[Show abstract][Hide abstract] ABSTRACT: Background
Novel synthesized analogs of Aplidin, PM01215 and PM02781, were tested for antiangiogenic effects on primary human endothelial cells in vitro and for inhibition of angiogenesis and tumor growth in vivo.
Antiangiogenic activity of both derivatives was evaluated by real-time cell proliferation, capillary tube formation and vascular endothelial growth factor (VEGF)-induced spheroid sprouting assays. Distribution of endothelial cells in the different phases of the cell cycle was analyzed by flow cytometry. Aplidin analogs were tested in vivo in chicken chorioallantoic membrane (CAM) assays.
Both derivatives inhibited angiogenic capacities of human endothelial cells (HUVECs) in vitro at low nanomolar concentrations. Antiangiogenic effects of both analogs were observed in the CAM. In addition, growth of human multiple myeloma xenografts in vivo in CAM was significantly reduced after application of both analogs. On the molecular level, both derivatives induced cell cycle arrest in G1 phase. This growth arrest of endothelial cells correlated with induction of the cell cycle inhibitor p16 INK4A and increased senescence-associated beta galactosidase activity. In addition, Aplidin analogs induced oxidative stress and decreased production of the vascular maturation factors Vasohibin-1 and Dickkopf-3.
From these findings we conclude that both analogs are promising agents for the development of antiangiogenic drugs acting independent on classical inhibition of VEGF signaling.
BMC Cancer 10/2015; 2015(15):738. DOI:10.1186/s12885-015-1729-4 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the function of epidermal Langerhans cells (LCs) has been extensively scrutinized, little is known about their localization in epidermal appendages. We studied sebaceous glands in dermal sheet preparations of murine ear skin. In both BALB/c and C57BL/6 mice, a similar density of glands was found (~65/mm(2) ). However, the occurrence of LCs associated to sebaceous glands was clearly strain-dependent. Approximately 70% of sebaceous glands included LCs in BALB/c mice, as compared to only 18% in C57BL/6. LCs consistently co-expressed MHCII and Langerin/CD207 and were negative for the maturation marker CD86. LCs associated with sebaceous glands, which belong to the epidermal compartment, accounted for 46.5% (BALB/c) and 16.2% (C57BL/6) of all Langerin+ cells detectable in dermal sheets. Since sebaceous glands are considered as entry portals for microorganisms, the presence of LCs is likely to be related to immunosurveillance. Therefore, our results represent a relevant insight for future functional investigations on LCs. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Total pancreatectomy is a last option for patients in whom all other efforts for managing intractable pain caused by chronic pancreatitis have failed. When performed with a simultaneous islet autotransplantation, endogenous insulin production can be preserved to some extent. Although it does not necessarily prevent any future need for exogenous insulin, the diabetic state of the patient is less severe compared to pancreatectomy alone. In contrast to islet allotransplantation, the patients do not require immunosuppressive drugs and are not at risk for autoimmune destruction or alloimmune rejection. Consequently, insulin independence is more likely to be preserved in autoislet-transplanted patients than in those patients who received alloislet transplants. Therefore, the setting of islet autotransplantation allows the study of the transplanted islets without interference from medication or immunological variables.
Islets of Langerhans, 01/2015: pages 1229-1243; , ISBN: 978-94-007-6685-3
[Show abstract][Hide abstract] ABSTRACT: Objective
Aim of this study was to identify the nitric oxide synthase (NOS) isoform involved in early microcirculatory derangements following solid organ transplantation.
Tetrahydrobiopterin donor treatment has been shown to specifically attenuate these derangements following pancreas transplantation, and tetrahydrobiopterin-mediated protective effects to rely on its NOS-cofactor activity, rather than on its antioxidant capacity. However, the NOS-isoform mainly involved in this process has still to be defined.
Using a murine pancreas transplantation model, grafts lacking one of the three NOS-isoforms were compared to grafts from wild-type controls. Donors were treated with either tetrahydrobiopterin or remained untreated. All grafts were subjected to 16 h cold ischemia time and transplanted into wild-type recipients. Following 4 h graft reperfusion, microcirculation was analysed by confocal intravital fluorescence microscopy. Recipient survival was monitored for 50 days.
Transplantation of the pancreas from untreated wild-type donor mice resulted in microcirculatory damage of the transplanted graft and no recipient survived more than 72 h. Transplanting grafts from untreated donor mice lacking either endothelial or inducible NOS led to similar outcomes. In contrast, donor treatment with tetrahydrobiopterin prevented microcirculatory breakdown enabling long-term survival. Sole exception was transplantation of grafts from untreated donor mice lacking neuronal NOS. It resulted in intact microvascular structure and long-term recipient survival, either if donor mice were untreated or treated with tetrahydrobiopterin.
We demonstrate for the first time the crucial involvement of neuronal NOS in early microcirculatory derangements following solid organ transplantation. In this model, protective effects of tetrahydrobiopterin are mediated by targeting this isoform.
PLoS ONE 11/2014; 9(11):e112570. DOI:10.1371/journal.pone.0112570 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Type 2 diabetes is associated with pancreatic α cell dysfunction, characterized by elevated fasting plasma glucagon concentrations and inadequate postprandial glucose- and insulin-induced suppression of glucagon secretion. The cause and the underlying mechanisms of α cell dysfunction are unknown.
American Journal of Clinical Nutrition 11/2014; 100(5):1222-31. DOI:10.3945/ajcn.114.092023 · 6.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Mitochondrial reactive oxygen species (ROS) produced during
early reperfusion are important triggers for the development of ischemia/
reperfusion injury (IRI) in the course of solid organ transplantation. However,
antioxidants yielded little clinical benefit in the prevention of IRI, most likely
because of the failure to timely and efficiently target to the site of ROS
production and action. In our work we are pursuing strategies to limit or prevent
the early increase in ROS by targeting intracellular signaling pathways.
Cytosolic p66SHC, which translocates to the mitochondria under stress to
, is one of the main sources of ROS under stress
conditions. Gene ablation experiments in mice also confirmed its important role
in the development of IRI. Here we identified pathways essential for the
activation of p66SHC, which may be targeted for therapeutic purposes.
HL-1 cardiomyocytes and mouse embryonic fibroblasts (MEFs)
were exposed to hypoxia/reoxygenation (HR) or pro-oxidant treatment.
Intracellular signaling was monitored by phosphorylation-specific antibodies.
ROS were detected by MitoTracker Red CM-H2XRos, DCF-DA, or protein-
based ROS probes
phosphorylation of p66SHC was analyzed by mass
Using MEFs and HL-1 cardiomyocytes we failed to detect the
previously reported PKCß-dependent phosphorylation of p66SHC on S36 and
instead identified S139 as critical target under oxidative stress. In p66SHC-
deficient MEFs reconstituted with the S139A mutant of p66SHC we observed
significantly reduced ROS levels. Also MAPK activities (ERK, JNK, p38),
p66SHC S36 phosphorylation and ROS production were increased under
prooxidant treatment and HR. Inhibition of signaling through MAPKs with
specific inhibitors showed a pronounced decrease in p66SHC S36 phosphor-
ylation only for JNK, which also directly interacted with p66SHC. Moreover,
JNK inhibition resulted in decreased ROS production and reduced DNA
damage. We further confirmed JNK-dependent regulation of p66SHC S36
phosphorylation, ROS production and cell death using JNK 1, 2 deficient MEFs.
Finally, the low ROS phenotype of JNK 1, 2 knockout MEFs was reversed by
introducing p66SHC mutated in S36E.
Interference with p66SHC activation by targeting upstream
pathways (PKCß, JNK) thus may provide a therapeutic approach for decreas-
ing damage to cells and organs during ischemia/reperfusion.
[Show abstract][Hide abstract] ABSTRACT: FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.
The effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.
We report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.
The combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.
Molecular Cancer 09/2014; 13(1):224. DOI:10.1186/1476-4598-13-224 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Premature birth represents a clinical situation of risk for brain injury. The diversity of pathophysiological processes complicates efforts to find effective therapeutic strategies. Excitotoxicity is one important factor in the pathogenesis of preterm brain injury. The observation that sigma-1 receptor agonists possess neuroprotective potential, at least partly mediated by a variety of anti-excitotoxic mechanisms, has generated great interest in targeting those receptors to counteract brain injury. The objective of this study was to evaluate the effect of the highly specific sigma-1 receptor agonist, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) to protect against excitotoxic developmental brain injury in vivo and in vitro. Primary hippocampal neurons were pre-treated with PPBP before glutamate was applied and subsequently analysed for cell death (PI/calcein AM), mitochondrial activity (TMRM) and morphology of the neuronal network (WGA) using confocal microscopy. Using an established neonatal mouse model we also determined whether systemic injection of PPBP significantly attenuates excitotoxic brain injury. PPBP significantly reduced neuronal cell death in primary hippocampal neurons exposed to glutamate. Neurons treated with PPBP showed a less pronounced loss of mitochondrial membrane potential and fewer morphological changes after glutamate exposure. A single intraperitoneal injection of PPBP given one hour after the excitotoxic insult significantly reduced microglial cell activation and lesion size in cortical gray and white matter. The present study provides strong support for the consideration of sigma-1 receptor agonists as a candidate therapy for the reduction of neonatal excitotoxic brain lesions and might offer a novel target to counteract developmental brain injury.
[Show abstract][Hide abstract] ABSTRACT: Background:
The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model.
Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy.
Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr.
Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.
[Show abstract][Hide abstract] ABSTRACT: Prolonged Ischemia (I) times caused by organ procurement and transport are main contributors to a decrease in organ function, which is further enhanced during early reperfusion (R). This combined damage, referred to as ischemia-reperfusion injury (IRI), is a main contributor to delayed graft function, which leads to costly and lengthy follow-up treatments or even organ loss. Methods to monitor the status of a graft prior to transplantation are, therefore, highly desirable to optimize the clinical outcome. Here we propose the use of fine needle biopsies, which are analyzed by real time live confocal microscopy. Such a combination provides information about the functional and structural integrity of an organ within a few minutes. To confirm the feasibility of this approach, we obtained fine needle biopsies from rodent kidneys and exposed them to various stress conditions. Following the addition of a range of live stains, biopsies were monitored for mitochondrial function, cell viability and tissue integrity using confocal live cell imaging. Our data demonstrate that this procedure requires minimal time for sample preparation and data acquisition and is well suitable to record organ damage resulting from unphysiological stress. This article is protected by copyright. All rights reserved.
Transplant International 04/2014; 27(8). DOI:10.1111/tri.12338 · 2.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many diseases and pathological conditions are characterized by transient or constitutive overproduction of reactive oxygen species (ROS). ROS are causal for ischemia/reperfusion (IR)-associated tissue injury (IRI), a major contributor to organ dysfunction or failure. Preventing IRI with antioxidants failed in the clinic, most likely due to the difficulty to timely and efficiently target them to the site of ROS production and action. IR is also characterized by changes in the activity of intracellular signaling molecules including the stress kinase p38MAPK. While ROS can cause the activation of p38MAPK, we recently obtained in vitro evidence that p38MAPK activation is responsible for elevated mitochondrial ROS levels, thus suggesting a role for p38MAPK upstream of ROS and their damaging effects.
Here we identified p38MAPKalpha as the predominantly expressed isoform in HL-1 cardiomyocytes and siRNA-mediated knockdown demonstrated the pro-oxidant role of p38MAPKalpha signaling. Moreover, the knockout of the p38MAPK effector MAPKAP kinase 2 (MK2) reproduced the effect of inhibiting or knocking down p38MAPK. To translate these findings into a setting closer to the clinic a stringent kidney clamping model was used. p38MAPK activity increased upon reperfusion and p38MAPK inhibition by the inhibitor BIRB796 almost completely prevented severe functional impairment caused by IR. Histological and molecular analyses showed that protection resulted from decreased redox stress and apoptotic cell death.
These data highlight a novel and important mechanism for p38MAPK to cause IRI and suggest it as a potential therapeutic target for prevention of tissue injury.
Cell Communication and Signaling 01/2014; 12(1):6. DOI:10.1186/1478-811X-12-6 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Islet transplantation is a valid treatment option for patients suffering from type 1 diabetes mellitus. To assure optimal islet cell quality, specialized islet isolation facilities have been developed. Utilization of such facilities necessitates transportation of islet cells to distant institutions for transplantation. Despite its importance, a clinically feasible solution for the transport of islets has still not been established. We here compare the functionality of isolated islets from C57BL/6 mice directly after the isolation procedure as well as after two simulated transport conditions, static versus rotation. Islet cell quality was assessed using real-time live confocal microscopy. In vivo islet function after syngeneic transplantation was determined by weight and blood sugar measurements as well as by intraperitoneal glucose tolerance tests. Vascularization of islets was documented by fluorescence microscopy and immunohistochemistry. All viability parameters documented comparable cell viability in the rotary group and the group transplanted immediately after isolation. Functional parameters assessed in vivo displayed no significant difference between these two groups. Moreover, vascularization of islets was similar in both groups. In conclusion, rotary culture conditions allows the maintenance of highest islet quality for at least 15 h, which is comparable to that of freshly isolated islets.
[Show abstract][Hide abstract] ABSTRACT: Langerhans cells (LCs) are dendritic cells (DCs) residing in epithelia, where they critically regulate immunity and tolerance. The p14 adaptor molecule is part of the late endosomal/lysosomal adaptor and MAPK and mTOR activator/regulator (LAMTOR) complex, thereby contributing to the signal transduction of the "extracellular signaling-regulated kinase" (ERK) and the "mammalian target of rapamycin" (mTOR) cascade. Furthermore, p14 represents an important regulator for endosomal sorting processes within the cell. Mutated, dysfunctional p14 leads to a human immunodeficiency disorder with endosomal/lysosomal defects in immune cells. Since p14 participates in the regulation of endosomal trafficking, growth factor signaling and cell proliferation, we investigated the role of p14 in mouse DCs/LCs using a conditional knock out mouse model. p14-deficient animals displayed a virtually complete loss of LCs in the epidermis early after birth due to impaired proliferation and increased apoptosis of LCs. Repopulation analysis after application of contact sensitizer leads to the recruitment of a transient LC population, predominantly consisting of short-term LCs. The underlying molecular mechanism involves the p14-mediated disruption of the LAMTOR complex which results in the malfunction of both ERK and mTOR signal pathways. Hence, we conclude that p14 acts as a novel and essential regulator of LC homeostasis in vivo.
[Show abstract][Hide abstract] ABSTRACT: Ischemia-reperfusion injury is a primarily non-allospecific event leading to the depletion of the essential nitric oxide synthase cofactor and potent antioxidant tetrahydrobiopterin. Suboptimal concentrations of tetrahydrobiopterin result in a reduced biosynthesis of nitric oxide leading to vascular endothelial dysfunction. Tetrahydrobiopterin supplementation has been shown to protect from this pathological state in a plethora of cardiovascular diseases including transplant-related ischemia-reperfusion injury. Even though still controversially discussed, there is increasing evidence emerging from both human as well as animal studies that tetrahydrobiopterin-mediated actions rely on its nitric oxide synthase cofactor activity rather than on its antioxidative properties. Herein, we review the current literature regarding the role of tetrahydrobiopterin in ischemia-reperfusion injury including our experience acquired in a murine pancreas transplantation model.
[Show abstract][Hide abstract] ABSTRACT: Dysregulation of the insulin-like growth factor (IGF) axis is implicated in the development of benign prostatic hyperplasia (BPH) and prostate cancer (PCa), two of the most common diseases affecting elderly males. PCa is the second leading cause of male-related cancer death in Western societies. Although distinct pathologies, BPH and PCa are both characterized by extensive stromal remodeling, in particular fibroblast-to-myofibroblast differentiation thought to be induced by elevated local production of transforming growth factor beta 1 (TGFβ1). We previously showed that TGFβ1-mediated fibroblast-to-myofibroblast differentiation of primary human prostatic stromal cells (PrSCs) resulted in dsyregulation of several components of the IGF axis, including induction of IGF binding protein 3 (IGFBP3). Using isoform-specific lentiviral-mediated knockdown, we demonstrate herein that IGFBP3 is essential for TGFβ1-mediated differentiation. Whilst recombinant human IGFBP3 alone was not sufficient to induce differentiation, IGFBP3 synergistically potentiated TGFβ1-mediated stromal remodeling predominantly via an IGF-independent mechanism. Consistent with these in vitro findings, IGFBP3 immunohistochemistry revealed elevated levels of IGFBP3 in the hyperplastic fibromuscular stroma of BPH specimens and in the tumor-adjacent stroma of high-grade PCa. Collectively, these data indicate that dysregulation of the stromal IGF axis, in particular elevated IGFBP3, plays a crucial role in fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma and indicate the therapeutic potential of inhibiting stromal remodeling and the resulting dysregulation of the stromal IGF axis as a novel strategy for the treatment of advanced PCa and BPH.
[Show abstract][Hide abstract] ABSTRACT: Infections with enterohemorrhagic Escherichia coli (EHEC) are a primary cause of hemolytic uremic syndrome (HUS). Recently, Shiga toxin 2 (Stx2), the major virulence factor of EHEC, was reported to interact with complement, implying that the latter is involved in the pathogenesis of EHEC-induced HUS.The aim of the present study was to investigate the effect of Stx2 on the expression of membrane-bound complement regulators CD46, CD55 and CD59 on proximal tubular epithelial (HK-2) and glomerular endothelial (GEnC) cells, derived from human kidney cells that are involved in HUS.Incubation with Stx2 did not influence the amount of CD46 and CD55 on the surface of HK-2 and GEnC cells, as determined by FACS. In contrast, CD59 was significantly reduced by half on GEnC cells, but less pronounced on HK-2 cells. With increasing amounts of Stx2 reduction of CD59 also reached significance in HK-2 cells. ELISA analyses showed that CD59 was not present in supernatant of Stx2-treated cells, implying that CD59 reduction was not caused by cleavage from the cell surface. In fact, RT-qPCR analyses showed downregulation of CD59 mRNA as likely reason for CD59 cell surface reduction. In addition, a significant increase in terminal complement complex deposition on HK-2 cells was observed after treatment with Stx2, as possible consequence of CD59 downregulation.In summary, Stx2 downregulates CD59 on mRNA and protein levels on tubular epithelial and glomerular endothelial cells, which likely contributes to complement activation and kidney destruction in EHEC-associated HUS.
Infection and immunity 05/2013; 81(8). DOI:10.1128/IAI.01079-12 · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipocalin-2 (Lcn2) expression contributes to ischemia and reperfusion injury (IRI) by enhancing pro-inflammatory responses. The aim of this work was to elucidate the regulation of Lcn2 during hypoxia and its effects on the expression of key chemokines and adhesion molecules. Lcn2 wt and Lcn2(-/-) mice were used in a heterotopic heart transplantation model. Quantitative RT-PCR was applied for chemokine gene expression analysis. Reporter gene studies were used to elucidate the regulation of the Lcn2 promoter by hypoxia. HIF-1β expression led to a 2.4-fold induction of the Lcn2 promoter. Apart from an earlier onset of granulocyte infiltration in the Lcn2 wt setting after 2 h of reperfusion compared with the Lcn2(-/-) setting (P < 0.013), exogenous application of recombinant Lcn2 revealed a trend toward increase of granulocyte infiltration. Analyzed chemokines were expressed significantly higher in the Lcn2 wt setting at 2 h of reperfusion (P ≤ 0.05). The number of apoptotic cells observed in Lcn2(-/-) grafts was significantly higher than in the Lcn2 wt setting. Our results indicate that Lcn2 affects granulocyte infiltration in the reperfused graft by modulating the expression of chemokines, their receptors and the apoptotic rate.
Transplant International 05/2013; 26(7). DOI:10.1111/tri.12116 · 2.60 Impact Factor