Sex-dependent aortic valve pathology in patients with rheumatic heart disease

Figures

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  Fig 1. Increased collagen expression in aortic valves of RHD patients. (A-B’): H&E staining shows hyaline degeneration and fibrotic infiltration, consistent with fibromyxomatous degeneration, in aortic valves of male and female RHD patients. (C-F): Masson’s Trichrome staining reveals significantly increased collagen expression (blue staining) in the fibrosa (f) and ventricularis (v) of aortic valves of female patients, while no significant difference in muscle fibers (red staining) is seen between the two groups. (G): RT-qPCR analysis shows increased COL1A1 and COL2A1 expression in aortic valves of female patients. GAPDH expression is used to normalize the expression level. n = 5 patients/group for H&E and Masson staining, n = 8 patients/group for RT-qPCR, *p < 0.05, scale bar = 100μm.

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  Fig 2. Calcification and neovascularization in aortic valves of RHD patients. (A, B, E): Von Kossa staining (n = 8 patients/group) shows a similar level of granular calcium deposits (asterisk) in aortic valves of male and female patients. (C, D, F): PECAM1 immunostaining (n = 5 patients/group) shows significantly increased neovascularization (arrows) in aortic valves of female patients. *p < 0.05, scale bar = 50μm or 100μm.

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  Fig 3. Different cell fate in aortic valves between male and female RHD patients. (A-C): TUNEL assay shows a significant decrease in the intensity and density of apoptotic cells (brown staining) in aortic valves of female patients. (D-F): Ki67 immunostaining indicates no difference in proliferative cells (brown staining) aortic valves between male and female patients. (G): RT-qPCR analysis showing increased BCL2 expression and decreased expression of BAX and CASP10 in aortic valves of female patients. GAPDH expression is used to normalize the expression level. (H): Decreased expression of autophagy genes, BECN1 and LC3A, in aortic valves of female patients. n = 8 patients/group for TUNEL assay and Ki67 staining, n = 5 for RT-qPCR, *p < 0.05, **p < 0.01, scale bar = 100μm.

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  Fig 4. Increased monocyte/macrophage infiltration in aortic valves of female RHD patients. (A-C): CD68 staining shows significantly increased infiltration by monocytes/macrophages in aortic valves of female RHD patients as compared to male patients. (D-F): CD163 staining reveals increased monocytes/macrophage infiltration in the valves of female RHD patients. (G-I): CD4 staining shows an increase trend of CD4+ T lymphocytes in female patients. n = 5 patients/group for CD68 and CD163 staining, n = 8 for CD4 staining, *p < 0.05, bar = 100μm.

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  Fig 5. Expression of inflammatory genes in aortic valves of male and female RHD patients. (A-C): Phospho-p65 (S536) immunostaining shows significantly increased NFKB activity in aortic valves of female RHD patients. (D-F): IFNG immunostaining reveals significant upregulation of IFN level in aortic valves of female RHD patients. (G-I): IL10 immunostaining indicates a similar IL10 level in aortic valves between the two groups. (J): RT-qPCR analysis shows increased expression of NFKB pathway genes (p65, IL8, and NOS3) in aortic valves of female patients, whereas the expression of NFKB inhibitor, IKBA, is downregulated. (K): RT-qPCR analysis indicates increased expression of Th1 cytokines (IFNG and IL12B) in aortic valves of female patients, but no difference in the expression of Th2 cytokines between the two groups. The expression of inflammatory related genes (TGFB1 and PTGS2) is also significantly increased in aortic valves of female patients. GAPDH expression is used to normalize the expression level. n = 8 patients/group for all immunostaining, n = 5 or 8 for RT-qPCR, *p < 0.05, **p < 0.01, scale bar = 100μm.

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  Fig 6. Working model: Upregulation cytokines and NFKB in aortic valve of female RHD patients. Increased monocyte/macrophage infiltration and production of proinflammatory cytokines, especially Th1 cytokines, in aortic valve of female RHD patients activate NFKB. The activated phosphorylated p65 enters nuclei and induces transcription of inflammatory genes, further accelerating the inflammatory process in the diseased aortic valves.