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Rapamycin impairs endothelial cell function in human internal thoracic arteries.

Reineke DC, Müller-Schweinitzer E, Winkler B, Kunz D, Konerding MA, Grussenmeyer T, Carrel TP, Eckstein FS, Grapow MT - Eur. J. Med. Res. (2015)

Bottom Line: Comparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 μM rapamycin.Western blots after 20-h incubation with rapamycin (10 nM-1 μM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1.Furthermore, 1 μM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, University Hospital Berne, Bern, CH-3010, Switzerland.

ABSTRACT

Background: Definitive fate of the coronary endothelium after implantation of a drug-eluting stent remains unclear, but evidence has accumulated that treatment with rapamycin-eluting stents impairs endothelial function in human coronary arteries. The aim of our study was to demonstrate this phenomenon on functional, morphological and biochemical level in human internal thoracic arteries (ITA) serving as coronary artery model.

Methods: After exposure to rapamycin for 20 h, functional activity of ITA rings was investigated using the organ bath technique. Morphological analysis was performed by scanning electron microscopy and evaluated by two independent observers in blinded fashion. For measurement of endothelial nitric oxide synthase (eNOS) release, mammalian target of rapamycin (mTOR) and protein kinase B (PKB) (Akt) activation, Western blotting on human mammary epithelial cells-1 and on ITA homogenates was performed.

Results: Comparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 μM rapamycin. Electron microscopic evaluation of the endothelial layer showed no differences between controls and samples exposed to 10 μM rapamycin. Western blots after 20-h incubation with rapamycin (10 nM-1 μM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1. Furthermore, 1 μM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged.

Conclusions: The present data suggests that 20-h exposure of ITA rings to rapamycin reduces endothelium-mediated relaxation through down-regulation of Akt-phosphorylation via the mTOR signalling axis within the ITA tissue without injuring the endothelial cell layer.

No MeSH data available.


Related in: MedlinePlus

Left-hand traces. Top: Representative Western blot bands for phospho (Ser 2481)-mTOR (p-mTOR) and mTOR (anti-c-terminal antibody) on internal thoracic artery tissue. Bottom: Columns indicate repression of p-mTOR and mTOR on ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation. Each test was performed and normalized in six experiments, and the bars represent SEM. Right-hand traces. Top: Representative Western blot bands for phospho (Ser 473)-Akt (p-Akt) and Akt on internal thoracic artery tissue. Bottom: Columns indicate changes of p-Akt (n = 24) and Akt (n = 19) in ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation, and the bars represent SEM
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Fig4: Left-hand traces. Top: Representative Western blot bands for phospho (Ser 2481)-mTOR (p-mTOR) and mTOR (anti-c-terminal antibody) on internal thoracic artery tissue. Bottom: Columns indicate repression of p-mTOR and mTOR on ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation. Each test was performed and normalized in six experiments, and the bars represent SEM. Right-hand traces. Top: Representative Western blot bands for phospho (Ser 473)-Akt (p-Akt) and Akt on internal thoracic artery tissue. Bottom: Columns indicate changes of p-Akt (n = 24) and Akt (n = 19) in ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation, and the bars represent SEM

Mentions: When the effect of rapamycin was tested in homogenates from ITA segments, a significant (p < 0.001) reduction of the protein phosphorylation on Ser473 (p-Akt) indicated substantial down-regulation of Akt phosphorylation, whereas Akt protein levels remained unchanged (p = 0.59, Fig. 4, right).Fig. 4


Rapamycin impairs endothelial cell function in human internal thoracic arteries.

Reineke DC, Müller-Schweinitzer E, Winkler B, Kunz D, Konerding MA, Grussenmeyer T, Carrel TP, Eckstein FS, Grapow MT - Eur. J. Med. Res. (2015)

Left-hand traces. Top: Representative Western blot bands for phospho (Ser 2481)-mTOR (p-mTOR) and mTOR (anti-c-terminal antibody) on internal thoracic artery tissue. Bottom: Columns indicate repression of p-mTOR and mTOR on ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation. Each test was performed and normalized in six experiments, and the bars represent SEM. Right-hand traces. Top: Representative Western blot bands for phospho (Ser 473)-Akt (p-Akt) and Akt on internal thoracic artery tissue. Bottom: Columns indicate changes of p-Akt (n = 24) and Akt (n = 19) in ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation, and the bars represent SEM
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4502526&req=5

Fig4: Left-hand traces. Top: Representative Western blot bands for phospho (Ser 2481)-mTOR (p-mTOR) and mTOR (anti-c-terminal antibody) on internal thoracic artery tissue. Bottom: Columns indicate repression of p-mTOR and mTOR on ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation. Each test was performed and normalized in six experiments, and the bars represent SEM. Right-hand traces. Top: Representative Western blot bands for phospho (Ser 473)-Akt (p-Akt) and Akt on internal thoracic artery tissue. Bottom: Columns indicate changes of p-Akt (n = 24) and Akt (n = 19) in ITA tissue exposed for 20 h to 1 μM rapamycin in relation to controls (Ctl). Asterisk indicates statistical significance for down-regulation, and the bars represent SEM
Mentions: When the effect of rapamycin was tested in homogenates from ITA segments, a significant (p < 0.001) reduction of the protein phosphorylation on Ser473 (p-Akt) indicated substantial down-regulation of Akt phosphorylation, whereas Akt protein levels remained unchanged (p = 0.59, Fig. 4, right).Fig. 4

Bottom Line: Comparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 μM rapamycin.Western blots after 20-h incubation with rapamycin (10 nM-1 μM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1.Furthermore, 1 μM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Surgery, University Hospital Berne, Bern, CH-3010, Switzerland.

ABSTRACT

Background: Definitive fate of the coronary endothelium after implantation of a drug-eluting stent remains unclear, but evidence has accumulated that treatment with rapamycin-eluting stents impairs endothelial function in human coronary arteries. The aim of our study was to demonstrate this phenomenon on functional, morphological and biochemical level in human internal thoracic arteries (ITA) serving as coronary artery model.

Methods: After exposure to rapamycin for 20 h, functional activity of ITA rings was investigated using the organ bath technique. Morphological analysis was performed by scanning electron microscopy and evaluated by two independent observers in blinded fashion. For measurement of endothelial nitric oxide synthase (eNOS) release, mammalian target of rapamycin (mTOR) and protein kinase B (PKB) (Akt) activation, Western blotting on human mammary epithelial cells-1 and on ITA homogenates was performed.

Results: Comparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 μM rapamycin. Electron microscopic evaluation of the endothelial layer showed no differences between controls and samples exposed to 10 μM rapamycin. Western blots after 20-h incubation with rapamycin (10 nM-1 μM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1. Furthermore, 1 μM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged.

Conclusions: The present data suggests that 20-h exposure of ITA rings to rapamycin reduces endothelium-mediated relaxation through down-regulation of Akt-phosphorylation via the mTOR signalling axis within the ITA tissue without injuring the endothelial cell layer.

No MeSH data available.


Related in: MedlinePlus