In-Vitro Assessment of Endothelial and Arterial Smooth Muscle Function in a Marfan Mouse Model

$40,000 funded by TFF & CMA

Dr. Cornelis (Casey) Van Breemen

Cardiac Sciences Research Laboratory, BC Research Institute for Children

Loss of Elastic Fiber Integrity and Reduction of Vascular Smooth Muscle Contraction Resulting From the Upregulated Activities of Matrix Metalloproteinase-2 and -9 in the Thoracic Aortic Aneurysm in Marfan Syndrome

Ada W.Y. Chung, Karen Au Yeung, George G.S. Sandor, Daniel P. Judge, Harry C. Dietz, Cornelis van Breemen

Circulation Research 2007;101:512-522


Thoracic aortic aneurysm (TAA) is the life-threatening complication of Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 gene. TAA is characterized by degradation of elastic fiber, suggesting the involvement of matrix metalloproteinase (MMP)-2 and -9, the activation of which is regulated by TIMP (tissue inhibitor of MMP) types 1 and 2. We hypothesized that MMP-2 and -9 were upregulated during TAA formation in Marfan syndrome, causing loss of elastic fibers and structural integrity. We studied mice, from 3 to 12 months, heterozygous for a mutant Fbn1 allele encoding a cysteine substitution in fibrillin-1 (Fbn1C1039G/+, designated as "Marfan" mice) (n=120), the most common class of mutation in Marfan syndrome. The littermates, Fbn1+/+ served as controls (n=120). In Marfan aneurysmal thoracic aorta, mRNA and protein expression of MMP-2 and -9 were detected at 3 months and peaked at 6 months of age, accompanied by severe elastic fiber fragmentation and degradation. From 3 to 9 months, the MMP-2/TIMP-2 ratio increased by 43% to 63% compared with the controls. Dilated thoracic aorta demonstrated increased elasticity but distention caused a pronounced loss of contraction, suggesting weakening of the aortic wall. Breaking stress of the aneurysmal aorta was 70% of the controls. Contraction in response to depolarization and receptor stimulation decreased in the aneurysmal thoracic aorta by 50% to 80%, but the expression of-smooth muscle actin between the 2 strains was not significantly different. This report demonstrates the upregulation of MMP-2 and -9 during TAA formation in Marfan syndrome. The resulting elastic fiber degeneration with deterioration of the aortic contraction and mechanical properties may explain the pathogenesis of TAA.

Endothelial dysfunction and compromised eNOS/Akt signaling in the thoracic aorta during the progression of Marfan syndrome

A W Y Chung1, K Au Yeung1, S F Cortes2, G G S Sandor3, D P Judge4, H C Dietz4 and C van Breemen1
British Journal of Pharmacology (2007) 150, 1075–1083.

Background and purpose: Aortic complications account for the major mortality in Marfan syndrome (MFS), a connective tissue disorder caused by mutations in FBN1 encoding fibrillin-1. We hypothesized that MFS impaired endothelial function and nitric oxide (NO) production in the aorta.

Experimental approach
Mice (at 3, 6, 9 and 12 months of age) heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1 C1039G/+, Marfan mice, n=75), the most common class of mutation in MFS, were compared with age-matched control littermates (n=75). Thoracic and abdominal aortas from the two groups were studied.

Key results
Isometric force measurements revealed that relaxation in response to ACh (but not to sodium nitroprusside) was diminished in the phenylephrine-precontracted Marfan thoracic aorta at 6 months of age (pEC50=6.12 0.22; maximal response, Emax=52.7 6.8%; control: pEC50=7.34 0.19; Emax=84.8 2.2%). At one year, both inhibition of NO production with N -nitro-L-arginine methyl ester, or denudation of endothelium increased the phenylephrine-stimulated contraction in the control thoracic aorta by 35%, but had no effect in the Marfan aorta, indicating a loss of basal NO production in the Marfan vessel. From 6 months, a reduced phosphorylation of endothelial NOS (eNOS)Ser1177 and AktThr308 detected by Western blotting was observed in the Marfan thoracic aorta, which was accompanied by decreased levels of cGMP. Expressions of Akt and eNOS in the abdominal aorta were not different between the two groups.

Conclusions and Implications
MFS impairs endothelial function and signaling of NO production in the thoracic aorta, suggesting the importance of NO in the age-related progression of thoracic aortic manifestations.

Overall we draw two important conclusions related to future management of Marfan disease: first, inhibition of metalloproteinases may prevent weakening of the aorta and second upregulation of endothelial nitricoxide-synthase is likely to improve vascular fuction.