The matrix proteins aggrecan and fibulin-1 play a key role in determining aortic stiffness

Figures

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  Figure 1. Study Flow. Design and samples investigated.

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  Figure 2. ACAN Gene. Genomic map and protein domains with linkage disequilibrium (LD) plot and tagSNPs genotyped.

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  Figure 3. FBLN-1 Gene. Genomic map with linkage disequilibrium (LD) plot showing tagSNPs genotyped.

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  Figure 4. The aPWV is associated with ACAN and FBLN-1 gene polymorphisms. There is a significant allele dose-effect for each of the ACAN and FBLN-1 SNPs, with homozygotes for CC (A), GG (B), GG (C), AA (D) and AA (E) having the largest aPWV. *P < 0.001.

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  Figure 5. ACAN gene polymorphisms associate with aortic stiffness and gene expression. (A), (C) Elastic modulus compared to rs2882676 and rs2293087 genotypes. CC and GG donors had stiffer arteries compared to AA and TT ones (*P = 0.024, **P > 0.05). (B), (D) Transcript expression pattern of rs2882676 and rs2293087 across the three genotypes (*P = 0.04, **P > 0.05). Bar graph represents the mean ± SEM of ACAN transcript, normalized against GAPDH.

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  Figure 6. FBLN-1 gene polymorphisms relationship with aortic stiffness and gene expression. (A), (C) Elastic modulus compared to rs2018279 and rs2238823 genotypes. (B), (D) Transcript expression level of rs2018279 and rs2238823 polymorphisms. Bar graph represents the mean ± SEM of FBLN-1 gene expression normalized against GAPDH. *P > 0.05.

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  Figure 7. Transcript levels. (A) ACAN and (B) FBLN-1 in donor aortas (n = 52) that showed either low or high elastic modulus (EM) ex vivo. Bars represent mean ± SEM of ACAN and FBLN-1 gene expression normalized against GAPDH. *P < 0.05.

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  Figure 8. Aggrecan tissue and protein distribution in young (<32 y) versus older (>60 y) aortic tissue donors. (A) Representative sections showing immunohistochemical staining for the G1-G2 domain of aggrecan in the medial layer. Scale bar, 50 μm. (B) Top panel: Quantification plot shows aggrecan protein levels are down-regulated in older donors as compared to younger donors. Each dot on the scatter plot exemplifies the signal intensities of individual donor samples. Bars represent mean signal intensity normalized against β-actin. *P = 0.026. Middle panel: Representative western blots showing the expression of the chondroitin-sulfate (CS) attachment region of aggrecan in young versus older donors. Bottom panel: Schematic diagram illustrates the degradation of C-terminal aggrecan fragments and their diffusion out of the vessel wall with increasing age, accompanied by an accumulation of G1 fragments. CS, chondroitin-sulfate; LP, link protein. (C) Representative immunofluorescent staining of aortic tissue shows uniform expression of aggrecan G3 domain (green) across the vessel wall of younger donors that is largely absent from older ones. Scale bar, 50 μm.

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  Figure 9. Aggrecan protein domain distribution in young versus old aortas and in high versus low EM donors. (A) Representative immunohistochemical staining for G1 domain comparing young (<32 y) versus old donors (>60 y), and in aortas with low and high EM. (B), (C) Representative western blots showing quantitative analysis of aggrecan-CS signal intensities, normalized against β-actin in independent young and old donor samples with high EM (n = 5, 2 young and 3 old) and low EM (n = 5, 2 young and 3 old) values. Each dot on the scatter plot exemplifies the signal intensities of individual donor samples. Solid bars are the mean signal intensity in the plot. *P < 0.05.

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  Figure 10. Heat-map distribution of aggrecan protein G3 domain immunostaining in young (<32 y) versus old donors (>60 y), and in low versus high elastic modulus donor aortas. Representative immunofluorescence staining shows higher G3 deposits in young aortas with low EM compared to high EM samples; and in old aortas with low EM compared to high EM group. Heat map scale bar 0 to 1 demonstrates the aggrecan protein distribution and intensity in each sample and in each group. Scale bar, 50 μm.

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  Figure 11. Distribution of elastin fibers in young (<32 y) versus old (>60 y), and in low versus high EM donor aortas. With aging and increased stiffness, elastic fibers are frayed and fragmented. Scale bar, 50 μm.

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  Figure 12. Fibulin-1 tissue and protein distribution in young versus older donor aortas. (A) Representative immunohistochemical staining showing the pattern of FBLN-1 distribution in aortic sections from a young (<32 y) versus an old donor (>60 y). Fibulin-1 is expressed throughout the vessel wall but appears denser in the intima and adventitial side of media in young aorta compared to the more even distribution in the older aorta. Scale bar, 50 μm. (B) Representative western blot comparing fibulin-1 protein expression in young and old donors. (C) Quantitative analysis of fibulin-1 signal intensities, normalized against β-Actin in young (n = 6) versus old donor tissues (n = 7). Each dot on the scatter plot exemplifies the signal intensities of individual donor samples. Solid bar is the mean intensity. *P = 0.008.