Hypoxia inducible factor-1α (HIF-1α) and some HIF-1 target genes are elevated in experimental glaucoma

Abstract

Low levels of hypoxia have been suggested to be a mechanism of retinal damage in glaucoma. To test the hypothesis that the activation of the hypoxia-responsive transcription factor hypoxia inducible factor-1alpha (HIF-1alpha) is involved in the pathophysiology of glaucoma, we used a rat model of glaucoma to study (1) HIF-1alpha retinal protein levels by immunoblot analysis, (2) cellular localization of HIF-1alpha in the retina by immunohistochemistry, and (3) expression of retinal HIF-1 gene targets by quantitative real-time polymerase chain reaction. Glaucoma was unilaterally induced in rats by injecting hypertonic saline in episcleral veins. We find that HIF-1alpha protein was increased in the retina following elevation of intraocular pressure, specifically in Müller glia and astrocytes but not in activated microglia. Eight established HIF-1 target genes were measured in experimental glaucoma. Retinal Epo, Flt-1, Hsp-27, Pai-1, and Vegfa mRNA levels were increased and Et-1, Igf2, and Tgfbeta3 levels were decreased in the glaucomatous retinas. Thus, the increase in HIF-1alpha levels in Müller glia and astrocytes is accompanied by a marked up regulation of some, but not all, HIF-1 transcriptional targets. These data support the hypothesis that HIF-1alpha becomes transcriptionally active in astrocytes and Müller cells but not microglia or neurons in glaucoma, arguing against a global hypoxia stimulus to the retina.

Figure 1

Western blotting: HIF-1α levels are increased in the glaucomatous retina compared to controls. a HIF-1α expression after 5 days of elevated IOP (n=6). A band for HIF-1α was detected between 100 and 150 kDa in the control and glaucomatous retinas as well as in the positive control brain. HIF-1α levels were elevated in the glaucomatous retinas compared to controls. b HIF-1α expression after 10 days of elevated IOP (n=6). Similarly, HIF-1α was detected between 100 and 150 kDa in the control and glaucomatous retinas. HIF-1α expression remained elevated in the glaucomatous retinas compared to controls. c Glaucomatous/control ratios of HIF-1α levels in the retina. HIF-1α levels were significantly increased in the glaucomatous retinas compared to controls (P=0.009 for the 5-day group and P=0.021 for the 10-day group). Fold increase was (mean ± SEM) 1.37±0.11 and 1.34±0.13 after 5 and 10 days of elevated IOP, respectively. Positive control: brain, loading control: α-tubulin

Figure 2

HIF-1α staining is not present in RGC. RGC were previously backlabeled with Fluoro-Gold (a, d, and g). Fluoro-Gold images were converted to gray scale to optimize visualization to determine colocalization with HIF-1α. b No nonspecific staining was observed when sections were incubated with secondary antibody alone. Subtle HIF-1α staining was observed in the inner layers of the normal retina (e) and was increased under conditions of high IOP (h). HIF-1α staining (red) did not colocalize with the RGC marker Fluoro-Gold (white) in the normal retina (f) or in the retina under conditions of high IOP (i). Insets are higher magnification views of the RGC indicated by arrows

Figure 3

HIF-1α expression is increased in Müller glia in the glaucomatous retina. Sections were double stained with an anti-HIF-1α antibody (red) followed by staining with an antivimentin antibody (green). a, b No nonspecific staining was observed when sections were incubated with their respective secondary antibodies alone. As shown in the same sections in Fig. 2, there is subtle HIF-1α staining in the inner layers of the normal retina (d) that increase under conditions of high IOP (g). d Low levels of HIF-1α staining colocalized (yellow) with the Müller glia marker vimentin under conditions of normal IOP. i Elevated HIF-1α expression was observed primarily in Müller glia as the staining colocalized with vimentin (yellow)

Figure 4

HIF-1α is also expressed by astrocytes but not by activated microglia in the glaucomatous retina. a–c and j–l There was no nonspecific staining in the negative control sections which were incubated only with the secondary antibodies. d–i To examine whether astrocytes express HIF-1α, retinal sections were double stained with anti-HIF-1α and anti-GFAP primary antibodies. e In the normal retina, expression of the astrocyte marker GFAP was observed in the RGC layer and INL. d–f GFAP expression (green) did not colocalize with HIF-1α (red) staining in the normal retina. h In the glaucomatous retina, increased GFAP expression was present in the RGC layer and INL. g–i GFAP staining colocalized with HIF-1α expression in the glaucomatous retina. m–r To test whether HIF-1α was expressed by activated microglia, retinal sections were double labeled with anti-HIF-1α and anti-IBA1 primary antibodies. n Expression of the activated microglia marker IBA1 (green) was minimal in the normal retina. m–o IBA1 expression did not colocalize with HIF-1α (red) staining. q versus n In the glaucomatous retina, stronger IBA1 staining was observed in the RGC layer and INL compared with the normal retina. p–r IBA1 staining did not colocalize with HIF-1α staining in the glaucomatous retina (see arrowheads and insets)