Trimethylamine N-Oxide (TMAO) Impairs Purinergic Induced Intracellular Calcium Increase and Nitric Oxide Release in Endothelial Cells

Figure 1. TMAO does not impact BAE-1 viability. BAE-1 were treated with different concentrations of TMAO that showed no cytotoxic effect after (a) 24 h, (b) 48 h, and (c) 72 h (n = 3 independent experiments).

Figure 2. TMAO does not impact mitochondrial membrane potential. Bar graphs of 568/488 nm ratio related to control condition (CTRL) showing that TMAO was not able to induce mitochondrial membrane depolarization after (a) 1 h and (b) 24 h of treatment and it acted as a neutral factor in presence of a stressor, as MEN, that on the contrary induced a reduction of 568/488 nm ratio. Number of cells for each condition in 1 h treatment: CTRL: 180; TMAO: 180; MEN: 180; TMAO + MEN: 180 (n = 3 independent experiments). Number of cells for each condition in 24 h treatment: CTRL: 83; TMAO: 83; MEN: 83; TMAO+MEN: 83 (n = 3 independent experiments). * p < 0.05, ** p < 0.01 (vs. CTRL); # p < 0.05, ## p < 0.01, ### p < 0.001 (vs. TMAO). (c) Representative confocal microscopy images of BAE-1 treated for 1 h with TMAO 100 μM, MEN 100 μM, MEN 100 μM + TMAO 100 μM. Merged images at 568 nm and 488 nm (magnification 60X).

Figure 3. TMAO does not impact reactive oxygen species (ROS) increase. Bar graphs of mean fluorescence (Ex/Em 485 nm/520 nm) related to control condition (CTRL) showing that TMAO 100 μM was not able to induce ROS production in basal condition and it acted as a neutral factor in presence of a stressor, MEN, after (a) 1 h and (b) 24 h of treatment (n = 3 independent experiments). * p < 0.05 (vs. CTRL); # p < 0.05, ## p < 0.01 (vs. TMAO).

Figure 4. TMAO treatment alters intracellular calcium in purinergic response to ATP. (a) Bar graph representing the Fmax-Ft/2 fluorescence after ATP stimulation in basal condition (ATP) or after treatment with TMAO 100 μM for 1 h or 24 h. Number of cells for each experimental condition: ATP: 47; TMAO 1 h + ATP: 47; TMAO 24 h + ATP: 47 (n = 4 independent experiments). * p < 0.05 (b) Mean fluorescence variations of normalized curves aligned with respect to the peak value (Fmax) after ATP stimulation in a single representative experiment. Dotted line indicates the t/2 at which curve differences were considered.

Figure 5. TMAO influences nitric oxide (NO) release in purinergic response to ATP. (a) Bar graph showing fluorescence variations before and after ATP stimulation related to basal treatment (CTRL). Treatment with TMAO for 24 h reduced NO release during purinergic stimulation. Number of cells for each experimental condition: CTRL: 63; TMAO 1 h + ATP: 63; TMAO 24 h + ATP: 57 (n = 4 independent experiments). Statistical differences were evaluated with ANOVA and Kruskal–Wallis test. * p < 0.05. (b) Normalized traces of fluorescence variations (568 nm) of single cells stimulated with ATP in basal condition and after 1 h or 24 h of TMAO pretreatment.

Figure 6. TMAO impacts on eNOS phosphorylation at Ser1179 in purinergic stimulation with ATP. (a) Basal treatment with TMAO for 1 h and 24 h did not induce any variation in eNOS phosphorylation, while treatment for 24 h induced a reduction of eNOSSer1179/eNOS ratio in purinergic response to ATP. (n = 4 independent experiments). **** p < 0.0001; ** p < 0.01. (b) Representative Western blot showing the effect of TMAO treatment for 1 h and 24 h in eNOS phosphorylation at Ser1179 after ATP stimulation.