Smimmo, Martina (2024) Role of Transsulfuration pathway in vascular complications associated with Metabolic Syndrome:H2S donors as a new potential therapeutic strategy. [Tesi di dottorato]

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Abstract

Embarking on a journey to unravel the intricate mechanisms underlying vascular complications in metabolic disorders, during my Ph.D. program I was devoted to a comprehensive exploration of Metabolic Syndrome (MS), Type 1 Diabetes (T1D), and coronary atherosclerosis. This ambitious endeavor was divided into three distinct but interconnected projects, each shedding light on novel aspects of these complex pathologies. I concluded my Ph.D. program at Neilos s.r.l company where I delved into the nutraceutical market, assessing the positioning of a potential nutraceutical product rooted in my research. In the first part of my Ph.D. at University of Naples Federico II, by using in vitro and ex vivo approaches, I elucidated the impairment of the hydrogen sulfide (H2S) pathway in vascular complications associated with Metabolic Syndrome. In the in vitro model simulating hyperlipidemic/hyperglycemic conditions, several hallmarks of endothelial dysfunction were observed, including eNOS/NO signaling impairment, ROS overproduction, and a reduction in CSE-derived H2S. Transitioning to an ex vivo model using db/db mice, decreased CBS and CSE expression in the aorta were found, leading to reduced L-cysteine-induced vasorelaxation. Molecular analysis revealed altered eNOS/NO signaling with changes in the eNOS/Cav-1 ratio, along with reduced Ach- and Iso-induced vasorelaxation. In vivo treatment with the H2S donors NaHS and Erucin ameliorated vascular dysfunction observed in db/db mice without impacting eNOS/NO signaling, further highlighting a specific action on smooth muscle component rather than the endothelium. In db/db aortas, reduced cGMP levels were also detected, implicating a defective sGC/cGMP signaling. NaHS and Erucin administration restored cGMP content. This beneficial effect involves an increased sGC activity, due to enzyme persulfidation, coupled with PDE5 inhibition, demonstrated in sGC overexpressing cells. Collectively, these results demonstrate a pivotal role of reduced cGMP levels in impaired vasorelaxation in a murine model of MS involving an impairment of both H2S and NO signaling. In the second part of my Ph.D. at University of Naples Federico II, I shifted focus to Type 1 Diabetes (T1D) and its cardiovascular complications, using Non-Obese Diabetic (NOD) mice, which exhibit spontaneous type 1 diabetes. The characterization of vascular dysfunction was performed, noting a marked reduction in Ach- and L-cys induced vasorelaxation. Treatment with H2S donors (Erucin and NaHS) positively impacted endothelial-dependent vasodilation, specifically targeting the eNOS/NO pathway. Remarkably, this positive effect on vascular function was achieved without altering blood glucose levels, underscoring the specific targeting of H2S donors to endothelial pathways. Expanding the scope of our study, we investigated the immune profile in diabetic mice, finding elevated pro-inflammatory T-helper (Th) cells, specifically Th1 and Th17, in diabetic condition. Interestingly, treatment with H2S donors effectively attenuated this immune imbalance, suggesting a broader immunomodulatory role for H2S in Type 1 diabetes. The key findings emphasized the versatility of H2S donors in ameliorating vascular dysfunction and influencing the immune profile, presenting a potential therapeutic way for both MS and T1D. In the third part of my Ph.D., at Weill Cornell University of New York, I studied the role of sphingolipid pathway in the onset and progression of atherosclerosis. The ER membrane protein Nogo-B has been identified as a key negative regulator of SLs synthesis, however its role in macrophages is unclear. Therefore, here, I investigated how the loss of Nogo-B impacts macrophage functions in atherosclerosis. In vivo data showed that the loss of Nogo-B specifically in macrophage cells, enhanced atherosclerotic lesions increasing necrotic core and plaque vulnerability elevating the risk of plaque rupture, a critical consequence in coronary artery disease (CAD). The in vitro model, using primary peritoneal macrophages isolated from Nogo-Bf/f ApoE-/- and NgKO-ApoE-/- mice, revealed that the absence of Nogo-B led to an accumulation of total ceramides and hexosilceramides, supporting Nogo-B's inhibitory role on serine palmitoyltransferase (SPT). Notably, lipidomic analysis indicated increased cholesterol ester (CE) levels in MΦ Nogo-B KO ApoE-/- macrophages, couple to a heightened expression of cholesterol biosynthetic genes. Linked to the specific depletion of Nogo-B in macrophages, an overactivation of the inflammasome, a key event in the inflammatory response associated with atherosclerosis, was observed. Intriguingly, this overactivation appeared to be independent of Nogo-B's canonical function as an SPT enzyme regulator. These findings unveil a novel and critical role of Nogo-B in macrophages during the development of atherosclerosis. During the latter part of my Ph.D., I spent six months at Neilos S.r.l, where my focus was on positioning a novel formulation based on Eruca sativa Mill. extract as a supportive treatment for Metabolic Syndrome. Market analysis identified six existing formulations for MS, primarily targeting glucose and/or cholesterol management. In contrast to current formulations, which focus on glucose and cholesterol, Eruca sativa Mill. uniquely addresses cardiovascular complications, filling a critical gap in the market.

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