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 unbi...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. c RNAs were made from the isolated total RNAs and the labeled c RNA 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, IL15 RA, IL6, IL8 and CXCL3 that were up-regulated. The transcripts for C3, CCL2, IL11 RA 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.展开更多
基金Supported by NIH Heart,Lung,and Blood Institute(No.HLO74815)Institute of Neurological Disorders and Stroke(No.NS-084823)
文摘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. c RNAs were made from the isolated total RNAs and the labeled c RNA 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, IL15 RA, IL6, IL8 and CXCL3 that were up-regulated. The transcripts for C3, CCL2, IL11 RA 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.
