Regnase-1 overexpression as a therapeutic approach of Marfan syndrome

Figures

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  Figure 1. Decreased regnase-1 expression in aortic SMCs of mgR/mgR mice and Marfan patients (A) Representative images showing regnase-1 expression in murine thoracic aortic tissue (red). Nuclei were stained with DAPI (blue), and the autofluorescence of elastic fibers was visualized on the green channel. The scale bar represents 50 μm, and asterisks show the aortic lumen). (B) Statistical quantification of mean red fluorescence intensity in immunohistochemistry pictures (n = 5, 15 images analyzed/group). Asterisks show the aortic lumen. (C) Illustrative images showing regnase-1 expression in human thoracic aortic tissue from controls and Marfan patients. The scale bar represents 50 μm. (D) Statistical quantification of red mean fluorescence intensity in the media of human samples (n = 4, 16 images analyzed/group). (E) Illustrative immunohistochemistry image showing MALT-1 expression (magenta) in murine thoracic aortic cryosections. Elastin fluorescence was visualized on the green channel. DAPI (blue) was used to mark cell nuclei. Asterisks show the aortic lumen. (F) Corresponding quantification of mean fluorescence intensity as a marker for MALT-1 expression (n = 5, 15 images analyzed/group).

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  Figure 2. Regnase-1 overexpression decreases proinflammatory markers and elastin degradation in mouse SMCs in vitro (A) Illustrative images of western blots showing protein levels of regnase-1 and β-actin as loading control in cell lysates of murine SMCs following AAV9SLR transduction. (B) Corresponding statistical quantification of regnase-1 levels in treated cells. Values were normalized to EGFP-treated SMCs (3 independent experiments). (C) Illustrative images of western blots showing protein levels of VCAM-1 and β-actin as loading control in cell lysates of murine SMCs in the depicted treatment groups. (D) Corresponding statistical quantification of VCAM-1 level. EGFP-transduced cells served as controls (3 independent experiments). (E) Degree of macrophage migration toward the supernatant of SMCs in the depicted treatment groups, indicating different levels of promigratory cytokines (3 independent experiments). (F) Assessment of NF-κB activation in murine SMC in different treatment groups (3 independent experiments). (G) Degree of elastin degradation in treated murine SMCs measured fluorometrically (3 independent experiments). (H and I) Statistical quantification of relative mRNA levels…

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  Figure 3. Regnase-1 overexpression decreases MMP9 expression and activity in mouse SMCs in vitro (A) Illustrative images of western blots showing protein levels of MMP9 and β-actin, used as loading control in cell lysates of murine SMCs in different treatment groups. (B) Corresponding quantification of relative MMP9 protein levels following transduction (3 independent experiments). (C) Expression levels of MMP9 in murine SMCs in different treatment groups. GAPDH was used as a housekeeping gene (3 independent experiments). (D) Degree of gelatin degradation in murine SMCs in different treatment groups (3 independent experiments).

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  Figure 4. Targeted transgene overexpression following AAV injection in male mgR/mgR mice (A) Schematic representation of the experimental design of the in vivo study. (B) Representative immunohistochemistry images showing EGFP expression in murine thoracic aortic tissue (red) for mice injected with AAV9-G2CNN-EGFP (upper line) and AAV9-G2CNN-regnase-1 (lower line). Boxed images show successful transduction of both endothelial and SMCs. Nuclei were stained with DAPI (blue). The autofluorescence of elastic fibers was visualized on the green channel. Asterisks show the aortic lumen. The scale bar represents 25 μm. (C) Statistical quantification of mean red fluorescence intensity showing EGFP levels within the aortic wall following injection of AAV9-G2CNN (n = 3, 9 images analyzed/group). (D) Illustrative images of western blots showing protein levels of regnase-1 and β-actin as loading control in tissue lysates of murine aortas following transduction. Tissue isolated from WT mice served as control. (E) Corresponding statistical quantification of protein levels of regnase-1 in depicted treatment groups and WT mice (n = 5 for WT mice, n = 6 for AAV-EGFP injected mice, n = 7 for AAV-regnase-1…

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  Figure 5. Regnase-1 overexpression improves elastin architecture and decreases aortic aneurysm size in male mgR/mgR mice (A) Representative images of thoracic aortic cryosections subjected to elastic van Gieson staining. Elastic fibers appear black and collagen appears red. Arrows indicate islands of damage. The scale bar represents 25 μm. (B) Statistical quantification of the number of islands of damage in the media in different treatment groups of mgR/mgR mice and WT mice as controls (n = 7). (C) Representative echocardiographic images of the thoracic aorta for different treatment groups and WT mice used as controls. (D) Illustrative image showing areas where aortic size was measured in an ultrasound image. (E) Statistical quantification of aortic diameter at aortic annulus (L1), sinotubular junction (L2), aortic arch (L3), and proximal descending aorta (L4) in mice subjected to the designed gene therapy approach and WT mice (n = 7 for WT mice and AAV-EGFP-injected mice, n = 10 for AAV-regnase-1-injected mice).

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  Figure 6. Aortic regnase-1 overexpression decreases MMP expression and inflammation in male mgR/mgR mice (A) Representative images showing MMP9 expression in murine thoracic aortic tissue (red) for the depicted treatment groups and WT mice. Nuclei were stained with DAPI (blue). The autofluorescence of elastic fibers was visualized on the green channel. Asterisks show the aortic lumen. The scale bar represents 50 μm. (B) Statistical quantification of mean red fluorescence intensity in immunohistochemical analyses as a measure of MMP9 expression in the aortic wall of different treatment groups and WT mice (n = 7 for WT mice, n = 6 for AAV-EGFP-injected mice, n = 8 for AAV-regnase-1-injected mice, 3 images/section. (C) Illustrative images showing VCAM-1 expression (red) in thoracic aortic sections from mice in the depicted groups. DAPI (blue) was used as a nuclear counterstaining, and elastin autofluorescence is shown in green. The scale bar represents 50 μm, and asterisks depict the aortic lumen. (D) Statistical quantification of VCAM-1 protein level in thoracic aortic tissue. WT mice served as controls (n = 5 for WT mice and AAV-regnase-1-injected mice, n = 4 for AAV-EGFP-injected mice, 3…