Fig 2: Folic acid diet reduces superoxide production, restores NO bioavailability and recouples eNOS in Fbn1C1039G/+mice.Fbn1+/+ and Fbn1C1039G/+ mice of 4 weeks age were fed FA diet (15 mg/kg/day) for 4 and 8 weeks, after which aortas were freshly harvested for electron spin resonance (ESR) determination of superoxide production, NO bioavailability and eNOS uncoupling activity. (A) Superoxide production was significantly elevated at 4, 8 and 12 weeks of age in Fbn1C1039G/+ mice, which was completely attenuated by FA diet after oral FA administration for 4 and 8 weeks. Data are presented as Mean ± SEM, n = 4–6. (B) NO bioavailability was time-dependently decreased at 8 and 12 weeks of age in Fbn1C1039G/+ mice, which was completely or largely restored by FA diet. Data are presented as Mean ± SEM, n = 7–12. (C) Total superoxide production in the presence or absence of l-NAME was measured by ESR for determination of eNOS uncoupling activity (l-NAME-inhibitable superoxide production) in Fbn1C1039G/+ mice. eNOS was uncoupled at 4, 8 and 12 weeks of age in Fbn1C1039G/+ mice, which was completely attenuated by FA diet. Data are presented as Mean ± SEM, n = 5–7. *p < 0.05, **p < 0.01.

Fig 3: TGFß blocking antibody recouples eNOS and attenuates ROS production via inhibition of NOX4 and restoration of DHFR expression in Fbn1C1039G/+mice. The aortas were isolated from 12 weeks old Fbn1+/+ and Fbn1C1039G/+ mice and subjected to Western blotting, immunohistochemistry (IHC) and electron spin resonance (ESR) analyses. (A) Representative Western blots and grouped densitometric data of aortic protein expression levels of inactive and mature TGFß indicating upregulation of mature TGFß in Fbn1C1039G/+ mice. Data are presented as Mean ± SEM, n = 5–9. (B) Representative Western blots and grouped densitometric data of aortic NOX4 protein expression indicating upregulation of NOX4 in Fbn1C1039G/+ mice. Data are presented as Mean ± SEM, n = 4. (C) Representative Western blots and grouped densitometric data of endothelial DHFR protein expression indicating downregulation of DHFR in Fbn1C1039G/+ mice. Data are presented as Mean ± SEM, n = 5. (D) Representative IHC images of aortic roots, indicating downregulation of endothelial DHFR in 12 weeks Fbn1C1039G/+ mice. Arrows indicating highly expressed DHFR in the endothelial layer of the control animals, which was however reduced in Fbn1C1039G/+ mice. In parallel experiments, 4 weeks old Fbn1C1039G/+ mice were treated with TGFß blocking antibody for 4 weeks prior to analyses of NOX4 expression and eNOS uncoupling activity. (E) Representative Western blots and grouped densitometric data of aortic NOX4 protein expression in Fbn1C1039G/+ mice treated with TGFß blocking antibody, indicating abrogated NOX4 expression. Data are presented as Mean ± SEM, n = 4. (F) Representative Western blots and grouped densitometric data of endothelial DHFR protein expression in Fbn1C1039G/+ mice treated with TGFß blocking antibody, indicating restored DHFR expression. Data are presented as Mean ± SEM, n = 5. G) Superoxide production was determined by ESR in the aortic tissues of Fbn1C1039G/+ mice after TGFß blocking antibody treatment for 4 weeks. The results indicate that TGFß blocking antibody reduced ROS production. Data are presented as Mean ± SEM, n = 5–7. (H) Total superoxide production in the presence or absence of l-NAME was determined by ESR in the aortic tissues of Fbn1C1039G/+ mice after TGFß blocking antibody treatment for 4 weeks. The results indicate recoupling of eNOS by treatment with TGFß blocking antibody. Data are presented as Mean ± SEM, n = 5. Bar = 30 µm, *p < 0.05, **p < 0.01.

Fig 4: TGFß-dependent GTPCHI downregulation contributes to baseline deficiencies of H4B and eNOS uncoupling in Fbn1C1039G/+animals. The aortas were isolated from 12 weeks old Fbn1+/+ and Fbn1C1039G/+ and subjected to Western blotting analysis to examine GTPCHI expression. (A) Representative Western blots and grouped densitometric data of aortic GTPCHI expression, indicating downregulation of GTPCHI in Fbn1C1039G/+ mice. Data are presented as Mean ± SEM, n = 4–6. In parallel experiments, human aortic endothelial cells (HAECs) were treated by TGFß blocking antibody with or without fibrillin siRNA transfection, after which cells were lysed for determination of GTPCHI protein expression by Western blotting. (B) Representative Western blots and grouped densitometric data of GTPCHI protein expression, indicating restoration of GTPCHI protein abundance with TGFß blocking antibody. Data are presented as Mean ± SEM, n = 5. (C) Representative Western blots and grouped densitometric data of Fibrillin expression, confirming efficacy in silencing fibrillin expression with fibrillin siRNA. Data are presented as Mean ± SEM, n = 5. (D) Representative Western blots and grouped densitometric data of GTPCHI protein expression, indicating downregulation of GTPCHI by fibrillin deficiency, which was reversed by TGFß blocking antibody. Data are presented as Mean ± SEM, n = 5. *p < 0.05, ***p < 0.001.

Fig 5: Recoupling of eNOS with folic acid diet abrogates expansion of abdominal aortas in Fbn1C1039G/+animals.Fbn1+/+ and Fbn1C1039G/+ mice of 4 weeks age were fed FA diet (15 mg/kg/day) for 4 and 8 weeks. (A) Representative images of abdominal aortas by echocardiography. (B) Echo-defined areas of abdominal aortas indicating time-dependent expansion of abdominal aortas in Fbn1C1039G/+ mice, which was attenuated by FA diet. Data are presented as Mean ± SEM, n = 6–38. (C) Representative H&E images of abdominal aortas, indicating features of expansion and remodeling in Fbn1C1039G/+ mice that were alleviated by FA diet. (D) Representative VVG images of abdominal aortas. Arrows indicate flattening and degradation of elastin fibers, which were attenuated by FA diet. Bar = 100 µm, ***p < 0.001.