Targeting feed-forward signaling of TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ axis with anti-TGFβ and folic acid attenuates formation of aortic aneurysms: Novel mechanisms and therapeutics

In the present study we aimed to identify novel mechanisms and therapeutics for thoracic aortic aneurysm (TAA) in Fbn1(C1039G/+) Marfan Syndrome (MFS) mice. The expression of mature/active TGFβ and its downstream effector NOX4 were upregulated while tetrahydrobiopterin (H(4)B) salvage enzyme dihydrofolate reductase (DHFR) was downregulated in Fbn1(C1039G/+) mice. In vivo treatment with anti-TGFβ completely attenuated NOX4 expression, restored DHFR protein abundance, reduced ROS production, recoupled eNOS and attenuated aneurysm formation. Intriguingly, oral administration with folic acid (FA) to recouple eNOS markedly alleviated expansion of aortic roots and abdominal aortas in Fbn1(C1039G/+) mice, which was attributed to substantially upregulated DHFR expression and activity in the endothelium to restore tissue and circulating levels of H(4)B. Notably, circulating H(4)B levels were accurately predictive of tissue H(4)B bioavailability, and negatively associated with expansion of aortic roots, indicating a novel biomarker role of circulating H(4)B for TAA. Furthermore, FA diet abrogated TGFβ and NOX4 expression, disrupting the feed-forward loop to inactivate TGFβ/NOX4/DHFR/eNOS uncoupling axis in vivo and in vitro, while PTIO, a NO scavenger, reversed this effect in cultured human aortic endothelial cells (HAECs). Besides, expression of the rate limiting H(4)B synthetic enzyme GTP cyclohydrolase 1 (GTPCHI), was downregulated in Fbn1(C1039G/+) mice at baseline. In cultured HAECs, RNAi inhibition of fibrillin resulted in reduced GTPCHI expression, while this response was abrogated by anti-TGFβ, indicating TGFβ-dependent downregulation of GTPCHI in response to fibrillin deficiency. Taken together, our data for the first time reveal that uncoupled eNOS plays a central role in TAA formation, while anti-TGFβ and FA diet robustly abolish aneurysm formation via inactivation of a novel TGFβ/NOX4/DHFR/eNOS uncoupling/TGFβ feed-forward pathway. Correction of fibrillin deficiency is additionally beneficial via preservation of GTPCHI function.