Innovative Therapy with Stem Cell-Derived Extracellular Vesicles on Cardiac Hypertrophy in an Animal Model of Atherosclerosis; Elucidation of the Molecular Mechanisms Involved in the Repair Process

Figure 1. Analysis of body (A) and heart weights (B) collected at the end of the diet and treatment period for the seven experimental groups. Values are expressed as mean ± standard deviation (SD). The values were calculated by two-way ANOVA, Bonferroni post-test.

Figure 2. Representative anatomical observations of the abdominal and thoracic cavities in experimental groups of animals subjected to different dietary and therapeutic conditions. The first panel (24C) illustrates the control group with normal liver morphology and organ appearance, indicative of a standard physiological state. The second row (81HH) displays a subject from the hypertensive–hyperlipidemic (HH) group, with pathological changes including an enlarged, yellowish, and fatty liver consistent with hepatic steatosis, together with a more congested appearance of the visceral tissues. In the third row, a subject from one of the treated groups (54HH-Smad2/3 siRNA) shows visible improvements in liver morphology, with a reduction in the yellowish discoloration and fatty texture. The overall appearance of the abdominal and thoracic organs in the treated animals appears to be more consistent with that of the control group, indicating a therapeutic effect of the administered treatment.

Figure 3. Left ventricular structural and functional parameters assessed across experimental groups following dietary and therapeutic interventions. Left ventricular cavity size (mm2) (B), relative wall thickness (RWT) (D.1), and left ventricular fractional shortening (%) (D.2) were evaluated to assess cardiac remodeling and function. (A) Left ventricle—internal cavity—representative recordings, long axis, in B-mode; (B) graphical representation of internal cavity (mm2); (C) left ventricle—diameter in diastole and systole/anterior and posterior wall thickness—representative recordings in M-mode; (D.1) graphical representation of relative wall thickness; (D.2) graphical representation of left ventricular shortening fraction. The data presented herein are expressed as means ± standard deviation (SD), based on duplicate measurements. Statistical significance is indicated as follows: ** p < 0.01, and * p < 0.05 compared to the control group; ## p < 0.01 compared to the HH group. Two-way ANOVA, Bonferroni post-test was applied.

Figure 4. ELISA quantification of plasma levels for TGF-β1 and AngII for the seven experimental groups is presented as means ± standard deviation (SD) based on triplicate measurements. Statistical significance is indicated as follows: ** p < 0.01, *** p < 0.005 compared to the control group; # p < 0.05, and ### p < 0.005 compared to the HH group. Two-way ANOVA, Bonferroni post-test was applied.

Figure 5. Assessment of reactive oxygen species production (ROS) in the left ventricle of the seven experimental groups at the end of 4 months of diet and/or treatment. (A) Representative fluorescence and phase-contrast images taken under an inverted microscope (20× magnification) to highlight the level of reactive oxygen species in the left ventricle of the C, HH, HH-EVs (ADSCs), HH-EVs (BMMSCs), HH-EVs (ADSCs) + Smad2/3 siRNA, HH-EVs (BMMSCs) + Smad2/3 siRNA, HH-Smad2/3 siRNA. (B) Graphical representation of the levels of reactive oxygen species in the left ventricle. Data are presented as mean ± standard deviation (SD) based on duplicate measurements. Statistical significance is indicated as *** p < 0.005, ** p < 0.01 compared to the control group; ### p < 0.005, ## p < 0.01, compared to the HH group. Two-way ANOVA, Bonferroni post-test was applied.

Figure 6. Illustrative immunofluorescence images showing inflammatory markers and cell types associated with cardiac hypertrophy and atherosclerosis at the end of the diet and/or treatment. Thin cryosections of the left ventricle collected from the seven experimental groups were labeled as follows: (A.1) MIF, (B.1) ANF, (C.1) COL1A, (D.1) Cx43, (E.1) CTN1, (F.1) α-SMA, (G.1) T cells, (H.1) macrophages, (I.1) M1 macrophages, and (J.2) M2 macrophages. Nuclei were visualized by DAPI staining in blue fluorescence. Images were taken at a total magnification of 20×. (A.2–J.2)—graphical representation of the levels of the assessed markers in the left ventricle. Data are presented as mean ± standard deviation (SD) based on triplicate measurements. Statistical significance is indicated as *** p < 0.005, ** p < 0.01, and * p < 0.05 compared to the control group; ### p < 0.005, compared to the HH group. Two-way ANOVA, Bonferroni post-test was applied.

Figure 7. Western blot analysis results and representative images of the blots for the proteins analyzed, as follows: (A.1,A.2)—SMAD2, (B.1,B.2)—Nf-κB p50 and GAPDH in the left ventricle of the seven experimental hamster groups. The charts summarize protein levels in all investigated groups based on four independent experiments conducted after four months of diet and/or treatment. Data are presented as mean ± standard deviation (SD). Protein expression levels were normalized to GAPDH. Statistical significance is indicated as * p < 0.05 compared to the control group; and # p < 0.05 compared to the HH group. Two-way ANOVA, Bonferroni post-test was applied.