Targetable cellular signaling events mediate vascular pathology in vascular Ehlers-Danlos syndrome

Abstract

Vascular Ehlers-Danlos syndrome (vEDS) is an autosomal-dominant connective tissue disorder caused by heterozygous mutations in the COL3A1 gene, which encodes the pro-α 1 chain of collagen III. Loss of structural integrity of the extracellular matrix is believed to drive the signs and symptoms of this condition, including spontaneous arterial dissection and/or rupture, the major cause of mortality. We created 2 mouse models of vEDS that carry heterozygous mutations in Col3a1 that encode glycine substitutions analogous to those found in patients, and we showed that signaling abnormalities in the PLC/IP3/PKC/ERK pathway (phospholipase C/inositol 1,4,5-triphosphate/protein kinase C/extracellular signal-regulated kinase) are major mediators of vascular pathology. Treatment with pharmacologic inhibitors of ERK1/2 or PKCβ prevented death due to spontaneous aortic rupture. Additionally, we found that pregnancy- and puberty-associated accentuation of vascular risk, also seen in vEDS patients, was rescued by attenuation of oxytocin and androgen signaling, respectively. Taken together, our results provide evidence that targetable signaling abnormalities contribute to the pathogenesis of vEDS, highlighting unanticipated therapeutic opportunities.

Keywords: Cardiology; Collagens; Genetic diseases; Mouse models; Vascular Biology.

Figure 1. Col3a1^G209S/+ and Col3a1^G938D/+ mice recapitulate vEDS phenotypes.

(A) Sanger sequencing of genomic DNA confirmed the intended Col3a1 c.625_626GG>TC corresponding to G209S. (B) Sanger sequencing of genomic DNA confirmed the intended Col3a1 c.2813G>A corresponding to G938D. (C) Kaplan-Meier survival curve for comparing Col3a1^+/+ (n = 53) to Col3a1^G209S/+ mice (n = 79), which died from vascular rupture or dissection. Significant differences were calculated using log-rank (Mantel-Cox) analysis. (D) Kaplan-Meier survival curve for comparing Col3a1^+/+ (n = 78) to Col3a1^G938D/+ mice (n = 51), which died from vascular rupture or dissection. Significant differences were calculated using log-rank (Mantel-Cox) analysis. (E) Quantification of collagen content in aortic cross sections, as measured by normalized PSR intensity. Error bars show mean ± SEM. Asterisks signify significant differences using 1-way ANOVA with Dunnett’s multiple comparisons post hoc test. ****P < 0.0001, DF = 2, F = 13.97. (F) Quantification of elastin breaks in VVG-stained aortic cross sections. Error bars show mean ± SEM. Asterisks signify significant differences using Kruskal-Wallis with Dunn’s multiple comparisons post hoc test. *P < 0.05, **P < 0.01. (G) Quantification of aortic wall thickness in aortic cross sections. Error bars show mean ± SEM. Asterisks signify significant differences using 1-way ANOVA with Dunnett’s multiple comparisons post hoc test. **P < 0.01, DF = 2, F = 10.16. (H) Histological staining (H&E = hematoxylin & eosin, VVG = Verhoeff Van Gieson, Masson’s Trichrome, and PSR = Picrosirius Red) of wild-type and vEDS aortic cross sections. Scale bars: 50 μm.

Figure 2. vEDS aortas display a molecular signature for excessive PKC/ERK signaling.

(A) Unsupervised hierarchical clustering using the most differentially expressed genes from RNAseq was performed, and vEDS samples clustered separately from controls. (B) Upstream analysis based on differentially expressed genes. Significant enrichment was determined using Fisher’s exact test. (C) Representative Western blot analysis of pPKCβ and pERK comparing Col3a1^+/+ to Col3a1^G209S/+ proximal descending aortas. (D) Representative Western blot analysis of pPKCβ and pERK comparing Col3a1^+/+ to Col3a1^G938D/+ proximal descending aortas. (E) Quantification of pPKCβ and pERK levels normalized to β-actin loading control comparing Col3a1^+/+ (n = 6) to Col3a1^G209S/+ (n = 6) and Col3a1^G938D/+ (n = 8) aortas. Error bars show mean ± SEM. Asterisks signify significant differences using 2-tailed Student’s t test (pERK/G209S T = 2.053, DF = 16; pPKCβ/G209S T = 2.950, DF = 10; pERK/G938D T = 2.770, DF = 13; *P < 0.05) or Mann-Whitney test (G938D/pPKCβ; *P < 0.05) depending on Shapiro-Wilk normality tests.

Figure 3. Inhibition of excessive PKCβ or ERK signaling prevents death due to aortic dissection.

(A) Kaplan-Meier survival curve comparing Col3a1^G938D/+ (n = 93) to Col3a1^G938D/+ (n = 16) mice receiving ruboxistaurin in the diet starting at weaning and continuing for 40 days. Significant differences were calculated using log-rank (Mantel-Cox) analysis. P21 = postnatal day 21. (B) Representative Western blot and analysis of pPKCβ and pERK comparing Col3a1^+/+ (n = 6) to Col3a1^G938D/+ (n = 6) proximal descending aortas and quantification of pPKCβ and pERK levels normalized to β-actin loading control for vEDS aortas. Error bars show mean ± SEM. Asterisks signify significant differences using 1-way ANOVA with Dunnett’s multiple comparisons post hoc test. **P < 0.01, ***P < 0.001, DF = 3, F = 13. (C) Kaplan-Meier survival curve comparing Col3a1^G938D/+ (n = 93) to Col3a1^G938D/+ (n = 20) mice receiving cobimetinib in the drinking water starting at weaning and continuing for 40 days. Significant differences were calculated using log-rank (Mantel-Cox) analysis. P21 = postnatal day 21. All findings from drug trials are based on analyses using a universal control group with n = 93 across all drug tests that started at P21. FDR-adjusted P values are presented in Supplemental Table 3.

Figure 4. Androgen signaling at puberty increases the risk of aortic rupture.

(A) Kaplan-Meier survival curve comparing Col3a1^G938D/+ (n = 51) to Col3a1^G938D/+ (n = 41) mice receiving hydralazine from birth. (B) Kaplan-Meier survival curve comparing female Col3a1^G938D/+ (n = 24) to female Col3a1^G938D/+ (n = 23) mice receiving hydralazine starting from birth and bicalutamide starting from weaning (n = 9). P values shown are between untreated mice and mice receiving hydralazine (lower) and mice receiving hydralazine and mice receiving hydralazine and bicalutamide (upper). (C) Kaplan-Meier survival curve comparing male Col3a1^G938D/+ (n = 27) to male Col3a1^G938D/+ mice receiving hydralazine starting from birth (n = 18) and male Col3a1^G938D/+ mice receiving hydralazine starting from birth and bicalutamide starting from weaning and continuing for 2 months (n = 18). P values shown are between untreated mice and mice receiving hydralazine (lower) and mice receiving hydralazine and mice receiving hydralazine and bicalutamide (upper). (D) Kaplan-Meier survival curve comparing Col3a1^G938D/+ (n = 93) mice to Col3a1^G938D/+ mice receiving bicalutamide (n = 20). (E) Kaplan-Meier survival curve comparing Col3a1^G938D/+ (n = 93) mice to Col3a1^G938D/+ mice receiving hydralazine and spironolactone (n = 16). (F) Representative Western blot analysis of pERK comparing Col3a1^+/+ (n = 9) to Col3a1^G938D/+ (n = 7) mice, Col3a1^G938D/+ mice on hydralazine sampled at age P40 (Hydral (E), n = 5), Col3a1^G938D/+ mice on hydralazine and spironolactone sampled at age P70 (Hydral (L)+spiro, n = 5), and Col3a1^G938D/+ mice on hydralazine sampled at age P70 (Hydral (L), n = 5) proximal descending aortas. (G) Quantification of pERK levels normalized to β-actin–loading control for aortas. Error bars show mean ± SEM. Asterisks signify significant differences using 1-way ANOVA with Dunnett’s multiple comparisons post hoc test. **P < 0.01, *P < 0.05, DF = 4, F = 7.07. For all survival curves, significant differences were calculated using log-rank (Mantel-Cox) analysis. P0, postnatal day 0; P21, postnatal day 21; hydral, hydralazine; bical, bicalutamide; spiro, spironolactone. All findings from drug trials are based on analyses using a control group with n = 93 across all drug tests. FDR-adjusted P values are presented in Supplemental Table 3.

Figure 5. Oxytocin signaling during lactation increases the risk of aortic rupture.

(A) Kaplan-Meier survival curve for lactating Col3a1^G209S/+ mice (n = 22) compared with never-pregnant female (n = 55) Col3a1^G209S/+ mice. (B) Kaplan-Meier survival curve comparing Col3a1^G209S/+ lactating (n = 22) mice to Col3a1^G209S/+ females with pups removed on the day of delivery thereby preventing lactation and eliminating the lactation-induced prolonged elevation of oxytocin (n = 13). (C) Kaplan-Meier survival curve comparing Col3a1^G209S/+ lactating mice (n = 22) to Col3a1^G209S/+ females with oxytocin receptor antagonist (OTA) administered via a continuous subcutaneous infusion pump implanted at the end of the third week of gestation and continued through the 4 weeks of lactation, for a total of 5 weeks of treatment (n = 10). (D) Kaplan-Meier curve demonstrating the survival of Col3a1^G209S/+ lactating mice treated with trametinib (n = 21), a MEK inhibitor, initiated at the start of the third week of pregnancy and continued through 4 weeks of lactation, in comparison to untreated lactating Col3a1^G209S/+ mice (n = 22). (E) Kaplan-Meier curve demonstrating the survival of Col3a1^G209S/+ lactating mice treated with hydralazine (n = 23) initiated at the start of the third week of pregnancy and continued through 4 weeks of lactation, in comparison to lactating untreated Col3a1^G209S/+ mice (n = 22). (F) Kaplan-Meier curve demonstrating the survival of Col3a1^G209S/+ lactating mice treated with propranolol (n = 8), initiated at the start of the third week of pregnancy and continued through 4 weeks of lactation, in comparison to lactating untreated Col3a1^G209S/+ mice (n = 22). For all survival curves, significant differences were calculated using log-rank (Mantel-Cox) analysis, and controls are pooled analyses of n = 22 mice. FDR-adjusted P values are presented in Supplemental Table 3.

Figure 6. Inhibition of oxytocin signaling decreases ERK activation and ERK target gene expression.

(A) Representative Western blot analysis of pERK1/2 in the proximal descending aorta comparing Col3a1^+/+ never-pregnant, Col3a1^+/+ lactating, Col3a1^G209S/+ never-pregnant, and Col3a1^G209S/+ lactating mice, Col3a1^G209S/+ mice with pups removed thereby preventing lactation, and Col3a1^G209S/+ mice treated with trametinib (MEKi), oxytocin receptor antagonist (OTA), hydralazine, or propranolol. (B) Quantification of Western blot analysis of pERK1/2 in the proximal descending aorta in Col3a1^+/+ never-pregnant (n = 5), Col3a1^+/+ lactating (n = 5), Col3a1^G209S/+ never-pregnant (n = 5), and Col3a1^G209S/+ lactating mice (n = 5), Col3a1^G209S/+ mice with pups removed thereby preventing lactation (n = 5), and Col3a1^G209S/+ mice treated with trametinib (n = 5, MEKi), oxytocin receptor antagonist (n = 5, OTA), hydralazine (n = 3), or propranolol (n = 5). Error bars show mean ± SEM. Asterisks signify significant differences of log-transformed data using 1-way ANOVA with Dunnett’s multiple comparisons post hoc test. ***P < 0.001. DF = 8, F = 8.06. (C) qPCR analysis of Fos normalized to Gapdh in the proximal descending aorta comparing wild-type never-pregnant (n = 6, NP), wild-type lactating (n = 4, LAC), and Col3a1^G209S/+ never-pregnant mice (n = 8, NP), Col3a1^G209S/+ lactating mice (n = 10), and Col3a1^G209S/+ mice treated with trametinib (n = 5, LAC + MEKi), oxytocin receptor antagonist (n = 4, LAC + OTA), or hydralazine (n = 3, LAC + hyd). Error bars show mean ± SEM. Asterisks signify significant differences of log-transformed data using 1-way ANOVA with Dunnett’s multiple comparisons post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. DF = 6, F = 13.84. (D) qPCR analysis of Egr1 normalized to Gapdh in the proximal descending aorta comparing Col3a1^+/+ never-pregnant (n = 10, NP), Col3a1^+/+ lactating (n = 7, LAC), Col3a1^G209S/+ never-pregnant (n = 5, NP), and Col3a1^G209S/+ lactating mice (n = 10, LAC), and Col3a1^G209S/+ mice treated with trametinib (n = 5, LAC + MEKi), oxytocin receptor antagonist (n = 4, LAC + OTA), or hydralazine (n = 3, LAC + hyd). Error bars show mean ± SEM. Asterisks signify significant differences using 1-way ANOVA with Dunnett’s multiple comparisons post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DF = 6, F = 14.84.