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Gene expression profiling of vasoregression in the retina--involvement of microglial cells.

Feng Y, Wang Y, Li L, Wu L, Hoffmann S, Gretz N, Hammes HP - PLoS ONE (2011)

Bottom Line: Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression.Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression.Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.

View Article: PubMed Central - PubMed

Affiliation: 5 Medical Department, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.

ABSTRACT
Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.

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Confirmation of gene expression profiles in SD and TGR retinas at 1- and 3-month.Transcriptional analysis of genes was performed by quantitative real-time PCR in rats of 1-month SD (white bars), 1-month TGR (gray bars), 3-month SD (striped bars) and 3-month TGR (black bars). The values at 1-month are standardized to 1. *p<0.05, **p<0.01, ***p<0.001: 3-month TGR compared with 1-month TGR. #p<0.05, ###p<0.001: 3-month TGR compared with 3-month SD.
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pone-0016865-g004: Confirmation of gene expression profiles in SD and TGR retinas at 1- and 3-month.Transcriptional analysis of genes was performed by quantitative real-time PCR in rats of 1-month SD (white bars), 1-month TGR (gray bars), 3-month SD (striped bars) and 3-month TGR (black bars). The values at 1-month are standardized to 1. *p<0.05, **p<0.01, ***p<0.001: 3-month TGR compared with 1-month TGR. #p<0.05, ###p<0.001: 3-month TGR compared with 3-month SD.

Mentions: To validate the differential transcriptional gene expression observed on microarray analysis, we carried out TaqMan probe based on quantitative real-time PCR with independently retrieved TGR and SD retinas at 1 and 3 months of age. As shown in Figure 4, mRNA levels of CD74 were high in TGR retinas at 3 months, whereas expression of CD74 was low in TGR retinas at 1 month and SD retinas at 1 and 3 months. Similarly, A2M (alpha-2-macroglobulin), B2M (beta-2-macroglobulin), CEBPB, CFB (complement factor B), CTSS (cathapsin S), RT1-DA (RT1 class II, locus Da), SERPING1 (serine/cysteine peptidase inhibitor, clade G, member 1), C1qa (complement component 1, q subcomponent, alpha polypeptide) and TNFRSF1A (Tumor necrosis factor receptor superfamily member 1A) were expressed significantly higher in 3-month TGR retinas than in other three groups. These ten genes retrieved from microarray analysis were fully confirmed in quantitative real time PCR. Expression of CD74 detected was 19-fold higher in 3-month TGR retinas compared with age-matched SD rats in real time PCR analysis. Upregulated transcripts of B2M (3-fold), RT1-DA (23-fold) and CTSS (3-fold) which participate in antigen processing and presentation were confirmed by quantitative real-time PCR. Furthermore, complement regulation mediated by CFB (16-fold), SERPING1 (7-fold), and C1qa (3-fold) was also confirmed by real time PCR. Upregulation in inflammatory responses via A2M (4-fold), CEBPB (3-fold), TNFRSF1A (3-fold) and IL-1b (2-fold) were also confirmed. Thus, the expression levels of regulated genes from microarray analysis were fully confirmed by quantitative real time PCR.


Gene expression profiling of vasoregression in the retina--involvement of microglial cells.

Feng Y, Wang Y, Li L, Wu L, Hoffmann S, Gretz N, Hammes HP - PLoS ONE (2011)

Confirmation of gene expression profiles in SD and TGR retinas at 1- and 3-month.Transcriptional analysis of genes was performed by quantitative real-time PCR in rats of 1-month SD (white bars), 1-month TGR (gray bars), 3-month SD (striped bars) and 3-month TGR (black bars). The values at 1-month are standardized to 1. *p<0.05, **p<0.01, ***p<0.001: 3-month TGR compared with 1-month TGR. #p<0.05, ###p<0.001: 3-month TGR compared with 3-month SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040753&req=5

pone-0016865-g004: Confirmation of gene expression profiles in SD and TGR retinas at 1- and 3-month.Transcriptional analysis of genes was performed by quantitative real-time PCR in rats of 1-month SD (white bars), 1-month TGR (gray bars), 3-month SD (striped bars) and 3-month TGR (black bars). The values at 1-month are standardized to 1. *p<0.05, **p<0.01, ***p<0.001: 3-month TGR compared with 1-month TGR. #p<0.05, ###p<0.001: 3-month TGR compared with 3-month SD.
Mentions: To validate the differential transcriptional gene expression observed on microarray analysis, we carried out TaqMan probe based on quantitative real-time PCR with independently retrieved TGR and SD retinas at 1 and 3 months of age. As shown in Figure 4, mRNA levels of CD74 were high in TGR retinas at 3 months, whereas expression of CD74 was low in TGR retinas at 1 month and SD retinas at 1 and 3 months. Similarly, A2M (alpha-2-macroglobulin), B2M (beta-2-macroglobulin), CEBPB, CFB (complement factor B), CTSS (cathapsin S), RT1-DA (RT1 class II, locus Da), SERPING1 (serine/cysteine peptidase inhibitor, clade G, member 1), C1qa (complement component 1, q subcomponent, alpha polypeptide) and TNFRSF1A (Tumor necrosis factor receptor superfamily member 1A) were expressed significantly higher in 3-month TGR retinas than in other three groups. These ten genes retrieved from microarray analysis were fully confirmed in quantitative real time PCR. Expression of CD74 detected was 19-fold higher in 3-month TGR retinas compared with age-matched SD rats in real time PCR analysis. Upregulated transcripts of B2M (3-fold), RT1-DA (23-fold) and CTSS (3-fold) which participate in antigen processing and presentation were confirmed by quantitative real-time PCR. Furthermore, complement regulation mediated by CFB (16-fold), SERPING1 (7-fold), and C1qa (3-fold) was also confirmed by real time PCR. Upregulation in inflammatory responses via A2M (4-fold), CEBPB (3-fold), TNFRSF1A (3-fold) and IL-1b (2-fold) were also confirmed. Thus, the expression levels of regulated genes from microarray analysis were fully confirmed by quantitative real time PCR.

Bottom Line: Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression.Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression.Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.

View Article: PubMed Central - PubMed

Affiliation: 5 Medical Department, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.

ABSTRACT
Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.

Show MeSH
Related in: MedlinePlus