Blood coagulation is vital for life by reducing blood loss upon injury. One of the key players in the initial steps of coagulation is Von Willebrand Factor (VWF). VWF is mainly produced and stored... Show moreBlood coagulation is vital for life by reducing blood loss upon injury. One of the key players in the initial steps of coagulation is Von Willebrand Factor (VWF). VWF is mainly produced and stored by the cells that line the blood vessels and functions upon secretion by creating a platelet plug at the side of injury. Mutations in VWF can disturb this process, for instance through insufficient VWF secretion, which causes Von Willebrand disease, a hereditary bleeding disorder characterized by prolonged bleeding. As the morphology of VWF is highly related to its functionality, we aimed to obtain more knowledge on the mechanisms that can be affected in Von Willebrand disease by studying the normal lifecycle of VWF using correlative light and electron microscopy (CLEM). CLEM combines fluorescent light microscopy with electron microscopy on the same sample to obtain structural information of specific biological events that are difficult to study with electron microscopy alone. Using CLEM, novel information was obtained regarding the formation of the VWF storage organelle, in addition we also discovered novel pathophysiological mechanisms acting during VWF secretion that may be effectuated in Von Willebrand disease. Show less
Aims High-fat diet-induced obesity (DIO) is amajor contributor to type II diabetes and micro-andmacro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of... Show moreAims High-fat diet-induced obesity (DIO) is amajor contributor to type II diabetes and micro-andmacro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stemcell-derived endothelial cells (iPSC-ECs) fromobese individuals could potentially limit their therapeutic efficacy forPVD. The aimof this studywas to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays.Methods and results Six-week-old C57Bl/6 micewere fed with a normal or high-fat diet. At 24weeks, iPSCs were generated fromtail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayerapproach. In vitro functional analysis revealed that iPSCECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusionwas observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, Nv-nitro-L-arginine methyl ester.Conclusion This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population. Show less
