Identification of type IV collagen exposure as a molecular imaging target for early detection of thoracic aortic dissection

Figure 1. Pathological features of TAD in different stages. Mice were fed BAPN for 0, 1, 2, 3, and 4 weeks, respectively, to simulate the different stages of TAD development. (A) Staining of elastin in the aortic arch in mice. High-power view (20X) of the rectangular areas is shown in the lower rows in each panel (10X). Scale bars on the upper and lower panels, 100 μm and 50 μm, respectively. (B) Representative photographs of macroscopic features of TAD in different groups. (C) Ultrasound of the aortic arch from different groups (scale bar, 2 mm). (D) Evans blue staining of the aortic arch from different groups. Arrow indicates endothelial injury (scale bar, 5 mm). (E) Vessel diameters in mice from different groups. Data are presented as median ± interquartile range (IQR), n = 6 for each time point. The probability value was calculated with K-W test. (F) Rupture rates in mice in progressive TAD models, n = 128. The normality of each group was validated with Kolmogorov-Smirnov test (K-S test).

Figure 2. Collagen exposure after endothelial injury as an early signal of TAD development. (A) Frozen sections of the aortic arch in control mice were stained for Col-I/III/IV (scale bar, 25 μm). (B) Sirius Red staining of the aortic arch at different time points (red arrow, Col-I; green arrow, Col-III; faint yellow arrow, Col-IV; white dashed line, intima; scale bar, 25 μm). (C) Frozen sections of the aortic arch from different groups, stained for Col-IV (scale bar, 10 μm). (D) Correlation between percentage Col-IV-positive area in the aortic arch and stage of TAD development (r2 = 0.9624, p < 0.0001, n = 10 for each time point). The correlation coefficient and probability value were calculated with Spearman's correlation test. (E) Immunohistochemistry for Col-IV in normal and TAD human tissues (scale bar, 25 μm). (F) Percentage Col-IV-positive area in D (median ± IQR, n = 6, p = 0.002). The probability value was calculated with Mann-Whitney U test. * indicate the vascular lumen. The normality of each group was validated with Kolmogorov-Smirnov test (K-S test).

Figure 3. Characterization of Col-IV-DOTA-Gd-RhB molecular probes. (A) HPLC for Col-IV and CDR. (B) Ultraviolet absorption spectrum for CDR. (C) Fluorescence curve for CDR at different concentrations. (D) Fluorescence intensities of CDR in SMCs at different concentrations (r2 = 0.9590, p < 0.0001, n = 4). The Correlation coefficient and probability value were calculated with Spearman's correlation test. (E) Unbound relaxivity of CDR at 7 T. The relaxivity of unbound CDR at 7 T was obtained by measuring T1 at concentrations of 0-0.25 mM in PBS. (F) R1 value measured by MRI was linearly correlated with Gd3+ content (r2 = 0.9920, p < 0.0001). The Correlation coefficient and probability value were calculated with Pearson's correlation test. (G) Bound relaxivity of CDR at 7 T. The relaxivity of bound CDR in SMCs at 7 T was measured by incubating SMCs with CDR at 0-0.30 μM. (H) R1 value measured by MRI was linearly correlated with Gd3+ content (r2 = 0.9930, p < 0.0001). The Correlation coefficient and probability value were calculated with Pearson's correlation test. The normality of each group was validated with Kolmogorov-Smirnov test (K-S test).

Figure 4. Target specificity of CDR in vitro. (A) Immunofluorescence staining in VSMCs, macrophages, and ECs after incubation with CDR for 24 h (red, CDR; green, Col-IV, scale bar, 25 μm). (B) Percentage of positive cells (CDR+) in the three groups after incubation with CDR for 24 h. The probability value of each point was calculated with K-W test. The correlation coefficient was calculated with Spearman's correlation test (r2 = 0.9643, p < 0.0001; median ± IQR, n = 4). (C) Cytotoxicity (Hoechst- cells / Total cells) calculated by flow cytometry after incubation with CDR for 24 h (median ± IQR, n = 4). The probability value of each point was calculated with K-W test. The normality of each group was validated with Kolmogorov-Smirnov test (K-S test).

Figure 5. MRI and BLI of TAD in mice. (A) MRI, BLI and Pathological imaging of TAD in mice administered BAPN. (B) Line indicates cross-sectional MRI slice of descending aorta where T1 signal enhancement was acquired. (C) Representative MRI images of mice 6 h after injection with CG and CDR, following BAPN administration for 0, 2, and 4 weeks. Mice were examined by BL after MR imaging. The red arrows for the CG or CDR groups indicate the same position. (D) Quantification of fluorescence efficiency in thoracic aorta and kidney after CDR injection at different stages of TAD (mean ± SD, n = 4). The probability value of each group was calculated with one way ANOVA and adjusted with the post hoc test. (E) MRI (upper), fluorescence (middle) and histology (bottom) images of the aortas from WT mice treated with BAPN for different times (arrow indicates tear or elastin disorder; scale bar, 100 μm). (F) R1 value of descending aorta wall in mice administered BAPN for different times (median ± IQR, n = 6). The probability value of each group was calculated with Mann-Whitney U test and adjusted with the Bonferroni correction. (G) Quantification of percentage RhB-positive area at different times (median…

Figure 6. Correlation between %NSE calculated by CDR-enhanced MRI and severity of TAD. (A) CDR-enhanced MRI after 2 weeks of BAPN administration, and severity of dissection detected by elastin staining after 4 weeks of BAPN administration. (B) MRI (upper) and corresponding elastin-staining (bottom) images of ascending aorta and descending aorta, respectively, at different stages of TAD development (white dashed line indicates ROIs). MRI was performed after 2 weeks of BAPN administration and elastin staining was performed after death or 4 weeks of BAPN administration in mice. (C) Survival curves for two groups (%NSE ≥ median and %NSE < median) (p = 0.0043, n = 10). (D) Quantitative analysis of %NSE in both groups (median ± IQR, p = 0.008, n = 5). The probability value of each point was calculated with Mann-Whitney U test. (E) Correlation between %NSE and rupture time in all mice (r2 = 0.8482, p = 0.001, n = 10). The correlation coefficient was calculated with Spearman's correlation test. (F) Correlation between %NSE and rupture time in the death group (r2 = 0.8281, p = 0.032, n = 5). The correlation coefficient was calculated with Pearson's correlation test. The normality of each group was…