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Osteoporosis, a complex chronic disease with increasing prevalence, is characterised by reduced bone mineral density (BMD) and increased fracture risk. The high heritability of BMD suggests substantial impact of the individual genetic disposition on bone phenotypes and the development of osteoporosis. In the past years, genome-wide association studies (GWAS) identified hundreds of genetic variants associated with BMD or osteoporosis. Here, we analysed 1103 single nucleotide polymorphisms (SNPs), previously identified as associated with estimated BMD (eBMD) in the UK Biobank. We assessed whether these SNPs are related to heel stiffness index obtained by quantitative ultrasound in 5665 adult participants of the Study of Health in Pomerania (SHIP). We confirmed 45 significant associations after correction for multiple testing. Next, we analysed six selected SNPs in 631 patients evaluated for osteoporosis [rs2707518 (CPED1/WNT16), rs3779381 (WNT16), rs115242848 (LOC101927709/EN1), rs10239787 (JAZF1), rs603424 (PKD2L1) and rs6968704 (JAZF1)]. Differences in minor allele frequencies (MAF) of rs2707518 and rs3779381 between SHIP participants (higher MAF) and patients evaluated for osteoporosis (lower MAF) indicated a protective effect of the minor allele on bone integrity. In contrast, differences in MAF of rs603424 indicated a harmful effect. Co-localisation analyses indicated that the rs603424 effect may be mediated via stearoyl-CoA desaturase (SCD) expression, an enzyme highly expressed in adipose tissue with a crucial role in lipogenesis. Taken together, our results support the role of the WNT16 pathway in the regulation of bone properties and indicate a novel causal role of SCD expression in adipose tissue on bone integrity.
Exogenous glucocorticoids increase the risk for osteoporosis, but the role of endogenous glucocorticoids remains elusive. Here, we describe the generation and validation of a loss- and a gain-of-function model of the cortisol producing enzyme 11β-HSD1 (HSD11B1) to modulate the endogenous glucocorticoid conversion in SCP-1 cells — a model for human mesenchymal stem cells capable of adipogenic and osteogenic differentiation. CRISPR-Cas9 was successfully used to generate a cell line carrying a single base duplication and a 5 bp deletion in exon 5, leading to missense amino acid sequences after codon 146. These inactivating genomic alterations were validated by deep sequencing and by cloning with subsequent capillary sequencing. 11β-HSD1 protein levels were reduced by 70% in the knockout cells and cortisol production was not detectable. Targeted chromosomal integration was used to stably overexpress HSD11B1. Compared to wildtype cells, HSD11B1 overexpression resulted in a 7.9-fold increase in HSD11B1 mRNA expression, a 5-fold increase in 11β-HSD1 protein expression and 3.3-fold increase in extracellular cortisol levels under adipogenic differentiation. The generated cells were used to address the effects of 11β-HSD1 expression on adipogenic and osteogenic differentiation. Compared to the wildtype, HSD11B1 overexpression led to a 3.7-fold increase in mRNA expression of lipoprotein lipase (LPL) and 2.5-fold increase in lipid production under adipogenic differentiation. Under osteogenic differentiation, HSD11B1 knockout led to enhanced alkaline phosphatase (ALP) activity and mRNA expression, and HSD11B1 overexpression resulted in a 4.6-fold and 11.7-fold increase in mRNA expression of Dickkopf-related protein 1 (DKK1) and LPL, respectively. Here we describe a HSD11B1 loss- and gain-of-function model in SCP-1 cells at genetic, molecular and functional levels. We used these models to study the effects of endogenous cortisol production on mesenchymal stem cell differentiation and demonstrate an 11β-HSD1 dependent switch from osteogenic to adipogenic differentiation. These results might help to better understand the role of endogenous cortisol production in osteoporosis on a molecular and cellular level.
Postoperative restenosis in patients with external ear canal (EEC) atresia or stenosis is a common complication following canaloplasty. Our aim in this study was to explore the feasibility of using a three dimensionally (3D)-printed, patient-individualized, drug ((dexamethasone (DEX)), and ciprofloxacin (cipro))-releasing external ear canal implant (EECI) as a postoperative stent after canaloplasty. We designed and pre-clinically tested this novel implant for drug release (by high-performance liquid chromatography), biocompatibility (by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay), bio-efficacy (by the TNF-α (tumor necrosis factor-alpha)-reduction test (DEX) and inhibition zone test (for cipro)), and microbial contamination (formation of turbidity or sediments in culture medium). The EECI was implanted for the first time to one patient with a history of congenital EEC atresia and state after three canaloplasties due to EEC restenosis. The preclinical tests revealed no cytotoxic effect of the used materials; an antibacterial effect was verified against the bacteria Staphylococcus aureus and Pseudomonas aeruginosa, and the tested UV-irradiated EECI showed no microbiological contamination. Based on the test results, the combination of silicone with 1% DEX and 0.3% cipro was chosen to treat the patient. The EECI was implantable into the EEC; the postoperative follow-up visits revealed no otogenic symptoms or infections and the EECI was explanted three months postoperatively. Even at 12 months postoperatively, the EEC showed good epithelialization and patency. Here, we report the first ever clinical application of an individualized, drug-releasing, mechanically flexible implant and suggest that our novel EECI represents a safe and effective method for postoperatively stenting the reconstructed EEC.
OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.
This communication introduces the first-time application of high-resolution continuum-source molecular absorption spectrometry (HR CS MAS) for the quantification of a peptide. The graphite furnace technique was employed and the tripeptide glutathione (GSH) served as a model compound. Based on measuring sulfur in terms of carbon monosulfide (CS), a method was elaborated to analyze aqueous solutions of GSH. The most prominent wavelength of the CS molecule occurred at 258.0560 nm and was adduced for monitoring. The methodological development covered the optimization of the pyrolysis and vaporization temperatures. These were found optimally to be 250 °C and 2250 °C, respectively. Moreover, the effect of modifiers (zirconium, calcium, magnesium, palladium) on the absorption signals was investigated. The best results were obtained after permanent coating of the graphite tube with zirconium (total amount of 400 μg) and adding a combination of palladium (10 µL, 10 g L−1) and calcium (2 µL, 1 g L−1) as a chemical modifier to the probes (10 µL). Aqueous standard samples of GSH were used for the calibration. It showed a linear range of 2.5–100 µg mL−1 sulfur contained in GSH with a correlation coefficient R2 > 0.997. The developed method exhibited a limit of detection (LOD) and quantification (LOQ) of 2.1 µg mL−1 and 4.3 µg mL−1 sulfur, respectively. The characteristic mass accounted for 5.9 ng sulfur. The method confirmed the general suitability of MAS for the analysis of an oligopeptide. Thus, this study serves as groundwork for further development in order to extend the application of classical atomic absorption spectrometry (AAS).
Transmembrane drug transport in hepatocytes is one of the major determinants of drug pharmacokinetics. In the present study, ABC transporters (P-gp, MRP1, MRP2, MRP3, MRP4, BCRP, and BSEP) and SLC transporters (MCT1, NTCP, OAT2, OATP1B1, OATP1B3, OATP2B1, OCT1, and OCT3) were quantified for protein abundance (LC-MS/MS) and mRNA levels (qRT-PCR) in hepatitis C virus (HCV)-infected liver samples from the Child–Pugh class A (n = 30), B (n = 21), and C (n = 7) patients. Protein levels of BSEP, MRP3, MCT1, OAT2, OATP1B3, and OCT3 were not significantly affected by HCV infection. P-gp, MRP1, BCRP, and OATP1B3 protein abundances were upregulated, whereas those of MRP2, MRP4, NTCP, OATP2B1, and OCT1 were downregulated in all HCV samples. The observed changes started to be seen in the Child–Pugh class A livers, i.e., upregulation of P-gp and MRP1 and downregulation of MRP2, MRP4, BCRP, and OATP1B3. In the case of NTCP, OATP2B1, and OCT1, a decrease in the protein levels was observed in the class B livers. In the class C livers, no other changes were noted than those in the class A and B patients. The results of the study demonstrate that drug transporter protein abundances are affected by the functional state of the liver in hepatitis C patients.
Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting the oral cavity. It is characterized by high morbidity and very few therapeutic options. Angiotensin (Ang)-(1-7) is a biologically active heptapeptide, generated predominantly from AngII (Ang-(1-8)) by the enzymatic activity of angiotensin-converting enzyme 2 (ACE 2). Previous studies have shown that Ang-(1-7) counterbalances AngII pro-tumorigenic actions in different pathophysiological settings, exhibiting antiproliferative and anti-angiogenic properties in cancer cells. However, the prevailing effects of Ang-(1-7) in the oral epithelium have not been established in vivo. Here, we used an inducible oral-specific mouse model, where the expression of a tamoxifen-inducible Cre recombinase (CreERtam), which is under the control of the cytokeratin 14 promoter (K14-CreERtam), induces the expression of the K-ras oncogenic variant KrasG12D (LSLK-rasG12D). These mice develop highly proliferative squamous papilloma in the oral cavity and hyperplasia exclusively in oral mucosa within one month after tamoxifen treatment. Ang-(1-7) treated mice showed a reduced papilloma development accompanied by a significant reduction in cell proliferation and a decrease in pS6 positivity, the most downstream target of the PI3K/Akt/mTOR signaling route in oral papilloma. These results suggest that Ang-(1-7) may be a novel therapeutic target for OSCC.
Background: Unwanted drug-drug interactions (DDIs), as caused by the upregulation of clinically relevant drug metabolizing enzymes and transporter proteins in intestine and liver, have the potential to threaten the therapeutic efficacy and safety of drugs. The molecular mechanism of this undesired but frequently occurring scenario of polypharmacy is based on the activation of nuclear receptors such as the pregnane X receptor (PXR) or the constitutive androstane receptor (CAR) by perpetrator agents such as rifampin, phenytoin or St. John’s wort. However, the expression pattern of nuclear receptors in human intestine and liver remains uncertain, which makes it difficult to predict the extent of potential DDIs. Thus, it was the aim of this study to characterize the gene expression and protein abundance of clinically relevant nuclear receptors, i.e., the aryl hydrocarbon receptor (AhR), CAR, farnesoid X receptor (FXR), glucocorticoid receptor (GR), hepatocyte nuclear factor 4 alpha (HNF4α), PXR and small heterodimer partner (SHP), in the aforementioned organs. Methods: Gene expression analysis was performed by quantitative real-time PCR of jejunal, ileal, colonic and liver samples from eight human subjects. In parallel, a targeted proteomic method was developed and validated in order to determine the respective protein amounts of nuclear receptors in human intestinal and liver samples. The LC-MS/MS method was validated according to the current bioanalytical guidelines and met the criteria regarding linearity (0.1–50 nmol/L), within-day and between-day accuracy and precision, as well as the stability criteria. Results: The developed method was successfully validated and applied to determine the abundance of nuclear receptors in human intestinal and liver samples. Gene expression and protein abundance data demonstrated marked differences in human intestine and liver. On the protein level, only AhR and HNF4α could be detected in gut and liver, which corresponds to their highest gene expression. In transfected cell lines, PXR and CAR could be quantified. Conclusions: The substantially different expression pattern of nuclear receptors in human intestinal and liver tissue may explain the different extent of unwanted DDIs in the dependence on the administration route of drugs.
Sphingosine-1-phosphate (S1P) is a versatile signaling lipid involved in the regulation of numerous cellular processes. S1P regulates cellular proliferation, migration, and apoptosis as well as the function of immune cells. S1P is generated from sphingosine (Sph), which derives from the ceramide metabolism. In particular, high concentrations of S1P are present in the blood. This originates mainly from erythrocytes, endothelial cells (ECs), and platelets. While erythrocytes function as a storage pool for circulating S1P, platelets can rapidly generate S1P de novo, store it in large quantities, and release it when the platelet is activated. Platelets can thus provide S1P in a short time when needed or in the case of an injury with subsequent platelet activation and thereby regulate local cellular responses. In addition, platelet-dependently generated and released S1P may also influence long-term immune cell functions in various disease processes, such as inflammation-driven vascular diseases. In this review, the metabolism and release of platelet S1P are presented, and the autocrine versus paracrine functions of platelet-derived S1P and its relevance in various disease processes are discussed. New pharmacological approaches that target the auto- or paracrine effects of S1P may be therapeutically helpful in the future for pathological processes involving S1P.
Organic cation transporter 1 (OCT1) is a membrane transporter that affects hepatic uptake of cationic and weakly basic drugs. OCT1 transports structurally highly diverse substrates. The mechanisms conferring this polyspecificity are unknown. Here, we analyzed differences in transport kinetics between human and mouse OCT1 orthologs to identify amino acids that contribute to the polyspecificity of OCT1. Following stable transfection of HEK293 cells, we observed more than twofold differences in the transport kinetics of 22 out of 28 tested substrates. We found that the β2-adrenergic drug fenoterol was transported with eightfold higher affinity but at ninefold lower capacity by human OCT1. In contrast, the anticholinergic drug trospium was transported with 11-fold higher affinity but at ninefold lower capacity by mouse Oct1. Using human–mouse chimeric constructs and site-directed mutagenesis, we identified nonconserved amino acids Cys36 and Phe32 as responsible for the species-specific differences in fenoterol and trospium uptake. Substitution of Cys36 (human) to Tyr36 (mouse) caused a reversal of the affinity and capacity of fenoterol but not trospium uptake. Substitution of Phe32 to Leu32 caused reversal of trospium but not fenoterol uptake kinetics. Comparison of the uptake of structurally similar β2-adrenergics and molecular docking analyses indicated the second phenol ring, 3.3 to 4.8 Å from the protonated amino group, as essential for the affinity for fenoterol conferred by Cys36. This is the first study to report single amino acids as determinants of OCT1 polyspecificity. Our findings suggest that structure–function data of OCT1 is not directly transferrable between substrates or species.
The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.
Course of disease and risk factors for hospitalization in outpatients with a SARS-CoV-2 infection
(2022)
We analyzed symptoms and comorbidities as predictors of hospitalization in 710 outpatients in North-East Germany with PCR-confirmed SARS-CoV-2 infection. During the first 3 days of infection, commonly reported symptoms were fatigue (71.8%), arthralgia/myalgia (56.8%), headache (55.1%), and dry cough (51.8%). Loss of smell (anosmia), loss of taste (ageusia), dyspnea, and productive cough were reported with an onset of 4 days. Anosmia or ageusia were reported by only 18% of the participants at day one, but up to 49% between days 7 and 9. Not all participants who reported ageusia also reported anosmia. Individuals suffering from ageusia without anosmia were at highest risk of hospitalization (OR 6.8, 95% CI 2.5–18.1). They also experienced more commonly dyspnea and nausea (OR of 3.0, 2.9, respectively) suggesting pathophysiological connections between these symptoms. Other symptoms significantly associated with increased risk of hospitalization were dyspnea, vomiting, and fever. Among basic parameters and comorbidities, age > 60 years, COPD, prior stroke, diabetes, kidney and cardiac diseases were also associated with increased risk of hospitalization. In conclusion, due to the delayed onset, ageusia and anosmia may be of limited use in differential diagnosis of SARS-CoV-2. However, differentiation between ageusia and anosmia may be useful for evaluating risk for hospitalization.
The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM).
In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans.
Dynamics of Vascular Protective and Immune Supportive Sphingosine-1-Phosphate During Cardiac Surgery
(2021)
Introduction
Sphingosine-1-phosphate (S1P) is a signaling lipid and crucial in vascular protection and immune response. S1P mediated processes involve regulation of the endothelial barrier, blood pressure and S1P is the only known inducer of lymphocyte migration. Low levels of circulatory S1P correlate with severe systemic inflammatory syndromes such as sepsis and shock states, which are associated with endothelial barrier breakdown and immunosuppression. We investigated whether S1P levels are affected by sterile inflammation induced by cardiac surgery.
Materials and Methods
In this prospective observational study we included 46 cardiac surgery patients, with cardiopulmonary bypass (CPB, n=31) and without CPB (off-pump, n=15). Serum-S1P, S1P-sources and carriers, von-Willebrand factor (vWF), C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured at baseline, post-surgery and at day 1 (POD 1) and day 4 (POD 4) after surgical stimulus.
Results
Median S1P levels at baseline were 0.77 nmol/mL (IQR 0.61-0.99) and dropped significantly post-surgery. S1P was lowest post-surgery with median levels of 0.37 nmol/mL (IQR 0.31-0.47) after CPB and 0.46 nmol/mL (IQR 0.36-0.51) after off-pump procedures (P<0.001). The decrease of S1P was independent of surgical technique and observed in all individuals. In patients, in which S1P levels did not recover to preoperative baseline ICU stay was longer and postoperative inflammation was more severe. S1P levels are associated with its sources and carriers and vWF, as a more specific endothelial injury marker, in different phases of the postoperative course. Determination of S1P levels during surgery suggested that also the anticoagulative effect of heparin might influence systemic S1P.
Discussion
In summary, serum-S1P levels are disrupted by major cardiac surgery. Low S1P levels post-surgery may play a role as a new marker for severity of cardiac surgery induced inflammation. Due to well-known protective effects of S1P, low S1P levels may further contribute to the observed prolonged ICU stay and worse clinical status. Moreover, we cannot exclude a potential inhibitory effect on circulating S1P levels by heparin anticoagulation during surgery, which would be a new pro-inflammatory pleiotropic effect of high dose heparin in patients undergoing cardiac surgery.
PIM1 Inhibition Affects Glioblastoma Stem Cell Behavior and Kills Glioblastoma Stem-like Cells
(2021)
Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.
Doxorubicin is a frequently used anticancer drug to treat many types of tumors, such as breast cancer or bronchial carcinoma. The clinical use of doxorubicin is limited by its poorly predictable cardiotoxicity, the reasons of which are so far not fully understood. The drug is a substrate of several efflux transporters such as P-gp or BCRP and was recently reported to be a substrate of cation uptake transporters. To evaluate the potential role of transporter proteins in the accumulation of doxorubicin at its site of action (e.g., mammary carcinoma cells) or adverse effects (e.g., heart muscle cells), we studied the expression of important uptake and efflux transporters in human breast cancer and cardiac tissue, and investigated the affinity of doxorubicin to the identified transporters. The cellular uptake studies on doxorubicin were performed with OATP1A2*1, OATP1A2*2, and OATP1A2*3-overexpressing HEK293 cells, as well as OCT1-, OCT2-, and OCT3- overexpressing MDCKII cells. To assess the contribution of transporters to the cytotoxic effect of doxorubicin, we determined the cell viability in the presence and absence of transporter inhibitors in different cell lines. Several transporters, including P-gp, BCRP, OCT1, OCT3, and OATP1A2 were expressed in human heart and/or breast cancer tissue. Doxorubicin could be identified as a substrate of OCT1, OCT2, OCT3, and OATP1A2. The cellular uptake into cells expressing genetic OATP1A2 variants was markedly reduced and correlated well with the increased cellular viability. Inhibition of OATP1A2 (naringin) and OCT transporters (1-methyl-4-phenylpyridinium) resulted in a significant decrease of doxorubicin-mediated cytotoxicity in cell lines expressing the respective transporters. Similarly, the excipient Cremophor EL significantly inhibited the OCT1-3- and OATP1A2-mediated cellular uptake and attenuated the cytotoxicity of doxorubicin. In conclusion, genetic and environmental-related variability in the expression and function of these transporters may contribute to the substantial variability seen in terms of doxorubicin efficacy and toxicity.
The tricyclic antidepressant amitriptyline is frequently prescribed but its use is limited by its narrow therapeutic range and large variation in pharmacokinetics. Apart from interindividual differences in the activity of the metabolising enzymes cytochrome P450 (CYP) 2D6 and 2C19, genetic polymorphism of the hepatic influx transporter organic cation transporter 1 (OCT1) could be contributing to interindividual variation in pharmacokinetics. Here, the impact of OCT1 genetic variation on the pharmacokinetics of amitriptyline and its active metabolite nortriptyline was studied in vitro as well as in healthy volunteers and in depressive disorder patients. Amitriptyline and nortriptyline were found to inhibit OCT1 in recombinant cells with IC50 values of 28.6 and 40.4 µM. Thirty other antidepressant and neuroleptic drugs were also found to be moderate to strong OCT1 inhibitors with IC50 values in the micromolar range. However, in 35 healthy volunteers, preselected for their OCT1 genotypes, who received a single dose of 25 mg amitriptyline, no significant effects on amitriptyline and nortriptyline pharmacokinetics could be attributed to OCT1 genetic polymorphism. In contrast, the strong impact of the CYP2D6 genotype on amitriptyline and nortriptyline pharmacokinetics and of the CYP2C19 genotype on nortriptyline was confirmed. In addition, acylcarnitine derivatives were measured as endogenous biomarkers for OCT1 activity. The mean plasma concentrations of isobutyrylcarnitine and 2-methylbutyrylcarnitine were higher in participants with two active OCT1 alleles compared to those with zero OCT1 activity, further supporting their role as endogenous in vivo biomarkers for OCT1 activity. A moderate reduction in plasma isobutyrylcarnitine concentrations occurred at the time points at which amitriptyline plasma concentrations were the highest. In a second, independent study sample of 50 patients who underwent amitriptyline therapy of 75 mg twice daily, a significant trend of increasing amitriptyline plasma concentrations with decreasing OCT1 activity was observed (p = 0.018), while nortriptyline plasma concentrations were unaffected by the OCT1 genotype. Altogether, this comprehensive study showed that OCT1 activity does not appear to be a major factor determining amitriptyline and nortriptyline pharmacokinetics and that hepatic uptake occurs mainly through other mechanisms.
Intestinal transporter proteins are known to affect the pharmacokinetics and in turn the efficacy and safety of many orally administered drugs in a clinically relevant manner. This knowledge is especially well-established for intestinal ATP-binding cassette transporters such as P-gp and BCRP. In contrast to this, information about intestinal uptake carriers is much more limited although many hydrophilic or ionic drugs are not expected to undergo passive diffusion but probably require specific uptake transporters. A transporter which is controversially discussed with respect to its expression, localization and function in the human intestine is the organic cation transporter 1 (OCT1). This review article provides an up-to-date summary on the available data from expression analysis as well as functional studies in vitro, animal findings and clinical observations. The current evidence suggests that OCT1 is expressed in the human intestine in small amounts (on gene and protein levels), while its cellular localization in the apical or basolateral membrane of the enterocytes remains to be finally defined, but functional data point to a secretory function of the transporter at the basolateral membrane. Thus, OCT1 should not be considered as a classical uptake transporter in the intestine but rather as an intestinal elimination pathway for cationic compounds from the systemic circulation.
Genome-wide association studies have identified an association between isobutyrylcarnitine (IBC) and organic cation transporter 1 (OCT1) genotypes. Higher IBC blood concentrations in humans with active OCT1 genotypes and experimental studies with mouse OCT1 suggested an OCT1-mediated efflux of IBC. In this study, we wanted to confirm the suggested use of IBC as an endogenous biomarker of OCT1 activity and contribute to a better understanding of the mechanisms behind the association between blood concentrations of carnitine derivatives and OCT1 genotype. Blood and urine IBC concentrations were quantified in healthy volunteers regarding intra- and interindividual variation and correlation with OCT1 genotype and with pharmacokinetics of known OCT1 substrates. Furthermore, IBC formation and transport were studied in cell lines overexpressing OCT1 and its naturally occurring variants. Carriers of high-activity OCT1 genotypes had about 3-fold higher IBC blood concentrations and 2-fold higher amounts of IBC excreted in urine compared to deficient OCT1. This was likely due to OCT1 function, as indicated by the fact that IBC correlated with the pharmacokinetics of known OCT1 substrates, like fenoterol, and blood IBC concentrations declined with a 1 h time delay following peak concentrations of the OCT1 substrate sumatriptan. Thus, IBC is a suitable endogenous biomarker reflecting both, human OCT1 (hOCT1) genotype and activity. While murine OCT1 (mOCT1) was an efflux transporter of IBC, hOCT1 exhibited no IBC efflux activity. Inhibition experiments confirmed this data showing that IBC and other acylcarnitines, like butyrylcarnitine, 2-methylbutyrylcarnitine, and hexanoylcarnitine, showed reduced efflux upon inhibition of mOCT1 but not of hOCT1. IBC and other carnitine derivatives are endogenous biomarkers of hOCT1 genotype and phenotype. However, in contrast to mice, the mechanisms underlying the IBC-OCT1 correlation in humans is apparently not directly the OCT1-mediated efflux of IBC. A plausible explanation could be that hOCT1 mediates cellular concentrations of specific regulators or co-substrates in lipid and energy metabolism, which is supported by our in vitro finding that at baseline intracellular IBC concentration is about 6-fold lower alone by OCT1 overexpression.
Organic cation transporter 1 (OCT1, SLC22A1) is localized in the sinusoidal membrane of human hepatocytes and mediates hepatic uptake of weakly basic or cationic drugs and endogenous compounds. Common amino acid substitutions in OCT1 were associated with altered pharmacokinetics and efficacy of drugs like sumatriptan and fenoterol. Recently, the common splice variant rs35854239 has also been suggested to affect OCT1 function. rs35854239 represents an 8 bp duplication of the donor splice site at the exon 7-intron 7 junction. Here we quantified the extent to which this duplication affects OCT1 splicing and, as a consequence, the expression and the function of OCT1. We used pyrosequencing and deep RNA-sequencing to quantify the effect of rs35854239 on splicing after minigene expression of this variant in HepG2 and Huh7 cells and directly in human liver samples. Further, we analyzed the effects of rs35854239 on OCT1 mRNA expression in total, localization and activity of the resulting OCT1 protein, and on the pharmacokinetics of sumatriptan and fenoterol. The 8 bp duplication caused alternative splicing in 38% (deep RNA-sequencing) to 52% (pyrosequencing) of the minigene transcripts when analyzed in HepG2 and Huh7 cells. The alternatively spliced transcript encodes for a truncated protein that after transient transfection in HEK293 cells was not localized in the plasma membrane and was not able to transport the OCT1 model substrate ASP+. In human liver, however, the alternatively spliced OCT1 transcript was detectable only at very low levels (0.3% in heterozygous and 0.6% in homozygous carriers of the 8 bp duplication, deep RNA-sequencing). The 8 bp duplication was associated with a significant reduction of OCT1 expression in the human liver, but explained only 9% of the general variability in OCT1 expression and was not associated with significant changes in the pharmacokinetics of sumatriptan and fenoterol. Therefore, the rs35854239 variant only partially changes splicing, causing moderate changes in OCT1 expression and may be of only limited therapeutic relevance.
(1) Background: Sepsis is a leading cause of death and a global public health problem. Accordingly, deciphering the underlying molecular mechanisms of this disease and the determinants of its morbidity and mortality is pivotal. This study examined the effect of the rs951818 SNP of the negative costimulatory lymphocyte-activation gene 3 (LAG-3) on sepsis mortality and disease severity. (2) Methods: 707 consecutive patients with sepsis were prospectively enrolled into the present study from three surgical ICUs at University Medical Center Goettingen. Both 28- and 90-day mortality were analyzed as the primary outcome, while parameters of disease severity served as secondary endpoints. (3) Results: In the Kaplan–Meier analysis LAG-3 rs951818 AA-homozygote patients showed a significantly lower 28-day mortality (17.3%) compared to carriers of the C-allele (23.7%, p = 0.0476). In addition, these patients more often received invasive mechanical ventilation (96%) during the course of disease than C-allele carriers (92%, p = 0.0466). (4) Conclusions: Genetic profiling of LAG-3 genetic variants alone or in combination with other genetic biomarkers may represent a promising approach for risk stratification of patients with sepsis. Patient-individual therapeutic targeting of immune checkpoints, such as LAG-3, may be a future component of sepsis therapy. Further detailed investigations in clinically relevant sepsis models are necessary.
Gene Expression and Protein Abundance of Hepatic Drug Metabolizing Enzymes in Liver Pathology
(2021)
Pentathiepins are polysulfur-containing compounds that exert antiproliferative and cytotoxic activity in cancer cells, induce oxidative stress and apoptosis, and inhibit glutathione peroxidase (GPx1). This renders them promising candidates for anticancer drug development. However, the biological effects and how they intertwine have not yet been systematically assessed in diverse cancer cell lines. In this study, six novel pentathiepins were synthesized to suit particular requirements such as fluorescent properties or improved water solubility. Structural elucidation by X-ray crystallography was successful for three derivatives. All six underwent extensive biological evaluation in 14 human cancer cell lines. These studies included investigating the inhibition of GPx1 and cell proliferation, cytotoxicity, and the induction of ROS and DNA strand breaks. Furthermore, selected hallmarks of apoptosis and the impact on cell cycle progression were studied. All six pentathiepins exerted high cytotoxic and antiproliferative activity, while five also strongly inhibited GPx1. There is a clear connection between the potential to provoke oxidative stress and damage to DNA in the form of single- and double-strand breaks. Additionally, these studies support apoptosis but not ferroptosis as the mechanism of cell death in some of the cell lines. As the various pentathiepins give rise to different biological responses, modulation of the biological effects depends on the distinct chemical structures fused to the sulfur ring. This may allow for an optimization of the anticancer activity of pentathiepins in the future.
Purpose: Periodontitis is an inflammatory disease of the oral cavity with an alarmingly high prevalence within the adult population. The signaling lipid sphingosine-1-phosphate (S1P) plays a crucial role in inflammatory and immunomodulatory responses. In addition to cardiovascular disease, sepsis and tumor entities, S1P has been recently identified as both mediator and biomarker in osteoporosis. We hypothesized that S1P may play a role in periodontitis as an inflammation-prone bone destructive disorder. The goal of our study was to evaluate associations between periodontitis and S1P serum concentrations in the Study of Health in Pomerania (SHIP)-Trend cohort. In addition, we investigated the expression of S1P metabolizing enzymes in inflamed gingival tissue.
Patients and Methods: We analyzed data from 3371 participants (51.6% women) of the SHIP-Trend cohort. Periodontal parameters and baseline characteristics were assessed. Serum S1P was measured by liquid chromatography tandem mass spectrometry. The expression of S1P metabolizing enzymes was determined by immunofluorescence staining of human gingival tissue.
Results: S1P serum concentrations were significantly increased in subjects with both moderate and severe periodontitis, assessed as probing depth and clinical attachment loss. In contrast, no significant association of S1P was seen with caries variables (number and percentage of decayed or filled surfaces). S1P concentrations significantly increased with increasing high-sensitivity C-reactive protein (hs-CRP) levels. Interestingly, inflamed compared to normal human gingival tissue exhibited elevated expression levels of the S1P-generating enzyme sphingosine kinase 1 (SphK1).
Conclusion: We report an intriguingly significant association of various periodontal parameters with serum levels of the inflammatory lipid mediator S1P. Our data point towards a key role of S1P during periodontitis pathology. Modulation of local S1P levels or its signaling properties may represent a potential future therapeutic strategy to prevent or to retard periodontitis progression and possibly reduce periodontitis-related tooth loss.
Abomasal emptying rate of diarrhoeic and healthy suckling calves fed with oral rehydration solutions
(2020)
Abstract
The aim of the study was to determine the abomasal emptying rate (AER) of calves suffering from naturally occurring diarrhoea compared with that of healthy calves. Furthermore, the effects of an oral rehydration solution (ORS) mixed into milk replacer on the AER were determined. Acetaminophen absorption test (APAT) was performed to estimate the AER. Sixty Holstein–Frisian calves (age < 14 days) were included in the study and divided into groups as follows: healthy calves (H; n = 16), healthy calves fed with ORS (HORS; n = 14), diarrhoeic calves (D; n = 15) and diarrhoeic calves fed with ORS (DORS; n = 15). For the APAT, the calves were fed 2 L of milk replacer containing 50 mg acetaminophen (AP)/kg body weight. Venous blood samples were collected before and after milk replacer and AP intake in 30–60 min intervals for 12 hr. During the APAT, no significant differences in median maximum acetaminophen concentration (Cmax) were observed among all groups. Time to reach maximum acetaminophen concentration (Tmax) in DORS (median 390 min, 25/75 quartiles: 300/480 min) was significantly higher compared with that in H (median: 270 min 25/75 quartiles: 210/315 min) and HORS (median: 300 min (25/75 quartiles: 240/360 min). Non‐linear regression revealed that the calculated abomasal half‐life (AP t1/2) tended to be delayed in DORS (median: 652 min, 25/75 quartiles: 445/795 min, p = .10). The area under the AP curve values (AUC) from 0 to 120 min and 0 to 240 min of the observation period were significantly higher in H than D and DORS. In conclusion, significant differences in the AER indices reflected delayed abomasal emptying in diarrhoeic calves. Furthermore, the hypertonic ORS tended to have an additive delaying impact on the AER, which needs attention for the feeding management of diarrhoeic calves.
Thiamine is substrate of the hepatic uptake transporter organic cation transporter 1 (OCT1), and pathological lipid metabolism was associated with OCT1‐dependent thiamine transport. However, it is unknown whether clinical pharmacokinetics of thiamine is modulated by OCT1 genotype. We analyzed thiamine transport in vitro, thiamine blood concentrations after high‐dose and low‐dose (nutritional) intake, and heritability of thiamine and thiamine‐phosphate blood concentrations. The variant OCT1*2 had reduced and OCT1*3 to OCT1*6 had deficient thiamine uptake activity. However, pharmacokinetics of thiamine did not differ depending on OCT1 genotype. Further studies in primary human hepatocytes indicated that several cation transporters, including OCT1, OCT3, and THTR‐2, contribute to hepatic uptake of thiamine. As much as 54% of the variation in thiamine and 75% in variation of thiamine monophosphate plasma concentrations was determined by heritable factors. Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research.
Previous studies have reported the fundamental role of immunoregulatory
proteins in the clinical phenotype and outcome of sepsis. This study investigated two functional single
nucleotide polymorphisms (SNPs) of T cell immunoglobulin and mucin domain-containing protein 3
(TIM-3), which has a negative stimulatory function in the T cell immune response. Methods: Patients
with sepsis (n = 712) were prospectively enrolled from three intensive care units (ICUs) at the University
Medical Center Goettingen since 2012. All patients were genotyped for the TIM-3 SNPs rs1036199 and
rs10515746. The primary outcome was 28-day mortality. Disease severity and microbiological findings
were secondary endpoints. Results: Kaplan–Meier survival analysis demonstrated a significantly
lower 28-day mortality for TIM-3 rs1036199 AA homozygous patients compared to C-allele carriers
(18% vs. 27%, p = 0.0099) and TIM-3 rs10515746 CC homozygous patients compared to A-allele
carriers (18% vs. 26%, p = 0.0202). The TIM-3 rs1036199 AA genotype and rs10515746 CC genotype
remained significant predictors for 28-day mortality in the multivariate Cox regression analysis after
adjustment for relevant confounders (adjusted hazard ratios: 0.67 and 0.70). Additionally, patients
carrying the rs1036199 AA genotype presented more Gram-positive and Staphylococcus epidermidis
infections, and rs10515746 CC homozygotes presented more Staphylococcus epidermidis infections.
Conclusion: The studied TIM-3 genetic variants are associated with altered 28-day mortality and
susceptibility to Gram-positive infections in sepsis.
Membrane monocarboxylate transporter 1 (SLC16A1/MCT1) plays an important role in
hepatocyte homeostasis, as well as drug handling. However, there is no available information
about the impact of liver pathology on the transporter levels and function. The study was aimed to
quantify SLC16A1 mRNA (qRT-PCR) and MCT1 protein abundance (liquid chromatography–tandem
mass spectrometry (LC¬¬–MS/MS)) in the livers of patients diagnosed, according to the standard
clinical criteria, with hepatitis C, primary biliary cirrhosis, primary sclerosing hepatitis, alcoholic liver
disease (ALD), and autoimmune hepatitis. The stage of liver dysfunction was classified according to
Child–Pugh score. Downregulation of SLC16A1/MCT1 levels was observed in all liver pathology
states, significantly for ALD. The progression of liver dysfunction, from Child–Pugh class A to C,
involved the gradual decline in SLC16A1 mRNA and MCT1 protein abundance, reaching a clinically
significant decrease in class C livers. Reduced levels of MCT1 were associated with significant
intracellular lactate accumulation. The MCT1 transcript and protein did not demonstrate significant
correlations regardless of the liver pathology analyzed, as well as the disease stage, suggesting
posttranscriptional regulation, and several microRNAs were found as potential regulators of MCT1
abundance. MCT1 membrane immunolocalization without cytoplasmic retention was observed in all
studied liver pathologies. Overall, the study demonstrates that SLC16A1/MCT1 is involved in liver
pathology, especially in ALD
Salivary glands provide secretory functions, including secretion of xenobiotics and among
them drugs. However, there is no published information about protein abundance of drug transporters
measured using reliable protein quantification methods. Therefore, mRNA expression and absolute
protein content of clinically relevant ABC (n = 6) and SLC (n = 15) family member transporters in the
human parotid gland, using the qRT-PCR and liquid chromatography-tandem mass spectrometry
(LC−MS/MS) method, were studied. The abundance of nearly all measured proteins ranged between
0.04 and 0.45 pmol/mg (OCT3 > MRP1 > PEPT2 > MRP4 > MATE1 > BCRP). mRNAs of ABCB1,
ABCC2, ABCC3, SLC10A1, SLC10A2, SLC22A1, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLCO1A2,
SLCO1B1, SLCO1B3 and SLCO2B1 were not detected. The present study provides, for the first time,
information about the protein abundance of membrane transporters in the human parotid gland,
which could further be used to define salivary bidirectional transport (absorption and secretion)
mechanisms of endogenous compounds and xenobiotics.
Renal drug transporters such as the organic cation transporters (OCTs), organic anion
transporters (OATs) and multidrug resistance proteins (MRPs) play an important role in the tubular
secretion of many drugs influencing their efficacy and safety. However, only little is known about
the distinct protein abundance of these transporters in human kidneys, and about the impact of
age and gender as potential factors of inter-subject variability in their expression and function.
The aim of this study was to determine the protein abundance of MDR1, MRP1-4, BCRP, OAT1-3,
OCT2-3, MATE1, PEPT1/2, and ORCTL2 by liquid chromatography-tandem mass spectrometry-based
targeted proteomics in a set of 36 human cortex kidney samples (20 males, 16 females; median age
53 and 55 years, respectively). OAT1 and 3, OCT2 and ORCTL2 were found to be most abundant
renal SLC transporters while MDR1, MRP1 and MRP4 were the dominating ABC transporters.
Only the expression levels of MDR1 and ORCTL2 were significantly higher abundant in older donors.
Moreover, we found several significant correlations between different transporters, which may
indicate their functional interplay in renal vectorial transport processes. Our data may contribute to
a better understanding of the molecular processes determining renal excretion of drugs.
Neurosteroids, comprising pregnane, androstane, and sulfated steroids can alter neuronal excitability through interaction with ligand-gated ion channels and other receptors and have therefore a therapeutic potential in several brain disorders. They can be formed in brain cells or are synthesized by an endocrine gland and reach the brain by penetrating the blood–brain barrier (BBB). Especially sulfated steroids such as pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) depend on transporter proteins to cross membranes. In this review, we discuss the involvement of ATP-binding cassette (ABC)- and solute carrier (SLC)-type membrane proteins in the transport of these compounds at the BBB and in the choroid plexus (CP), but also in the secretion from neurons and glial cells. Among the ABC transporters, especially BCRP (ABCG2) and several MRP/ABCC subfamily members (MRP1, MRP4, MRP8) are expressed in the brain and known to efflux conjugated steroids. Furthermore, several SLC transporters have been shown to mediate cellular uptake of steroid sulfates. These include members of the OATP/SLCO subfamily, namely OATP1A2 and OATP2B1, as well as OAT3 (SLC22A3), which have been reported to be expressed at the BBB, in the CP and in part in neurons. Furthermore, a role of the organic solute transporter OSTα-OSTβ (SLC51A/B) in brain DHEAS/PregS homeostasis has been proposed. This transporter was reported to be localized especially in steroidogenic cells of the cerebellum and hippocampus. To date, the impact of transporters on neurosteroid homeostasis is still poorly understood. Further insights are desirable also with regard to the therapeutic potential of these compounds.
The multifunctional sphingosine-1-phosphate (S1P) is a lipid signaling molecule and central
regulator in the development of several cancer types. In recent years, intriguing information has
become available regarding the role of S1P in the progression of Glioblastoma multiforme (GBM),
the most aggressive and common brain tumor in adults. S1P modulates numerous cellular processes
in GBM, such as oncogenesis, proliferation and survival, invasion, migration, metastasis and stem cell
behavior. These processes are regulated via a family of five G-protein-coupled S1P receptors (S1PR1-5)
and may involve mainly unknown intracellular targets. Distinct expression patterns and multiple
intracellular signaling pathways of each S1PR subtype enable S1P to exert its pleiotropic cellular
actions. Several studies have demonstrated alterations in S1P levels, the involvement of S1PRs
and S1P metabolizing enzymes in GBM pathophysiology. While the tumorigenic actions of S1P
involve the activation of several kinases and transcription factors, the specific G-protein (Gi, Gq,
and G12/13)-coupled signaling pathways and downstream mediated effects in GBM remain to be
elucidated in detail. This review summarizes the recent findings concerning the role of S1P and its
receptors in GBM. We further highlight the current insights into the signaling pathways considered
fundamental for regulating the cellular processes in GMB and ultimately patient prognosis.
Background: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been
shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell
migration by transforming growth factor (TGF)-β1. However, it is not known whether activation
of non-SMAD TGF-β signaling (e.g., RAS–RAF–MEK–extracellular signal-regulated kinase (ERK)
signaling) is required for cell migration and whether it is also dependent on PAR2. Methods: RNA
interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure
cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation
to detect a PAR2–ALK5 physical interaction. Results: Inhibition of ERK signaling with the MEK
inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells.
ERK activation in response to PAR2 agonistic peptide (PAR2–AP) was strong and rapid, while it was
moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD
activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is
downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT
cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10
and 100 µM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide
evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2–APand TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for
TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a
physical interaction between PAR2 and ALK5
The G protein-coupled receptor proteinase-activated receptor 2 (PAR2) has been implicated
in various aspects of cellular physiology including inflammation, obesity and cancer. In cancer,
it usually acts as a driver of cancer progression in various tumor types by promoting invasion and
metastasis in response to activation by serine proteinases. Recently, we discovered another mode
through which PAR2 may enhance tumorigenesis: crosstalk with transforming growth factor-β
(TGF-β) signaling to promote TGF-β1-induced cell migration/invasion and invasion-associated gene
expression in ductal pancreatic adenocarcinoma (PDAC) cells. In this chapter, we review what is
known about the cellular TGF-β responses and signaling pathways affected by PAR2 expression,
the signaling activities of PAR2 required for promoting TGF-β signaling, and the potential molecular
mechanism(s) that underlie(s) the TGF-β signaling–promoting effect. Since PAR2 is activated through
various serine proteinases and biased agonists, it may couple TGF-β signaling to a diverse range of
other physiological processes that may or may not predispose cells to cancer development such as
local inflammation, systemic coagulation and pathogen infection.
Heart Rate Reduction by Ivabradine Improves Aortic Compliance in Apolipoprotein E-Deficient Mice
(2012)
Background: Impaired vascular compliance is associated with cardiovascular mortality. The effects of heart rate on vascular compliance are unclear. Therefore, we characterized effects of heart rate reduction (HRR) by I(f) current inhibition on aortic compliance and underlying molecular mechanisms in apolipoprotein E-deficient (ApoE<sup>–</sup>/<sup>–</sup>) mice. Methods: ApoE<sup>–</sup>/<sup>–</sup> mice fed a high-cholesterol diet and wild-type (WT) mice were treated with ivabradine (20 mg/kg/d) or vehicle for 6 weeks. Compliance of the ascending aorta was evaluated by MRI. Results: Ivabradine reduced heart rate by 113 ± 31 bpm (∼19%) in WT mice and by 133 ± 6 bpm (∼23%) in ApoE<sup>–</sup>/<sup>–</sup> mice. Compared to WT controls, ApoE<sup>–</sup>/<sup>–</sup> mice exhibited reduced distensibility and circumferential strain. HRR by ivabradine increased distensibility and circumferential strain in ApoE<sup>–</sup>/<sup>–</sup> mice but did not affect both parameters in WT mice. Ivabradine reduced aortic protein and mRNA expression of the angiotensin II type 1 (AT1) receptor and reduced rac1-GTPase activity in ApoE<sup>–</sup>/<sup>–</sup> mice. Moreover, membrane translocation of p47<sup>phox</sup> was inhibited. In ApoE<sup>–</sup>/<sup>–</sup> mice, HRR induced anti-inflammatory effects by reduction of aortic mRNA expression of IL-6, TNF-alpha and TGF-beta. Conclusion: HRR by ivabradine improves vascular compliance in ApoE<sup>–</sup>/<sup>–</sup> mice. Contributing mechanisms include downregulation of the AT1 receptor, attenuation of oxidative stress and modulation of inflammatory cytokine expression.
Background: Chronic kidney disease (CKD) and low serum total testosterone (TT) concentrations are independent predictors of mortality risk in the general population, but their combined potential for improved mortality risk stratification is unknown. Methods: We used data of 1,822 men from the population-based Study of Health in Pomerania followed- up for 9.9 years (median). The direct effects of kidney dysfunction (estimated glomerular filtration rate <60 ml/min/ 1.73 m<sup>2</sup>), albuminuria (urinary albumin-creatinine ratio ≧2.5 mg/mmol) and their combination (CKD) on all-cause and cardiovascular mortality were analyzed using multivariable Cox regression models. Serum TT concentrations below the age-specific 10th percentile (by decades) were considered low and were used for further risk stratification. Results: Kidney dysfunction (hazard ratio, HR, 1.40; 95% confidence interval, CI, 1.02–1.92), albuminuria (HR, 1.38; 95% CI, 1.06–1.79), and CKD (HR, 1.42; 95% CI, 1.09–1.84) were associated with increased all-cause mortality risk, while only kidney dysfunction (HR, 2.01; 95% CI, 1.21–3.34) was associated with increased cardiovascular mortality risk after multivariable adjustment. Men with kidney dysfunction and low TT concentrations were identified as high-risk individuals showing a more than 2-fold increased all-cause mortality risk (HR, 2.52; 95% CI, 1.08–5.85). Added to multivariable models, nonsignificant interaction terms suggest that kidney dysfunction and low TT are primarily additive rather than synergistic mortality risk factors. Conclusion: In the case of early loss of kidney function, measured TT concentrations might help to detect high-risk individuals for potential therapeutic interventions and to improve mortality risk assessment and outcome.