Intravenous sildenafil citrate and post-cardiac surgery acute kidney injury: a double-blind, randomised, placebo-controlled trial.

BACKGROUND: This study assessed whether i.v. sildenafil citrate prevented acute kidney injury in at-risk patients undergoing cardiac surgery with cardiopulmonary bypass. METHODS: In a double-blind RCT, adults at increased risk of acute kidney injury undergoing cardiac surgery in a single UK tertiary centre were randomised to receive sildenafil citrate 12.5 mg kg-1 i.v. over 150 min or dextrose 5% at the commencement of surgery. The primary outcome was serum creatinine measured at six post-randomisation time points. The primary analysis used a linear mixed-effects model adjusted for the stratification variables, baseline estimated glomerular filtration rate, and surgical procedure. Secondary outcomes considered clinical events and potential disease mechanisms. Effect estimates were expressed as mean differences (MDs) or odds ratios with 95% confidence intervals. RESULTS: The analysis population comprised eligible randomised patients that underwent valve surgery or combined coronary artery bypass graft and valve surgery, with cardiopulmonary bypass, between May 2015 and June 2018. There were 60 subjects in the sildenafil group and 69 in the placebo control group. The difference between groups in creatinine concentration was not statistically significant (MD: 0.88 µmol L-1 [-5.82, 7.59]). There was a statistically significant increase in multiple organ dysfunction scores in the sildenafil group (MD: 0.54 [0.02, 1.07]; P=0.044). Secondary outcomes, and biomarkers of kidney injury, endothelial function, and inflammatory cell activation, were not significantly different between the groups. CONCLUSIONS: These results do not support the use of i.v. sildenafil citrate for kidney protection in adult cardiac surgery. CLINICAL TRIAL REGISTRATION: ISRCTN18386427.

Biomarkers of Inflammation and Lung Recovery in Extracorporeal Membrane Oxygenation Patients With Persistent Pulmonary Hypertension of the Newborn: A Feasibility Study.

OBJECTIVES: Extracorporeal membrane oxygenation is a treatment for Persistent Pulmonary Hypertension of the Newborn with high mortality. HYPOTHESIS: the extracorporeal membrane oxygenation circuit results in inflammatory responses that mitigate against successful weaning. DESIGN: Single-center prospective observational feasibility study. SETTING: PICU. PATIENTS: Twenty-four neonates requiring extracorporeal membrane oxygenation support for Persistent Pulmonary Hypertension of the Newborn. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The reference outcome was death or more than 7 days of extracorporeal membrane oxygenation support. Other outcomes included serial measures of plasma-free hemoglobin and markers of its metabolism, leucocyte, platelet and endothelial activation, and biomarkers of inflammation. Of 24 participants recruited between February 2016 and June 2017, 10 died or required prolonged extracorporeal membrane oxygenation support. These patients were sicker at baseline with higher levels of plasma-free hemoglobin within 12 hours of cannulation (geometric mean ratio, 1.92; 95% CIs, 1.00-3.67; p = 0.050) but not thereafter, versus those requiring less than 7 days extracorporeal membrane oxygenation. Serum haptoglobin concentrations were significantly elevated in both groups. Patients who died or required prolonged extracorporeal membrane oxygenation support demonstrated elevated levels of platelet-leucocyte aggregation, but decreased concentrations of mediators of the inflammatory response: interleukin-8, C-reactive protein, and tumor necrosis factor α. CONCLUSIONS: Clinical status at baseline and not levels of plasma-free hemoglobin or the systemic inflammatory response may determine the requirement for prolonged extracorporeal membrane oxygenation support in neonates.

Extracellular vesicles released by CD40/IL-4-stimulated CLL cells confer altered functional properties to CD4+ T cells.

The complex interplay between cancer cells, stromal cells, and immune cells in the tumor microenvironment (TME) regulates tumorigenesis and provides emerging targets for immunotherapies. Crosstalk between CD4(+) T cells and proliferating chronic lymphocytic leukemia (CLL) tumor B cells occurs within lymphoid tissue pseudofollicles, and investigating these interactions is essential to understand both disease pathogenesis and the effects of immunotherapy. Tumor-derived extracellular vesicle (EV) shedding is emerging as an important mode of intercellular communication in the TME. In order to characterize tumor EVs released in response to T-cell-derived TME signals, we performed microRNA (miRNA [miR]) profiling of EVs released from CLL cells stimulated with CD40 and interleukin-4 (IL-4). Our results reveal an enrichment of specific cellular miRNAs including miR-363 within EVs derived from CD40/IL-4-stimulated CLL cells compared with parental cell miRNA content and control EVs from unstimulated CLL cells. We demonstrate that autologous patient CD4(+) T cells internalize CLL-EVs containing miR-363 that targets the immunomodulatory molecule CD69. We further reveal that autologous CD4(+) T cells that are exposed to EVs from CD40/IL-4-stimulated CLL cells exhibit enhanced migration, immunological synapse signaling, and interactions with tumor cells. Knockdown of miR-363 in CLL cells prior to CD40/IL-4 stimulation prevented the ability of CLL-EVs to induce increased synapse signaling and confer altered functional properties to CD4(+) T cells. Taken together, these data reveal a novel role for CLL-EVs in modifying T-cell function that highlights unanticipated complexity of intercellular communication that may have implications for bidirectional CD4(+) T-cell:tumor interactions within the TME.

An Observational Cohort Feasibility Study to Identify Microvesicle and Micro-RNA Biomarkers of Acute Kidney Injury Following Pediatric Cardiac Surgery.

OBJECTIVES: Micro-RNA, small noncoding RNA fragments involved in gene regulation, and microvesicles, membrane-bound particles less than 1 µm known to regulate cellular processes including responses to injury, may serve as disease-specific biomarkers of acute kidney injury. We evaluated the feasibility of measuring these signals as well as other known acute kidney injury biomarkers in a mixed pediatric cardiac surgery population. DESIGN: Single center prospective cohort feasibility study. SETTING: PICU. PATIENTS: Twenty-four children (≤ 17 yr) undergoing cardiac surgery with cardiopulmonary bypass without preexisting inflammatory state, acute kidney injury, or extracorporeal life support. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Acute kidney injury was defined according to modified Kidney Diseases Improving Global Outcomes criteria. Blood and urine samples were collected preoperatively and at 6-12 and 24 hours. Microvesicles derivation was assessed using flow cytometry and NanoSight analysis. Micro-RNAs were isolated from plasma and analyzed by microarray and quantitative real-time polymerase chain reaction. Data completeness for the primary outcomes was 100%. Patients with acute kidney injury (n = 14/24) were younger, underwent longer cardiopulmonary bypass, and required greater inotrope support. Acute kidney injury subjects had different fractional content of platelets and endothelial-derived microvesicles before surgery. Platelets and endothelial microvesicles levels were higher in acute kidney injury patients. A number of micro-RNA species were differentially expressed in acute kidney injury patients. Pathway analysis of candidate target genes in the kidney suggested that the most often affected pathways were phosphatase and tensin homolog and signal transducer and activator of transcription 3 signaling. CONCLUSIONS: Microvesicles and micro-RNAs expression patterns in pediatric cardiac surgery patients can be measured in children and potentially serve as tools for stratification of patients at risk of acute kidney injury.

Orai/CRACM1 and KCa3.1 ion channels interact in the human lung mast cell plasma membrane.

BACKGROUND: Orai/CRACM1 ion channels provide the major Ca(2+) influx pathway for FcεRI-dependent human lung mast cell (HLMC) mediator release. The Ca(2+)-activated K(+) channel KCa3.1 modulates Ca(2+) influx and the secretory response through hyperpolarisation of the plasma membrane. We hypothesised that there is a close functional and spatiotemporal interaction between these Ca(2+)- and K(+)-selective channels. RESULTS: Activation of FcεRI-dependent HLMC KCa3.1 currents was dependent on the presence of extracellular Ca(2+), and attenuated in the presence of the selective Orai blocker GSK-7975A. Currents elicited by the KCa3.1 opener 1-EBIO were also attenuated by GSK-7975A. The Orai1 E106Q dominant-negative mutant ablated 1-EBIO and FcεRI-dependent KCa3.1 currents in HLMCs. Orai1 but not Orai2 was shown to co-immunoprecipitate with KCa3.1 when overexpressed in HEK293 cells, and Orai1 and KCa3.1 were seen to co-localise in the HEK293 plasma membrane using confocal microscopy. CONCLUSION: KCa3.1 activation in HLMCs is highly dependent on Ca(2+) influx through Orai1 channels, mediated via a close spatiotemporal interaction between the two channels.

Mammalian SUN protein interaction networks at the inner nuclear membrane and their role in laminopathy disease processes.

The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209-228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.

TGFß1 induces resistance of human lung myofibroblasts to cell death via down-regulation of TRPA1 channels.

BACKGROUND AND PURPOSE: TGFß1-mediated myofibroblast activation contributes to pathological fibrosis in many diseases including idiopathic pulmonary fibrosis (IPF), where myofibroblast resistance to oxidant-mediated apoptosis is also evident. We therefore investigated the involvement of redox-sensitive TRPA1 ion channels on human lung myofibroblasts (HLMFs) cell death and TGFß1-mediated pro-fibrotic responses. EXPERIMENTAL APPROACH: The effects of TGFß1 stimulation on TRPA1 expression and cell viability was studied in HLMFs derived from IPF patients and non-fibrotic patients. We also examined a model of TGFß1-dependent fibrogenesis in human lung. We used qRT-PCR, immunofluorescent assays, overexpression with lentiviral vectors and electrophysiological methods. KEY RESULTS: TRPA1 mRNA, protein and ion currents were expressed in HLMFs derived from both non-fibrotic patient controls and IPF patients, and expression was reduced by TGFß1. TRPA1 mRNA was also down-regulated by TGFß1 in a model of lung fibrogenesis in human lung. TRPA1 over-expression or activation induced HLMF apoptosis, and activation of TRPA1 channel activation by H2 O2 induced necrosis. TRPA1 inhibition following TGFß1 down-regulation or pharmacological inhibition, protected HLMFs from both apoptosis and necrosis. Lentiviral vector mediated TRPA1 expression was also found to induce sensitivity to H2 O2 induced cell death in a TRPA1-negative HEK293T cell line. CONCLUSION AND IMPLICATIONS: TGFß1 induces resistance of HLMFs to TRPA1 agonist- and H2 O2 -mediated cell death via down-regulation of TRPA1 channels. Our data suggest that therapeutic strategies which prevent TGFß1-dependent down-regulation of TRPA1 may reduce myofibroblast survival in IPF and therefore improve clinical outcomes.

Lamin A-linked progerias: is farnesylation the be all and end all?

HGPS (Hutchinson-Gilford progeria syndrome) is a severe childhood disorder that appears to mimic an accelerated aging process. The disease is most commonly caused by gene mutations that disrupt the normal post-translational processing of lamin A, a structural component of the nuclear envelope. Impaired processing results in aberrant retention of a farnesyl group at the C-terminus of lamin A, leading to altered membrane dynamics. It has been widely proposed that persistence of the farnesyl moiety is the major factor responsible for the disease, prompting clinical trials of farnesyltransferase inhibitors to prevent lamin A farnesylation in children afflicted with HGPS. Although there is evidence implicating farnesylation in causing some of the cellular defects of HGPS, results of several recent studies suggest that aberrant lamin A farnesylation is not the only determinant of the disease. These findings have important implications for the design of treatments for this devastating disease.

SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton.

Nuclear migration and positioning within cells are critical for many developmental processes and are governed by the cytoskeletal network. Although mechanisms of nuclear-cytoskeletal attachment are unclear, growing evidence links a novel family of nuclear envelope (NE) proteins that share a conserved C-terminal SUN (Sad1/UNC-84 homology) domain. Analysis of Caenorhabditis elegans mutants has implicated UNC-84 in actin-mediated nuclear positioning by regulating NE anchoring of a giant actin-binding protein, ANC-1. Here, we report the identification of SUN1 as a lamin A-binding protein in a yeast two-hybrid screen. We demonstrate that SUN1 is an integral membrane protein located at the inner nuclear membrane. While the N-terminal domain of SUN1 is responsible for detergent-resistant association with the nuclear lamina and lamin A binding, lamin A/C expression is not required for SUN1 NE localization. Furthermore, SUN1 does not interact with type B lamins, suggesting that NE localization is ensured by binding to an additional nuclear component(s), most likely chromatin. Importantly, we find that the luminal C-terminal domain of SUN1 interacts with the mammalian ANC-1 homologs nesprins 1 and 2 via their conserved KASH domain. Our data provide evidence of a physical nuclear-cytoskeletal connection that is likely to be a key mechanism in nuclear-cytoplasmic communication and regulation of nuclear position.

Tensin1 expression and function in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) constitutes a major cause of morbidity and mortality. Genome wide association studies have shown significant associations between airflow obstruction or COPD with a non-synonymous SNP in the TNS1 gene, which encodes tensin1. However, the expression, cellular distribution and function of tensin1 in human airway tissue and cells are unknown. We therefore examined these characteristics in tissue and cells from controls and people with COPD or asthma. Airway tissue was immunostained for tensin1. Tensin1 expression in cultured human airway smooth muscle cells (HASMCs) was evaluated using qRT-PCR, western blotting and immunofluorescent staining. siRNAs were used to downregulate tensin1 expression. Tensin1 expression was increased in the airway smooth muscle and lamina propria in COPD tissue, but not asthma, when compared to controls. Tensin1 was expressed in HASMCs and upregulated by TGFß1. TGFß1 and fibronectin increased the localisation of tensin1 to fibrillar adhesions. Tensin1 and α-smooth muscle actin (αSMA) were strongly co-localised, and tensin1 depletion in HASMCs attenuated both αSMA expression and contraction of collagen gels. In summary, tensin1 expression is increased in COPD airways, and may promote airway obstruction by enhancing the expression of contractile proteins and their localisation to stress fibres in HASMCs.