Simvastatin restores pulmonary endothelial function in the setting of pulmonary over-circulation

Figures

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  Fig. 1.. Simvastatin enhances endothelial function in Shunt lambs. Changes in Western blot analysis shows that total eNOS protein levels are unchanged between vehicle- and simvastatin-treated shunt lambs (N = 4) (A). A representative image is shown, β-actin was used to normalize for protein loading. NOS activity in lung tissue is higher in simvastatin-treated than vehicle-treated shunt lambs (B). Conversely, both total lung superoxide production (C), as well as NOS-derived superoxide (D), a measure of eNOS uncoupling, are decreased in simvastatin-treated shunt lambs (N = 6). NOx levels from plasma are significantly higher in simvastatin-treated than vehicle-treated shunt lambs (D). PAP and PVR are analyzed as percent change from immediate pre-treatment baseline, in response to acetylcholine (ACh, 1 μg/kg), an endothelium-dependent vasodilator, in vehicle- (N = 7) and simvastatin-treated (N = 6) shunt lambs (F). Values are mean ± SD or SEM. *P < 0.05 by two tailed t-test comparing percent reduction in physiologic parameters from baseline (A). *P < 0.05 by two tailed t-test comparing samples from vehicle-treated to statin-treated Shunt lambs (B–F).

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  Fig. 2.. Simvastatin enhances eNOS coupling in control pulmonary arterial endothelial cells. Simvastatin (5 μM for 1 h) increases NO levels in PAECs derived from control lambs by both DAF fluorescence intensity and bioavailable NOx in cell media (A, B) and decreases NOS-derived superoxide levels from treated PAECs (C). Western blot analysis shows that simvastatin decreases eNOS phosphorylation at S1177 (D) and S617 (E) relative to total eNOS. This corresponds to a decrease in Akt1 activity as determined by reductions in pS473 levels relative to total Akt (F). Simvastatin decreases the levels of eNOS localized to the mitochondria as determined by colocalization of eNOS and mitochondria by comparing mean Pearson coefficient derived from fluorescence microscopy (G) or presence of eNOS in mitochondrial protein fractions by Western blotting (H). Representative images are shown for each Western blot and for the fluorescence microscopy, β-actin (D–F) or VDAC (H) was used to normalize for protein loading. Duplicate blots were run or re-probed with eNOS or Akt1 to allow data to be presented as a ratio of phospho to total protein. Values are mean ± SEM. N = 6 apart from Fig. 2G (N = 3). *P < 0.05…

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  Fig. 3.. Increases in eNOS phosphorylation correlate with increased Akt1 activity in pulmonary arterial endothelial cells isolated from shunt lambs. Western blot analysis shows that Akt1 activity is increased in Shunt PAECs as determined by increased total pS473 levels (A). This corresponds to increased eNOS phosphorylation at S1177 (B) and S617 (C). Representative images are shown for each Western blot, β-actin was used to normalize protein loading. The levels of eNOS localized to the mitochondria is increased in Shunt PAECs as determined by colocalization of eNOS and mitochondria comparing mean Pearson coefficients derived from fluorescence microscopy (D). Representative images are shown. NO levels are decreased in shunt PAECs by both DAF fluorescence intensity and bioavailable NOx in cell media (E, F) and NOS-derived superoxide from PAECs are enhanced (G). Values are mean ± SEM. N = 3 Control and N = 3 Shunt PAEC. *P < 0.05 by two tailed t-test comparing shunt to control samples.

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  Fig. 4.. Simvastatin reduces mitochondrial eNOS levels and enhances coupling in pulmonary arterial endothelial cells isolated from shunt lambs. Western blot analysis shows that simvastatin (5 μM treatment for 24 h) decreases Akt1 activity in Shunt PAECs as determined by decreased pS473 levels (N = 3) (A). Simvastatin also decreases the levels of eNOS localized to the mitochondria as determined by colocalization of eNOS and mitochondria comparing mean Pearson coefficients derived from fluorescence microscopy (B) (N = 3). Simvastatin also increases NO levels in shunt PAECs by both DAF fluorescence intensity and bioavailable NOx in cell media (C, D) (N = 6) and decreases NOS-derived superoxide levels from PAECs (E) (N = 6). Values are mean ± SEM. *P < 0.05 by two tailed t-test comparing statin treated to vehicle treated samples.

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  Fig. 5.. Simvastatin decreases mitochondrial eNOS levels in shunt lambs. Western blot analysis of peripheral lung tissue shows that Akt1 activity is decreased in simvastatin-compared to vehicle-treated Shunt lambs as determined by decreased pS473 levels (A). Western blot analysis also shows that eNOS phosphorylation at S1177 (B) and S617 (C) as well as the levels of eNOS localized to the mitochondria is decreased in simvastatin-compared to vehicle-treated shunt lambs. Representative images are shown for each Western blot. β-actin (A–C) or VDAC (D) was used to normalize for protein loading. Duplicate blots were run or re-probed with eNOS or Akt1 to allow data to be presented as a ratio of phospho to total protein. N = 4 or the simvastatin-treated group. N = 4 for vehicle-treated group. Values are mean ± SEM. *P < 0.05 by two tailed t-test comparing statin treated to vehicle treated samples.

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  Fig. 6.. Simvastatin increases the level of the endogenous Akt1 inhibitor, CTMP. Western blot analysis shows that CTMP levels are decreased in Shunt PAECs compared to control PAECs (A). Simvastatin (5 μm) increases CTMP levels in both Control- (B) and shunt-PAECs (C) relative to their respective matched vehicle treated PAECs, and in peripheral lung tissue of statin compared to vehicle treated shunt lambs (D). Values are mean ± SEM. *P < 0.05 by two tailed t-test comparing shunt to control samples (A) or statin treated to vehicle treated samples (B–D). N = 6 for simvastatin-treated PAEC. N = 6 for the simvastatin and vehicle-treated lambs.

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  Fig. 7.. CTMP over-expression decreases mitochondrial eNOS and restores NO signaling in pulmonary arterial endothelial cells isolated from shunt lambs. PAECs were transduced with increasing multiplicity of infection (MOI) from 1 to 20 viral particles/cell exposure of an adenovirus expressing CTMP and Western blot analysis was used to determine the increase in CTMP protein levels (A). An MOI of 10 was then used to transduce shunt PAECs for 48 h. CTMP over-expression increases NO generation in shunt PAECs by both DAF fluorescence intensity and bioavailable NOx in cell media (B, C). The increase in NO generation correlates with a decrease in the levels of eNOS localized to the mitochondria as determined by colocalization of eNOS and mitochondria comparing mean Pearson coefficients derived from fluorescence microscopy (D) and reduced NOS-derived superoxide from PAECs (E). Values are mean ± SEM. N = 6 *P < 0.05 by two tailed t-test comparing AdCTMP to vehicle treated controls.