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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.

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Sensorgrams of BIAcore measurements. Binding of CTGF at the indicated concentrations to immobilized BMP-2 was recorded at a flow-rate of 10 μl/min at 25 °C. The association phase was set to five and the dissociation phase to three min. Specific binding of hCTGF to immobilized BMP-2 reveals an apparent KD value of ~400 nM.
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f2: Sensorgrams of BIAcore measurements. Binding of CTGF at the indicated concentrations to immobilized BMP-2 was recorded at a flow-rate of 10 μl/min at 25 °C. The association phase was set to five and the dissociation phase to three min. Specific binding of hCTGF to immobilized BMP-2 reveals an apparent KD value of ~400 nM.

Mentions: To study the biologic effects on human tenon fibroblasts (HTFs) recombinant hCTGF was prepared as described above. Purity and homogeneity of the protein was analyzed by SDS–PAGE and western blot (Figure 1A,B). Recombinant CTGF appears as two bands with an electrophoretic mobility corresponding to 36 kDa (unglycosylated) and 38 kDa (glycosylated isoform) in gel electrophoresis analysis as reported [4]. Biologic activity of recombinant hCTGF was tested by binding to immobilized BMP-2 using BiacoreTM technology. Specific binding of hCTGF to immobilized BMP-2 revealed an apparent KD value of 400 nM consistent with previous results obtained for binding of CTGF [5] to the BMP-2 homolog BMP-4 (Figure 2).


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)

Sensorgrams of BIAcore measurements. Binding of CTGF at the indicated concentrations to immobilized BMP-2 was recorded at a flow-rate of 10 μl/min at 25 °C. The association phase was set to five and the dissociation phase to three min. Specific binding of hCTGF to immobilized BMP-2 reveals an apparent KD value of ~400 nM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Sensorgrams of BIAcore measurements. Binding of CTGF at the indicated concentrations to immobilized BMP-2 was recorded at a flow-rate of 10 μl/min at 25 °C. The association phase was set to five and the dissociation phase to three min. Specific binding of hCTGF to immobilized BMP-2 reveals an apparent KD value of ~400 nM.
Mentions: To study the biologic effects on human tenon fibroblasts (HTFs) recombinant hCTGF was prepared as described above. Purity and homogeneity of the protein was analyzed by SDS–PAGE and western blot (Figure 1A,B). Recombinant CTGF appears as two bands with an electrophoretic mobility corresponding to 36 kDa (unglycosylated) and 38 kDa (glycosylated isoform) in gel electrophoresis analysis as reported [4]. Biologic activity of recombinant hCTGF was tested by binding to immobilized BMP-2 using BiacoreTM technology. Specific binding of hCTGF to immobilized BMP-2 revealed an apparent KD value of 400 nM consistent with previous results obtained for binding of CTGF [5] to the BMP-2 homolog BMP-4 (Figure 2).

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