Manzo, Onorina Laura (2021) Role of gasotransmitters and non canonical mediators in inflammatory-based vascular diseases (IBVD). [Tesi di dottorato]

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Abstract Background and purpose "The novo biosynthesis" is the most relevant pathway of SLs synthesis, where serine-palmitoyltransferase (SPT) is the rate limiting enzyme of the entire pathway. Its activity is negatively regulated by Nogo-B, a resident protein. Within vasculature, the physiological SPT/Nogo-B ratio modulates the SLs production rate 3. Inflammatory-based vascular diseases (IBVD) are characterized by chronic inflammatory vascular process. Although they have different etiology, they have a as a common feature the endothelial dysfunction, defined as reduction of NO biosynthesis. Despite SLs homeostasis has been altered in diabetic condition 5, their involvement in IBVD is not well-clarified. Among anti-diabetic drugs metformin is one of the most used worldwide. Increasing clinical data support the beneficial role metformin on the endothelial function in diabetic conditions. Based on these findings, the aims of this study were: 1) to investigate a possible involvement of SLs in endothelial dysfunction associated to type 1 diabetes 2) to investigate the possible protective action of metformin on hyperglycemia-induced endothelium dysfunction through SLs signaling. Experimental approach Non-obese diabetic mice (NOD) were divided in two groups according to glycemia levels, normoglycemic or NODI and diabetic or NODIII mice and treated with Myriocin (0.3 mg/Kg), a pharmacological inhibitor of SPT, or vehicle. After 24 h, aortas were harvested and used for testing vascular reactivity. Molecular analysis and mass spectrometry-based metabolomics was performed for the evaluation of SLs content. In a separate set of experiments, NODIII mice were treated with Metformin (300 mg/Kg/Daily) and mesenteric arteries harvested to evaluate vascular function. Ex vivo and in vitro studies on microsomal fractions from mouse lung and human umbilical vein endothelial cells (HUVEC) have also been performed to confirm the data obtained in vivo and ex vivo. Key results and conclusions De novo biosynthesis is altered during development of endothelial dysfunction-induced by type 1 diabetes, demonstrated by a significant increase of both SPT expression and activity, in ex vivo and in vitro. Determination of SLs by metabolomic approach revealed an accrual of both ceramides in diabetic aortas suggesting that in diabetic condition the over-expression of SPT leads to ceramide accumulation within the vasculature altering its function and reactivity. Metformin administration is largely associated to beneficial effect in endothelial function 9 but the exact mechanisms of action is still unknown. Metformin treatment of NODIII mice induced an improvement of vascular reactivity in mesenteric arteries (MA) due to a recover of eNOS/NO signaling. Incubation of both aorta and MA from healthy mouse with ceramide recapitulated the impairment observed in diabetic mice confirming the detrimental action of ceramide accrual induced by diabetes. The take home messages of this project are as follows: 1) de novo biosynthesis of SLs is altered in T1D due to an increase of SLs metabolism and accumulation of ceramide in vasculature. 2) Metformin treatment in T1D restored endothelial function, improved significantly NO availability and reduced SPT activity leading to physiological extent independently to its anti-glycemic activity. According to this study, the effect of metformin on SLs homeostasis could be accreditable as a new mechanism of action leads to protect against endothelial dysfunction associated to type 1 diabetes mellitus.

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