Genetic ablation of TRPC6 facilitated age-dependent atherosclerosis progression in an ApoE-/- mouse model

Figures

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  Figure 1. Body weights and serum total cholesterol levels in TRPC6-/- and TRPC6+/+ mice. (A) Body weight comparison of mature and aged TRPC6+/+ (n=6 and n=14, respectively) and TRPC6-/- (n=9 and n=26, respectively) mice. (B, C) Sex-stratified comparison of body weights in both genotypes in aged male (B, n=9 for TRPC6+/+ and n=16 for TRPC6-/-) and female (C, n=5 for TRPC6+/+ and n=10 for TRPC6-/-) mice. (D, E) Serum total cholesterol levels in mature and aged TRPC6+/+ (n=3 and n=8, respectively) and TRPC6-/- (n=3 and n=22, respectively) mice. Two-tailed t-tests were used for normal data sets, and the Mann-Whitney Rank Sum Test for non-normal data sets. Black-filled circles show individual weight measurements, and the red-filled circle corresponds to the mean value ± SEM. **p < 0.01. ***p < 0.001. n.d. = no significant difference.

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  Figure 2. TRPC6 deletion increases atheroma burden without affecting calcification in an age-dependent manner. (A–D). Representative Von Kossa-stained aortic sections from mature mice with both TRPC6+/+ (A) and TRPC6-/- (B) genotypes and aged mice with both TRPC6+/+ (C) and TRPC6-/- (D) genotypes. (E, F) Quantification of atheroma burden and calcification in aortic sections from mature mice shows no significant differences between TRPC6-/- and TRPC6+/+ groups (in (E), TRPC6+/+: n=6; TRPC6-/-: n=8; in (F), TRPC6+/+: n=6; TRPC6-/-: n=8, respectively). (G) In aged mice, TRPC6-/- mice exhibit increased atheroma burden compared to controls (TRPC6+/+: n=13; TRPC6-/-: n=25), even among sex-stratified male (H), TRPC6+/+: n=8; TRPC6-/-: n=16) and female (I), TRPC6+/+: n=5; TRPC6-/-: n=9) groups. (J) The analysis of Von Kossa-stained aortic sections from aged mice shows that calcification remains unchanged (TRPC6+/+: n=13; TRPC6-/-: n=25). Two-tailed t-tests were used for normal data sets, and the Mann-Whitney Rank Sum Test for non-normal data. Data are presented as mean ± SEM (red-filled circle and red error bars) with individual data points shown as black-filled circles. M, tunica media; Ca,…

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  Figure 3. TRPC6 deletion increases fibrous cap formation in an age-dependent manner (SMA staining). (A–D) Representative SMA-stained aortic sections from TRPC6-/- and TRPC6+/+ mice in mature (A, B) and aged groups (C, D, E-L). Quantification of atheroma burden (E, H), sex-stratified %atheroma. (I, J), fibrous cap length (K), and fibrous cap thickness (L) in aortic sections from mature (E-G) and aged (H-L) in TRPC6-/- and TRPC6+/+ groups (in (E), TRPC6+/+: n=5 and TRPC6-/-: n=8; in (F), TRPC6+/+: n=6 and TRPC6-/-: n=9; in (G), TRPC6+/+: n=6 and TRPC6-/-: n=9; in (H), TRPC6+/+: n=14 and TRCP6-/-: n=18; in (I), TRPC6+/+: n=9 and TRPC6-/-: n=13; in (J), TRPC6+/+: n=5 and TRPC6-/-: n=5; in (K), TRPC6 +/+: n=14 and TRPC6-/-: n=18; in (L), TRPC6+/+; n=14 and TRPC6-/-: n=18). Two-tailed t-tests were used for normally distributed data, and the Mann-Whitney Rank Sum Test for non-normally distributed data. M, tunica media; FC, fibrous cap; A, atheroma. Data are presented as mean ± SEM (red-filled circle and red error bars) with individual data points shown as black-filled circles. **p < 0.01. ***p < 0.001. n.d. = no significant difference.

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  Figure 4. TRPC6 deletion alters fibrous cap formation without affecting collagen or smooth muscle cell content (MTC staining). (A–D) Representative Masson’s Trichrome (MTC)-stained aortic sections from mature and aged TRPC6+/+ and TRPC6-/- mice. (E, F, K–M) Quantification of atheroma burden (E), TRPC6+/+: n= 6; TRPC6-/-: n= 7), fibrous cap length (% of atheroma length, (F), TRCP6+/+: n=6; TRPC6-/-: n=7), fibrous cap thickness (K), TRPC6+/+: n= 6; TRPC6-/-: n= 7), smooth muscle cell (SMC, (L), TRPC6+/+: n= 6; TRPC6-/-: n= 5) content, and collagen content (M), TRPC6+/+: n= 6; TRPC6-/-: n= 5) in aortic sections from mature mice in both TRPC6-/- and TRPC6+/+ groups. (G–J). Quantification of atheroma burden (G), TRPC6+/+: n= 12; TRPC6-/-: n= 19), sex-stratified %atheroma (in (H), TRPC6+/+: n= 7 and TRPC6-/-: n= 15; in (I), TRPC6+/+: n= 5; TRPC6-/-: n= 4), fibrous cap length (% of atheroma length, (J), TRPC6+/+: n= 12; TRPC6-/-: n= 19, fibrous cap thickness (N), TRPC6+/+: n= 10; TRPC6-/-: n= 19), smooth muscle cell (SMC) content (O), TRPC6+/+: n= 12; TRPC6-/-: n= 19), and collagen content (P), TRPC6+/+: n= 12; TRPC6-/-: n= 16) in aortic sections from aged mice in both genotypes. Two-tailed…