Figure 1.
Saturated fatty acids (SFAs), but not unsaturated fatty acids (UFAs), cause mitochondrial damage and ROS elevation in human endothelial cells. (A) Chemical structures of common free fatty acids (FFAs) in plasma, including the SFAs palmitic acid (PA) and stearic acid (SA), as well as the UFAs oleic acid (OA), arachidonic acid (ArA), and eicosapentaenoic acid (EPA). (B) Effects of FFA (100 μM, 6 h) treatments on intracellular ROS levels, as assessed by 2′7′-DCFDA assays, in human umbilical vein endothelial cells (HUVECs). FFAs were conjugated to bovine serum albumin (BSA) to facilitate intracellular delivery; BSA alone served as a vehicle control (CTL). (C) Summary of effects of FFA treatments on intracellular ROS levels in HUVECs shown in B. For each experimental condition, ≥50 randomly selected cells from at least three independent experiments were analyzed. Relative ROS level was determined by normalizing DCFDA intensity to that of the CTL group. Mean (±SEM) and individual-cell (blue dots) data are shown. (D) Dose-dependent effects of PA on intracellular ROS levels in HUVECs. Antioxidant NAC (5 mM) co-treatment effects were examined. (E) Summary of PA effects on intracellular ROS levels of HUVECs. (F) Subcellular localization of fluorescently labeled PA analog in HUVECs treated with BODIPY-FL-C16 (C16 FL, 10 μM) for 6 h. SIM images of mitochondria immunolabeled with anti-Tomm20 antibody are shown. (G) Effects of PA and OA on the morphology of MitoTracker-labeled mitochondria (green). (H) Summary of PA and OA effects on mitochondrial fragmentation (quantitated as mean length of mitochondria). For each experimental condition, ≥50 randomly selected cells from at least three independent repeats were analyzed. Mitochondrial length was normalized to that of the control group. (I) The effects of PA and OA on the morphology of mitochondria immunolabeled with anti-Tomm20 antibody. (J) SIM images showing PA and OA effects on mitochondrial morphology. (K) Summary of PA and OA effects on mitochondrial morphology, as shown in I. (L) Effects of PA and OA on mitochondria membrane potential monitored with JC-1 labeling. (M) Summary of PA and OA effects on mitochondrial membrane potential determined by the relative ratio of red (JC-1 monomer) versus green (JC-1 aggregate) signal, as shown in L. (N) Effects of PA and OA treatment on mitochondria membrane potential monitored with TMRE labeling. (O) Quantitative analysis of data in N. Scale bar = 10 µm. All data are presented as means ± SEMs; *P < 0.05, **P < 0.01, ***P < 0.001.

Saturated fatty acids (SFAs), but not unsaturated fatty acids (UFAs), cause mitochondrial damage and ROS elevation in human endothelial cells. (A) Chemical structures of common free fatty acids (FFAs) in plasma, including the SFAs palmitic acid (PA) and stearic acid (SA), as well as the UFAs oleic acid (OA), arachidonic acid (ArA), and eicosapentaenoic acid (EPA). (B) Effects of FFA (100 μM, 6 h) treatments on intracellular ROS levels, as assessed by 2′7′-DCFDA assays, in human umbilical vein endothelial cells (HUVECs). FFAs were conjugated to bovine serum albumin (BSA) to facilitate intracellular delivery; BSA alone served as a vehicle control (CTL). (C) Summary of effects of FFA treatments on intracellular ROS levels in HUVECs shown in B. For each experimental condition, ≥50 randomly selected cells from at least three independent experiments were analyzed. Relative ROS level was determined by normalizing DCFDA intensity to that of the CTL group. Mean (±SEM) and individual-cell (blue dots) data are shown. (D) Dose-dependent effects of PA on intracellular ROS levels in HUVECs. Antioxidant NAC (5 mM) co-treatment effects were examined. (E) Summary of PA effects on intracellular ROS levels of HUVECs. (F) Subcellular localization of fluorescently labeled PA analog in HUVECs treated with BODIPY-FL-C16 (C16 FL, 10 μM) for 6 h. SIM images of mitochondria immunolabeled with anti-Tomm20 antibody are shown. (G) Effects of PA and OA on the morphology of MitoTracker-labeled mitochondria (green). (H) Summary of PA and OA effects on mitochondrial fragmentation (quantitated as mean length of mitochondria). For each experimental condition, ≥50 randomly selected cells from at least three independent repeats were analyzed. Mitochondrial length was normalized to that of the control group. (I) The effects of PA and OA on the morphology of mitochondria immunolabeled with anti-Tomm20 antibody. (J) SIM images showing PA and OA effects on mitochondrial morphology. (K) Summary of PA and OA effects on mitochondrial morphology, as shown in I. (L) Effects of PA and OA on mitochondria membrane potential monitored with JC-1 labeling. (M) Summary of PA and OA effects on mitochondrial membrane potential determined by the relative ratio of red (JC-1 monomer) versus green (JC-1 aggregate) signal, as shown in L. (N) Effects of PA and OA treatment on mitochondria membrane potential monitored with TMRE labeling. (O) Quantitative analysis of data in N. Scale bar = 10 µm. All data are presented as means ± SEMs; *P < 0.05, **P < 0.01, ***P < 0.001.

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