Limits...
Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro.

Seher A, Nickel J, Mueller TD, Kneitz S, Gebhardt S, ter Vehn TM, Schlunck G, Sebald W - Mol. Vis. (2011)

Bottom Line: This study specifies the important role of hCTGF for primary tenon fibroblast function.The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes.The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.

View Article: PubMed Central - PubMed

Affiliation: Universitätsklinikum Würzburg, Abteilung für Molekulare Innere Medizin, Würzburg, Germany. axel.seher@biozentrum.uni-wuerzburg.de

ABSTRACT

Purpose: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix(TM) oligonucleotide array technology to identify genes that are regulated by hCTGF.

Methods: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix(TM) oligonucleotide array technology. Results were validated by real time RT-PCR.

Results: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology.

Conclusions: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.

Show MeSH

Related in: MedlinePlus

Expression level of inflammatory genes in human tenon fibroblasts after CTGF stimulation. HTFs were stimulated with 6 μg/ml CTGF for 48 h. Total RNA was isolated and gene expression was analyzed via affymetrix array. Selected genes out of the catagory “inflammation” were validated using real time RT–PCR. The figure shows the fold induction measured by affymetrix arrays compared to the results of real time RT–PCR (RT) for every single donor using the RNA used in array analysis. The results were additionally validated for every donor in an independent experiment (donors were stimulated again with CTGF). Isolated RNA was analyzed via RT–PCR (Repeat). RT–PCR was performed twice for every single experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3021567&req=5

f5: Expression level of inflammatory genes in human tenon fibroblasts after CTGF stimulation. HTFs were stimulated with 6 μg/ml CTGF for 48 h. Total RNA was isolated and gene expression was analyzed via affymetrix array. Selected genes out of the catagory “inflammation” were validated using real time RT–PCR. The figure shows the fold induction measured by affymetrix arrays compared to the results of real time RT–PCR (RT) for every single donor using the RNA used in array analysis. The results were additionally validated for every donor in an independent experiment (donors were stimulated again with CTGF). Isolated RNA was analyzed via RT–PCR (Repeat). RT–PCR was performed twice for every single experiment.

Mentions: Array results were validated by analyzing six selected genes of the category “inflammatory and wounding response” (TNF-alpha, IL-1, IL-6, IL-8, CXCL-1, and CXCL-6). On the one hand we tested the RNA probes measured by affymetrixTM oligonucleotide arrays and on the other hand we analyzed the RNA from one additional experiment in each case for all three donors. The array results could be confirmed for all six genes (Figure 5).


Gene expression profiling of connective tissue growth factor (CTGF) stimulated primary human tenon fibroblasts reveals an inflammatory and wound healing response in vitro.

Seher A, Nickel J, Mueller TD, Kneitz S, Gebhardt S, ter Vehn TM, Schlunck G, Sebald W - Mol. Vis. (2011)

Expression level of inflammatory genes in human tenon fibroblasts after CTGF stimulation. HTFs were stimulated with 6 μg/ml CTGF for 48 h. Total RNA was isolated and gene expression was analyzed via affymetrix array. Selected genes out of the catagory “inflammation” were validated using real time RT–PCR. The figure shows the fold induction measured by affymetrix arrays compared to the results of real time RT–PCR (RT) for every single donor using the RNA used in array analysis. The results were additionally validated for every donor in an independent experiment (donors were stimulated again with CTGF). Isolated RNA was analyzed via RT–PCR (Repeat). RT–PCR was performed twice for every single experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3021567&req=5

f5: Expression level of inflammatory genes in human tenon fibroblasts after CTGF stimulation. HTFs were stimulated with 6 μg/ml CTGF for 48 h. Total RNA was isolated and gene expression was analyzed via affymetrix array. Selected genes out of the catagory “inflammation” were validated using real time RT–PCR. The figure shows the fold induction measured by affymetrix arrays compared to the results of real time RT–PCR (RT) for every single donor using the RNA used in array analysis. The results were additionally validated for every donor in an independent experiment (donors were stimulated again with CTGF). Isolated RNA was analyzed via RT–PCR (Repeat). RT–PCR was performed twice for every single experiment.
Mentions: Array results were validated by analyzing six selected genes of the category “inflammatory and wounding response” (TNF-alpha, IL-1, IL-6, IL-8, CXCL-1, and CXCL-6). On the one hand we tested the RNA probes measured by affymetrixTM oligonucleotide arrays and on the other hand we analyzed the RNA from one additional experiment in each case for all three donors. The array results could be confirmed for all six genes (Figure 5).

Bottom Line: This study specifies the important role of hCTGF for primary tenon fibroblast function.The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes.The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.

View Article: PubMed Central - PubMed

Affiliation: Universitätsklinikum Würzburg, Abteilung für Molekulare Innere Medizin, Würzburg, Germany. axel.seher@biozentrum.uni-wuerzburg.de

ABSTRACT

Purpose: The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix(TM) oligonucleotide array technology to identify genes that are regulated by hCTGF.

Methods: Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix(TM) oligonucleotide array technology. Results were validated by real time RT-PCR.

Results: hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology.

Conclusions: This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.

Show MeSH
Related in: MedlinePlus