Influence of the Anesthetic Technique on Circulating Extracellular Vesicles in Bladder Cancer Patients Undergoing Radical Cystectomy: A Prospective, Randomized Trial.

Anesthetics have been shown to alter tumor progression and seem to influence surgical cancer outcome. Circulating extracellular vesicles as mediators of intercellular communication are involved in cancer progression and may be influenced by anesthetics. In this prospective, randomized study, effects of anesthetics on extracellular vesicles and associated micro-RNAs in bladder cancer patients undergoing radical cystectomy were tested. Extracellular vesicles from 51 patients at four perioperative time points receiving Propofol or Sevoflurane were extracted with polymer-based methods and quantified with a nanoparticle-tracking analysis. Vesicle-associated micro-RNAs were analyzed with a real-time polymerase chain reaction using array cards and single assays for tumor-associated miR-21-5p, miR-15a-5p, miR-17-5p and miR-451a. Plasma extracellular vesicle concentration (suture: fold change (fc) in Propofol at 4.1 ± 3.9 vs. Sevoflurane at 0.8 ± 0.5; p = 0.003) and associated miRNAs increased significantly (+30% post induction, +9% 30 Min surgery) in the Propofol group. Tumor-associated miRNAs increased during surgery in both groups (fc in miR-21-5p: 24.3 ± 10.2, p = 0.029; fc in miR-15a-5p: 9.7 ± 3.8, p = 0.027; fc in miR-17-5p: 5.4 ± 1.7, p = 0.014), whereas antitumor miR-451a increased in the Propofol group only (fc: 2.5 ± 0.6 vs. 1.0 ± 0.2; p = 0.022). Anesthetics influence extracellular vesicles and associated micro-RNAs of bladder cancer patients during surgery. Increased expression of antitumor micro-RNA may be an explanatory approach for decreased tumor cell viability after Propofol.

Polymorphic Variants in the GRK5 Gene Promoter Are Associated With Diastolic Dysfunction in Coronary Artery Bypass Graft Surgery Patients.

BACKGROUND: The G-protein-coupled receptor kinase 5 (GRK5) is a mediator of cardiovascular homeostasis and participates in inflammation and cardiac fibrosis, both being involved in the development of diastolic dysfunction (DD). While mechanisms of transcriptional regulation of the GRK5 promoter are unclear, we tested the hypotheses, that (1) GRK5 expression varies depending on functional single nucleotide polymorphisms (SNPs) in the GRK5 promoter and (2) this is associated with DD in patients undergoing coronary artery bypass graft (CABG) surgery. METHODS: We amplified and sequenced the GRK5 promoter followed by cloning, reporter assays, and electrophoretic mobility shift assays (EMSA). GRK5 messenger ribonucleic acid (mRNA) expression was determined in right atrial tissue sampled from 50 patients undergoing CABG surgery. In another prospective study, GRK5 genotypes were associated with determinants of diastolic function using transesophageal echocardiography in 255 patients with CABG with normal systolic left ventricular (LV) function. Specifically, we measured ejection fraction (EF), transmitral Doppler early filling velocity (E), tissue Doppler early diastolic lateral mitral annular velocity (E' lateral), and calculated E/E', E' norm and the difference of E' lateral and E' norm to account for age-related changes in diastolic function. RESULTS: We identified 6 SNPs creating 3 novel haplotypes with the greatest promoter activation in haplotype tagging (ht) SNP T(-678)C T-allele constructs (P < .001). EMSAs showed allele-specific transcription factor binding proving functional activity. GRK5 mRNA expression was greatest in TT genotypes (TT: 131 fg/µg [95% CI, 108-154]; CT: 109 [95% confidence interval {CI}, 93-124]; CC: 83 [95% CI, 54-112]; P = .012). Moreover, GRK5 genotypes were significantly associated with determinants of diastolic function. Grading of DD revealed more grade 3 patients in TT compared to CT and CC genotypes (58% vs 38% vs 4%; P = .023). E´ lateral was lowest in TT genotypes (P = .007) and corresponding E/E' measurements showed 1.27-fold increased values in TT versus CC genotypes (P = .01), respectively. While E' norm values were not different between genotypes (P = .182), the difference between E' lateral and E' norm was significantly higher in TT genotypes compared to CC and CT genotypes (-1.2 [interquartile range {IQR}, 2.7], -0.5 [IQR, 3.4], and -0.4 [IQR, 4.2; P = .035], respectively). CONCLUSIONS: A functional GRK5 SNP results in allele-dependent differences in GRK5 promoter activity and mRNA expression. This is associated with altered echocardiographic determinants of diastolic function. Thus, SNPs in the GRK5 promoter are associated with altered perioperative diastolic cardiac function. In the future, preoperative testing for these and other SNPs might allow to initiate more specific diagnostic and perioperative pathways to benefit patients at risk.

Enhanced antitumoral activity of TLR7 agonists via activation of human endogenous retroviruses by HDAC inhibitors.

In this work, we are reporting that "Shock and Kill", a therapeutic approach designed to eliminate latent HIV from cell reservoirs, is extrapolatable to cancer therapy. This is based on the observation that malignant cells express a spectrum of human endogenous retroviral elements (HERVs) which can be transcriptionally boosted by HDAC inhibitors. The endoretroviral gene HERV-V2 codes for an envelope protein, which resembles syncytins. It is significantly overexpressed upon exposure to HDAC inhibitors and can be effectively targeted by simultaneous application of TLR7/8 agonists, triggering intrinsic apoptosis. We demonstrated that this synergistic cytotoxic effect was accompanied by the functional disruption of the TLR7/8-NFκB, Akt/PKB, and Ras-MEK-ERK signalling pathways. CRISPR/Cas9 ablation of TLR7 and HERV-V1/V2 curtailed apoptosis significantly, proving the pivotal role of these elements in driving cell death. The effectiveness of this new approach was confirmed in ovarian tumour xenograft studies, revealing a promising avenue for future cancer therapies.

Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site.

BACKGROUND: G protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression. METHODS: Five deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots. RESULTS: Investigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression. CONCLUSION: We characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

Extracellular vesicles isolated from patients undergoing remote ischemic preconditioning decrease hypoxia-evoked apoptosis of cardiomyoblasts after isoflurane but not propofol exposure.

Remote ischemic preconditioning (RIPC) can evoke cardioprotection following ischemia/reperfusion and this may depend on the anesthetic used. We tested whether 1) extracellular vesicles (EVs) isolated from humans undergoing RIPC protect cardiomyoblasts against hypoxia-induced apoptosis and 2) this effect is altered by cardiomyoblast exposure to isoflurane or propofol. EVs were isolated before and 60 min after RIPC or Sham from ten patients undergoing coronary artery bypass graft surgery with isoflurane anesthesia and quantified by Nanoparticle Tracking Analysis. Following EV-treatment for 6 hours under exposure of isoflurane or propofol, rat H9c2 cardiomyoblasts were cultured for 18 hours in normoxic or hypoxic atmospheres. Apoptosis was detected by flow cytometry. Serum nanoparticle concentrations in patients had increased sixty minutes after RIPC compared to Sham (2.5x1011±4.9x1010 nanoparticles/ml; Sham: 1.2x1011±2.0x1010; p = 0.04). Hypoxia increased apoptosis of H9c2 cells (hypoxia: 8.4%±0.6; normoxia: 2.5%±0.1; p<0.0001). RIPC-EVs decreased H9c2 cell apoptosis compared to control (apoptotic ratio: 0.83; p = 0.0429) while Sham-EVs showed no protection (apoptotic ratio: 0.97). Prior isoflurane exposure in vitro even increased protection (RIPC-EVs/control, apoptotic ratio: 0.79; p = 0.0035; Sham-EVs/control, apoptotic ratio:1.04) while propofol (50µM) abrogated protection by RIPC-EVs (RIPC-EVs/control, Apoptotic ratio: 1.01; Sham-EVs/control, apoptotic ratio: 0.94; p = 0.602). Thus, EVs isolated from patients undergoing RIPC under isoflurane anesthesia protect H9c2 cardiomyoblasts against hypoxia-evoked apoptosis and this effect is abrogated by propofol. This supports a role of human RIPC-generated EVs in cardioprotection and underlines propofol as a possible confounder in RIPC-signaling mediated by EVs.

Remote ischaemic preconditioning increases serum extracellular vesicle concentrations with altered micro-RNA signature in CABG patients.

BACKGROUND: Remote ischaemic preconditioning (RIPC) can attenuate myocardial ischaemia/reperfusion injury but its underlying mechanisms remain largely unknown. Recently, extracellular vesicles (EVs) containing microRNAs (miRNAs) were shown to mediate distant intercellular communication that may be involved in cardioprotection. We tested the hypothesis that RIPC in anaesthetized patients undergoing coronary artery bypass (CABG) surgery results in the release of EVs from the ischaemic/reperfused arm into the blood stream harbouring cardioprotective miRNAs. METHODS: In 58 patients randomised to RIPC (three 5/5 minutes episodes of left arm ischaemia/reperfusion by suprasystolic blood pressure cuff inflations/deflations) or Sham, a subprotocol comprising of parallel right radial artery and regional (left subclavian) venous blood sampling before (awake) and 5 and 60 minutes after RIPC/Sham during isoflurane/sufentanil anaesthesia could be completed. EVs were extracted by polymer-based precipitation methods, their concentrations measured, and their miRNA signature analysed. RESULTS: Five minutes after RIPC, regional venous EV concentrations downstream from the cuff increased and arterial concentrations increased after 60 minutes (fold change [fc]: RIPC: 1.33 ± 0.5, Sham: 0.91 ± 0.31; P = 0.003 for interaction). Already 5 minutes after RIPC, expression of 26 miRNAs (threshold fc: 3.0, P < 0.05) isolated from EVs including the cardioprotective miR-21 had increased. RIPC also decreased postoperative Troponin I concentrations (AUC RIPC: 336 ng/mL × 72 hours ± 306 vs Sham: 713 ± 1013; P  = â€Š0.041). CONCLUSIONS: Remote ischaemic preconditioning increases serum EV concentrations, most likely by early EV release from the patients' left (RIPC) arm, alters their miRNA signature, and is associated with myocardial protection. Thus, an increased EV concentration with an altered miR-signature may mediate the RIPC effect.

The GRK2 Promoter Is Regulated by Early-Growth Response Transcription Factor EGR-1.

The G-protein-coupled receptor kinase 2 (GRK2) plays a major role in cardiovascular diseases, and its expression is increased in heart failure. However, only little is known about factors being involved in up-regulation of GRK2 expression through transcriptional regulation of its promoter. Since the transcription factor early-growth response 1 (EGR-1) is also up-regulated in patients with heart failure, we tested the hypothesis that EGR-1 regulates GRK2 transcription. Stimulation of immortalized rat cardiomyocytes (H9c2) with phorbol 12-myristate 13-acetate (PMA) resulted in up-regulation of Egr-1 and subsequently of Grk2 mRNA expression, with maximum Grk2 expression (p = 0.008) 5 hr after PMA stimulation and being abolished by actinomycin D, indicating a transcriptional mechanism. To identify naturally occurring variants affecting promoter transcriptional activity, we identified a novel G(-43)A polymorphism (rs182084609), which surrounded a putative EGR-1-binding site. While the minor A allele frequency was rare (0.02), this variant was used to explore regulation by EGR-1 and promoter construct with altered alleles at nt-43 were subjected of reporter assays in human embryonic kidney cells (Hek293). Here, EGR-1 over-expression resulted in a more than twofold increase in GRK2 promoter activity but only in the presence of the G-allele (p = 0.04). In electrophoretic mobility shift assays, EGR-1 over-expression resulted in a specific binding of transcription factors only to the G oligonucleotide. Finally, EGR-1 over-expression resulted in increased GRK2 mRNA expression (p = 0.03). We identified EGR-1 as a regulator of GRK2 transcription. Suppression of GRK2 expression by inhibition of EGR-1 binding to GRK2 might be a promising approach to mitigate adrenergic desensitization.

Simple methods for the detection of HLA-G variants in coding and non-coding regions.

The non-classical human leukocyte antigen (HLA)-G plays a crucial role in the induction of tolerance at the feto-maternal interface as well as in transplantation, cancer, inflammation, and autoimmune diseases. To understand gene regulation and the impact of polymorphic sites on the function, simple and easy feasible approaches are needed for the detection of HLA-G variants in coding and non-coding regions. Here we summarize a set of methods for the identification of variants in the exon 2-4, in the 3' untranslated region and in the gene promoter region of the HLA-G gene.