The mTORC2/SGK1/NDRG1 Signaling Axis Is Critical for the Mesomesenchymal Transition of Pleural Mesothelial Cells and the Progression of Pleural Fibrosis.

Progressive lung scarring because of persistent pleural organization often results in pleural fibrosis (PF). This process affects patients with complicated parapneumonic pleural effusions, empyema, and other pleural diseases prone to loculation. In PF, pleural mesothelial cells undergo mesomesenchymal transition (MesoMT) to become profibrotic, characterized by increased expression of α-smooth muscle actin and matrix proteins, including collagen-1. In our previous study, we showed that blocking PI3K/Akt signaling inhibits MesoMT induction in human pleural mesothelial cells (HPMCs) (1). However, the downstream signaling pathways leading to MesoMT induction remain obscure. Here, we investigated the role of mTOR complexes (mTORC1/2) in MesoMT induction. Our studies show that activation of the downstream mediator mTORC1/2 complex is, likewise, a critical component of MesoMT. Specific targeting of mTORC1/2 complex using pharmacological inhibitors such as INK128 and AZD8055 significantly inhibited transforming growth factor ß (TGF-ß)-induced MesoMT markers in HPMCs. We further identified the mTORC2/Rictor complex as the principal contributor to MesoMT progression induced by TGF-ß. Knockdown of Rictor, but not Raptor, attenuated TGF-ß-induced MesoMT in these cells. In these studies, we further show that concomitant activation of the SGK1/NDRG1 signaling cascade is essential for inducing MesoMT. Targeting SGK1 and NDRG1 with siRNA and small molecular inhibitors attenuated TGF-ß-induced MesoMT in HPMCs. Additionally, preclinical studies in our Streptococcus pneumoniae-mediated mouse model of PF showed that inhibition of mTORC1/2 with INK128 significantly attenuated the progression of PF in subacute and chronic injury. In conclusion, our studies demonstrate that mTORC2/Rictor-mediated activation of SGK1/NDRG1 is critical for MesoMT induction and that targeting this pathway could inhibit or even reverse the progression of MesoMT and PF.

Arterial oxygen pressure during veno-venous extracorporeal membrane oxygenation may be increased by advancing the tip of the drainage cannula into the superior vena cava: a case report.

A simple and robust method for veno-venous extracorporeal membrane oxygenation (V-V ECMO) involves a drainage cannula into the inferior vena cava via the femoral vein (FV) and a reinfusion cannula into the right atrium (RA) via the internal jugular vein (IJV) (F-J configuration). However, with this method, the arterial oxygen (PaO2) is said to remain below 100 mmHg.Since recently, in our ICU, to prevent drainage failure, we apply a modification from the commonly practiced F-J configuration by advancing the tip of the drainage cannula inserted via the FV into the superior vena cava (SVC) and crossing the reinfusion cannula inserted via the IJV in the RA (F(SVC)-J(RA) configuration). We experienced that this modification can be associated with unexpectedly high PaO2 values, which here we investigated in detail.Veno-arteriovenous ECMO was induced in a 65-year-old male patient who suffered from repeated cardiac arrest due to acute respiratory distress syndrome. His chest X-ray images showed white-out after lung rest setting, consistent with near-absence of self-lung ventilation. Cardiac function recovered and the system was converted to F(SVC)-J(RA) configuration, after which both PaO2 and partial pressure of pulmonary arterial oxygen values remained high above 200 mmHg. Transesophageal echocardiography could not detect right-to-left shunt, and more efficient drainage of the native venous return flow compared to common F-J configuration may explain the increased PaO2.Although the F(SVC)-J(RA) configuration is a small modification of the F-J configuration, it seems to provide a revolutionary improvement in the ECMO field by combining robustness/simplicity with high PaO2 values.

Abnormal stenosis of a drainage cannula due to excessive negative pressure during venovenous extracorporeal membrane oxygenation management: a case report.

We report a case in which excessive negative pressure may have been applied to the proximal side hole of a drainage cannula during venovenous extracorporeal membrane oxygenation (V-V ECMO), resulting in abnormal stenosis of the drainage cannula. V-V ECMO was introduced in a 71-year-old male patient who was transferred from another hospital for severe respiratory failure associated with varicella pneumonia and acute respiratory distress syndrome. Drainage was performed using a PCKC-V™ 24Fr (MERA, Japan) cannula via the right femoral vein with the tip of the cannula near the level of the diaphragm under fluoroscopy. Reinfusion was performed via the right internal jugular vein. Due to poor systemic oxygenation, the drainage cannula was withdrawn caudally and refixed to reduce the effect of recirculation. Two days later, drainage pressure dropped rapidly, and frequent ECMO flow interruption occurred due to poor drainage. An abdominal X-ray revealed abnormal stenosis of the proximal side hole site of the drainage cannula. We diagnosed that the drainage cannula was damaged, and it was replaced with another, namely a Medtronic Bio-Medicus™ 25 Fr (GETINGE, Sweden) cannula. However, the removed drainage cannula was not damaged, suggesting that the cannula was temporarily stenosed by momentary excessive negative pressure. In a multi-stage drainage cannula, the main drainage site is the proximal side hole, with little negative pressure applied at the apical foramen in a mock experimental ex vivo drainage test in a water tank. Hence, improvement of a multi-stage drainage cannula is recommended, such as adequate reinforcement of the side hole site with a wire.

Investigation of factors affecting COVID-19 pancreatic injury: a single-center, retrospective study.

Pancreatic injury is considered an organ-related complication in patients with coronavirus disease 2019 (COVID-19). However, it is unclear whether COVID-19 status affects pancreatic injury. This retrospective study aimed to determine whether COVID-19 affects the occurrence of pancreatic injuries. Consecutive patients diagnosed with sepsis admitted to the ICU between March 2020 and September 2021 were included. The primary endpoint was a pancreatic injury, which was defined as amylase or lipase levels > 3 times the upper limit of the normal range. Among the 177 patients included in the analysis, 40 (23%) were COVID-19 patients, and 54 (31%) had pancreatic injuries. Of these three patients, acute pancreatitis was diagnosed based on computed tomography. The pancreatic injury was significantly more common among COVID-19 patients (75 vs. 18%, p < 0.001). Multivariate analysis showed that COVID-19 and steroid use were independent risk factors for pancreatic injury (Odds Ratio (OR) 4.79 [95% confidence interval (CI) 1.48-15.5], p = 0.009; OR 4.02 [95% CI 1.42-11.4], p = 0.009). This study revealed that the proportion of pancreatic injury in septic patients with COVID-19 was significantly higher than in those without COVID-19. It may be difficult to diagnose pancreatitis based on amylase and lipase levels in COVID-19 patients.

Calponin 1 contributes to myofibroblast differentiation of human pleural mesothelial cells.

Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, how these transformed mesothelial cells contribute to lung fibrosis remains unclear. Here, we investigated the mechanism of contractile myofibroblast differentiation of PMCs. Transforming growth factor-ß (TGF-ß) induced marked upregulation of calponin 1 expression, which was correlated with notable cytoskeletal rearrangement in human PMCs (HPMCs) to produce stress fibers. Downregulation of calponin 1 expression reduced stress fiber formation. Interestingly, induced stress fibers predominantly contain α-smooth muscle actin (αSMA) associated with calponin 1 but not ß-actin. Calponin 1-associated stress fibers also contained myosin II and α-actinin. Furthermore, focal adhesions were aligned with the produced stress fibers. These results suggest that calponin 1 facilitates formation of stress fibers that resemble contractile myofibrils. Supporting this notion, TGF-ß significantly increased the contractile activity of HPMCs, an effect that was abolished by downregulation of calponin 1 expression. We infer that differentiation of HPMCs to contractile myofibroblasts facilitates stiffness of scar tissue in pleura to promote pleural fibrosis (PF) and that upregulation of calponin 1 plays a central role in this process.

Caveolin-1-Derived Peptide Reduces ER Stress and Enhances Gelatinolytic Activity in IPF Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by an excess deposition of extracellular matrix in the pulmonary interstitium. Caveolin-1 scaffolding domain peptide (CSP) has been found to mitigate pulmonary fibrosis in several animal models. However, its pathophysiological role in IPF is obscure, and it remains critical to understand the mechanism by which CSP protects against pulmonary fibrosis. We first studied the delivery of CSP into cells and found that it is internalized and accumulated in the Endoplasmic Reticulum (ER). Furthermore, CSP reduced ER stress via suppression of inositol requiring enzyme1α (IRE1α) in transforming growth factor ß (TGFß)-treated human IPF lung fibroblasts (hIPF-Lfs). Moreover, we found that CSP enhanced the gelatinolytic activity of TGFß-treated hIPF-Lfs. The IRE1α inhibitor; 4µ8C also augmented the gelatinolytic activity of TGFß-treated hIPF-Lfs, supporting the concept that CSP induced inhibition of the IRE1α pathway. Furthermore, CSP significantly elevated expression of MMPs in TGFß-treated hIPF-Lfs, but conversely decreased the secretion of collagen 1. Similar results were observed in two preclinical murine models of PF, bleomycin (BLM)- and adenovirus expressing constitutively active TGFß (Ad-TGFß)-induced PF. Our findings provide new insights into the mechanism by which lung fibroblasts contribute to CSP dependent protection against lung fibrosis.

NOX1 Promotes Mesothelial-Mesenchymal Transition through Modulation of Reactive Oxygen Species-mediated Signaling.

Pleural organization may occur after empyema or complicated parapneumonic effusion and can result in restrictive lung disease with pleural fibrosis (PF). Pleural mesothelial cells (PMCs) may contribute to PF through acquisition of a profibrotic phenotype, mesothelial-mesenchymal transition (MesoMT), which is characterized by increased expression of α-SMA (α-smooth muscle actin) and other myofibroblast markers. Although MesoMT has been implicated in the pathogenesis of PF, the role of the reactive oxygen species and the NOX (nicotinamide adenine dinucleotide phosphate oxidase) family in pleural remodeling remains unclear. Here, we show that NOX1 expression is enhanced in nonspecific human pleuritis and is induced in PMCs by THB (thrombin). 4-Hydroxy-2-nonenal, an indicator of reactive oxygen species damage, was likewise increased in our mouse model of pleural injury. NOX1 downregulation blocked THB- and Xa (factor Xa)-mediated MesoMT, as did pharmacologic inhibition of NOX1 with ML-171. NOX1 inhibition also reduced phosphorylation of Akt, p65, and tyrosine 216-GSK-3ß, signaling molecules previously shown to be implicated in MesoMT. Conversely, ML-171 did not reverse established MesoMT. NOX4 downregulation attenuated TGF-ß- and THB-mediated MesoMT. However, NOX1 downregulation did not affect NOX4 expression. NOX1- and NOX4-deficient mice were also protected in our mouse model of Streptococcus pneumoniae-mediated PF. These data show that NOX1 and NOX4 are critical determinants of MesoMT.

A case of spontaneous rectus sheath hematoma induced by lateral semi-prone positional changes during extracorporeal membrane oxygenation.

Spontaneous abdominal wall hematomas are relatively rare and mainly attributed to anticoagulation and severe cough. Despite the high incidence of anticoagulation-related bleeding complications, there are no reports of spontaneous abdominal wall hematomas during extracorporeal membrane oxygenation (ECMO). We report a case of a spontaneous rectus sheath hematoma caused by alternation of the lateral semi-prone position during ECMO in a 76-year-old female patient with severe acute respiratory distress syndrome. Unfractionated heparin 12,000-14,000 units/day was administered for anticoagulation during ECMO. From Day 6 of ECMO, the patient who was under deep sedation was alternately placed in the left and right lateral semi-prone positions every 4 h, for approximately 20 h per day. On Day 12 of ECMO, the patient developed hypotension with anemia and a palpable mass in the right lower abdomen. Abdominal ultrasonographic imaging revealed a huge echo-free space centered in the right lower abdomen. Emergency contrast-enhanced computed tomography (CT) scanning showed extravasation from the superior and inferior epigastric arteries as well as a rectus sheath hematoma. Despite no apparent contrast leakage, an inferior epigastric artery embolization was undertaken because the patient was on ECMO. On Day 13 after ECMO initiation, ECMO and anticoagulation were discontinued. On CT scanning a week later, the hematoma had reduced. In conclusion, spontaneous abdominal wall hematoma is a rare and important complication that might occur during ECMO. Thus, careful physical examination should be routinely conducted when the patient is semi-prone during ECMO.

p116Rip promotes myosin phosphatase activity in airway smooth muscle cells.

Myosin phosphatase-Rho interacting protein (p116Rip ) was originally found as a RhoA-binding protein. Subsequent studies by us and others revealed that p116Rip facilitates myosin light chain phosphatase (MLCP) activity through direct and indirect manners. However, it is unclear how p116Rip regulates myosin phosphatase activity in cells. To elucidate the role of p116Rip in cellular contractile processes, we suppressed the expression of p116Rip by RNA interference in human airway smooth muscle cells (HASMCs). We found that knockdown of p116Rip in HASMCs led to increased di-phosphorylated MLC (pMLC), that is phosphorylation at both Ser19 and Thr18. This was because of a change in the interaction between MLCP and myosin, but not an alteration of RhoA/ROCK signaling. Attenuation of Zipper-interacting protein kinase (ZIPK) abolished the increase in di-pMLC, suggesting that ZIPK is involved in this process. Moreover, suppression of p116Rip expression in HASMCs substantially increased the histamine-induced collagen gel contraction. We also found that expression of the p116Rip was decreased in the airway smooth muscle tissue from asthmatic patients compared with that from non-asthmatic patients, suggesting a potential role of p116Rip expression in asthma pathogenesis.

Myocardin Is Involved in Mesothelial-Mesenchymal Transition of Human Pleural Mesothelial Cells.

Pleural fibrosis is characterized by severe inflammation of the pleural space and pleural reorganization. Subsequent thickening of the visceral pleura contributes to lung stiffness and impaired lung function. Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, the mechanisms that underlie MesoMT remain unclear. Here, we investigated the role of myocardin in the induction of MesoMT. Transforming growth factor ß (TGF-ß) and thrombin induced MesoMT and markedly upregulated the expression of myocardin, but not myocardin-related transcription factor A (MRTF-A) or MRTF-B, in human PMCs (HPMCs). TGF-ß stimulation notably induced the nuclear translocation of myocardin in HPMCs, whereas nuclear translocation of MRTF-A and MRTF-B was not observed. Several genes under the control of myocardin were upregulated in cells undergoing MesoMT, an effect that was accompanied by a dramatic cytoskeletal reorganization of HPMCs consistent with a migratory phenotype. Myocardin gene silencing blocked TGF-ß- and thrombin-induced MesoMT. Although myocardin upregulation was blocked, MRTF-A and MRTF-B were unchanged. Myocardin, α-SMA, calponin, and smooth muscle myosin were notably upregulated in the thickened pleura of carbon black/bleomycin and empyema mouse models of fibrosing pleural injury. Similar results were observed in human nonspecific pleuritis. In a TGF-ß mouse model of pleural fibrosis, PMC-specific knockout of myocardin protected against decrements in lung function. Further, TGF-ß-induced pleural thickening was abolished by PMC-specific myocardin knockout, which was accompanied by a marked reduction of myocardin, calponin, and α-SMA expression compared with floxed-myocardin controls. These novel results show that myocardin participates in the development of MesoMT in HPMCs and contributes to the pathogenesis of pleural organization and fibrosis.

Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s-1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s-1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells.

Visualization of stimulus-specific heterogeneous activation of individual vascular smooth muscle cells in aortic tissues.

Intercellular communication among autonomic nerves, endothelial cells (ECs), and vascular smooth muscle cells (VSMCs) plays a central role in an uninterrupted regulation of blood flow through vascular contractile machinery. Impairment of this communication is linked to development of vascular diseases such as hypertension, cerebral/coronary vasospasms, aortic aneurism, and erectile dysfunction. Although the basic concept of the communication as a whole has been studied, the spatiotemporal correlation of ECs/VSMCs in tissues at the cellular level is unknown. Here, we show a unique VSMC response to ECs during contraction and relaxation of isolated aorta tissues through visualization of spatiotemporal activation patterns of smooth muscle myosin II. ECs in the intimal layer dictate the stimulus-specific heterogeneous activation pattern of myosin II in VSMCs within distinct medial layers. Myosin light chain (MLC) phosphorylation (active form of myosin II) gradually increases towards outer layers (approximately threefold higher MLC phosphorylation at the outermost layer than that of the innermost layer), presumably by release of an intercellular messenger, nitric oxide (NO). Our study also demonstrates that the MLC phosphorylation at the outermost layer in spontaneously hypertensive rats (SHR) during NO-induced relaxation is quite high and approximately 10-fold higher than that of its counterpart, the Wister-Kyoto rats (WKY), suggesting that the distinct pattern of myosin II activation within tissues is important for vascular protection against elevated blood pressure.

Inhibition of Glycogen Synthase Kinase 3ß Blocks Mesomesenchymal Transition and Attenuates Streptococcus pneumonia-Mediated Pleural Injury in Mice.

Pleural loculation affects about 30,000 patients annually in the United States and in severe cases can resolve with restrictive lung disease and pleural fibrosis. Pleural mesothelial cells contribute to pleural rind formation by undergoing mesothelial mesenchymal transition (MesoMT), whereby they acquire a profibrotic phenotype characterized by increased expression of α-smooth muscle actin and collagen 1. Components of the fibrinolytic pathway (urokinase plasminogen activator and plasmin) are elaborated in pleural injury and strongly induce MesoMT in vitro. These same stimuli enhance glycogen synthase kinase (GSK)-3ß activity through increased phosphorylation of Tyr-216 in pleural mesothelial cells and GSK-3ß mobilization from the cytoplasm to the nucleus. GSK-3ß down-regulation blocked induction of MesoMT. Likewise, GSK-3ß inhibitor 9ING41 blocked induction of MesoMT and reversed established MesoMT. Similar results were demonstrated in a mouse model of Streptococcus pneumoniae-induced empyema. Intraperitoneal administration of 9ING41, after the induction of pleural injury, attenuated injury progression and improved lung function (lung volume and compliance; P < 0.05 compared with untreated and vehicle controls). MesoMT marker α-smooth muscle actin was reduced in 9ING41-treated mice. Pleural thickening was also notably reduced in 9ING41-treated mice (P < 0.05). Collectively, these studies identify GSK-3ß as a newly identified target for amelioration of empyema-related pleural fibrosis and provide a strong rationale for further investigation of GSK-3ß signaling in the control of MesoMT and pleural injury.

Phosphorylation of myosin II regulatory light chain by ZIP kinase is responsible for cleavage furrow ingression during cell division in mammalian cultured cells.

Zipper-interacting protein kinase (ZIPK) is known to regulate several functions such as apoptosis, smooth muscle contraction, and cell migration. While exogenously expressed GFP-ZIPK localizes to the cleavage furrow, role of ZIPK in cytokinesis is obscure. Here, we show that ZIPK is a major MRLC kinase during mitosis. Moreover, ZIPK siRNA-mediated knockdown causes delay of cytokinesis. The delay in cytokinesis of ZIPK-knockdown cells was rescued by the exogenous diphosphorylation-mimicking MRLC mutant. Taken together, these findings suggest that ZIPK plays a role in the progression and completion of cytokinesis through MRLC phosphorylation.

ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosin II isoforms.

Migration of medial vascular smooth muscle cells (VSMCs) into the intimal layer contributes to pathological remodeling of the blood vessel in arterial hypertension and atherosclerosis. It is well established that reorganization of cytoskeletal networks is an essential component of cellular motile events. However, there is currently a lack of insight into the cellular characteristics of VSMC migration under three-dimensional environments. Here, we investigated the mechanisms of VSMC migration and remodeling using two different collagen matrix assays as in vitro models: migration of VSMCs within a collagen matrix for VSMC invasion and contraction of a collagen gel by VSMCs for VSMC remodeling and contraction. We found that nonmuscle myosin IIA (NMIIA) and nonmuscle myosin IIB (NMIIB) differentially contribute to the migratory capacity of VSMCs via NMII isoform-dependent cytoskeletal reorganization. Depletion of NMIIA by short hairpin RNA revealed a unique interplay between actomyosin and microtubules during VSMC migration. On the other hand, NMIIB was required for the structural maintenance of migrating VSMC. Interestingly, there was a significant difference between NMIIA and NMIIB knockdown in the VSMC migration but not in the VSMC-mediated collagen gel contraction. Furthermore, depletion of zipper-interacting protein kinase by short hairpin RNA resulted in an impairment of VSMC migration and a substantial decrease of VSMC-mediated collagen gel contraction. These results suggest that NMIIA and NMIIB uniquely control VSMC migration and may contribute to vascular remodeling, which are both regulated by zipper-interacting protein kinase.

Promotion of a venous thromboembolism prevention protocol at a perioperative management center.

Objectives: Perioperative venous thromboembolism (VTE) is a potentially fatal complication, making preoperative VTE diagnosis and secondary thromboprophylaxis important. This study was performed to investigate the impact of promotion of a preoperative VTE protocol at a perioperative management center (PMC) on detecting the preoperative VTE rate and subsequent treatment. Methods: This retrospective study involved patients aged ≥20 years who underwent elective anesthesia. The patients were divided into two groups: the pre-PMC group (January to October 2014, before the opening of the PMC) and the post-PMC group (January to December 2019, after the opening of the PMC). The rates of preoperative lower-limb compression ultrasonography (CUS), VTE detection, anticoagulation therapy, and new postoperative pulmonary embolism (PE) were compared between the two groups. Results: The pre-PMC and post-PMC groups comprised 3737 and 5388 patients, respectively. The preoperative CUS and VTE detection rates were significantly higher in the post-PMC than pre-PMC group (7.2% and 1.43% vs. 25.6% and 3.93%, respectively; P<0.001). There was no significant difference in the rate of anticoagulation therapy in patients with preoperative VTE (88.9% vs. 84.7%, P=0.43). Heparin and direct oral anticoagulants were primarily used in the pre-PMC and post-PMC groups, respectively. The efficacy and safety were comparable between the two groups. No new postoperative PE was detected in either group. Conclusions: Promotion of the preoperative VTE protocol led by the PMC increased the rates of preoperative CUS and preoperative VTE detection. This may aid in secondary thromboprophylaxis in the preoperative period and prevention of postoperative PE.

Crosstalk between adenosine A1 and ß1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes.

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in ß(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by ß(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of ß(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and ß-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The ß(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/ß-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing ß-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and ß(1)-adrenergic agonists on function are complex in the heart.

Prkg2 regulates alveolar type 2-mediated re-alveolarization.

BACKGROUND: The cGMP-dependent type 2 protein kinase, encoded by the prkg2 gene, is highly expressed in alveolar type 2 epithelial (AT2) cells. It is unclear whether prkg2 regulates AT2 cell homeostasis and re-alveolarization of injured lungs. This study aimed to investigate the role of prkg2 in the regulation of the fate of AT2 in vitro. METHODS: Primary AT2 cells of wild-type (wt) and prkg2-/- mice were co-cultured with fibroblasts as three-dimensional organoids. The colony formation was analyzed between days 4 and 12 post-seeding. EdU assay was used to detect cells with active DNA synthesis. AT1 and AT2 cells in organoids were visualized with anti-podoplanin and anti-surfactant protein C antibodies, respectively. RESULTS: Prkg2-/- AT2 cells developed a greater number of organoids than wt controls. However, compared to wt organoids, a lower number of AT2 but a greater number of AT1 cells were visualized. In addition, a lower number of proliferated cells (EdU+) were observed in prkg2-/- organoids compared to wt controls. The numbers of organoids and EdU+ cells were significantly reduced in protein kinase A (PKA) inhibitor H89-treated wt and prkg2-/- cultures. Organoids and EdU+ cells were increased by lipopolysaccharides (LPS) in both wt and prkg2-/- groups. The increase in the proportion of AT1 and AT2 cells in organoids was only seen in wt controls. CONCLUSIONS: Prkg2 may regulate the lineage of AT2 cells, which is affected by endotoxins and the interactive PKA signaling pathway.

Effect of multidisciplinary interventions in perioperative management center on duration of preoperative fasting: A single-center before-and-after study.

Objectives: Our aims were to clarify the actual situation regarding preoperative fasting and determine whether multidisciplinary interventions in a perioperative management center shorten the duration of preoperative fasting. Methods: The cohort of this before-and-after study comprised patients undergoing elective surgery aged 18 years or older who underwent general anesthesia at one of three stages: after starting a short preoperative fasting protocol (Group A), after the anesthesiologist started explaining the protocol (Group B), and after the start of the perioperative management center (Group C). Instructions on drinking clear fluids were given up to 2 h and 4 h before the start of elective surgery to the first patient on the list (on-time) and to the second and subsequent patients (on-call), respectively. Data were collected retrospectively in Groups A and B and prospectively in Group C. Results: The study cohort comprised 89 patients in Group A (50 on-time, 39 on-call), 108 in Group B (65 on-time, 43 on-call), and 284 in Group C (182 on-time, 102 on-call). The difference between the instructed and last drinking time was significantly shorter in Group C than Group A (30 [10, 140] vs. 30 [10, 60] vs. 20 [0, 50] min, p=0.003). The duration of fasting was significantly shorter in Group C than Group B (243 [150, 395] vs. 213 [151, 323] vs. 180 [146, 280] min, p=0.01). Conclusions: Multidisciplinary interventions at the perioperative management center tended to reduce the duration of fasting, suggesting that this approach may contribute to improved compliance.

A Larger Membrane Area Increases Cytokine Removal in Polymethyl Methacrylate Hemofilters.

Blood purification is performed to control cytokines in critically ill patients. The relationship between the clearance (CL) and the membrane area during adsorption is not clear. We hypothesized that the CL increases with the hydrophobic area when hydrophobic binding contributes to cytokine adsorption. We investigated the relationship between the hemofilter membrane area and the CL of the high mobility group box 1 protein (HMGB-1) and interleukin-6 (IL-6). We performed experimental hemofiltration in vitro using polymethyl methacrylate membranes CH-1.8W (1.8 m2) and CH-1.0N (1.0 m2), as well as polysulfone membrane NV-18X (1.8 m2). After adding 100 mg of HMGB1 or 10 µg of IL-6 into the test solution, experimental hemofiltration was conducted for 360 min in a closed-loop circulation system, and the same amount of HMGB1 and IL-6 was added after 180 min. With CH-1.8W and CH-1.0N, both HMGB-1 and IL-6 showed a rapid concentration decrease of more than 70% at 180 min and 360 min after the re-addition. At 15 min, the CL of HMGB-1 was CH-1.8W: 28.4 and CH-1.0N: 19.8, and that of IL-6 was CH-1.8W: 41.1 and CH-1.0N: 25.4. CH-1.8W and CH-1.0N removed HMGB1 and IL-6 by adsorption and CH-1.8W was superior in CL, which increased with a greater membrane area.