Il ruolo della via del mevalonato nelle cellule endoteliali e nell'angiogenesi

Angiogenesis is an essential cellular process implicated in the remodeling of the vasculature, allowing the formation of a new network of vessels from pre-existing vessels. Cellular metabolism plays a critical role during the quiescence and angiogenic state of the endothelial cells (ECs). Nowadays, it is clear how EC metabolism is a driver of angiogenesis and how ECs adapt their metabolic state when they switch from a quiescence to an angiogenic phenotype, and vice versa. In this context, the role of the mevalonate (MVA) pathway has been left behind. This metabolic pathway is involved in the production of isopentenyl-diphosphate (IPP). Then, isopentenyl pyrophosphate isomerase 1 (IDI1) catalyzes the isomerization of IPP to DMAPP. Two molecules of IPP are condensed with DMAPP through farnesyl pyrophosphate synthase (FDPS) activity forming farnesyl pyrophosphate (FPP). To elucidate the role of the MVA pathway during angiogenesis, we took advantage of genetic loss-of-function approaches targeting IDI1 gene. Zebrafish homozygous knock-out (KO) for IDI1 exhibits severe cardiovascular defects and in angiogenesis process. To unravel the correlation between MVA pathway, IDI1 and EC homeostasis during angiogenesis, we exploited a IDI1 tissue-specific conditional KO mouse model. Neonatal retinal angiogenesis assay revealed a significant decrease of different angiogenesis parameters in IDI1ΔEC compared to the control. The decrease in pHH3+ nuclei of the retinal ECs in IDI1ΔEC mice confirmed a decline in the ECs proliferation rate. Block of cell proliferation was also observed in HUVECs, followed by caspase 3-independent cell death, suggesting an alternative cell death process. Moreover, we found a decrease in the proliferation rate upon IDI1 silencing (IDI1KD) in human umbilical vein endothelial cells (HUVECs). In this condition we also detected a decrease of cyclin A and RRM2 protein levels, markers of the cell cycle. Interestingly, upon tamoxifen-induced IDI1 ablation, mice exhibited loss of body weight and, lastly, death. Our in vitro data were further corroborated by the analysis of kidney vessels: in this organ we noted retraction and collapse of the capillary tuft in the glomeruli in IDI1ΔEC mice. We performed proteomic analysis to identify the molecular process inducing cell death upon IDI1 silencing. Intriguingly, we found a remarkable increase of ICAM1 and CDH13, an event occurring under oxidative stress conditions. These results prompted us to evaluate the levels of Reactive Oxygen Species (ROS) in our model. We detected a significant increase of general oxidative stress and lipid peroxidation. We also found a decrease of specific antioxidant enzymes involved in the antioxidant response. Furthermore, IDI1KD cells displayed an increased sensitivity to H2O2 and cumene treatments. Taken together, these data suggest the involvement of an oxidative stress-induced cell death. To verify our hypothesis, we treated IDI1KD cells with antioxidant compounds which were able to partially rescue cell viability upon IDI1 silencing in vitro. Furthermore, in vivo treatments with ferrostatin-1 and liproxstatin-1 rescued intussusceptive and sprouting angiogenesis in zebrafish and mouse models, respectively. We performed the genetic silencing of FDPS in HUVECs. Differently from IDI1KD, FDPSKD cells showed an increase in cell proliferation and resistance from cumene-induced oxidative stress. The opposite outcomes obtained by the silencing of these two consecutive enzymes of the MVA pathway led us to suppose the contribution of distinct metabolites accumulation. In line with our hypothesis, mass spectrometry analysis revealed an accumulation of IPP upon IDI1 silencing and an accumulation of DMAPP upon FDPS silencing. We then investigated whether DMAPP accumulation is linked to a cell survival phenotype. Accordingly, we treated IDIKD cells with DMAPP rescuing cell viability in IDI1KD cells as well as increasing cell cycle markers.