Elastin Insufficiency Confers Proximal and Distal Pulmonary Vasculopathy in Mice, Partially Remedied by the KATP Channel Opener Minoxidil: Considerations and Cautions for the Treatment of People With Williams-Beuren Syndrome

Figures

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  Figure 1. Residual increased right ventricular pressure estimate in WBS participants with history of pulmonary artery intervention. (A) shows the TRVmax recorded by echocardiogram. RVSPe can be approximated from TRVmax using Bernoulli's equation as RVSPe=4*(TRVmax)2 + right atrial (RA) pressure. (B) illustrates Z-scored measures of main (MPA), left (LPA) and right (RPA) pulmonary artery lumen diameter. In both figures, WBS participants with a history of PA intervention are red, WBS participants with no PA intervention are black and controls are gray. A mean line +/- SD line is shown for each group. Open circles represent individuals who had SVAS repair. Closed circles are used for those not requiring SVAS repair. Multiple comparison testing (after ANOVA) by Tukey. * p < 0.05, ** p < 0.01.

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  Figure 2. Sex and Eln genotype differences in hemodynamic parameters. (A) shows RVSP in closed circles and RVDP in open circles in male and female Eln+/+ and Eln+/− mice. (B) depicts HR in the same groups. (C–E) report heart mass as normalized to tibial length findings. (C): RVHM/TL, (D): LVHM/TL (this measure includes the interventricular septum and the LV in the LVHM measure), (E): tibial length. Eln+/+ data are presented in black and Eln+/− in red in all figures. A mean line +/- SD is shown for each sex/genotype combination. Multiple comparison testing (after ANOVA) by Tukey. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns is not significant.

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  Figure 3. Sex and Eln genotype differences in pulmonary artery morphology. (A) reports the relationship between intravascular pressure (x axis) and LPA diameter (y axis). (B–E) show VVG stained images of male Eln+/+ (B), male Eln+/− (C), female Eln+/+ (D), and female Eln+/− (E) main pulmonary artery. (F) shows lamellar number. (G) is the MPA internal medial circumference and (H) shows the medial thickness for the various sex-genotype groups. Eln+/+ data are presented in black and Eln+/− in red in all figures. In (A), mean +/- SD are shown for all points. Males are shown as dashed lines and females as solid lines. For (F–H), a mean line +/- SD is shown for each sex/genotype combination. Multiple comparison testing (after ANOVA) by Tukey. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns is not significant.

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  Figure 4. Minoxidil increases pulmonary artery caliber. (A) reports the relationship between intravascular pressure (x axis) and LPA diameter (y axis) for treated and untreated Eln+/− mice. (B–D) depict measures collected from unloaded histological sections of main pulmonary arteries. (B) shows the internal medial circumference. (C) is lamellar number and (D) shows the medial thickness. (E–G) show in vivo arterial diameter measures for the main (E), left (F) and right (G) pulmonary arteries. Eln+/+ data are presented in black and Eln+/− in red. Untreated mice are depicted with closed circles and treated (Tx) mice are shown with open circles. In (A), mean +/- SD are shown for all points. A mean line +/- SD is shown for each treatment/genotype combination in (B–G). Unpaired t-tests were used in (B–D). Multiple comparison testing (after ANOVA) by Tukey [(A) and (E–G)]. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns is not significant.

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  Figure 5. Minoxidil reduces right ventricular pressure and increases heart size in Eln+/− mice. (A) shows RVSP in circles and RVDP in squares in treated and untreated Eln+/− mice. (B) depicts HR in the same groups. (C,D) report heart mass as normalized to tibial length findings. (C): RVHM/TL and (D): LVHM/TL (this measure includes the interventricular septum and the LV in the LVHM measure). (E) depicts Estimated Left Ventricular Mass Index (LVMI) assessed using echocardiogram. Eln+/+ data are presented in black and Eln+/− are in red. Untreated mice are depicted with closed circles and minoxidil treated (Tx) mice are shown with open circles. A mean line +/- SD is shown for each Tx/genotype combination. Unpaired t-test was used for (A) and (B), Mann-Whitney for (C) and (D), and one-way ANOVA for (E). Multiple comparison testing (after ANOVA) by Tukey. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns is not significant.

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  Figure 6. Eln+/− mice display evidence of increased vascular resistance, not impacted by minoxidil. Panels A-C show pulmonary artery pulse wave Doppler. The typical parabolic trace is seen in the Eln+/+ (A), while a shelf-like systolic notch is present in the majority of treated and untreated Eln+/− mice (B) white arrow. In some cases, a second distinct systolic inflection, consistent with human systolic notching, becomes apparent (C) red arrow. Chi-square analysis (D) reports the frequency of notching in this cohort. (E) shows pulmonary artery acceleration time (PAAT). PAAT is associated with elevated pulmonary pressure and vascular resistance. In (D), Eln+/+ data are presented in black, untreated Eln+/− in red, and treated Eln+/− in blue. Mice without a notch are shown in solid bars and those with systolic notching have open bars. In (E), Eln+/+ data are presented in black and Eln+/− are in red. Untreated mice are depicted with closed circles and minoxidil treated (Tx) mice are shown with open circles. A mean line +/- SD is shown for each Tx/genotype combination. Multiple comparison testing (after ANOVA) by Tukey. *** p < 0.001 and ns is not significant.

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  Figure 7. Elastin insufficiency reduces PA branching angle and increases proximal arterial segment length. (A) (Eln+/+) and (B) (Eln+/−) contain a volume reconstruction of a lung set generated through ex vivo vascular casting and microCT. (C) (Eln+/+) and (D) (Eln+/−) are the same lung sets thresholded for large vessel analysis. The LPLA (asterisk denotes fourth branch) and RPLA (star marks the sixth branch) are noted with red lines to illustrate a typical path length measured by the software for each vessel. An arrowhead notes the position of the LULFA for small vessel analysis (see Figure 8) and yellow lines are an approximation of the angle formed between the LPA and RPA. This branch angle is evaluated in (E) at P1, P7, P30, and P90. The LPLA length (F) and tortuosity (G) are shown at the same time points. In (E–G), Eln+/+ data are presented in black and Eln+/− are in red. A mean line +/- SD are shown for each age/genotype combination. Multiple comparison testing (after ANOVA) by Sidak. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns is not significant (n = 5–8/group).

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  Figure 8. Elastin insufficiency shows diminished vascular branching early in development giving rise to increased branching in adults. Generation-specific arcade length [GSAL; (A–D)] and generation-specific branch number [GSBN; (E–H)] were evaluated at P1, P7, P30, and P90. Eln+/+ data are presented in black and Eln+/− are in red. Mean +/- SD are shown for each age/genotype combination. Multiple comparison testing (after ANOVA) by Sidak. * p < 0.05, *** p < 0.001, and ns is not significant (n = 5–8/group).

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  Figure 9. Minoxidil does not impact post-natal branching patterns in Eln+/− mice. A lack of treatment effect for minoxidil is evident in P90 left pulmonary lobar artery (LPLA) length (A), tortuosity (B) and LPA-RPA branching angle (C). Similarly, no treatment differences are noted in generation-specific arcade length (D) or branch number (E). Untreated Eln+/− values are in closed circles and treated Eln+/− values are depicted with open circles. In (A–C), a mean line +/- SD are shown for each timepoint/genotype combination. In (D,E), mean +/- SD are shown for all points. ns is not significant and n = 5–6/group for (D,E).

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  Figure 10. Developmental time-course of Elastin haploinsufficient vasculature. Early (P1 and P7) in development, distal Eln+/− PAs exhibit less branching than the Eln+/+. As the lungs grow and mature, the distal vasculature in the Eln+/− branch more - catching and eventually surpassing the Eln+/+ counterpart later (P30 and P90) in development. This suggests a possible compensatory response to elastin insufficiency mediated changes in pulmonary physiology that occur over a wide developmental window.