Abstract Body (Do not enter title and authors here): OBJECTIVES: Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Augmenting angiogenesis is one of the treatment innovations. The present study investigates the pro-angiogenic potential and mechanism of tryptophan-kynurenine pathway activation in a mouse model of hindlimb ischemia.

APPROACH AND RESULTS: Indoleamine 2,3-dioxygenase (IDO)1 is the first and rate-limiting enzyme in tryptophan-kynurenine metabolism while kynureninase (KYNU) catalyzed the production of 3-Hydroxyanthranilic Acid (HAA). IDO and KYNU expressions were markedly induced in endothelial cells (ECs) of the ischemic hindlimbs after femoral atery ligation in IDO^f/f mice, as well as 3-HAA generation. However, endothelial-specific depletion of IDO or KYNU evidently reduced blood flow recovery and impaired angiogenesis in the ischemic limbs along with diminished 3-HAA production. Nevertheless, intramuscular injection of 3-HAA to the ischemic limbs not only accelerated the blood flow recovery and angiogenesis in IDO^f/f mice, but also restored the impaired angiogenesis and blood flow recovery in IDO^f/fCDH5^cre mice. Concomitantly, interferon (IFN)-γ which induced the expression of IDO and KYNU further increased 3-HAA levels in ECs of the ischemic limbs in IDO^f/f and wild type (WT) mice and substantially improved the blood flow recovery and promoted angiogenesis. But these pro-angiogenic effects were abrogated by IDO or KYNU deficiency in ECs. Of note, both exogenous and endogenous 3-HAA significantly promoted tube formation and cell proliferation in human aortic endothelial cells (HAECs). Transcriptomic analyses and IDO or KYNU ablation in mouse lung endothelial cells (MLECs) revealed the induction of fibroblast growth factor (FGF) 9 by exogenous and endogenous 3-HAA. Moreover, inhibition of FGF9 expression in HAECs abolished 3-HAA-induced tube formation and cell proliferation although recombinant human FGF9 facilitated tube formation in HAECs. Accordantly, ECs in the ischemic limbs of WT mice exhibited evident increases of FGF9 expression which were exaserbated by IFN-γ and 3-HAA injection with boosted angiogenesis. However, endothelial-specific KYNU depletion repressed FGF9 levels along with impaired angiogenesis in the ischemic limbs.

CONCLUSIONS: These data underline the pro-angiogenic property of tryptophan-derived 3-HAA in the ischemic hindlimbs by promoting FGF9 secretion, and therefore unravel a therapeutic potential of 3-HAA in peripheral artery disease.