Cytoprotective Activated Protein C Averts Nlrp3 Inflammasome–Induced Ischemia-Reperfusion Injury Via Mtorc1 Inhibition

Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI

Activated Protein C Ameliorates Tubular Mitochondrial Reactive Oxygen Species and Inflammation in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is an emerging pandemic, paralleling the worldwide increase in obesity and diabetes mellitus. DKD is now the most frequent cause of end-stage renal disease and is associated with an excessive risk of cardiovascular morbidity and mortality. DKD is a consequence of systemic endothelial dysfunction. The endothelial-dependent cytoprotective coagulation protease activated protein C (aPC) ameliorates glomerular damage in DKD, in part by reducing mitochondrial ROS generation in glomerular cells. Whether aPC reduces mitochondrial ROS generation in the tubular compartment remains unknown. Here, we conducted expression profiling of kidneys in diabetic mice (wild-type and mice with increased plasma levels of aPC, APChigh mice). The top induced pathways were related to metabolism and in particular to oxidoreductase activity. In tubular cells, aPC maintained the expression of genes related to the electron transport chain, PGC1-α expression, and mitochondrial mass. These effects were associated with reduced mitochondrial ROS generation. Likewise, NLRP3 inflammasome activation and sterile inflammation, which are known to be linked to excess ROS generation in DKD, were reduced in diabetic APChigh mice. Thus, aPC reduces mitochondrial ROS generation in tubular cells and dampens the associated renal sterile inflammation. These studies support approaches harnessing the cytoprotective effects of aPC in DKD

p45 NF-E2 regulates syncytiotrophoblast differentiation by post-translational GCM1 modifications in human intrauterine growth restriction

AbstractPlacental insufficiency jeopardizes prenatal development, potentially leading to intrauterine growth restriction (IUGR) and stillbirth. Surviving fetuses are at an increased risk for chronic diseases later in life. IUGR is closely linked with altered trophoblast and placental differentiation. However, due to a paucity of mechanistic insights, suitable biomarkers and specific therapies for IUGR are lacking. The transcription factor p45 NF-E2 (nuclear factor erythroid derived 2) has been recently found to regulate trophoblast differentiation in mice. The absence of p45 NF-E2 in trophoblast cells causes IUGR and placental insufficiency in mice, but mechanistic insights are incomplete and the relevance of p45 NF-E2 for human syncytiotrophoblast differentiation remains unknown. Here we show that p45 NF-E2 negatively regulates human syncytiotrophoblast differentiation and is associated with IUGR in humans. Expression of p45 NF-E2 is reduced in human placentae complicated with IUGR compared with healthy controls. Reduced p45 NF-E2 expression is associated with increased syncytiotrophoblast differentiation, enhanced glial cells missing-1 (GCM1) acetylation and GCM1 desumoylation in IUGR placentae. Induction of syncytiotrophoblast differentiation in BeWo and primary villous trophoblast cells with 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) reduces p45 NF-E2 expression. Of note, p45 NF-E2 knockdown is sufficient to increase syncytiotrophoblast differentiation and GCM1 expression. Loss of p45 NF-E2 using either approach resulted in CBP-mediated GCM1 acetylation and SENP-mediated GCM1 desumoylation, demonstrating that p45 NF-E2 regulates post-translational modifications of GCM1. Functionally, reduced p45 NF-E2 expression is associated with increased cell death and caspase-3 activation in vitro and in placental tissues samples. Overexpression of p45 NF-E2 is sufficient to repress GCM1 expression, acetylation and desumoylation, even in 8-Br-cAMP exposed BeWo cells. These results suggest that p45 NF-E2 negatively regulates differentiation and apoptosis activation of human syncytiotrophoblast by modulating GCM1 acetylation and sumoylation. These studies identify a new pathomechanism related to IUGR in humans and thus provide new impetus for future studies aiming to identify new biomarkers and/or therapies of IUGR.</jats:p

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Micelle formation of Boc-Val-Val-Ile-OMe tripeptide in chloroform and its conformational analysis

The tripeptide derivative 1(Boc-Val-Val-Ile-OMe) forms micelles in chloroform as confirmed by UV–VIS, fluorescence and NMR spectroscopic techniques; the conformational analysis of the tripeptide 1 was determined by ¹H NMR spectroscopy

Aggregation, hydrogen bonding and thermodynamic studies on Boc-Val-Val-Ile-OMe tripeptide micelles in chloroform

Evidence for micelle formation of Boc-Val-Val-Ile-OMe (Boc =tert-butyloxycarbonyl) tripeptide (1), in chloroform has been obtained from IR and Raman scatter fluorescence spectroscopies. The critical micelle concentrations (c.m.c.s) of this peptide, obtained by these techniques, correlate well. It has been found that the micelle formation of the peptide in chloroform is hindered by increasing temperature. The aggregation numbers of the peptide have also been determined to be almost independent of temperature. The Δ<SUB>m</SUB>G<SUP>θ</SUP>, Δ<SUB>m</SUB>H<SUP>θ</SUP>, Δ<SUB>m</SUB>S<SUP>θ</SUP> and Δ<SUB>m</SUB>C<SUB>p</SUB> values have been estimated. Results from the above thermodynamic parameters indicate that the driving force for micellization of the tripeptide 1 in chloroform is entirely enthalpic in nature and the aggregates of the peptide in chloroform are ordered. The IR spectra of the peptide in the pre- and post-micellar regions were analysed; there is no change in the intensity of the intermolecular hydrogen-bonding pattern for the peptide in the monomeric and micellar states. However, the intensity of the solvent-exposed -N-H stretching band increased as a function of peptide concentration after attaining c.m.c

Surface active peptide-mediated porphyrin aggregation

Surface active pentapeptide [2(HCOO−). Lys-Ala-Ala-Lys(Z)-Tyr-OCH3] has been synthesized and its micelle formation investigated using conductometric, pH metric, and UV spectroscopic techniques. The double head double tail peptide molecules are shown to interact with water soluble meso-tetrakis (4-sulfonatophenyl)-porphyrin [TPPS]H2to form characteristic H-type aggregate at low concentrations, as evidenced by UV–Vis and fluorescence spectroscopic techniques. Spectroscopic analysis reveals that the aggregate contains 1:2 porphyrin–peptide combination. The equilibrium constant for the formation of peptide–porphyrin complex has been obtained by using absorption spectral data. The present studies provide new insight into the peptide–porphyrin interaction

3₁₀ helix formation in protected tripeptide

The conformational analysis of a synthetic peptide Boc-Lys(Z)-Gly-Val-NHMe has been carried out, as a model for nucleating segment in helix formation. 1H NMR studies (270 MHz) suggested that the Gly (2) NH, Val (3) NH and NHMe groups are solvent shielded. Conformational energy calculations and intramolecular hydrogen bonding constrains favour 310 helix structure for the peptide. Theoretical and spectroscopic results are consistent with the presence of a transannular 4 -> 1 hydrogen bond between Lys (1) CO and NHMe with Gly (2) NH and Val (3) being sterically shielded from the solvent environment

Performance analysis and enhancement of brain emotion-based intelligent controller and its impact on PMBLDC motor drive for electric vehicle applications

Growing awareness of electric vehicles insists on the necessity of Permanent Magnet Brushless Direct Current Motor (PMBLDCM) worldwide. This paper involves building a simulation model of the complete PMBLDCM drive system using Matlab/Simulink to control the speed and Torque. Using the Simulink model, speed parameters with various controllers of the drive system are studied and analyzed. The conventional PI controller is capable of controlling the speed of the PMBLDC motor. H1owever, it cannot give assurance of the stability of the motor throughout the load variation. In a speed loop, Improved Brain Emotional Learning Based Intelligent Controller (IBELBIC) is proposed in this paper. The proposed IBELBIC-based PMBLDC motor drive system with a rating of 3 phase, 30 V, 400 W, 3000 RPM is implemented using Spartan 3 Field Programmable Gate Array (FPGA) from Xilinx. This paper presents an IBELBIC design for enhancing the speed performance of the drive system at various set speeds and different load conditions of 1.2 Nm load, 0.6 Nm load, and no load. The proposed PMBLDCM drive system is realized with the help of a Very high speed integrated circuit Hardware Description Language (VHDL) programming algorithm of digital Pulse Width Modulation (PWM) generator topology. Various test cases are evaluated under different operating conditions to demonstrate the learning capability and the applicability of the proposed controller. In this paper, simulation validation is done using hardware setup, and experimental results are monitored on a computer using a customized program developed using LabVIEW (Laboratory Virtual Instrumentation Engineering Workbench). Instead of a Data Acquisition (DAQ) card, the Virtual Instrument Software Architecture (VISA) tool of LabVIEW software is used in this work and results in cost minimization of the experimental setup.Scopu

Hypercoagulability Impairs Plaque Stability in Diabetes-Induced Atherosclerosis

Diabetes mellitus, which is largely driven by nutritional and behavioral factors, is characterized by accelerated atherosclerosis with impaired plaque stability. Atherosclerosis and associated complications are the major cause of mortality in diabetic patients. Efficient therapeutic concepts for diabetes-associated atherosclerosis are lacking. Atherosclerosis among diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we demonstrate that atherosclerotic plaque stability is reduced in hyperglycemic mice expressing dysfunctional TM (TMPro/Pro mice), which have a pro-coagulant phenotype due to impaired thrombin inhibition and markedly reduced aPC generation. The vessel lumen and plaque size of atherosclerotic lesions in the truncus brachiocephalic were decreased in diabetic TMPro/Pro ApoE-/- mice compared to diabetic ApoE-/- mice. While lipid accumulation in lesions of diabetic TMPro/Pro ApoE-/- mice was lower than that in diabetic ApoE-/- mice, morphometric analyses revealed more prominent signs of instable plaques, such as a larger necrotic core area and decreased fibrous cap thickness in diabetic TMPro/Pro ApoE-/- mice. Congruently, more macrophages and fewer smooth muscle cells were observed within lesions of diabetic TMPro/Pro ApoE-/- mice. Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis