Single Cell Isolation from Surgically Resected Tissue Via Mechanical Dissociation Using TissueGrinder.

Primary cells form the basis of modern-day in vitro research analysis tools. Many conventional procedures for generating single-cell suspensions from solid tissue are neither robust nor reproducible. Here we describe primary cells isolation from surgically resected tumor tissue via enzyme-free mechanical dissociation using TissueGrinder, a novel semi-automated benchtop device. The isolated cells can be used for any downstream biochemical or cell-based analytic assay.

Adipose-Derived Mesenchymal Stem Cells Protect Endothelial Cells from Hypoxic Injury by Suppressing Terminal UPR In Vivo and In Vitro.

Adipose-derived stem cells (ASCs) have been used as a therapeutic intervention for peripheral artery disease (PAD) in clinical trials. To further explore the therapeutic mechanism of these mesenchymal multipotent stromal/stem cells in PAD, this study was designed to test the effect of xenogeneic ASCs extracted from human adipose tissue on hypoxic endothelial cells (ECs) and terminal unfolded protein response (UPR) in vitro and in an atherosclerosis-prone apolipoprotein E-deficient mice (ApoE-/- mice) hindlimb ischemia model in vivo. ASCs were added to Cobalt (II) chloride-treated ECs; then, metabolic activity, cell migration, and tube formation were evaluated. Fluorescence-based sensors were used to assess dynamic changes in Ca2+ levels in the cytosolic- and endoplasmic reticulum (ER) as well as changes in reactive oxygen species. Western blotting was used to observe the UPR pathway. To simulate an acute-on-chronic model of PAD, ApoE-/- mice were subjected to a double ligation of the femoral artery (DLFA). An assessment of functional recovery after DFLA was conducted, as well as histology of gastrocnemius. Hypoxia caused ER stress in ECs, but ASCs reduced it, thereby promoting cell survival. Treatment with ASCs ameliorated the effects of ischemia on muscle tissue in the ApoE-/- mice hindlimb ischemia model. Animals showed less muscle necrosis, less inflammation, and lower levels of muscle enzymes after ASC injection. In vitro and in vivo results revealed that all ER stress sensors (BIP, ATF6, CHOP, and XBP1) were activated. We also observed that the expression of these proteins was reduced in the ASCs treatment group. ASCs effectively alleviated endothelial dysfunction under hypoxic conditions by strengthening ATF6 and initiating a transcriptional program to restore ER homeostasis. In general, our data suggest that ASCs may be a meaningful treatment option for patients with PAD who do not have traditional revascularization options.

Comparison of Tumour-Specific Phenotypes in Human Primary and Expandable Pancreatic Cancer Cell Lines.

There is an ongoing need for patient-specific chemotherapy for pancreatic cancer. Tumour cells isolated from human tissues can be used to predict patients' response to chemotherapy. However, the isolation and maintenance of pancreatic cancer cells is challenging because these cells become highly vulnerable after losing the tumour microenvironment. Therefore, we investigated whether the cells retained their original characteristics after lentiviral transfection and expansion. Three human primary pancreatic cancer cell lines were lentivirally transduced to create expandable (Ex) cells which were then compared with primary (Pri) cells. No obvious differences in the morphology or epithelial-mesenchymal transition (EMT) were observed between the primary and expandable cell lines. The two expandable cell lines showed higher proliferation rates in the 2D and 3D models. All three expandable cell lines showed attenuated migratory ability. Differences in gene expression between primary and expandable cell lines were then compared using RNA-Seq data. Potential target drugs were predicted by differentially expressed genes (DEGs), and differentially expressed pathways (DEPs) related to tumour-specific characteristics such as proliferation, migration, EMT, drug resistance, and reactive oxygen species (ROS) were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We found that the two expandable cell lines expressed similar chemosensitivity and redox-regulatory capability to gemcitabine and oxaliplatin in the 2D model as compared to their counterparts. In conclusion, we successfully generated expandable primary pancreatic cancer cell lines using lentiviral transduction. These expandable cells not only retain some tumour-specific biological traits of primary cells but also show an ongoing proliferative capacity, thereby yielding sufficient material for drug response assays, which may provide a patient-specific platform for chemotherapy drug screening.

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.

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.

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

Introduction: Recent 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. Methods: Thirty-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. Results: TissueGinder 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. Discussion: Our 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.

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.

Concurrent stimulation of monocytes with CSF1 and polarizing cytokines reveals phenotypic and functional differences with classical polarized macrophages.

In atherosclerotic lesions, macrophages are exposed to CSFs and various microenvironmental cues, which ultimately drive their polarization state. We studied the expression of different CSFs in artery specimen and cultured vascular cells and assessed whether concurrent stimulation (CS) of monocytes with CSF1 and polarizing cytokines generated macrophages (CSM1 and CSM2) that were phenotypically and functionally different from classically polarized M1 and M2 macrophages. We also assessed the influence of acetylsalicylic acid (ASA) on the capacity of polarized macrophages to stimulate T-cell proliferation. CSF1 was the most prominent CSF expressed in arteries and cultured vascular cells. M1 and CSM1 macrophages differed in CD86 and CD14 expression, which was up-regulated respectively down-regulated by LPS. M2 and CSM2 macrophages were phenotypically similar. Cyclooxygenase expression was different in CSM1 (COX-1- and COX-2+ after LPS stimulation) and CSM2 (COX-1+ and COX-2- ) macrophages. TNFα production was more pronounced in CSM1 macrophages, whereas IL-10 was produced at higher levels by CSM2 macrophages. Proliferation of allogeneic T cells was strongly supported by CSM2, but not by CSM1 polarized macrophages. Although ASA did not affect anti-CD3/CD28-mediated proliferation, it significantly reduced CSM2 and CSM1-mediated T-cell proliferation. Supernatants of LPS-stimulated CSM2 but not of CSM1 macrophages could overcome the inhibition by ASA. Hence, we demonstrate that CSM1 and CSM2 macrophages are phenotypically and to some extent functionally distinct from classically polarized M1 and M2 macrophages. CSM2 macrophages produce a COX-1-dependent soluble factor that supports T-cell proliferation, the identity hereof is still elusive and warrants further studies.

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.

Weighted Gene Co-Expression Network Analysis Reveals Key Genes and Potential Drugs in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a prevalent aortic disease that causes high mortality due to asymptomatic gradual expansion and sudden rupture. The underlying molecular mechanisms and effective pharmaceutical therapy for preventing AAA progression have not been fully identified. In this study, we identified the key modules and hub genes involved in AAA growth from the GSE17901 dataset in the Gene Expression Omnibus (GEO) database through the weighted gene co-expression network analysis (WGCNA). Key genes were further selected and validated in the mouse dataset (GSE12591) and human datasets (GSE7084, GSE47472, and GSE57691). Finally, we predicted drug candidates targeting key genes using the Drug-Gene Interaction database. Overall, we identified key modules enriched in the mitotic cell cycle, GTPase activity, and several metabolic processes. Seven key genes (CCR5, ADCY5, ADCY3, ACACB, LPIN1, ACSL1, UCP3) related to AAA progression were identified. A total of 35 drugs/compounds targeting the key genes were predicted, which may have the potential to prevent AAA progression.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision.

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories. Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 ± 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

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 µM of MGO for 5 h induced p53 phosphorylation. This was paralleled by an increase in TUNEL and γ-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.

First report on establishment and characterization of a carcinosarcoma tumour cell line model of the bladder.

Carcinosarcoma of the urinary bladder is a very rare and aggressive subtype of bladder cancer with poor prognosis. Characteristically carcinosarcomas exhibit biphasic nature with both epithelial and mesenchymal differentiation. Limited information is available regarding its clinical features and appropriate treatments due to its rarity. Development of tumour models can further our understanding of bladder carcinosarcoma. We report establishment and characterization of the first-ever bladder carcinosarcoma cell line MaS-3. It is established by the outgrow method from 86 year-old caucasian male who underwent a radical pelvic resection after neoadjuvant radiotherapy. MaS-3 showed carcinosarcoma profile with high conformity with to the original tumour in terms of immunocytochemistry. Proteome analysis also aligned the MaS-3 cell line with the carcinosarcoma specimen rather than corresponding non-malignant tissue. Chemotherapy sensitivity testing revealed a great sensitivity of MaS-3 growth to 5-Fluorouracil, Gemcitabine and Cisplatin, with almost no impact of Irinotecan. Additionally, the suitability of MaS-3 for 3D in vitro experiments was also demonstrated. The newly established cell line MaS-3 shows typical characteristics of the tumour and may thus be a useful in vitro model system for studying the tumour biology and developing future of treatments of this rare but very aggressive entity.

N-Octanoyl-Dopamine inhibits cytokine production in activated T-cells and diminishes MHC-class-II expression as well as adhesion molecules in IFNγ-stimulated endothelial cells.

IFNγ enhances allograft immunogenicity and facilitates T-cell mediated rejection. This may cause interstitial fibrosis and tubular atrophy (IFTA), contributing to chronic allograft loss. We assessed if inhibition of T-cell activation by N-octanoyl dopamine (NOD) impairs adherence of activated T-cells to endothelial cells and the ability of activated T-cells to produce IFNγ. We also assessed if NOD affects IFNγ mediated gene expression in endothelial cells. The presence of NOD during T-cell activation significantly blunted their adhesion to unstimulated and cytokine stimulated HUVEC. Supernatants of these T-cells displayed significantly lower concentrations of TNFα and IFNγ and were less capable to facilitate T-cell adhesion. In the presence of NOD VLA-4 (CD49d/CD29) and LFA-1 (CD11a/CD18) expression on T-cells was reduced. NOD treatment of IFNγ stimulated HUVEC reduced the expression of MHC class II transactivator (CIITA), of MHC class II and its associated invariant chain CD74. Since IFTA is associated with T-cell mediated rejection and IFNγ to a large extent regulates immunogenicity of allografts, our current data suggest a potential clinical use of NOD in the treatment of transplant recipients. Further in vivo studies are warranted to confirm these in vitro findings and to assess the benefit of NOD on IFTA in clinically relevant models.

Analyses of Synthetic N-Acyl Dopamine Derivatives Revealing Different Structural Requirements for Their Anti-inflammatory and Transient-Receptor-Potential-Channel-of-the-Vanilloid-Receptor-Subfamily-Subtype-1 (TRPV1)-Activating Properties.

We studied the chemical entities within N-octanoyl dopamine (NOD) responsible for the activation of transient-receptor-potential channels of the vanilloid-receptor subtype 1 (TRPV1) and inhibition of inflammation. The potency of NOD in activating TRPV1 was significantly higher compared with those of variants in which the ortho-dihydroxy groups were acetylated, one of the hydroxy groups was omitted ( N-octanoyl tyramine), or the ester functionality consisted of a bulky fatty acid ( N-pivaloyl dopamine). Shortening of the amide linker (ΔNOD) slightly increased its potency, which was further increased when the carbonyl and amide groups (ΔNODR) were interchanged. With the exception of ΔNOD, the presence of an intact catechol structure was obligatory for the inhibition of VCAM-1 and the induction of HO-1 expression. Because TRPV1 activation and the inhibition of inflammation by N-acyl dopamines require different structural entities, our findings provide a framework for the rational design of TRPV1 agonists with improved anti-inflammatory properties.

Radiofluorinated N-Octanoyl Dopamine ([18F]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 [18F]F-NOD [18F]5 for in vivo assessment of NOD's elimination kinetics by means of PET imaging. [18F]5 was synthesized in reproducibly high radiochemical yields and purity (>98%) as well as high specific activities (>20 GBq/µmol). Stability tests showed no decomposition of [18F]5 over a period of 120 min in rat plasma. In vitro, low cell association was found for [18F]5, indicating no active transport mechanism into cells. In vivo, [18F]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.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

N-acyl dopamine derivates as lead compound for implementation in transplantation medicine.

Conjugates of fatty acids with ethanolamine, amino acids or monoamine neurotransmitters occur widely in nature giving rise to so-called endocannabinoids. Anandamide and 2-arachidonoyl glycerol are the best characterized endocannabinoids activating both cannabinoid receptors (CB1 and CB2) and transient receptor potential vanilloid type 1 (TRPV1) channels (anandamide) or activating cannabinoid receptors only (2-arachidonoyl glycerol). TRPV1 is also activated by vanilloids, such as capsaicin, and endogenous neurolipins, e.g. N-arachidonoyl dopamine (NADA) and N-oleoyl dopamine (OLDA). Because donor dopamine treatment has shown to improve transplantation outcome in renal and heart recipients, this review will mainly focus on the biological activities of N-acyl dopamine derivates (NADD) as potential non-hemodynamic alternative for implementation in transplantation medicine. Hence the influence of NADD on transplantation relevant entities, i.e. cold inflicted injury, cytoprotection, I/R-injury, immune-modulation and inflammation will be summarized. The cytoprotective properties of endogenous endocannabinoids in this context will be briefly touched upon.

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.