IFN-γ and TNF-α synergize to inhibit CTGF expression in human lung endothelial cells.

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-β, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-γ) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-γ in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-α) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-γ and TNF-α on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-γ and TNF-α down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-γ on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-γ and TNF-α alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-γ and TNF-α. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-γ in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-γ and TNF-α on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-γ and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-γ production might be beneficial to avoid airway remodeling in asthma

Akv murine leukemia virus enhances bone tumorigenesis in hMT-c-fos-LTR transgenic mice

AbstracthMt-c-fos-LTR transgenic mice (U. Rüther, D. Komitowski, F. R. Schubert, and E. F. Wagner. Oncogene 4, 861–865, 1989) developed bone sarcomas in 20% (3/15) of females at 448 ± 25 days and in 8% (1/12) of males at 523 days. After infection of newborns with Akv, an infectious retrovirus derived from the ecotropic provirus of the AKR mouse, 69% (20/28) of female animals and 83% (24/29) of males developed malignant fibrous-osseous tumors. The tumors in infected transgenics developed with higher frequency and a 200-days shorter mean tumor latency period. The hMt-c-fos-LTR transgene was expressed in all the fibrous-osseous tumors. They also showed newly integrated Akv proviruses, but in most tumors Akv was detected and expressed in only a small number of the tumor cells. Wild-type C3H mice infected with Akv developed benign osteomas with an incidence of 33% and a latency period of 474 days. The data indicate that Akv exerts distinct pathogenic effects on the skeleton. In hMt-c-fos-LTR transgenic mice, predisposed to bone sarcomagenesis, Akv acts synergistically with the fos transgene, resulting in the development of fibrous-osseous tumors

Interactions of Human Endothelial and Multipotent Mesenchymal Stem Cells in Cocultures

Current strategies for tissue engineering of bone rely on the implantation of scaffolds, colonized with human mesenchymal stem cells (hMSC), into a recipient. A major limitation is the lack of blood vessels. One approach to enhance the scaffold vascularisation is to supply the scaffolds with endothelial cells (EC)

IFN-gamma and TNF-alpha synergize to inhibit CTGF expression in human lung endothelial cells

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-b, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-c) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-c in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-a) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-c and TNF-a on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-c and TNF-a down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-c on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-c and TNF-a alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-c and TNF-a. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-c in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-c and TNF-a on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-c and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-c production might be beneficial to avoid airway remodeling in asthma

Effect of rCTGF on proliferation of LEC.

<p><b>A.</b> Proliferation in LEC was assessed 3 days after the addition of rCTGF at the indicated concentration. <b>B.</b> Proliferation in LEC was assessed after the addition of 100 ng/ml CTGF at the indicated time points. Values are means ± SEMs for 3 replicate experiments. Significant differences (p<0.05) compared with untreated cells are marked by *. RLU/s (relative light unit/second).</p

Effect of CTGF silencing on the proliferation of LEC and IFN-γ and TNF-α mediated inhibition. A.

<p>Proliferation of LEC, in which CTGF was down-regulated by CTGF shRNA transfection (right side; “CTGF shRNA”) and of control cells, which were transfected with non-specific scrambled shRNA (left side; “control shRNA”), was assessed after the addition of 100 ng/ml rCTGF with or without the addition of IFN-γ (500 U/ml) at 72 h. Values are means ± SEMs for 3 replicate cultures. Significant differences to response to rCTGF are marked by * (p<0.05), significant differences of proliferation between CTGF shRNA and non-specific scrambled shRNA transfected cells are marked by # (p<0.05), and significant differences of proliferation between CTGF-induced proliferation and CTGF-induced proliferation with the addition of IFN-γ are marked by $ (p<0.05). <b>B.</b> Proliferation of LEC, in which CTGF was down-regulated by CTGF shRNA transfection (lined column; “CTGF shRNA”) and of control cells, transfected with non-specific scrambled shRNA (white column; “control shRNA”), was assessed 5 days after the addition of TNF-α (20 ng/ml), IFN-γ (500 U/ml), or TNF-α+IFN-γ. Values are means ± SEMs for 3 replicate experiments and present as % compared with non stimulated cells (100%; black column). Significant differences between TNF-α and/or IFN-γ treated cells to control cells are marked by * (p<0.05).</p

Effect of CTGF shRNA on basal and TGF-β1 mediated induction of CTGF mRNA- and protein-expression in LEC. A.

<p>Effect on CTGF mRNA level: Four days after downregulation of CTGF by specific shRNA LEC were cultured in serum reduced medium (2% FCS) for 24 h before treatment with TGF-ß1. RNA and protein was isolated after 24 h of incubation with 10 ng/ml TGF-ß1. CTGF RNA expression, compared to β-actin as control, was detected by RT-PCR (A). <b>B.</b> Effect on CTGF protein level: CTGF and ß-actin expression on protein level was detected by western immunoblotting (B). Significant regulations of CTGF by TGF-ß1 are marked by * (p<0.05), CTGF down-regulation by CTGF specific shRNA compared to control cells are marked by # (p<0.05).</p

Effect of the Jak1-inhibitor AG-490 on IFN-γ induced inhibition of CTGF expression in LEC.

<p>LEC were incubated for different time points with 500 U/ml IFN-γ (A), with different concentrations of INF-γ for 5 min (B), or with or without the Jak-inhibitor AG-490 (30 µM) and INF-γ (500 U/ml) for 5 min (C). Stat1 phosphorylation and Stat1 expression were detected by immunoblotting with anti-phospho-Stat1 and anti-Stat1 antibodies. A representative of 3 independent experiments is shown. <b>D.</b> Luminometric analysis of CTGF-Luc reporter transfected LEC: LEC were incubated with medium alone (control) or with IFN-γ without or with the Jak-inhibitor AG-490 (30 µM). * p<0.05 vs. medium alone; # p<0.05 vs IFN-γ alone. <b>E+F.</b> LEC were incubated with IFN-γ with or without the Jak-inhibitor AG-490 (30 µM). CTGF mRNA expression compared to β-actin was measured by real-time PCR (<b>E</b>). CTGF and β-actin protein expression was determined by immunoblotting (<b>F</b>). Significant differences (p<0.05) compared with untreated cells are marked by *, compared with IFN-γ are marked with #. A representative of 3 independent experiments is shown (<b>F</b>).</p

Additive effect of IFN-γ and TNF-α on the inhibition of CTGF expression. A.

<p>Luminometric analysis of CTGF-Luc reporter transfected HPMEC cells: LEC were incubated with medium alone (control), TNF-α, IFN-γ, or INF-γ and TNF-α. * p<0.05 vs medium alone; # p<0.05 vs INF-γ or TNF-α alone. <b>B+C.</b> LEC were incubated with TNF-α and/or IFN-γ. CTGF mRNA expression compared to β-actin was measured by real-time PCR (B). CTGF and β-actin protein expression was determined by immunoblotting (C). Significant differences (p<0.05) compared with untreated cells are marked by *, compared with INF-γ or TNF-α are marked with #. A representative of 3 independent experiments is shown (C).</p