Anti-endothelial cell antibody rich sera from rheumatic heart disease patients induces proinflammatory phenotype and methylation alteration in endothelial cells

Figures

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  Figure 1. (i) Anti-Endothelial Cell Antibody (AECA) titers in Control, ARF, and RHD sera (ii) Percentage of patients positive for AECA after normalization with the controls. Experiment was performed in triplicate and data is represented as mean ± SD. Statistical significance was calculated by Student's t-tests. ns = non-significant, ****p < 0.0001.

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  Figure 2. (A): Bright-field microscopic images of HUVECs stimulation with pooled RHD, control sera at 3h, 6h, 12h and 24h (10x). (B): Bright field microscopic images of HMVECs stimulation with pooled RHD and control sera at 3h, 6h, and 12h (10x). (C): Representative micrograph of ECs showing membrane blebbing and apoptotic bodies (arrow) upon RHD sera stimulation (i) HUVECs at 24 h, and (ii) HMVECs at 12 h (20x).

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  Figure 3. (A): Relative mRNA level of RHD and healthy control sera stimulated HUVEC at 3h, 6h, 12h and 24h. (B): DNA methylation (%) of promoter region in HUVECs stimulated by RHD and control sera. (C): Relative mRNA level of RHD and healthy control sera stimulated HMVEC at 3h, 6h, 12h and 24h. (D): DNA methylation (%) of promoter region in HMVECs stimulated by RHD and control sera. Experiments were performed in triplicate and data is represented as mean ± SD. Statistical significance was calculated by Student's t-tests or one way-ANOVA. ns = non significant,*p < 0.05, **p < 0.01, ***p < 0.001****p < 0.0001.

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  Figure 4. Relative gene expression of proinflammatory (TNFα, IL-6, IL-1β, IL-8, and IFNγ), adhesion (ICAM1, VCAM1, and E selectin), and matrix genes (Vimentin, laminin B) in RHD patients and controls (2-ΔCp). Experiment was performed in triplicate and data was represented as mean ± SD. Statistical significance was calculated by Student's t-tests or one way-ANOVA. ns = non-significant, *p < 0.05, **p < 0.01, **p < 0.001****p < 0.0001.

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  Figure 5. Promoter methylation (%) of proinflammatory, adhesion, and cytoskeletal gene from mitral valve tissues of RHD patients and control. Experiment was performed in triplicate and data is represented as mean ± SD. Statistical significance was calculated by Students t tests or one way-ANOVA. ns-non significant, *p < 0.05, **p < 0.01, ***p < 0.001****p < 0.0001.