Immunometabolic mechanisms in the arterial wall: new targets to combat vascular diseases

Cardiovascular diseases (CVDs), the leading cause of mortality and disability worldwide, are largely caused by manifestations of atherosclerosis, a chronic inflammatory disease involving maladaptive immune responses and the formation of plaques in the artery wall. Having atherosclerosis is an independent risk factor of the common CVD abdominal aortic aneurysm (AAA); a disease characterized by the dilatation of the abdominal aorta driven by strong inflammatory reactions and an abnormal vascular remodeling process. Both AAA and atherosclerosis have been linked to local metabolic alterations in the arterial wall, including increased glycolysis and diminished mitochondrial respiration. Interestingly, this metabolic pattern coincides with the intracellular metabolism commonly found during activation and differentiation of proinflammatory immune cells, which have been proposed to drive chronic inflammation in AAA and atherosclerosis. In the present thesis, it was hypothesized that a better understanding of immunometabolic reactions commonly involved in AAA and atherosclerosis can reveal novel diagnostic and therapeutic targets to combat these deadly CVDs. The thesis aimed at advancing our knowledge of the pathophysiological role of the PDK/PDH axis and succinate signaling in driving or increasing the risk of vascular diseases.

In Study I, we investigated the role of the pyruvate dehydrogenase kinase (PDK)/pyruvate dehydrogenase (PDH) axis in AAA disease. First, we found increased levels of phosphorylated PDH-Ser293 and lactate in human AAA tissues suggesting that the PDK/PDH axis is involved in the rewiring of cellular metabolism during disease development. Next, we tested whether preventing a skewed PDK/PDH axis could circumvent the deleterious metabolic reprogramming in AAA. Using dichloroacetate (DCA), a pan-inhibitor of PDK isoenzymes, in the PPE-induced AAA model, AAA expansion was reduced. Combined transcriptome, proteome, and histological analyses of mouse AAA tissues and in vitro experiments with vascular smooth muscle cells (VSMCs) indicated that the PDK/PDH axis is a key regulator of vascular responses in AAA, influencing inflammation and VSMC dedifferentiation. These findings highlight immunometabolic reprogramming, through PDK inhibition, as a promising therapeutic strategy to combat AAA disease.

In Study II, we investigated the biomarker potential of the metabolite succinate, which has been implicated in different inflammatory processes, in AAA using plasma samples from men with AAA and matched controls from the VIVA screening trial. Our analysis showed significantly higher succinate levels in AAA individuals compared to controls. However, succinate showed no predictive power towards future clinical complications of AAA disease, including first-year growth rate, need for surgical repair or risk of death. Nevertheless, analysis of a relevant public transcriptome AAA dataset showed that diseased tissue presents an increased expression of genes encoding enzymes involved in the γ-aminobutyric acid (GABA)-shunt pathway and decreased expression of succinate dehydrogenase (SDH) isoenzymes, indicating a mechanism of succinate accumulation locally in AAA. Corroborating the previous results, we showed that succinate levels are increased in AAA tissue homogenates, and that dedifferentiation of human VSCMs in culture downregulates SDH isoenzymes genes and promotes succinate accumulation in vitro. Altogether, our findings suggest that accumulation of succinate in AAA could be the result of the metabolic reprograming of VMSCs in the aortic wall, potentially playing a role in deleterious vascular remodelling mechanisms.

In Study III, we explored the role of GPR91, which transduces cell signalling upon succinate ligation, in atherosclerosis. Using GPR91 KO (Gpr91-/-) mice and wildtype (WT) littermates, made hyperlipidaemic with the injection of murine recombinant adeno-associated virus expressing a gain-of-function mutated Pcsk9 and western diet feeding, we showed that the global ablation of GPR91-mediated signalling did not influence plaque burden compared to WTs. In line with this, assessment of plaque cell composition and inflammatory status showed no major differences in the infiltration of immune cells and the expression of pro- and antiinflammatory markers, as well as the content of αSMA. Surprisingly, expression of the macrophage and M1-like markers, Cd68 and Cxcl10, respectively, were increased in spleens of Gpr91-/- mice compared to WT littermates, suggesting GPR91-mediated peripheral inflammatory responses may differ from those in the aorta.