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Differential Effects of IFN-β on the Survival and Growth of Human Vascular Smooth Muscle and Endothelial Cells.

Sano E, Tashiro S, Tsumoto K, Ueda T - Biores Open Access (2015)

Bottom Line: To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC).The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities.The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo , Chiba, Japan .

ABSTRACT
It has been documented that interferon (IFN)-β is effective against the genesis of atherosclerosis or hyperplastic arterial disease in animal model. The main mechanism of the efficacy was antiproliferative action on the growth of vascular smooth muscle cells (SMC). To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC). IFN-β enhanced SMC apoptosis in serum starved medium. Conversely, EC apoptosis induced by serum and growth factor deprivation was inhibited by IFN-β. The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities. Anti-apoptotic bcl-2 mRNA was also up-regulated in EC. IFN-β inhibited SMC growth in a dose dependent manner. However, the growth of EC was rather enhanced by a low dose of IFNs. The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells. The growth stimulation on EC was considered to link with increase of S and G2/M phase cells. SMC produced IFN-β in response to various stimulants. However, IFN-β was not induced in EC. These suggested that endogenous IFN-β from SMC may act on EC and affect to EC functions. In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth. These effects were considered to contribute to a cure against hyperplastic arterial diseases as the mechanisms in the efficacy of IFN-β.

No MeSH data available.


Related in: MedlinePlus

Effects of interferon beta (IFN-β) on the apoptosis. (A) Effects of IFNs on dead cell generation of human coronary arterial smooth muscle cell (HCASMC). Confluent cells grown in 24-well plates were treated with IFNs for 4 days in serum-starved medium. After the staining dead cells with 0.45% trypan-blue solution, the floating dead cells were counted by Coulter counter. The average cell number and the standard error (SE) of the mean in the four wells were calculated. The proportion (%) of dead cells was expressed as the ratio of dead cells against starting confluent cell number (1×105 cells per well). **p<0.01, compared with each IFN minus and plus. (B) Effects of IFNs on dead cell generation of human aortic endothelial cell (HAEC). Confluent cells proliferated in 24 well collagen coated plates were treated with IFNs without serum and growth factors. The dead cells were counted after 4 days of IFN-β treatment. **p<0.01, compared with IFN-β minus and plus. (C, D) Apoptosis analysis by flow cytometry. The confluent HCASMC and HAEC were treated with 1,000 IU/mL of IFN-β. After the fixation, the cells were stained with propidium iodide (PI) solution and the fluorescence of the cells was analyzed by flow cytometer. The proportion (%) of apoptotic cells with degraded DNA was shown in the histogram as (A).
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f1: Effects of interferon beta (IFN-β) on the apoptosis. (A) Effects of IFNs on dead cell generation of human coronary arterial smooth muscle cell (HCASMC). Confluent cells grown in 24-well plates were treated with IFNs for 4 days in serum-starved medium. After the staining dead cells with 0.45% trypan-blue solution, the floating dead cells were counted by Coulter counter. The average cell number and the standard error (SE) of the mean in the four wells were calculated. The proportion (%) of dead cells was expressed as the ratio of dead cells against starting confluent cell number (1×105 cells per well). **p<0.01, compared with each IFN minus and plus. (B) Effects of IFNs on dead cell generation of human aortic endothelial cell (HAEC). Confluent cells proliferated in 24 well collagen coated plates were treated with IFNs without serum and growth factors. The dead cells were counted after 4 days of IFN-β treatment. **p<0.01, compared with IFN-β minus and plus. (C, D) Apoptosis analysis by flow cytometry. The confluent HCASMC and HAEC were treated with 1,000 IU/mL of IFN-β. After the fixation, the cells were stained with propidium iodide (PI) solution and the fluorescence of the cells was analyzed by flow cytometer. The proportion (%) of apoptotic cells with degraded DNA was shown in the histogram as (A).

Mentions: At first, the effects of various IFNs on dead cell generation were examined. HCASMC and HAEC were cultured in each growth medium until confluent (1×105 cells/well). IFNs were treated in the medium without serum and growth factor. The floating cells were stained with 0.45% trypan-blue solution for the confirmation of cell death and counted by coulter counter. As shown in Fig. 1A, the dead cells of HCASMC increased by IFN treatment in a dose dependent manner. IFN-β was most effective to HCASMC death. Whereas IFN-α and IFN-β inhibited dead cell generation of HAEC in a dose dependent manner as shown in Fig. 1B, IFN-γ did not inhibit the cell death. Next, the effects of IFN-β on the death of these cells were investigated by flow cytometry. The confluent cultures of HCASMC and HAEC were treated with IFN-β of 1,000 IU/mL in the serum and growth factor starved medium. The degraded DNA of apoptotic cells were detected in the histogram (Fig. 1C, D). The apoptotic cells of HCASMC increased with time elapse by IFN-β treatment as shown in Fig. 1C, whereas HAEC apoptotic cells decreased by IFN-β in spite of the vigorous apoptosis of control cells cultured in serum and growth factor starved medium, as shown in Fig. 1D. These results indicate the opposite function of IFN-β on the apoptosis of HCASMC and HAEC.


Differential Effects of IFN-β on the Survival and Growth of Human Vascular Smooth Muscle and Endothelial Cells.

Sano E, Tashiro S, Tsumoto K, Ueda T - Biores Open Access (2015)

Effects of interferon beta (IFN-β) on the apoptosis. (A) Effects of IFNs on dead cell generation of human coronary arterial smooth muscle cell (HCASMC). Confluent cells grown in 24-well plates were treated with IFNs for 4 days in serum-starved medium. After the staining dead cells with 0.45% trypan-blue solution, the floating dead cells were counted by Coulter counter. The average cell number and the standard error (SE) of the mean in the four wells were calculated. The proportion (%) of dead cells was expressed as the ratio of dead cells against starting confluent cell number (1×105 cells per well). **p<0.01, compared with each IFN minus and plus. (B) Effects of IFNs on dead cell generation of human aortic endothelial cell (HAEC). Confluent cells proliferated in 24 well collagen coated plates were treated with IFNs without serum and growth factors. The dead cells were counted after 4 days of IFN-β treatment. **p<0.01, compared with IFN-β minus and plus. (C, D) Apoptosis analysis by flow cytometry. The confluent HCASMC and HAEC were treated with 1,000 IU/mL of IFN-β. After the fixation, the cells were stained with propidium iodide (PI) solution and the fluorescence of the cells was analyzed by flow cytometer. The proportion (%) of apoptotic cells with degraded DNA was shown in the histogram as (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4497630&req=5

f1: Effects of interferon beta (IFN-β) on the apoptosis. (A) Effects of IFNs on dead cell generation of human coronary arterial smooth muscle cell (HCASMC). Confluent cells grown in 24-well plates were treated with IFNs for 4 days in serum-starved medium. After the staining dead cells with 0.45% trypan-blue solution, the floating dead cells were counted by Coulter counter. The average cell number and the standard error (SE) of the mean in the four wells were calculated. The proportion (%) of dead cells was expressed as the ratio of dead cells against starting confluent cell number (1×105 cells per well). **p<0.01, compared with each IFN minus and plus. (B) Effects of IFNs on dead cell generation of human aortic endothelial cell (HAEC). Confluent cells proliferated in 24 well collagen coated plates were treated with IFNs without serum and growth factors. The dead cells were counted after 4 days of IFN-β treatment. **p<0.01, compared with IFN-β minus and plus. (C, D) Apoptosis analysis by flow cytometry. The confluent HCASMC and HAEC were treated with 1,000 IU/mL of IFN-β. After the fixation, the cells were stained with propidium iodide (PI) solution and the fluorescence of the cells was analyzed by flow cytometer. The proportion (%) of apoptotic cells with degraded DNA was shown in the histogram as (A).
Mentions: At first, the effects of various IFNs on dead cell generation were examined. HCASMC and HAEC were cultured in each growth medium until confluent (1×105 cells/well). IFNs were treated in the medium without serum and growth factor. The floating cells were stained with 0.45% trypan-blue solution for the confirmation of cell death and counted by coulter counter. As shown in Fig. 1A, the dead cells of HCASMC increased by IFN treatment in a dose dependent manner. IFN-β was most effective to HCASMC death. Whereas IFN-α and IFN-β inhibited dead cell generation of HAEC in a dose dependent manner as shown in Fig. 1B, IFN-γ did not inhibit the cell death. Next, the effects of IFN-β on the death of these cells were investigated by flow cytometry. The confluent cultures of HCASMC and HAEC were treated with IFN-β of 1,000 IU/mL in the serum and growth factor starved medium. The degraded DNA of apoptotic cells were detected in the histogram (Fig. 1C, D). The apoptotic cells of HCASMC increased with time elapse by IFN-β treatment as shown in Fig. 1C, whereas HAEC apoptotic cells decreased by IFN-β in spite of the vigorous apoptosis of control cells cultured in serum and growth factor starved medium, as shown in Fig. 1D. These results indicate the opposite function of IFN-β on the apoptosis of HCASMC and HAEC.

Bottom Line: To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC).The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities.The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo , Chiba, Japan .

ABSTRACT
It has been documented that interferon (IFN)-β is effective against the genesis of atherosclerosis or hyperplastic arterial disease in animal model. The main mechanism of the efficacy was antiproliferative action on the growth of vascular smooth muscle cells (SMC). To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC). IFN-β enhanced SMC apoptosis in serum starved medium. Conversely, EC apoptosis induced by serum and growth factor deprivation was inhibited by IFN-β. The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities. Anti-apoptotic bcl-2 mRNA was also up-regulated in EC. IFN-β inhibited SMC growth in a dose dependent manner. However, the growth of EC was rather enhanced by a low dose of IFNs. The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells. The growth stimulation on EC was considered to link with increase of S and G2/M phase cells. SMC produced IFN-β in response to various stimulants. However, IFN-β was not induced in EC. These suggested that endogenous IFN-β from SMC may act on EC and affect to EC functions. In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth. These effects were considered to contribute to a cure against hyperplastic arterial diseases as the mechanisms in the efficacy of IFN-β.

No MeSH data available.


Related in: MedlinePlus