The Long Non-Coding RNA HOTAIR Is a Critical Epigenetic Mediator of Angiogenesis in Diabetic Retinopathy

Abstract

Purpose: Diabetic retinopathy (DR) remains a pressing issue worldwide. Abnormal angiogenesis is a distinct vascular lesion in DR, and research has established that vascular endothelial growth factor A (VEGF-A) is a primary mediator of such changes. However, limitations in current anti-VEGF therapies suggest that our understanding of molecular networks underlying ocular angiogenesis remains far from complete. Based on our long non-coding RNA (lncRNA) array analyses, HOX antisense intergenic RNA (HOTAIR) was identified as one of the top upregulated lncRNAs in high glucose-cultured human retinal endothelial cells (HRECs). Given the well-documented roles of HOTAIR in cancer, no studies have examined the epigenetic implications of HOTAIR in DR, and we investigated such relationships herein.

Methods: We used HRECs exposed to various glucose concentrations and epigenetic modulators to examine HOTAIR, angiogenic, and DR-related molecular markers. Oxidative stress, angiogenesis, and mitochondrial dysfunction were assessed. Retinal tissues of diabetic rodents and the vitreous humor and serum of patients with proliferative DR were also investigated.

Results: Hyperglycemia significantly augmented HOTAIR expression in HRECs and promoted angiogenesis, oxidative damage, and mitochondrial aberrations. Similarly, vitreous humor and serum from proliferative DR patients and retinas from diabetic animals demonstrated increased HOTAIR expression compared to non-diabetic controls. HOTAIR knockdown protected against glucose-induced increases of angiogenic and diabetes-associated molecules in the retina. Mechanistically, we showed that HOTAIR exerts its capabilities by preventing oxidative stress and modulating epigenetic pathways involving histone methylation, histone acetylation, DNA methylation, and transcription factors.

Conclusions: Our findings suggest that HOTAIR is a critical lncRNA in the pathogenesis of DR and may potentially be important for diagnostic and therapeutic targeting.

Figure 1.

HOTAIR is localized in the nucleus and cytoplasm of retinal endothelial cells. (A) HOTAIR localization in HRECs at 48 hours as indicated by RNA FISH using Stellaris FISH probes for human HOTAIR. Cells were also counterstained with DAPI to visualize the nuclei. The upper panel inset demonstrates nuclear localization of HOTAIR, and the lower panel inset demonstrates perinuclear localization. Original magnification, 20×. Scale bars: 100 µm. (B) Mean integrated densities of HOTAIR expression calculated using ImageJ (normalized to NG). Statistical significance was assessed using two-tailed Student's t-test (****P < 0.0001). Data represent the mean ± SEM of 50 cells captured per sample (n = 4 or 5 independent samples per group).

Figure 2.

HOTAIR knockdown prevents glucose-mediated induction of several angiogenic factors and diabetes-related molecules in vitro. RT-qPCR analyses of expression of (A) HOTAIR, (B) VEGF-A, (C) ET-1, (D) ANGPTL4, (E) PGF, (F) IL-1β, (G) HIF-1α, (H) PARP1, and (I) cytochrome b following the administration of SCR siRNA or siHOTAIR in HRECs subjected to 48 hours of NG or HG culture. Data are expressed as a ratio to β-actin and normalized to SCR NG. Statistical significance was assessed using one-way ANOVA for multiple comparisons, followed by Tukey's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant). Data represent the mean ± SEM of three independent experiments (n = 6/group).

Figure 3.

HOTAIR mediates angiogenesis in vitro. (A) Endothelial tube formation assay at the 6-hour mark for HRECs treated with scrambled (SCR) siRNA, siHOTAIR, or exogenous VEGF and cultured in NG or HG conditions. WimTube Image analyzer software was used to calculate (B) the number of tubules and (C) the total branching points in each group. Glucose-induced angiogenesis was prevented by siHOTAIR, but only partially reversed by VEGF supplementation. Statistical significance was assessed using one-way ANOVA for multiple comparisons, followed by Tukey's post hoc test (****P < 0.0001). Data represent the mean ± SEM of three independent experiments (n = 8/group), and images were captured from at least two field views per well (original magnification, 40×).

Figure 4.

Knockdown of HOTAIR through intravitreal injections significantly prevents diabetes-induced retinal vascular permeability and early elevations of angiogenic and other molecules in the retina. RT-qPCR analyses of expression of (A) HOTAIR, (B) Vegf-a, (C) Et-1, (D) Angptl4, (E) Parp1, (F) Mcp-1, (G) Il-1β, and (H) Hif-1α in retinal tissues. Data are expressed as a ratio to β-actin and normalized to SCR control. Statistical significance was assessed using one-way ANOVA for multiple comparisons, followed by Tukey's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant). Data represent the mean ± SD (n = 6/group). Immunohistochemical staining of the mouse retina for IgG showed enhanced extravascular diffuse retinal stain, signifying increased extravasation in the diabetic mice with SCR siRNA (score = 3) (J) compared to non-diabetic controls with SCR siRNA (score = 1) (I). Photomicrographs for non-diabetic control mice (K) and diabetic mice administered siHOTAIR injections (L) showed positive IgG stains (arrows; score = 1) limited to the intravascular space. No changes in IgG leakage were demonstrated between control mice treated with SCR siRNA (I) and control mice treated with siHOTAIR (K). WT-C, wild-type non-diabetic control; WT-D, wild-type diabetic control; SCR, scrambled siRNA; siHOTAIR, siRNA targeting mouse HOTAIR. Original magnification, 200× (I to L).

Figure 5.

HOTAIR knockdown partially prevents glucose-induced mitochondrial depolarization/dysfunction. (A) Images captured from the JC-1 assay, where HRECs were pretreated with SCR siRNAs or siHOTAIR and subsequently cultured in NG or HG for 48 hours. JC-1 stains demonstrated HG-induced mitochondrial dysfunction (evidenced by low ΔΨM and more green and less red fluorescence when compared to SCR NG) and were partially corrected by HOTAIR knockdown (siHOTAIR). Cells were also counterstained with DAPI. Original magnification, 20×. Scale bars: 100 µm. (B) The JC-1 red/green fluorescence ratio was quantified using ImageJ. Statistical significance was assessed using one-way ANOVA for multiple comparisons, followed by Tukey's post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant). Data represent the mean ± SEM of 20 cells captured per sample (n = 8 independent samples per group).

Figure 6.

Knockdown of HOTAIR significantly prevents glucose-induced oxidative damage. (A) Images captured from the 8-OHdG assay, where HRECs were pretreated with SCR siRNAs or siHOTAIR and subsequently cultured in NG or HG for 48 hours. 8-OHdG stains showed that HG-induced nuclear and mitochondrial oxidative DNA damage (indicated by stronger green fluorescence) was prevented by siHOTAIR. Cells were also counterstained with DAPI. Original magnification, 20×. Scale bars: 100 µm. (B) Mean integrated densities of 8-OHdG expression were quantified using ImageJ. Statistical significance was assessed using one-way ANOVA for multiple comparisons, followed by Tukey's post hoc test (*P < 0.05, ****P < 0.0001). Data represent the mean ± SEM of 20 cells captured per sample (n = 8 independent samples per group).

Figure 7.

DNA methylation profiling of HRECs. (A) Human HOTAIR is located on chromosome 12:54,356,092–54,368,740 (hg19), and its approximate size (for transcript variant 1) is 12,649 nucleotides containing a total of six exons (UCSC database⁵⁴). (B) Unsupervised hierarchical clustering with heatmap using all the CpGs in HRECs that span across HOTAIR, which amounted to 59 probes. No distinctions were noted between cells treated with different concentrations of glucose (5 mM vs. 25 mM) or the duration of culture (2 vs. 7 days). Rows indicate CpGs and columns show the samples; the color scale from blue to red indicates the level of methylation from 0 to 1 (with 0 indicating no methylation and 1 indicating maximum methylation) (n = 3 independent samples per group, indicated by the top panel colors). HR5_2, HRECs cultured in 5-mM glucose for 2 days; HR25_2, HRECs cultured in 25-mM glucose for 2 days; HR5_7, HRECs cultured in 5-mM glucose for 7 days; HR25_7, HRECs cultured in 25-mM glucose for 7 days.

Figure 8.

Schematic of hyperglycemia-induced regulation of lncRNA HOTAIR in retinal endothelial cells. HOTAIR can regulate the transcription of several genes implicated in the pathogenesis of diabetic retinopathy through direct and indirect mechanisms involving alterations in the PRC2–P300–DNMT–CTCF epigenetic axis. HOTAIR can also contribute to both mitochondrial and DNA oxidative damage. ROS, reactive oxygen species.