Fig 2: Reversion of eNOS glutathionylation by DTT in placentas. A, Representative pictures of DTT-sensitive eNOS glutathionylation in placentas from one normal pregnancy (upper panel), and one PE patient (lower panel). Slides were preincubated with DTT (1 mM, 30 min room temperature), before immunofluorescence staining and confocal microscopy, as indicated in Fig. 1 (scale bar 20 µm). In insert, higher magnification picture (scale bar 5 µm). B, Immunoprecipitation and western-blot experiments showing the reversible eNOS glutathionylation in placentas, after DTT treatment of the protein extract. Left, representative western-blot picture; Right, expression of the data as median with interquartile range. The statistical analysis was assessed using a Mann-Whitney test. *p < 0.05.

Fig 3: TNF-a induces endothelial dysfunction in Eahy926 cells. (A) Eahy926 cells were treated with 0.1% PBS (control) and TNF-a (1, 5, 10 and 20 ng/ml). mRNA expression levels of eNOS were investigated using RT-qPCR. *P<0.05 and **P<0.01 vs. control. KLF15 mRNA and protein levels in Eahy926 cells transfected with human pTA2-KLF15 and negative control pTA2 were investigated using (B) RT-qPCR and (C) western blot analyses, respectively. ß-actin was used as an internal control. *P<0.05 and **P<0.01 vs. NC. NC, negative control pTA2 vector; KLF15, Krüppel-like factor 15; TNF-a, tumor necrosis factor-a; eNOS, endothelial nitric oxide synthase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Fig 4: Soluble aggregated tau induces microtubule instability, increases markers of cellular senescence, and blocks eNOS activation in brain microvasculature in 8-month-old male and female P301S(PS19) mice modeling tauopathy.A, B Decreased levels of acetylated tubulin in microvasculature of P301S(PS19) mice. A Representative immunofluorescent images of cortical brain sections from WT control and P301S(PS19) mice stained with DAPI (blue) and lectin (red) and immunostained for acetyl-a-tubulin (green); B Quantitation of acetyl-a-tubulin immunoreactive signal co-localized with lectin-reactive brain microvasculature (Unpaired, two-sided Student’s t-test, t(21) = 2.582, *, p = 0.018. Control, n = 12; PS19, n = 11 mice). C, D Decreased eNOS activation in microvasculature isolated from P301S(PS19) mice. C Representative electropherograms from capillary electrophoresis immunoassays of phospho-eNOS (S1176) and total eNOS in microvasculature purified from brains of WT and P301S(PS19) mice. D Quantitative analyses of phospho-eNOS (S1176) normalized to total eNOS levels in isolated brain vasculature of P301S(PS19) mice (Unpaired, two-sided Student’s t-test, t(6.248)=2.662, *p = 0.036. n = 6 mice per group). E, F Increased inhibition of eNOS in microvasculature isolated from P301S(PS19) mice. E Representative electropherograms from capillary electrophoresis immunoassays of phospho-eNOS (T495) and ß-actin in microvasculature isolated from brains of WT control and P301S(PS19) mice. F Quantitative analyses of data in E (Student’s t test with Welch’s correction, t(5.966) = 3.370, *, p = 0.0152, n = 6 mice per group). G, H Increased markers of senescence in microvasculature isolated from P301S(PS19) mice. G mRNA abundance for cell cycle arrest mediators cyclin dependent kinase inhibitor 2 A (Cdkn2a, p16, unpaired, two-sided Student’s t test with Welch’s correction, t(19) = 0.77, p = 0.45. WT control, n = 9; PS19, n = 12), cyclin dependent kinase inhibitor 1 A (Cdkn1a, p21, unpaired, two-sided Student’s t test with Welch’s correction (t(13.3) = 2.794, *, p = 0.015. WT control, n = 8; PS19, n = 13) and tumor protein 53 (Tp53, p53, unpaired, two-sided Student’s t test (t(19) = 0.5973, p = 0.56. WT control, n = 10; PS19, n = 11 mice); H Senescence-associated secretory phenotype components interleukin 6 (IL-6, unpaired two-sided Student’s t test, t(17)=1.023, p = 0.32. WT control, n = 9; PS19, n = 10 mice), interleukin 1ß (IL-1ß, unpaired, two-sided Student’s t test with Welch’s correction, t(12.87) = 2.497, *p = 0.027. WT control, n = 7; PS19, n = 12 mice), tumor necrosis factor a (TNFa, unpaired, two-sided Student’s t test with Welch’s correction, t(10.46) = 2.309, *, p = 0.043. WT control, n = 8; PS19, n = 10 mice), monocyte chemoattractant protein-1 (MCP-1, unpaired, two-sided Student’s t test with Welch’s correction, t(11.07) = 2.794, *, p = 0.018. WT control, n = 7; PS19, n = 12 mice), and plasminogen-activator inhibitor-1 (PAI-1, unpaired, two-sided Student’s t test with Welch’s correction, t(15.53) = 1.701, p = 0.11. WT control, n = 10; PS19, n = 12 mice), measured with quantitative real-time PCR (qRT-PCR) in isolated brain vasculature of WT and P301S(PS19) mice. Data are representative images and electropherograms, and means ± SEM. For post-hoc analyses, the lack of a specific P value in the legend reflects the information reported by GraphPad Prism Version 9.4.0.

Fig 5: pO2changes elicit eNOS S-glutathionylation and dysfunction in HTR8. A, Representative pictures of eNOS and glutathione expression in HTR8 incubated in low pO2 (O2 1%) (left panel), or pO2 changes (18 h O2 1%-2 h O2 20%-2 h O2 1%) before (middle panel), and after (right panel) DTT treatment (1 mM, 30 min, room temperature). The colocalization of eNOS and glutathione was studied as described in the legend to Fig. 2 and in the Method Section. Upper panels, scale-bar 10 µm, lower panels, scale-bar 5 µm. B, Immunoprecipitation and immunoblotting experiments showing the glutathionylation of eNOS in HTR8 submitted to low pO2 or pO2 changes. The reversibility of glutathionylation was studied by preincubating eNOS immunoprecipitates with DTT (1 mM, 30 min, room temperature), before immunoblotting. C, Measurement of NO production by HTR8 in low pO2 conditions or exposed to pO2 changes. DAF-FM diacetate probe (5 µM) was added to the HTR8 culture medium (5 µM) 30 min before the end of the incubation. The production of NO was fluorometrically recorded (exc/em 495/525 nm) for 60 min in the presence or absence of BH4 (10 µM). A negative control using L-NAME (100 µM) was done concomitantly, and the L-NAME-insensitive residual fluorescence was substracted to each point, for eliminating the non NO-specific DAF fluorescence. A positive control was done by stimulating HTR8 with VEGF (20 nM, 15 min). The results are expressed as % of NO produced by the control in low pO2, means ± SEM of 4 separate experiments, statistical analysis with a Student t-test. *p < 0.05; ns, non significant.