N-acyl Dopamines - renoprotective therapeutics, acting on TRPV1 signaling:Biological properties and molecular mechanisms

NOD is a synthetic N - acyl dopamine which exhibits anti - inflammatory, reno - protective and cytoprotective properties. NOD has become an interesting drug candidate for clinical use in the setting of ischemia - induced acute kidney injury and Transplantation. This thesis investigates the relevant chemical and biological properties that grant NOD reno - protective effects in these contexts. The experimental studies described in this thesis demonstrate firstly, that NOD conveys protection in the setting of ischemia induced AKI via TRPV1 activation. Secondly, different molecular entities within NOD are required for TRPV1 activation and for its anti - inflammatory property. Thirdly, in vivo tissue distribution and elimination kinetics of NOD using [18F]F – labelled NOD demonstrate a rapid renal and hepatobiliary clearance. Finally, NOD’s prominent redox active nature significantly affects the cell behaviour, i.e. NOD induces ER stress resulting in the induction of the unfolded protein response. This thesis provided insight in the biodistribution of NOD and pins down its renoprotective effect in vivo to TRPV1 activation. Although being a promising drug candidate for the treatment of acute kidney injury, detailed pharmacodynamic and pharmacokinetic studies are warranted before clinical implementation of NOD

ABCB5+ mesenchymal stromal cells therapy protects from hypoxia by restoring Ca2+ homeostasis in vitro and in vivo

Background: Hypoxia in ischemic disease impairs Ca2+ homeostasis and may promote angiogenesis. The therapeutic efficacy of mesenchymal stromal cells (MSCs) in peripheral arterial occlusive disease is well established, yet its influence on cellular Ca2+ homeostasis remains to be elucidated. We addressed the influence of ATP-binding cassette subfamily B member 5 positive mesenchymal stromal cells (ABCB5+ MSCs) on Ca2+ homeostasis in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Methods: Hypoxia was induced in HUVECs by Cobalt (II) chloride (CoCl2) or Deferoxamine (DFO). Dynamic changes in the cytosolic- and endoplasmic reticulum (ER) Ca2+ and changes in reactive oxygen species were assessed by appropriate fluorescence-based sensors. Metabolic activity, cell migration, and tube formation were assessed by standard assays. Acute-on-chronic ischemia in Apolipoprotein E knock-out (ApoE−/−) mice was performed by double ligation of the right femoral artery (DFLA). ABCB5+ MSC cells were injected into the ischemic limb. Functional recovery after DFLA and histology of gastrocnemius and aorta were assessed. Results: Hypoxia-induced impairment of cytosolic and ER Ca2+ were restored by ABCB5+ MSCs or their conditioned medium. Similar was found for changes in intracellular ROS production, metabolic activity, migratory ability and tube formation. The restoration was paralleled by an increased expression of the Ca2+ transporter Sarco-/endoplasmic reticulum ATPase 2a (SERCA2a) and the phosphorylation of Phospholamban (PLN). In acute-on-chronic ischemia, ABCB5+ MSCs treated mice showed a higher microvascular density, increased SERCA2a expression and PLN phosphorylation relative to untreated controls. Conclusions: ABCB5+ MSCs therapy can restore cellular Ca2+ homeostasis, which may beneficially affect the angiogenic function of endothelial cells under hypoxia in vitro and in vivo

TissueGrinder, a novel technology for rapid generation of patient-derived single cell suspensions from solid tumors by mechanical tissue dissociation

IntroductionRecent advances hold promise of making personalized medicine a step closer to implementation in clinical settings. However, traditional sample preparation methods are not robust and reproducible. In this study, the TissueGrinder, a novel mechanical semi-automated benchtop device, which can isolate cells from tissue in a very fast and enzyme-free way is tested for cell isolation from surgically resected tumor tissues.MethodsThirty-three surgically resected tumor tissues from various but mainly pancreatic, liver or colorectal origins were processed by both novel TissueGrinder and explant method. An optimized processing program for tumors from pancreatic, liver or colorectal cancer was developed. The viability and morphological characteristics of the isolated cells were evaluated microscopically. Expression of pancreatic cancer markers was evaluated in cells isolated from pancreatic tumors. Finally, the effect of mechanical stress on the cells was evaluated by assessing apoptosis markers via western blotting.ResultsTissueGinder was more efficient in isolating cells from tumor tissue with a success rate of 75% when compared to explant method 45% in terms of cell outgrowth six weeks after processing. Cells isolated with TissueGinder had a higher abundance and were more heterogeneous in composition as compared to explant method. Mechanical processing of the cells with TissueGrinder does not lead to apoptosis but causes slight stress to the cells.DiscussionOur results show that TissueGrinder can process solid tumor tissues more rapidly and efficiently and with higher success rate compared to the conventionally used explant method. The results of the study suggest that the TissueGrinder might be a suitable method for obtaining cells, which is important for its application in individualized therapy. Due to the great variance in different tumor entities and the associated individual tissue characteristics, a further development of the dissociation protocol for other types of tumors and normal tissue will be targeted

Methylglyoxal induces p53 activation and inhibits mTORC1 in human umbilical vein endothelial cells

Methylglyoxal (MGO), a precursor of advanced glycation end products (AGEs), is regarded as a pivotal mediator of vascular damage in patients with diabetes. We have previously reported that MGO induces transcriptional changes compatible with p53 activation in cultured human endothelial cells. To further substantiate this finding and to explore the underlying mechanisms and possible consequences of p53 activation, we aimed (1) to provide direct evidence for p53 activation in MGO-treated human umbilical vein endothelial cells (HUVECs), (2) to assess putative mechanisms by which this occurs, (3) to analyze down-stream effects on mTOR and autophagy pathways, and (4) to assess the potential benefit of carnosine herein. Exposure of HUVECs to 800 mu M of MGO for 5 h induced p53 phosphorylation. This was paralleled by an increase in TUNEL and gamma-H2AX positive cells, indicative for DNA damage. Compatible with p53 activation, MGO treatment resulted in cell cycle arrest, inhibition of mTORC1 and induction of autophagy. Carnosine co-treatment did not counteract MGO-driven effects. In conclusion, our results demonstrate that MGO elicits DNA damage and p53 activation in HUVECs, resulting in modulation of downstream pathways, e.g. mTORC1

N-Octanoyl Dopamine Treatment of Endothelial Cells Induces the Unfolded Protein Response and Results in Hypometabolism and Tolerance to Hypothermia

Aim: N-acyl dopamines (NADD) are gaining attention in the field of inflammatory and neurological disorders. Due to their hydrophobicity, NADD may have access to the endoplasmic reticulum (ER). We therefore investigated if NADD induce the unfolded protein response (UPR) and if this in turn influences cell behaviour. Methods: Genome wide gene expression profiling, confirmatory qPCR and reporter assays were employed on human umbilical vein endothelial cells (HUVEC) to validate induction of UPR target genes and UPR sensor activation by N-octanoyl dopamine (NOD). Intracellular ATP, apoptosis and induction of thermotolerance were used as functional parameters to assess adaptation of HUVEC. Results: NOD, but not dopamine dose dependently induces the UPR. This was also found for other synthetic NADD. Induction of the UPR was dependent on the redox activity of NADD and was not caused by selective activation of a particular UPR sensor. UPR induction did not result in cell apoptosis, yet NOD strongly impaired cell proliferation by attenuation of cells in the S-G2/M phase. Long-term treatment of HUVEC with low NOD concentration showed decreased intracellular ATP concentration paralleled with activation of AMPK. These cells were significantly more resistant to cold inflicted injury. Conclusions: We provide for the first time evidence that NADD induce the UPR in vitro. It remains to be assessed if UPR induction is causally associated with hypometabolism and thermotolerance. Further pharmacokinetic studies are warranted to address if the NADD concentrations used in vitro can be obtained in vivo and if this in turn shows therapeutic efficacy

Identification of the Key Genes and Potential Therapeutic Compounds for Abdominal Aortic Aneurysm Based on a Weighted Correlation Network Analysis

Background: There is still an unmet need for therapeutic drugs for patients with an abdominal aortic aneurysm (AAA), especially for candidates unsuitable for surgical or interventional repair. Therefore, the purpose of this in silico study is to identify significant genes and regulatory mechanisms in AAA patients to predicate the potential therapeutic compounds for significant genes. Methods: The GSE57691 dataset was obtained from Gene Expression Omnibus (GEO) and used to identify the differentially expressed genes (DEGs) and weighted correlation network analysis (WGCNA). The biological function of DEGs was determined using gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). AAA-related genes were obtained from the Comparative Toxicogenomics Database (CTD) using the keywords: aortic aneurysm and abdominal. The hub genes in AAA were obtained by overlapping DEGs, WGCNA-based hub genes, and CTD-based genes. The diagnostic values of hub genes were determined using ROC curve analysis. Hereby, a TF-miRNA-hub gene network was constructed based on the miRnet database. Using these data, potential therapeutic compounds for the therapy of AAA were predicted based on the Drug Gene Interaction Database (DGIdb). Results: A total of 218 DEGs (17 upregulated and 201 downregulated) and their biological function were explored; 4093 AAA-related genes were derived by text mining. Three hub modules and 144 hub genes were identified by WGCNA. asparagine synthetase (ASNS), axin-related protein 2 (AXIN2), melanoma cell adhesion molecule (MCAM), and the testis-specific Y-encoded-like protein 1 (TSPYL1) were obtained as intersecting hub genes and the diagnostic values were confirmed with ROC curves. As potential compounds targeting the hub genes, asparaginase was identified as the target compound for ASNS. Prednisolone and abiraterone were identified as compounds targeting TSPYL1. For MCAM and TSPYL1, no potential therapeutic compound could be predicted. Conclusion: Using WGCNA analysis and text mining, pre-existing gene expression data were used to provide novel insight into potential AAA-related protein targets. For two of these targets, compounds could be predicted

Biphasic Effects of Blue Light Irradiation on Human Umbilical Vein Endothelial Cells

Blue light regulates biological function in various cells, such as proliferation, oxidative stress, and cell death. We employed blue light illumination on human umbilical vein endothelial cells utilizing a LED device at 453 nm wavelength and revealed a novel biphasic response on human umbilical vein endothelial cells (HUVECs). The results showed that low fluence blue light irradiation promoted the fundamental cell activities, including cell viability, migration and angiogenesis by activating the angiogenic pathways such as the VEGF signaling pathway. In contrast, high fluence illumination caused the opposite effect on those activities by upregulating pro-apoptotic signaling cascades like ferroptosis, necroptosis and the p53 signaling pathways. Our results provide an underlying insight into photobiomodulation by blue light and may help to implement potential treatment strategies for treating angiogenesis-dependent diseases

Targeted and explorative profiling of kallikrein proteases and global proteome biology of pancreatic ductal adenocarcinoma, chronic pancreatitis, and normal pancreas highlights disease-specific proteome remodelling

Pancreatic ductal adenocarcinoma (PDAC) represents one of the most aggressive and lethal malignancies worldwide with an urgent need for new diagnostic and therapeutic strategies. One major risk factor for PDAC is the pre-indication of chronic pancreatitis (CP), which represents highly inflammatory pancreatic tissue. Kallikreins (KLKs) are secreted serine proteases that play an important role in various cancers as components of the tumor microenvironment. Previous studies of KLKs in solid tumors largely relied on either transcriptomics or immunodetection. We present one of the first targeted mass spectrometry profiling of kallikrein proteases in PDAC, CP, and normal pancreas. We show that KLK6 and KLK10 are significantly upregulated in PDAC (n=14) but not in CP (n=7) when compared to normal pancreas (n=16), highlighting their specific intertwining with malignancy. Additional explorative proteome profiling identified 5936 proteins in our pancreatic cohort and observed disease-specific proteome rearrangements in PDAC and CP. As such, PDAC features an enriched proteome motif for extracellular matrix (ECM) and cell adhesion while there is depletion of mitochondrial energy metabolism proteins, reminiscent of the Warburg effect. Although often regarded as a PDAC hallmark, the ECM fingerprint was also observed in CP, alongside with a prototypical inflammatory proteome motif as well as with an increased wound healing process and proteolytic activity, thereby possibly illustrating tissue autolysis. Proteogenomic analysis based on publicly accessible data sources identified 112 PDAC-specific and 32 CP-specific single amino acid variants, which among others affect KRAS and ANKHD1. Our study emphasizes the diagnostic potential of kallikreins and provides novel insights into proteomic characteristics of PDAC and CP

Radiofluorinated N-Octanoyl Dopamine ([F-18]F-NOD) as a Tool To Study Tissue Distribution and Elimination of NOD in Vitro and in Vivo

To mitigate pretransplantation injury in organs of potential donors, N-octanoyl dopamine (NOD) treatment might be considered as it does not affect hemodynamic parameters in braindead (BD) donors. To better assess optimal NOD concentrations for donor treatment, we report on the fast and facile radiofluorination of the NOD-derivative [F-18]F-NOD [F-18]5 for in vivo assessment of NOD's elimination kinetics by means of PET imaging. [F-18]5 was synthesized in reproducibly high radiochemical yields and purity (>98%) as well as high specific activities (>20 GBq/mu mol). Stability tests showed no decomposition of [F-18]5 over a period of 120 min in rat plasma. In-vitro, low cell association was found for [F-18]5, indicating no active transport mechanism into cells. In vivo, [F-18]5 exhibited a fast blood clearance and a predominant hepatobiliary elimination. As these data suggest that also NOD might be cleared fast, further pharmacokinetic evaluation is warranted

N-octanoyl dopamine treatment exerts renoprotective properties in acute kidney injury but not in renal allograft recipients

N-octanoyl dopamine (NOD) treatment improves renal function when applied to brain dead donors and in the setting of warm ischaemia-induced acute kidney injury (AKI). Because it also activates transient receptor potential vanilloid type 1 (TRPV1) channels, we first assessed if NOD conveys its renoprotective properties in warm ischaemia-induced AKI via TRPV1 and secondly, if renal transplant recipients also benefit from NOD treatment. We induced warm renal ischaemia in Lewis, wild-type (WT) and TRPV1(-/-) Sprague-Dawley (sd) rats by clamping the left renal artery for 45 min. Transplantations were performed in allogeneic and syngeneic donor-recipient combinations (Fisher to Lewis and Lewis to Lewis) with a cold ischaemia time of 20 h. Treatment was instituted directly after restoration of organ perfusion. Renal function, histology and perfusion were assessed by serum creatinine, microscopy and magnetic resonance imaging (MRI) using arterial spin labelling (ASL). NOD treatment significantly improved renal function in Lewis rats after warm ischaemia-induced AKI. It was, however, not effective after prolonged cold ischaemia. The renoprotective properties of NOD were only observed in Lewis or WT, but not in TRPV1(-/-) sd rats. Renal inflammation was significantly abrogated by NOD. MRI-ASL showed a significantly lower cortical perfusion in ischaemic when compared with non-ischaemic kidneys. No overall differences were observed in renal perfusion between NOD- and NaCl-treated rats. NOD treatment reduces renal injury in warm ischaemia, but is not effective in renal transplant in our experimental animal models. The salutary effect of NOD appears to be TPRV1-dependent, not involving large changes in renal perfusion