Deletion of vascular thromboxane A2 receptors and its impact on angiotensin II-induced hypertension and atherosclerotic lesion formation in the aorta of Ldlr-deficient mice.

The thromboxane A2 receptor (TP) has been shown to play a role in angiotensin II (Ang II)-mediated hypertension and pathological vascular remodeling. To assess the impact of vascular TP on Ang II-induced hypertension, atherogenesis, and pathological aortic alterations, i.e. aneurysms, we analysed Western-type diet-fed and Ang II-infused TPVSMC KO/Ldlr KO, TPEC KO/Ldlr KO mice and their respective wild-type littermates (TPWT/Ldlr KO). These analyses showed that neither EC- nor VSMC-specific deletion of the TP significantly affected basal or Ang II-induced blood pressure or aortic atherosclerotic lesion area. In contrast, VSMC-specific TP deletion abolished and EC-specific TP deletion surprisingly reduced the ex vivo reactivity of aortic rings to the TP agonist U-46619, whereas VSMC-specific TP knockout also diminished the ex vivo response of aortic rings to Ang II. Furthermore, despite similar systemic blood pressure, there was a trend towards less atherogenesis in the aortic arch and a trend towards fewer pathological aortic alterations in Ang II-treated female TPVSMC KO/Ldlr KO mice. Survival was impaired in male mice after Ang II infusion and tended to be higher in TPVSMC KO/Ldlr KO mice than in TPWT/Ldlr KO littermates. Thus, our data may suggest a deleterious role of the TP expressed in VSMC in the pathogenesis of Ang II-induced aortic atherosclerosis in female mice, and a surprising role of the endothelial TP in TP-mediated aortic contraction. However, future studies are needed to substantiate and further elucidate the role of the vascular TP in the pathogenesis of Ang II-induced hypertension, aortic atherosclerosis and aneurysm formation.

EGFR activation differentially affects the inflammatory profiles of female human aortic and coronary artery endothelial cells.

Endothelial cells (EC) are key players in vascular function, homeostasis and inflammation. EC show substantial heterogeneity due to inter-individual variability (e.g. sex-differences) and intra-individual differences as they originate from different organs or vessels. This variability may lead to different responsiveness to external stimuli. Here we compared the responsiveness of female human primary EC from the aorta (HAoEC) and coronary arteries (HCAEC) to Epidermal Growth Factor Receptor (EGFR) activation. EGFR is an important signal integration hub for vascular active substances with physiological and pathophysiological relevance. Our transcriptomic analysis suggested that EGFR activation differentially affects the inflammatory profiles of HAoEC and HCAEC, particularly by inducing a HCAEC-driven leukocyte attraction but a downregulation of adhesion molecule and chemoattractant expression in HAoEC. Experimental assessments of selected inflammation markers were performed to validate these predictions and the results confirmed a dual role of EGFR in these cells: its activation initiated an anti-inflammatory response in HAoEC but a pro-inflammatory one in HCAEC. Our study highlights that, although they are both arterial EC, female HAoEC and HCAEC are distinguishable with regard to the role of EGFR and its involvement in inflammation regulation, what may be relevant for vascular maintenance but also the pathogenesis of endothelial dysfunction.

Transcriptional impact of EGFR activation in human female vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) are critical for the vascular tone, but they can also drive the development of vascular diseases when they lose their contractile phenotype and de-differentiate. Previous studies showed that the epidermal growth factor receptor (EGFR) of VSMC is critical for vascular health, but most of the underlying mechanisms by which VSMC-EGFR controls vascular fate have remained unknown. We combined RNA-sequencing and bioinformatics analysis to characterize the effect of EGFR-activation on the transcriptome of human primary VSMC (from different female donors) and to identify potentially affected cellular processes. Our results indicate that the activation of human VSMC-EGFR is sufficient to trigger a phenotypical switch toward a proliferative and inflammatory phenotype. The extent of this effect is nonetheless partly donor-dependent. Our hypothesis-generating study thus provides a first insight into mechanisms that could partly explain variable susceptibilities to vascular diseases in between individuals.

The role of EGFR in vascular AT1R signaling: From cellular mechanisms to systemic relevance.

The epidermal growth factor receptor (EGFR) belongs to the ErbB-family of receptor tyrosine kinases that are of importance in oncology. During the last years, substantial evidence accumulated for a crucial role of EGFR concerning the action of the angiotensin II type 1 receptor (AT1R) in blood vessels, resulting form AT1R-induced EGFR transactivation. This transactivation occurs through the release of membrane-anchored EGFR-ligands, cytosolic tyrosine kinases, heterocomplex formation or enhanced ligand expression. AT1R-EGFR crosstalk amplifies the signaling response and enhances the biological effects of angiotensin II. Downstream signaling cascades include ERK1/2 and p38 MAPK, PLCγ and STAT. AT1R-induced EGFR activation contributes to vascular remodeling and hypertrophy via e.g. smooth muscle cell proliferation, migration and extracellular matrix production. EGFR transactivation results in increased vessel wall thickness and reduced vascular compliance. AT1R and EGFR signaling pathways are also implicated the induction of vascular inflammation. Again, EGFR transactivation exacerbates the effects, leading to endothelial dysfunction that contributes to vascular inflammation, dysfunction and remodeling. Dysregulation of the AT1R-EGFR axis has been implicated in the pathogenesis of various cardiovascular diseases and inhibition or prevention of EGFR signaling can attenuate part of the detrimental impact of enhanced renin-angiotensin-system (RAAS) activity, highlighting the importance of EGFR for the adverse consequences of AT1R activation. In summary, EGFR plays a critical role in vascular AT1R action, enhancing signaling, promoting remodeling, contributing to inflammation, and participating in the pathogenesis of cardiovascular diseases. Understanding the interplay between AT1R and EGFR will foster the development of effective therapeutic strategies of RAAS-induced disorders.

A current overview of RhoA, RhoB, and RhoC functions in vascular biology and pathology.

The Rho subfamily members of Rho GTPases, RhoA, RhoB, and RhoC, are key regulators of signal transduction in a variety of cellular processes, including regulation of actomyosin and microtubule dynamics, cell shape, cell adhesion, cell division, cell migration, vesicle/membrane trafficking, and cell proliferation. Traditionally, the focus of research on RhoA/B/C has been on tumor biology, as dysregulation of expression or function of these proteins plays an important role in the pathogenesis of various cancer entities. However, RhoA, RhoB, and RhoC are also important in the context of vascular biology and pathology because they influence endothelial barrier function, vascular smooth muscle contractility and proliferation, vascular function and remodelling as well as angiogenesis. In this context, RhoA/B/C exploit numerous effector molecules to transmit their signals, and their activity is regulated by a variety of guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) that enable precise spatiotemporal activation often in concert with other Rho GTPases. Although their protein structure is very similar, different mechanisms of regulation of gene expression, different localization, and to some extent different interaction with RhoGAPs and RhoGEFs have been observed for RhoA/B/C. In this review, we aim to provide a current overview of the Rho subfamily as regulators of vascular biology and pathology, analyzing database information and existing literature on expression, protein structure, and interaction with effectors and regulatory proteins. In this setting, we will also discuss recent findings on Rho effectors, RhoGEFs, RhoGAPs, as well as guanine nucleotide dissociation inhibitors (RhoGDIs).

Aspirin sensitivity of PIK3CA-mutated Colorectal Cancer: potential mechanisms revisited.

PIK3CA mutations are amongst the most prevalent somatic mutations in cancer and are associated with resistance to first-line treatment along with low survival rates in a variety of malignancies. There is evidence that patients carrying PIK3CA mutations may benefit from treatment with acetylsalicylic acid, commonly known as aspirin, particularly in the setting of colorectal cancer. In this regard, it has been clarified that Class IA Phosphatidylinositol 3-kinases (PI3K), whose catalytic subunit p110α is encoded by the PIK3CA gene, are involved in signal transduction that regulates cell cycle, cell growth, and metabolism and, if disturbed, induces carcinogenic effects. Although PI3K is associated with pro-inflammatory cyclooxygenase-2 (COX-2) expression and signaling, and COX-2 is among the best-studied targets of aspirin, the mechanisms behind this clinically relevant phenomenon are still unclear. Indeed, there is further evidence that the protective, anti-carcinogenic effect of aspirin in this setting may be mediated in a COX-independent manner. However, until now the understanding of aspirin's prostaglandin-independent mode of action is poor. This review will provide an overview of the current literature on this topic and aims to analyze possible mechanisms and targets behind the aspirin sensitivity of PIK3CA-mutated cancers.

The Functional Interaction of EGFR with AT1R or TP in Primary Vascular Smooth Muscle Cells Triggers a Synergistic Regulation of Gene Expression.

In vivo, cells are simultaneously exposed to multiple stimuli whose effects are difficult to distinguish. Therefore, they are often investigated in experimental cell culture conditions where stimuli are applied separately. However, it cannot be presumed that their individual effects simply add up. As a proof-of-principle to address the relevance of transcriptional signaling synergy, we investigated the interplay of the Epidermal Growth Factor Receptor (EGFR) with the Angiotensin-II (AT1R) or the Thromboxane-A2 (TP) receptors in murine primary aortic vascular smooth muscle cells. Transcriptome analysis revealed that EGFR-AT1R or EGFR-TP simultaneous activations led to different patterns of regulated genes compared to individual receptor activations (qualitative synergy). Combined EGFR-TP activation also caused a variation of amplitude regulation for a defined set of genes (quantitative synergy), including vascular injury-relevant ones (Klf15 and Spp1). Moreover, Gene Ontology enrichment suggested that EGFR and TP-induced gene expression changes altered processes critical for vascular integrity, such as cell cycle and senescence. These bioinformatics predictions regarding the functional relevance of signaling synergy were experimentally confirmed. Therefore, by showing that the activation of more than one receptor can trigger a synergistic regulation of gene expression, our results epitomize the necessity to perform comprehensive network investigations, as the study of individual receptors may not be sufficient to understand their physiological or pathological impact.

Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells.

Background The small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA-ROCK (rho associated coiled-coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)-mediated angiogenesis, whereas other work suggests VEGF-antagonistic effects of the RhoA-ROCK axis. Methods and Results To elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune-incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild-type RhoA but not dominant-negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA-related effects, but increased angiogenic sprouting and migration of control-treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR-2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression. Conclusions Our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.

Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells.

We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.

The F2-isoprostane 8-iso-PGF2α attenuates atherosclerotic lesion formation in Ldlr-deficient mice - Potential role of vascular thromboxane A2 receptors.

The F2-isoprostane 8-iso-PGF2α (also known as 15-F2t-isoprostane, iPF2α-III, 8-epi PGF2α, 15(S)-8-iso-PGF2α, or 8-Isoprostane), a thromboxane A2 receptor (TP) agonist, stable biomarker of oxidative stress, and risk marker of cardiovascular disease, has been proposed to aggravate atherogenesis in genetic mouse models of atherosclerotic vascular disease. Moreover, the TP plays an eminent role in the pathophysiology of endothelial dysfunction, atherogenesis, and cardiovascular disease. Yet it is unknown, how the TP expressed by vascular cells affects atherogenesis or 8-iso-PGF2α-related effects in mouse models of atherosclerosis. We studied Ldlr-deficient vascular endothelial-specific (EC) and vascular smooth muscle cell (VSMC)-specific TP knockout mice (TPECKO/Ldlr KO; TPVSMCKO/Ldlr KO) and corresponding wild-type littermates (TPWT/Ldlr KO). The mice were fed a Western-type diet for eight weeks and received either 8-iso-PGF2α or vehicle infusions via osmotic pumps. Subsequently, arterial blood pressure, atherosclerotic lesion formation, and lipid profiles were analyzed. We found that VSMC-, but not EC-specific TP deletion, attenuated atherogenesis without affecting blood pressure or plasma lipid profiles of the mice. In contrast to a previous report, 8-iso-PGF2α tended to reduce atherogenesis in TPWT/Ldlr KO and TPEC KO/Ldlr KO mice, again without significantly affecting blood pressure or lipid profiles of these mice. However, no further reduction in atherogenesis was observed in 8-iso-PGF2α-treated TPVSMC KO/Ldlr KO mice. Our work suggests that the TP expressed in VSMC but not the TP expressed in EC is involved in atherosclerotic lesion formation in Ldlr-deficient mice. Furthermore, we report an inhibitory effect of 8-iso-PGF2α on atherogenesis in this experimental atherosclerosis model, which paradoxically appears to be related to the presence of the TP in VSMC.

A Thromboxane A2 Receptor-Driven COX-2-Dependent Feedback Loop That Affects Endothelial Homeostasis and Angiogenesis.

BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.

Drug-Induced Idiosyncratic Agranulocytosis - Infrequent but Dangerous.

Drug-induced agranulocytosis is a life-threatening side effect that usually manifests as a severe form of neutropenia associated with fever or signs of sepsis. It can occur as a problem in the context of therapy with a wide variety of drug classes. Numerous drugs are capable of triggering the rare idiosyncratic form of agranulocytosis, which, unlike agranulocytosis induced by cytotoxic drugs in cancer chemotherapy, is characterised by "bizzare" type B or hypersensitivity reactions, poor predictability and a mainly low incidence. The idiosyncratic reactions are thought to be initiated by chemically reactive drugs or reactive metabolites that react with proteins and may subsequently elicit an immune response, particularly directed against neutrophils and their precursors. Cells or organs that exhibit specific metabolic and biotransformation activity are therefore frequently affected. In this review, we provide an update on the understanding of drug-induced idiosyncratic agranulocytosis. Using important triggering drugs as examples, we will summarise and discuss the chemical, the biotransformation-related, the mechanistic and the therapeutic basis of this clinically relevant and undesirable side effect.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

Angiotensin II receptor type 1 - An update on structure, expression and pathology.

The AT1 receptor, a major effector of the renin-angiotensin system, has been extensively studied in the context of cardiovascular and renal disease. Moreover, angiotensin receptor blockers, sartans, are among the most frequently prescribed drugs for the treatment of hypertension, chronic heart failure and chronic kidney disease. However, precise molecular insights into the structure of this important drug target have not been available until recently. In this context, seminal studies have now revealed exciting new insights into the structure and biased signaling of the receptor and may thus foster the development of novel therapeutic approaches to enhance the efficacy of pharmacological angiotensin receptor antagonism or to enable therapeutic induction of biased receptor activity. In this review, we will therefore highlight these and other seminal publications to summarize the current understanding of the tertiary structure, ligand binding properties and downstream signal transduction of the AT1 receptor.

The Role of ABCG2 in the Pathogenesis of Primary Hyperuricemia and Gout-An Update.

Urate homeostasis in humans is a complex and highly heritable process that involves i.e., metabolic urate biosynthesis, renal urate reabsorption, as well as renal and extrarenal urate excretion. Importantly, disturbances in urate excretion are a common cause of hyperuricemia and gout. The majority of urate is eliminated by glomerular filtration in the kidney followed by an, as yet, not fully elucidated interplay of multiple transporters involved in the reabsorption or excretion of urate in the succeeding segments of the nephron. In this context, genome-wide association studies and subsequent functional analyses have identified the ATP-binding cassette (ABC) transporter ABCG2 as an important urate transporter and have highlighted the role of single nucleotide polymorphisms (SNPs) in the pathogenesis of reduced cellular urate efflux, hyperuricemia, and early-onset gout. Recent publications also suggest that ABCG2 is particularly involved in intestinal urate elimination and thus may represent an interesting new target for pharmacotherapeutic intervention in hyperuricemia and gout. In this review, we specifically address the involvement of ABCG2 in renal and extrarenal urate elimination. In addition, we will shed light on newly identified polymorphisms in ABCG2 associated with early-onset gout.

A Combined Acceptor Photobleaching and Donor Fluorescence Lifetime Imaging Microscopy Approach to Analyze Multi-Protein Interactions in Living Cells.

Protein-protein interaction studies often provide new insights, i.e., into the formation of protein complexes relevant for structural oligomerization, regulation of enzymatic activity or information transfer within signal transduction pathways. Mostly, biochemical approaches have been used to study such interactions, but their results are limited to observations from lysed cells. A powerful tool for the non-invasive investigation of protein-protein interactions in the context of living cells is the microscopic analysis of Förster Resonance Energy Transfer (FRET) among fluorescent proteins. Normally, FRET is used to monitor the interaction state of two proteins, but in addition, FRET studies have been used to investigate three or more interacting proteins at the same time. Here we describe a fluorescence microscopy-based method which applies a novel 2-step acceptor photobleaching protocol to discriminate between non-interacting, dimeric interacting and trimeric interacting states within a three-fluorophore setup. For this purpose, intensity- and fluorescence lifetime-related FRET effects were analyzed on representative fluorescent dimeric and trimeric FRET-constructs expressed in the cytosol of HEK293 cells. In particular, by combining FLIM- and intensity-based FRET data acquisition and interpretation, our method allows to distinguish trimeric from different types of dimeric (single-, double- or triple-dimeric) protein-protein interactions of three potential interaction partners in the physiological setting of living cells.

Precursor-Directed Biosynthesis and Fluorescence Labeling of Clickable Microcystins.

Microcystins, cyclic nonribosomal heptapeptides, are the most well-known cyanobacterial toxins. They are exceptionally well studied, but open questions remain concerning their physiological role for the producing microorganism or their suitability as lead compounds for anticancer drug development. One means to study specialized metabolites in more detail is the introduction of functional groups that make a compound amenable for bioorthogonal, so-called click reactions. Although it was reported that microcystins cannot be derivatized by precursor-directed biosynthesis, we successfully used this approach to prepare clickable microcystins. Supplementing different azide- or terminal alkyne containing amino acid analogues into the cultivation medium of microcystin-producing cyanobacteria strains, we found that these strains differ strongly in their substrate acceptance. Exploiting this flexibility, we generated more than 40 different clickable microcystins. We conjugated one of these derivatives with a fluorogenic dye and showed that neither incorporation of the unnatural amino acid analogue nor attachment of the fluorescent label significantly affects the cytotoxicity against cell lines expressing the human organic anion transporting polypeptides 1B1 or 1B3. Using time-lapse microscopy, we observed that the fluorescent microcystin is rapidly taken up into eukaryotic cells expressing these transporters.

3D structure of the transporter ABCG2-What's new?

ABCG2 belongs to the ABC transporter superfamily and functions as a poly-specific efflux pump. As it can transport a broad spectrum of substrates out of cells, ABCG2 is thought to alter the pharmacokinetics of drugs applied to treat certain diseases. Especially, its potential to induce resistance to chemotherapy is currently the object of intense research. To foster understanding of mechanisms relevant for substrate recognition and selection of ABCG2 substrates and to finally develop selective therapeutic modulators (e.g. inhibitors) of ABCG2 transport activity, it is important to further explore the precise 3D structure of the transporter. While efforts to elucidate the three-dimensional structure of ABCG2 using X-ray crystal structure analysis have not been successful so far, high-resolution cryo-electron microscopy-based investigations have revealed exciting new insights into the structure and function of the transporter. In this review, we will focus on these seminal publications to summarize the current understanding of tertiary and quaternary structure, homodimerization or oligomerization, and functions of the ABCG2 transporter protein.

[Medical Cannabis-Related Relapse in a Patient with a History of Alcohol Abuse]. / Alkoholrückfall nach medizinisch indizierter Cannabistherapie.

HISTORY AND CLINICAL FINDINGS: In this report, a 60-year old patient with a history of mixed nociceptive and neuropathic chronic pain after successful removal of oral squamous cell cancer is described who received outpatient pain treatment in our clinic. Moreover, the patient presented with a history of alcohol abuse as well as anorexia and weight loss. EXAMINATIONS AND DIAGNOSIS: The patient was in reduced general condition and cachectic nutritional status. In addition, he suffered from oral pain that radiated to both ears and a related loss of appetite. TREATMENT: In the light of progressive cachexia, we started regular medical cannabis (Sativex®, contains i. e. delta-9-tetrahydrocannabinol and cannabidiol). Despite good initial tolerability, medical cannabis was stopped early due to alcohol relapse of the patient. After termination of medical cannabis, the patient regained control of alcohol consumption and achieved sobriety. CONCLUSIONS: We suggest that medical cannabis only be prescribed with particular caution in patients with a history of alcohol abuse.