Hemodynamic disturbance and mTORC1 activation: Unveiling the biomechanical pathogenesis of thoracic aortic aneurysms in Marfan syndrome

Abstract

Thoracic aortic aneurysm (TAA) significantly endangers the lives of individuals with Marfan syndrome (MFS), yet the intricacies of their biomechanical origins remain elusive. Our investigation delves into the pivotal role of hemodynamic disturbance in the pathogenesis of TAA, with a particular emphasis on the mechanistic contributions of the mammalian target of rapamycin (mTOR) signaling cascade. We uncovered that activation of the mTOR complex 1 (mTORC1) within smooth muscle cells, instigated by the oscillatory wall shear stress (OSS) that stems from disturbed flow (DF), is a catalyst for TAA progression. This revelation was corroborated through both an MFS mouse model (Fbn1 ^+/C1039G) and clinical MFS specimens. Crucially, our research demonstrates a direct linkage between the activation of the mTORC1 pathway and the intensity in OSS. Therapeutic administration of rapamycin suppresses mTORC1 activity, leading to the attenuation of aberrant SMC behavior, reduced inflammatory infiltration, and restoration of extracellular matrix integrity-collectively decelerating TAA advancement in our mouse model. These insights posit the mTORC1 axis as a strategic target for intervention, offering a novel approach to manage TAAs in MFS and potentially pave insights for current treatment paradigms.

Keywords: Biomechanicalpathogenesis; Marfan syndrome; Thoracic aortic aneurysm (TAA); Wall shear stress (WSS); mTORC1.

Biomechanical Approach of Thoracic Aortic Aneurysm Expansion

Fig. 1

Disturbed flow (DF) significantly upregulates the phosphatidylinositol 3-kinase-akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway in Marfan syndrome (MFS) patients. (A) Computed tomography angiography (CTA) reconstruction of a thoracic aortic aneurysm (TAA) in an MFS patient. (B, C) Computational fluid dynamic (CFD)-generated visualizations comparing DF and laminar flow (LF) in an MFS patient's TAA (B) and in a TAA of Fbn1^+/C1039G mice (C). Numbered boxes indicate representative areas under DF or LF patterns (boxes 1 and 2: DF; boxes 3 and 6: LF; boxes 4 and 5: DF). (D) A volcano plot highlights expression differences of differentially expressed genes (DEGs) between aortas under DF and LF from MFS patients, with 1,174 significant genes (260 upregulated in red, 914 downregulated in blue) between the DF (n = 2) and LF (n = 2) groups (P < 0.05). (E) A heatmap shows gene expression levels, with red indicating upregulation and blue indicating downregulation under LF (Control) and DF. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis from RNA-seq data contrasting DF with LF in MFS patient samples, where a false discovery rate (FDR) <0.01 denotes significant enrichment. (G) Gene Ontology (GO) enrichment analysis of DEGs between DF and LF. (H) Representative immunofluorescence images of phosphorylated S6 (p-S6, green) expression in the corresponding areas. (I) Representative western-blot images and analysis of P-S6 levels. Data are presented as mean ± standard error of the mean (SEM) for n = 6 per group. Statistical significance was determined by one-way analysis of variance (ANOVA), with ^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001, indicating significant differences; NS: not significant.

Fig. 2

Oscillatory shear stress (OSS) promotes mechanistic target of rapamycin complex 1 (mTORC1) pathway activation in a mouse model of Marfan syndrome (MFS). (A) Representative color Doppler ultrasound images illustrate aortic flow during systole in Fbn1^+/+ wild-type (WT) and Fbn1^+/C1039G mice at 30 week. Regions of oscillatory flow are indicated by white arrowheads. (B) Computational fluid dynamics (CFD) simulations visualize aortic velocity and flow streamlines, with disturbed flow patterns, indicated by red arrowheads, in both WT and Fbn1^+/C1039G mice. (C) Wall shear stress (WSS) distribution is mapped across the aorta, highlighting OSS regions, shown in dark blue and pointed out by red arrowheads. An area of representative OSS is delineated by a red dotted outline, contrasting with an area of normal WSS (NWSS) circumscribed by a yellow dotted outline. (D) En face immunofluorescence staining for phosphorylated ribosomal protein S6 (p-S6) is displayed in the aortic endothelium of both WT and Fbn1^+/C1039G mice. Areas subjected to OSS and NWSS are demarcated by red and yellow dotted outlines, respectively, with nuclei stained by 4′,6-diamidino-2-phenylindole (DAPI). (E, F) Magnified views of p-S6 staining within designated OSS (E) and NWSS (F) regions. (G) Quantitative assessment of p-S6 fluorescence intensity within OSS and NWSS areas demonstrates a significant upregulation of mTORC1 signaling in response to oscillatory shear. Data are mean ± standard error of the mean (SEM) for n = 6 mice per group. Statistical analyses were performed using one-way analysis of variance (ANOVA); significance is indicated by ^∗∗∗P < 0.001; NS: not significant.

Fig. 3

Mechanistic target of rapamycin complex 1 (mTORC1) pathway intensification in Fbn1^+/C1039G mouse aortas over time. (A) Morphology of ascending aortas at different growth stages. (B) Ultrasound showing aortic dilation (end-systolic diameter of the aorta) of Fbn1^+/C1039G mice at 8, 12, and 16 week. (C) The analysis of aortic root diameter tracking by ultrasound of Fbn1^+/+ wild-type (WT) and Fbn1^+/C1039G mice. (D) 3D reconstructions of aortic progression over time. (E) Computational fluid dynamic (CFD) analyses indicate increased disturbed flow correlating with aortic expansion. (F) Western blots and the relative protein level of phosphorylated S6 (p-S6), total S6 (t-S6), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the WT and Fbn1^+/C1039G mice at 8, 12, and 16 week. (G) Western blots and the relative protein level of p-S6, t-S6, and GAPDH of the WT, Fbn1^+/C1039G at 20 and 24 week. (H) Western blots and the relative protein level of phosphorylated S6 kinase (p-S6K), t-S6K, p-S6, t-S6, and β-actin of the WT and Fbn1^+/C1039G mice at 30 week. Data are presented as mean ± standard error of the mean (SEM) for n = 6 per group. Statistical significance was determined by one-way analysis of variance (ANOVA), with ^∗P < 0.05, and ^∗∗P < 0.01, indicating significant differences; NS: not significant.

Fig. 4

Rapamycin mitigates mechanistic target of rapamycin complex 1 (mTORC1) activation induced by oscillatory shear stress (OSS) in Fbn1^+/C1039G Mice. (A) Visualization of blood flow velocity and streamlines within the aortic lumen captures the dynamic hemodynamic alterations related to aneurysm progression and the disturbed flow of the three groups: wild-type (WT) littermates, Fbn1^+/C1039G mice, and Fbn1^+/C1039G mice subjected to long-term rapamycin treatment (Fbn1^+/C1039G + RA). (B) The presence of OSS across the aortic regions of the three groups: WT littermates, Fbn1^+/C1039G mice, and Fbn1^+/C1039G + RA, with OSS areas marked in gray. (C) Comparative aortic velocity mapping and cross-sectional OSS distribution among the three groups, with OSS zones delineated by white dashed lines. (D) Immunohistochemical detection of phosphorylated S6 (p-S6) in ascending aortic sections from the three groups. (E) Enlarged view of the OSS regions from the respective groups. (F) Quantification of p-S6 fluorescence intensity across the groups, indicating the modulation of mTORC1 signaling by rapamycin. (G–I) Western blot analysis profiling the expression of phosphorylated S6 kinase (p-S6K), total S6 kinase (t-S6K), phosphorylated S6 (p-S6), total S6 (t-S6), and statistical evaluation of protein expressions at 12 (G), 20 (H), and 30 week (I). Data expressed as mean ± standard error of the mean (SEM) for n = 6 per group. One-way analysis of variance (ANOVA) was utilized to assess statistical significance, denoted by ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001; NS: not significant.

Fig. 5

Rapamycin alleviates morphological and structural abnormalities in thoracic aortic aneurysm (TAA) of Fbn1^+/C1039G mice. (A) Ascending aortas from wild-type (WT), Fbn1^+/C1039G, and rapamycin-treated Fbn1^+/C1039G mice showcase morphological differences, with treatment durations noted. Fbn1^+/C1039G + RA: Fbn1^+/C1039G mice with long-term rapamycin treatment, starting from 8 and ended at 30 week; Fbn1^+/C1039G + RA (ST): Fbn1^+/C1039G mice with short-term rapamycin treatment, starting from 12 and ended at 16 week. (B) Representative ultrasound images showing the end-systolic diameter of the ascending aorta and arch from the WT, Fbn1^+/C1039G, and Fbn1^+/C1039G mice with long- and short-term rapamycin treatment. (C, D) The measurement of vessel diameters by serial ultrasound on aortic root (C) and ascending aorta (D) at 30 week. (E–G) Hematoxylin and eosin (H&E) (E), elastic van Gieson (EVG) (F), and Masson staining (G) of the aortas collected from 30-week-old mice. (H–K) Statistical analysis of the lumen area (H), media area (I), media thickness (J), and elastin degradation (K) among the four groups. The Mann-Whitney U test was used for evaluation of elastin degradation. Data are presented as mean ± standard error of the mean (SEM) for n = 6 per group. Statistical significance was determined by one-way analysis of variance (ANOVA), with ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 indicating significant differences; NS: not significant.

Fig. 6

Rapamycin attenuates vascular remodeling in the Fbn1^+/C1039G Marfan syndrome (MFS) mouse model. (A) mRNA expression of vascular smooth muscle cell (VSMC) markers α-smooth muscle actin (α-SMA) and smooth muscle 22 (SM22) quantified by real-time PCR in ascending aortas of wild-type (WT), Fbn1^+/C1039G, and long-term rapamycin-treated Fbn1^+/C1039G + RA mice. (B, C) Western blot (B) and analysis of relative protein levels (C) of smooth muscle myosin heavy chain (SM-MHC), α-SMA, SM22, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) across groups, with densitometry normalized to WT expression. (D) Immunofluorescence for phosphorylated S6 (p-S6) and α-SMA in aortic sections from each group, visualized with dual labeling (green and red, respectively). (E) Analysis of fluorescence intensity for p-S6 and α-SMA, highlighting the impact of rapamycin on molecular signaling and VSMC phenotype. (F) Further immunofluorescence for α-SMA and SM22, with quantification of fluorescence intensity. (G) Analysis of the fluorscence intensity of α-SMA and SM22. Data are presented as mean ± standard error of the mean (SEM) for n = 6 per group. Statistical significance was determined by one-way analysis of variance (ANOVA), with ^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001 indicating significant differences; NS: not significant.

Fig. 7

Rapamycin attenuates vascular remodeling by reducing inflammation, oxidative stress, and medial layer hyperplasia. (A) Expression levels of matrix metalloproteinases (MMP-2 and MMP-9) evaluated by real-time PCR, normalized to a reference gene. (B) Western blotting reveals differential expression of pro and active forms of MMP-9 and MMP-2 among the wild-type (WT), Fbn1^+/C1039G, and Fbn1^+/C1039G + RA mice. (C) Densitometry of MMPs, with relative band intensities compared to WT controls. (D–G) Immunofluorescent staining and fluorescence intensity for MMP-9, CD68 (D, E) and MMP-2 (F, G) in aortic sections. (H) Hematoxylin and eosin (H&E) and Sirius red staining of aortic sections, demonstrating decreased medial thickening and collagen deposition after rapamycin therapy. (I, J) Medial cellularity (I) and collagen content (J) quantified from stained sections, indicating reduced smooth muscle cell hyperplasia and collagen overaccumulation following treatment. Data are presented as mean ± standard error of the mean (SEM) for n = 6 per group. Statistical significance was determined by one-way analysis of variance (ANOVA), with ^∗P < 0.05, ^∗∗P < 0.01 and ^∗∗∗P < 0.001 indicating significant differences; NS: not significant.

Fig. 8

Schematic diagram of mechanistic target of rapamycin complex 1 (mTORC1) pathway-mediated thoracic aortic aneurysm expansion under oscillatory shear stress (OSS) in Marfan syndrome. TAA: thoracic aortic aneurysm; SMC: smooth muscle cell; ECM: extracellular matrix; MVs: micro vessels; MMPs: matrix metalloproteinases; FAK: focal adhesion kinase.