Homocysteine mediates transcriptional changes of the inflammatory pathway signature genes in human retinal pigment epithelial cells

Abstract

Aim: To test whether homocysteine (Hcy) can influence the transcriptional profile, we hypothesized that Hcy can lead to the induction of proinflammatory molecules in the retinal cells of aging people.

Methods: An unbiased in vitro inflammatory pathway focused study was designed employing retinal pigment epithelial (RPE) cell line, ARPE-19. Cells were cultured in the presence or absence of Hcy to capture target genes' expression profile. Three different concentrations of Hcy were added in the culture medium of confluent monolayers. cRNAs were made from the isolated total RNAs and the labeled cRNA probes were hybridized to microarrays specific for human disease pathway inflammatory cytokines, chemokines and their receptor gene micro-array panels as per manufacture's recommendations. Two Hcy up-regulated molecules: IL6 and CEBPB were further validated via Western blot analysis. Hcy's effect on ARPE-19 cellular morphology and genomic DNA integrity were also evaluated.

Results: Gene microarray analyses of RPE cells in response to Hcy treatment revealed alterations in the expressions of several inflammatory gene transcripts such as CCL5, CEBPB, IL13RA2, IL15RA, IL6, IL8 and CXCL3 that were up-regulated. The transcripts for C3, CCL2, IL11RA and IL18 genes exhibited down-regulation. The IL6 and CEBPB expressions were subsequently validated at the protein levels. Treatment of the retinal cells with increasing Hcy concentration influenced their density in culture however their morphology and DNA integrity remained unaffected.

Conclusion: These findings suggest that Hcy can potentially mediate the expression of chemokines, cytokines and interleukins receptors in the retinal cells without having any debilitating effects on their morphology and the genomic DNA integrity.

Keywords: chemokine; choroidal neovascularization; cytokine; gene expression; hyperhomocysteinemia; inflammation; macular degeneration; retinal remodeling.

Figure 1. Inflammatory cytokines and chemokines microarray analyses of ARPE-19 cells after Hcy treatment

In comparison to control (untreated), all Hcy (6, 30 and 150 µmol/L) treated cultures exhibited both up-regulation and down-regulation of inflammatory genes are shown by arrows.

Figure 2. Western blot assay for validation of microarray data at protein levels

CEBPB and IL-6 molecules were tested for further validation at protein levels where ARPE-19 cultures were treated with 30 or 150 µmol/L Hcy conc. The results showed a dose dependent Hcy effect on protein expression.

Figure 3. Detection of apoptosis via DNA laddering pattern of ARPE-19 cells after Hcy treatment

Different concentrations of Hcy: 0.10, 0.20, 0.50, 1.0 and 5.0 mmol/L were studied to gauge the effect on DNA degradation. None of the concentration induced apoptosis in this assay. Actinomycin-D served as a positive control for the induction of apoptosis. A: Kb plus marker; B: Actinomycin-D positive ctrl; C: 0.10 mmol/L Hcy; D: 0.20 mmol/L Hcy; E: 0.50 mmol/L Hcy; F: 1.00 mmol/L Hcy; G: 5.00 mmol/L Hcy.

Figure 4. Effect of Hcy on ARPE-19 cell morphology and density

A: Various concentrations of Hcy: 1.5, 5.0 and 15.0 mmol/L were used for studying the effects on ARPE-19 cells; B: Of 1.5, 5.0 and 15.0 mmol/L doses appear to be toxic for the ARPE-19 cells in culture. ^aP<0.05.

Figure 5. Schematic highlighting effects of Hcy on retinal pigment epithelium

A simplified scheme depicting the potentially damaging effects in retinal cells that can initiate degenerative changes in the retina during AMD disease process.