Background and aims:
Growing evidence suggest a critical role of distinct gut microbially produced metabolites in the development of cardiometabolic and -vascular diseases. The amino-acid derived metabolite imidazole propionate (ImP) is increased in patients with type II diabetes. However, its impact on endothelial cell physiology and vascular disease has not been examined, so far. Here, we aimed to study the effects of ImP on endothelial cell function and inflammatory activation as well as on endothelial regeneration after injury and development of atherosclerosis in athero-prone Apoe^-/- mice.

Methods and results:
Cell migration and angiogenic potential of primary human aortic endothelial cells (HAECs) were examined upon treatment with ImP at different concentrations (10 nM and 100 nM) using wound scratch and matrigel tube formation assays. ImP significantly reduced endothelial cell migration and the formation of tube-like structures on HAECs compared to control treatment. To identify the potential underlying mechanism of ImP mediated effects on endothelial cells, next-generation sequencing, western blotting and immunostaining were performed. Transcriptomic profiling of HAECs revealed altered regulation of a number of genes involved in angiogenesis in ImP treated cells. In particular, decreased PI3K/Akt signaling under pre-treatment of ImP followed by insulin receptor stimulation with IGF-1 was found. Moreover, ImP significantly led to upregulating expression of cellular adhesion-molecules ICAM-1, VCAM-1 and E-Selectin, consequently leading to enhanced adhesion of monocytes to HAECs under flow condition. Vascular regeneration in vivo was analyzed using a carotid artery injury model (CI) in C57BL/6N mice treated with ImP (400 µg) or vehicle for three weeks via drinking water. After treatment, CI was induced to the carotid artery using a bipolar microforceps. Re-endothelialization was determined by Evans blue staining 3 days post-CI. Our results showed that treatment with ImP significantly impaired wound healing as compared to control animals. Finally, atherosclerotic plaque formation was analyzed in vivo using Apolipoprotein E^-/- (ApoE^-/-) mice fed a high fat diet (HFD) for 12 weeks. Oil red O staining displayed increased atherosclerotic plaque formation in en face aorta and aortic root sections upon ImP treatment. CD68 expression was also enhanced in murine atherosclerotic lesions in response to ImP treatment indicative of increased macrophage accumulation.

Conclusion:
The gut microbially produced metabolite ImP impairs insulin receptor signaling pathway in endothelial cells, thereby affecting their functional properties such as migration, proliferation and angiogenic capacity. Moreover, ImP promotes inflammatory activation of endothelial cells leading to increased monocyte adhesion. These effects result in impaired vascular healing after injury and, ultimately, increased atherosclerosis in athero-prone Apoe^-/- mice.