Figure 1.
Increased mitochondrial biogenesis markers by LSS in HAECs.
(a) An overview of LSS protocol used. HAECs were exposed to exercise-mimicking LSS at 20 dyne/cm2 for 48 hrs, and then, recovery (Rec) LSS at 5 dyne/cm2 was followed for another 24 hrs. (b) Effect of LSS on the mRNA and protein expression of mitochondrial biogenesis markers. mRNA expression of NRF-1, SCO1, SCO2, TFAM, and COX IV were assessed by real-time PCR and protein contents of PGC-1α, VDAC, and p53R2 were analyzed by western blot. (c) Effect of LSS on the mRNA expression of mitochondrial dynamics markers. mRNA expression of Mfn1, Mfn2, OPA1, Fis1, and Drp1 were assessed by real-time PCR. (d) Effect of LSS on mtDNA contents. Relative mtDNA contents are expressed as a ratio of COX I and II to 18s rRNA. (e) Effect of LSS on mitochondrial mass. Mitochondria were labeled with MitoTracker Green in live HAECs. Representative fluorescence micrographs under STT (left panel) and after 48 hrs of LSS at 20 dyne/cm2 (right panel) are shown. Bar = 50 µm. The MitoTracker Green fluorescence intensities were analyzed using the Image J (NIH) software. All densitometry analyses values are shown as mean ± SE; * P<0.05 vs. STT; ** P<0.01 vs. STT.
Table 1.
Primer Sequences for Real-Time PCR.
Figure 2.
Effect of LSS on endothelial metabolism.
(a) Enhanced mitochondrial respiration in LSS-exposed HUVECs. Oxygen consumption of HUVECs was measured after the intermittent LSS exposure for up to 72 hours. Representative strips of the oxygen consumption measured (left panel). Normalized values to the number of cells (right panel). (b) Effect of LSS on ΔΨm in ECs. ΔΨm was estimated by using MitoTracker Red CMX Ros. Representative fluorescence micrographs for each condition are shown. Bar = 100 µm. The fluorescence intensities were analyzed using the Image J (NIH) software. (c) Heat map showing the expression of glycolysis markers by microarray analysis. Genes upregulated are presented in yellow and downregulated are in blue (upper panel). Average fold change of each of those glycolysis markers identified by microarray analysis are shown in a bar graph (lower panel). (d) Lactate concentration measured in cell culture medium at 12, 24, 36 hrs of post LSS or STT. Values were normalized to viable cell number. Data shown as means ± SE; * P<0.05 vs. STT; ** P<0.01 vs. STT.
Figure 3.
Effect of five weeks of voluntary wheel (VW) exercise on mitochondrial biogenesis markers in mice abdominal aorta (AA).
(a) Effect of VW running on mRNA expression of mitochondrial biogenesis markers in AA. mRNA expressions of PGC-1α, NRF1, TFAM, p53R2, SCO1, and SCO2 were examined by real-time PCR. Values were normalized to the level of housekeeping gene, TIF. (b) Effect of VW running on protein expression of mitochondrial biogenesis markers in AA. Tissue extracts of the AA from SED and VW group mice were subjected to western blot. The amount of phosphorylated- AMPKα was normalized by the amount of AMPKα protein. Protein content of mitochondrial biogenesis marker VDAC was also measured. The loading volume was normalized by the expression level of α-tubulin. (c) Effect of VW running on mtDNA content in AA. mtDNA contents were compared in between SED and VW run mice. Relative mtDNA content are expressed as a ratio of NADH dehydrogenase subunit 2 (ND II) to 18s rRNA. All densitometry analyses values are shown as means ± SE. Data shown represent results from a total of 5 mice per group; * P<0.05 vs. SED; ** P<0.01 vs. SED.
Figure 4.
Effect of five weeks of VW running on mitochondrial in mouse endothelium.
(A) Representative fluorescence micrographs of en face immunostaining. Endothelium of the greater curvature (GC), lesser curvature (LC), thoracic aorta (TA), femoral artery (FA), and mesenteric artery (MA) were stained in the sedentary (SED) and voluntary wheel (VW) run C57BL6 mice. The green fluorescent staining indicates mitochondrial density stained by VDAC, and the red color represents ECs stained by CD31 (an endothelial cell specific marker). Nuclei were counterstained with DAPI. Shown are representative images of en face staining labeled. (B) Illustration of mouse arterial tree. (C) Summary of densitometry analysis. Green fluorescence intensities by VDAC staining were analyzed using the Image J (NIH) software. Data shown as mean ± SE; Data shown represent results from a total of 10 mice per group * P<0.05 vs. SED. ** P<0.01 vs. SED.