Therapeutic drug monitoring (TDM) of calcineurin inhibitors (i.e., tacrolimus and cyclosporin A) is standard of care after solid organ transplantation. Although the incidence of acute rejection has... Show moreTherapeutic drug monitoring (TDM) of calcineurin inhibitors (i.e., tacrolimus and cyclosporin A) is standard of care after solid organ transplantation. Although the incidence of acute rejection has strongly decreased, there are still many patients who experience severe side effects or rejection after long-term treatment. In this healthy volunteer study we therefore aimed to identify biomarkers to move from a pharmacokinetic-based towards a pharmacodynamic-based monitoring approach for calcineurin inhibitor treatment. Healthy volunteers received a single dose of cyclosporine A (CsA) or placebo, after which whole blood samples were stimulated to measure ex vivo T cell functionality, including proliferation, cytokine production, and activation marker expression. The highest whole blood concentration of CsA was found at 2 h post-dose, which resulted in a strong inhibition of interferon gamma (IFNy) and interleukin-2 (IL-2) production and expression of CD154 and CD71 on T cells. Moreover, the in vitro effect of CsA was studied by incubation of pre-dose whole blood samples with a concentration range of CsA. The average in vitro and ex vivo CsA activity overlapped, making the in vitro dose-effect relationship an interesting method for prediction of post-dose drug effect. The clinical relevance of the results is to be explored in transplantation patients on calcineurin inhibitor treatment. Show less
In this thesis, we addressed the role of cellular metabolism in maturation of hiPSC-ECs. In Chapter 2, we compare hiPSC derived EC functionality and metabolism with primary human microvascular... Show moreIn this thesis, we addressed the role of cellular metabolism in maturation of hiPSC-ECs. In Chapter 2, we compare hiPSC derived EC functionality and metabolism with primary human microvascular endothelial cells (hMVEC) determining the presence of a luminal glycocalyx as a main functional target. This chapter presents new insights in mitochondrial dysfunction of hiPSC-EC, limiting their ability to produce sufficient glycocalyx and align to shear stress. Underlying the mitochondrial dysfunction, we found that hiPSC-EC have an open mitochondrial permeability transition pore (mPTP), indicating mitochondrial immaturity. By closing the mPTP during differentiation with Cyclosporin-A (CsA), binding to cyclophilin D of the mPTP, we were able to mature mitochondria and improve functionality of these cells, resulting in a reduction in ROS, increased glycocalyx production and the therefore providing iPSC-ECs the ability to align to shear stress. Chapter 3 Continues with the comparison of hiPSC-EC with hMVEC, focusing on von Willebrand Factor (VWF) and the production of Weibel Palade Bodies (WPB). Testing several differentiation protocols and even after addition of CsA, hiPSC-EC were found to lack mature WPBs. We showed that neither shear stress nor co-culture with pericytes could induce WPB formation. By further studying the metabolism with NMR we found that hiPSC-EC have a reduced glycolysis and lactate production and an increased intracellular pH (pHi). This coincides with a reduced expression of the proton coupled monocarboxylate transporter MCT1, which transports H+ and lactate into the cell to keep pHi in balance. Reducing the intracellular pH led to increased presence of functional VWF and maturation of WPB in hiPSC-ECs. In Chapter 4 we addressed the question how hiPSC-EC maintain their redoxbalance and produce enough ATP, since the vast majority of ATP and antioxidants of EC are obtained by glycolysis, which is significantly reduced in iPSC-ECs. We found that hiPSC-EC rely mainly on free fatty acid oxidation and presumably use NADPH do maintain their redox balance. This alternative metabolic state in hiPSCEC was found to be independent of the high expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which regulates fatty acid storage, glucose metabolism, lipid uptake and mitochondrial biogenesis. Since PGC1α is directly activated by ROS and is the master regulator of the energy metabolism, the high levels of PGC1α expression in hiPSCECcould also be a consequence instead of the cause of the observed differences in metabolism. In Chapter 5, we studied the immunogenic surface of hiPSC-ECs, since transplantation induced rejection by the host immune system is an essential hurdle in usage of IPSC derived tissue. Previous studies suggested that IPSC derived cell recognition by the host immune system is diminished compared to transplantation of allogenic human alternatives. On the other hand, endothelial surface MHC class 1 and 2 molecules do play an active, ‘APC like’, role in adult immunity. Therefore, we characterized hiPSC-EC surface immune complexes, unstimulated and after cytokine stimulation. In addition we tested how the observed difference in expression could influence CD8 T-cell activation. Furthermore, we characterized the expression of complement inhibitors on the cell surface, necessary for the protection against unprovoked complement activation in the blood. Chapter 6 provides a summary and discussion of the observations in this thesis, including future perspectives on studying metabolism and metabolepigenetics in iPSC derived kidney organoids by mass spectrometry imaging. Show less
