Loneliness associates with endothelial dysfunction in a community-based cohort: a pilot study and translational approach

Figures

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  Fig. 1. Association between soluble VE-cadherin and vascular inflammation in the DC-CNHA cohort, 2014–2017. Graphical display of the association between soluble VE-cadherin levels as detected by ELISA and Vascular Inflammation as detected by 18FDG-PET/CT. Data are shown as Forrest Blot with the standardized beta given, followed by the p-value in parentheses. Models are shown as unadjusted, BMI-adjusted, or BMI plus ASCVD 10-year risk score adjusted values. Significance is indicated by the asterisk and was assumed with a p-value reaching <0.05.

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  Fig. 2. Epinephrine and TNFα treatment combined exacerbate single-treatment effects on endothelial dysfunction. a, b Immunofluorescence staining of VE-cadherin (red) and labeling of F-actin (green) as well as nuclei (blue) with subsequent quantification of intercellular presence of gaps (6 h: n = 5, ON: n = 8; RM one-way ANOVA analysis with Tukey correction). Representative images are shown, and gaps are indicated by white arrows. The quantification of intercellular gaps is an indication of endothelial barrier dysfunction, subsequently allowing enhanced transport of blood components and cells into the arterial wall. c Images of VE-cadherin immunofluorescence (red) were taken at equal exposure time per experimental set, and the fluorescence intensity over the junctional space was measured as displayed in the histogram. Histogram quantification of VE-cadherin within the inter-endothelial junction allows for quantification of VE-cadherin re-distribution or loss; processes involved in endothelial barrier breakdown. Subsequent quantification of n = 5 is displayed as a graph (RM one-way ANOVA analysis with Tukey correction). Additionally, areas of widening and more diffuse VE-cadherin presence…

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  Fig. 3. Determination of potential synergistic activation of signaling pathways in Epi + TNF-treated HAoECs. a The NFκB pathway has previously been involved in chronic stress- and loneliness-related cell dysfunction, which is graphically summarized. b–h HAoECs were treated as shown for the indicated timepoints and subjected to western blot or immunofluorescence analysis. For each analyzed protein, a representative western blot image is shown with the molecular weight indicated on the right of the representative images in kDa. Western blot analysis results are displayed underneath the image. (n = 4–5; RM one-way ANOVA analysis with Tukey correction). b Western blot results for NFκB activation. c Display of Stat1 activation results for 15 and 60 min treatment timepoints. d Immunofluorescence staining of HAoECs for the phosphorylated Stat1 (red). Nuclei were labeled using DAPI (blue). (n = 3) e Presentation of Stat3 activation results for 15 and 60 min treatment timepoints. f Immunofluorescence staining of HAoECs for the phosphorylated Stat3 (red) with nuclei being labeled using DAPI in blue. (n = 3). g, h Western blot analysis of long-term activation of Stat1 or Stat3. Representative…

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  Fig. 4. Impact of JAK/Stat pathway inhibition on Epi + TNFα-induced endothelial dysfunction. HAoECs were treated with control, Epi+TNFα, or Epi+TNFα in the presence of tofacitinib, a JAK/Stat pathway inhibitor ON. a Summary of the results in this figure and the impact of JAK/Stat inhibition. (b) Display of Stat1 and Stat3 western blot results. A representative image of each analyzed protein is shown for each treatment time point. Western blot analysis results are displayed below. (60’: n = 4–6; Mixed-effects analysis with Sidak correction; 6 h and ON: n = 5; Stat1=RM one-way ANOVA analysis with Tukey correction; Stat3 = Friedman test with Dunn’s correction). c Immunofluorescence staining of VE-cadherin (red) and labeling of F-actin (green) as well as the nuclei (blue) with subsequent quantification of intercellular gap formation (n = 7; paired t-test between Epi+TNFα and Epi+TNFα + JAK inhibitor). White arrows indicated gaps between endothelial cells. d Images of VE-cadherin immunofluorescence after ON treatment were taken at equal exposure time per set, and the fluorescence intensity over the junctional space was measured as displayed in the histogram. Subsequent quantification of n = 5…