.002; ***P < 0.001). Data represent results in four independent experiments and are presented as the mean ± SD.

The regulation and predicted functions of Hb in the retina and optic nerve head. (A) Hypoxic stress leads to upregulation of HIF-1α and increased production and secretion of EPO by macroglial cells. EPO induces Hb synthesis through EPO-R signaling in autocrine and paracrine fashions. (B) Regulated expression of Hb in macroglial cells and RGCs likely provides an intrinsic protective mechanism against hypoxic/oxidative injury. Hb may facilitate tissue oxygen transport in the inner retina and optic nerve head to improve the oxygenation of RGCs and their axons. This oxygen-binding protein may also function in free radical (ROS) scavenging and nitric oxide (NO) detoxification.

Additional validation of rat retinal Hb expression by qRT-PCR and Western blot analyses. Whole retina or enriched cellular fractions were used for sampling. Enriched RGC mRNA and protein samples were obtained using RGCs selected from dissociated retinal cells through the two-step immunomagnetic selection process. Unselected fraction of retinal cells through this process, which contains macroglial cells, was also used for mRNA and protein sampling. (A) Summary of the results of qRT-PCR analysis using 40 amplification cycles, which support mRNA expression of Hb α and β side chains in the rat retina. All reactions were performed in triplicate and repeated three times using different samples. Presented are relative expression values (mean ± SD) calculated by normalizing the Ct values to β-actin using the 2^ΔΔCt method. In addition to control reactions using primers for β-actin, the specific cell markers GFAP and Thy-1 were also tested to validate enriched cellular fractions in samples. The actin-normalized Ct values for GFAP in the selected RGCs fraction (0.51 ± 0.4) and Thy-1 in the unselected glial fraction (0.64 ± 0.3) were too small to be seen in the graph, indicating little or no gene expression. (B) Western blot analysis further supporting Hb expression in rat retinal protein samples obtained from ocular hypertensive (OHT) or control eyes. Immunoblots were obtained using four different samples, and after normalization to β-actin, average band intensities were compared between control and ocular hypertensive samples. This comparison detected a less than two-fold (1.96 ± 0.2) increased expression of Hb in ocular hypertensive retinas relative to control retinas (Mann-Whitney rank sum test, P = 0.054). Quantitative mass spectrometric analysis similarly detected an approximate two-fold increase in expression of Hb in ocular hypertensive samples (2.02 ± 0.3-fold; Mann-Whitney rank sum test, P = 0.006).

Immunohistochemical analysis of the cellular localization of Hb expression in the human retina and optic nerve head. Consistent with proteomic findings, histologic sections of the retina and optic nerve head from human donor eyes exhibited immunolabeling with a specific Hb antibody. (A, B) Immunoperoxidase labeling of retina sections for Hb in glaucomatous donor eyes with moderate (A) or more advanced (B) damage and age-matched controls. (C) Immunoperoxidase labeling of optic nerve head sections in a glaucomatous donor with moderate damage and the age-matched control. The level of glaucomatous damage in these donor eyes was assessed based on the available clinical data, including optic disc assessments and visual field tests.¹⁹ An increase was detectable in the extent of Hb immunolabeling and the number of immunolabeled cells in most (31/38 eyes with glaucoma) but not all glaucomatous eyes, which also exhibited regional differences with an indistinct pattern. In addition to erythrocytes within blood vessels (v), intracellular Hb immunolabeling was most prominent in macroglial cells. Based on known morphologic features and retinal distributions of macroglial cell types, retinal Hb immunolabeling mainly corresponded to astrocytes (black arrow) characterized by smaller and oval shape nuclei and close localization to retinal vasculature in the RGC (white arrow) layer. In addition, some cells in the inner nuclear layer, likely corresponding to Müller cells, exhibited Hb immunolabeling. (C) Optic nerve head astrocytes also exhibited immunolabeling for Hb. (D) Double immunofluorescence labeling of the glaucomatous human retina using specific antibodies to Hb (green) and GFAP or Brn-3 (red) as marker for retinal macroglia or RGCs. In merged images, most of the Hb immunolabeling (yellow) was localized to GFAP-positive macroglial cells. However, some Brn-3-positive RGCs also exhibited Hb immunolabeling. rgc, retinal ganglion cell layer; in, inner nuclear layer; on, outer nuclear layer. Scale bar: (A, B) 100 μm; (C) 250 μm; (D) 150 μm.

Association of EPO and EPO-R with glaucoma. (A) Western blot analysis obtained with human retinal protein samples supports an over two-fold increased expression of EPO in glaucoma (Mann-Whitney rank sum test, P < 0.01). Data represent repeated immunoblots with protein samples from five human donor eyes with glaucoma and five control eyes without glaucoma. β-Actin serves as a loading and transfer control. Immunohistochemical analysis also determined EPO and EPO-R immunolabeling in the glaucomatous human retina. (B) Immunoperoxidase labeling of the retina sections obtained from human donor eyes with or without glaucoma. Based on morphologic assessment of cell types, retinal EPO immunolabeling predominantly localized to macroglial cells (black arrow), whereas EPO-R immunolabeling was detectable on both macroglia (black arrow) and RGCs (white arrow). (C, D) Double-immunofluorescence labeling of the glaucomatous human retina using specific antibodies to EPO or EPO-R (green) and GFAP or Hb (red). Merged images (yellow) in (C) and (D) support co-localization of EPO with GFAP and Hb in glaucomatous eyes. rgc, retinal ganglion cell layer; in, inner nuclear layer; on, outer nuclear layer. Scale bar, (B) 250 μm; (C, D) 150 μm.