Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis.

Introduction: Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, pro-healing phenotype, whereas OPN inhibition triggers a pro-inflammatory phenotype. However, the precise role of OPN in macrophage activation state is unknown. Methods: Here, we applied global proteome profiling via mass spectrometry (MS) analysis to gain a mechanistic understanding of OPN suppression versus induction in primary macrophage cultures. We analyzed protein networks and immune-related functional pathways in BMMΦ either with OPN knockout (OPNKO) or GA-mediated OPN induction compared with wild type (WT) macrophages. The most significant differentially expressed proteins (DEPs) were validated using immunocytochemistry, western blot, and immunoprecipitation assays. Results and discussion: We identified 631 DEPs in OPNKO or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPNKO macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (e.g., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages-inhibiting translation and protein turnover and inducing apoptosis-whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies.

Comparative Proteomic Analysis of HPV(+) Oropharyngeal Squamous Cell Carcinoma Recurrence.

Deintensification therapy for human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV(+) OPSCC) is under active investigation. An adaptive treatment approach based on molecular stratification could identify high-risk patients predisposed to recurrence and better select for appropriate treatment regimens. Collectively, 40 HPV(+) OPSCC FFPE samples (20 disease-free, 20 recurrent) were surveyed using mass spectrometry-based proteomic analysis via data-independent acquisition to obtain fold change and false discovery differences. Ten-year overall survival was 100.0 and 27.7% for HPV(+) disease-free and recurrent cohorts, respectively. Of 1414 quantified proteins, 77 demonstrated significant differential expression. Top enriched functional pathways included those involved in programmed cell death (73 proteins, p = 7.43 × 10-30), apoptosis (73 proteins, p = 5.56 × 10-9), ß-catenin independent WNT signaling (47 proteins, p = 1.45 × 10-15), and Rho GTPase signaling (69 proteins, p = 1.09 × 10-5). PFN1 (p = 1.0 × 10-3), RAD23B (p = 2.9 × 10-4), LDHB (p = 1.0 × 10-3), and HINT1 (p = 3.8 × 10-3) pathways were significantly downregulated in the recurrent cohort. On functional validation via immunohistochemistry (IHC) staining, 46.9% (PFN1), 71.9% (RAD23B), 59.4% (LDHB), and 84.4% (HINT1) of cases were corroborated with mass spectrometry findings. Development of a multilateral molecular signature incorporating these targets may characterize high-risk disease, predict treatment response, and augment current management paradigms in head and neck cancer.

Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids.

BACKGROUND: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. METHODS: Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. RESULTS: Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV <30%. The mid-throughput workflow had 67% (plasma), 90% (depleted), and 78% (dried blood) of peptides display an inter-day CV <30%. Lower limits of detection and quantification were determined for peptides and proteins observed in each biofluid and workflow. Based on each protein and peptide's analytical performance, we could describe the observable, reliable, reproducible, and quantifiable proteomes for each biofluid and workflow. CONCLUSION: The standardized workflows established here allows for reproducible and quantifiable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow should simplify discovery approaches and facilitate the translation of candidate markers into clinical use.

Profiling B-Type Natriuretic Peptide Cleavage Peptidoforms in Human Plasma by Capillary Electrophoresis with Electrospray Ionization Mass Spectrometry.

B-type Natriuretic Peptide (BNP) is a biologically active circulating hormone. Plasma concentrations of BNP are routinely used in the diagnosis of heart failure, and the intravenous infusion of recombinant BNP can be used for heart failure treatment. Like many bioactive polypeptides, multiple plasma enzymes are known to cleave circulating BNP, and as part of the CVD-B/D-HPP mandate, we sought to develop a technique capable of profiling these catabolic processes in plasma. We used a neutral-coated capillary electrophoresis-electrospray ionization (CESI) separation system coupled with high-resolution mass spectrometry to profile the proteolysis of exogenous recombinant BNP1-32 in plasma. Our method utilizes electrokinetic injection of minimally processed plasma samples to simultaneously monitor the dynamic generation and breakdown of at least five BNP peptidoforms in plasma. By integrating multisegment injection, our method can produce a multipoint BNP proteolytic profile for one sample within an hour. We envision applying this method to assess the potential relation between plasma-based BNP proteolysis and heart failure as well as a means of monitoring BNP bioavailability after therapeutic infusion.

Propofol cardioprotection for on-pump aortocoronary bypass surgery in patients with type 2 diabetes mellitus (PRO-TECT II): a phase 2 randomized-controlled trial.

PURPOSE: The efficacy of myocardial conditioning strategies is compromised in patients with advanced age, diabetes, or low ejection fraction. We conducted a single-centre parallel-arm blinded randomized-controlled trial to determine whether propofol provides perioperative myocardial protection. METHODS: Patients enrolled in this study were scheduled for primary aortocoronary bypass surgery utilizing normothermic cardiopulmonary bypass (CPB) with blood cardioplegia. The participants were stratified by diabetic status and left ventricular ejection fraction and randomly assigned to receive either an elevated dose of propofol -previously associated with experimental cardioprotection- or an isoflurane preconditioning regime. The primary endpoint was the coronary sinus (CS) concentration of 15-F2t-isoprostane (isoP). Secondary endpoints included in-hospital low cardiac output syndrome (LCOS) and major adverse cardiac events, 12- and 24-hr CS cardiac troponin I (cTnI) release, and myocardial B-cell lymphoma 2 (Bcl-2) protein expression. RESULTS: Data were analyzed from 125 of 137 randomized participants. Participants receiving propofol experienced a greater mean (SD) increase from baseline in CS 15-F2t-isoP levels compared with those receiving isoflurane [26.9 (10.9) pg·mL(-1) vs 12.1 (10.4) pg·mL(-1), respectively; mean difference, 14.8; 95% confidence interval (CI), 11.0 to 18.6; P < 0.001] but a decreased incidence of LCOS (20.9% vs 57.1%, respectively; relative risk [RR],0.37; 95% CI, 0.22 to 0.62; P < 0.001). The incidence of LCOS was similar between groups in participants without type 2 diabetes mellitus (DM2) (P = 0.382) but significantly decreased in the propofol DM2 subgroup compared with the isoflurane DM2 subgroup (17.9% vs 70.3%, respectively; RR, 0.26; 95% CI, 0.13 to 0.52; P < 0.001). Propofol was associated with an increase in myocardial Bcl-2 protein expression (P = 0.005), a lower incidence of a CS cTnI threshold for myocardial infarction (P = 0.014), and fewer heart failure events (P < 0.001). CONCLUSION: Propofol may be a preemptive intraoperative cardioprotectant for patients with DM2 under conditions of normothermic CPB and blood cardioplegic arrest. The study is registered at www.clinicaltrials.gov (NCT00734383) and www.controlled-trials.com (ISRCTN70879185).

Identification of a Set of Conserved Eukaryotic Internal Retention Time Standards for Data-independent Acquisition Mass Spectrometry.

Accurate knowledge of retention time (RT) in liquid chromatography-based mass spectrometry data facilitates peptide identification, quantification, and multiplexing in targeted and discovery-based workflows. Retention time prediction is particularly important for peptide analysis in emerging data-independent acquisition (DIA) experiments such as SWATH-MS. The indexed RT approach, iRT, uses synthetic spiked-in peptide standards (SiRT) to set RT to a unit-less scale, allowing for normalization of peptide RT between different samples and chromatographic set-ups. The obligatory use of SiRTs can be costly and complicates comparisons and data integration if standards are not included in every sample. Reliance on SiRTs also prevents the inclusion of archived mass spectrometry data for generation of the peptide assay libraries central to targeted DIA-MS data analysis. We have identified a set of peptide sequences that are conserved across most eukaryotic species, termed Common internal Retention Time standards (CiRT). In a series of tests to support the appropriateness of the CiRT-based method, we show: (1) the CiRT peptides normalized RT in human, yeast, and mouse cell lysate derived peptide assay libraries and enabled merging of archived libraries for expanded DIA-MS quantitative applications; (2) CiRTs predicted RT in SWATH-MS data within a 2-min margin of error for the majority of peptides; and (3) normalization of RT using the CiRT peptides enabled the accurate SWATH-MS-based quantification of 340 synthetic isotopically labeled peptides that were spiked into either human or yeast cell lysate. To automate and facilitate the use of these CiRT peptide lists or other custom user-defined internal RT reference peptides in DIA workflows, an algorithm was designed to automatically select a high-quality subset of datapoints for robust linear alignment of RT for use. Implementations of this algorithm are available for the OpenSWATH and Skyline platforms. Thus, CiRT peptides can be used alone or as a complement to SiRTs for RT normalization across peptide spectral libraries and in quantitative DIA-MS studies.

The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion.

Myocardial ischemia-reperfusion injury is an important cause of impaired heart function in the early postoperative period subsequent to cardiac surgery. Reactive oxygen species (ROS) generation increases during both ischemia and reperfusion and it plays a central role in the pathophysiology of intraoperative myocardial injury. Unfortunately, the cellular source of these ROS during ischemia and reperfusion is often poorly defined. Similarly, individual ROS members tend to be grouped together as free radicals with a uniform reactivity towards biomolecules and with deleterious effects collectively ascribed under the vague umbrella of oxidative stress. This review aims to clarify the identity, origin, and progression of ROS during myocardial ischemia and reperfusion. Additionally, this review aims to describe the biochemical reactions and cellular processes that are initiated by specific ROS that work in concert to ultimately yield the clinical manifestations of myocardial ischemia-reperfusion. Lastly, this review provides an overview of several key cardioprotective strategies that target myocardial ischemia-reperfusion injury from the perspective of ROS generation. This overview is illustrated with example clinical studies that have attempted to translate these strategies to reduce the severity of ischemia-reperfusion injury during coronary artery bypass grafting surgery.

Differences in myocardial PTEN expression and Akt signalling in type 2 diabetic and nondiabetic patients undergoing coronary bypass surgery.

OBJECTIVE: Patients with diabetes experience increased cardiovascular complications after cardiac surgery. Hyperglycaemia predicts increased mortality after myocardial infarction and may influence cardiovascular risk in humans. Impaired prosurvival phosphatase and tensin homologue on chromosome 10 (PTEN)-Akt signalling could be an important feature of the diabetic heart rendering it resistant to preconditioning. This study was designed to evaluate for differences and relationships of myocardial PTEN-Akt-related signalling and baseline glycaemic control marker in type 2 diabetic and nondiabetic patients undergoing coronary artery bypass surgery. METHODS: Right atrial biopsies and coronary sinus blood were obtained from 18 type 2 diabetic and 18 nondiabetic patients intraoperatively. Expression and phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), Bcl-2 and PTEN were evaluated by Western blot. Plasma 15-F(2t) -isoprostane concentrations were evaluated by liquid chromatography-mass spectrometry. RESULTS: PTEN expression and 15-F(2t) -isoprostane concentrations were significantly higher in diabetic patients. Increased fasting blood glucose levels correlated with increased coronary sinus plasma 15-F(2t) -isoprostane concentrations. Increased cardiac 15-F(2t) -isoprostane generation was highly correlated with myocardial PTEN expression. Bcl-2 expression and eNOS phosphorylation were significantly lower in diabetic compared with nondiabetic patients. Akt phosphorylation tended to be lower in diabetic patients; however, this tendency failed to reach statistical significance. CONCLUSION: The current results suggest that prosurvival PTEN-Akt signalling is impaired in the diseased diabetic myocardium. Hyperglycaemia and increased oxidative stress may contribute to this phenomenon. These findings strengthen the understanding of the underlying biologic mechanisms of cardiac injury in diabetic patients, which could facilitate development of new treatments to prevent cardiovascular complications in this high-risk population.

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation.

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H(2)O(2))-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H(2)O(2)-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H(2)O(2)-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H(2)O(2)-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.

Target-achieved propofol concentration during on-pump cardiac surgery: a pilot dose-finding study.

PURPOSE: Propofol concentrations that produce laboratory-based cardioprotective effects are generally greater than those produced under routine anesthesia during cardiac surgery. It is unknown whether experimental cardioprotective propofol concentrations can routinely be achieved during cardiopulmonary bypass (CPB) using continuous infusion. METHODS: Twenty-four patients scheduled for primary aortocoronary bypass grafting with CPB were allocated to receive one of three propofol infusion rates; 50, 100, or 150 microg x kg(-1) x min(-1) in an open-label pilot study. Data were described using a line of best fit to derive an experimental clinical maneuver predicted to produce a whole blood concentration of 5 microg x mL(-1) at reperfusion. A predetermined interim analysis of 30 patients who were receiving the derived maneuver in an ongoing study was used to evaluate the maneuver. Cardiac index (CI), systemic vascular resistance index (SVRI), and left ventricular stroke work index (LVSWI) were recorded. RESULTS: The infusion rate-concentration curve had an equation of y = 0.215e (0.0279x ), where y represents the whole blood concentration and x represents the infusion rate (r (2) = 0.781). The predicted infusion rate to achieve a mean concentration of 5 microg x mL(-1) was 113 microg x kg(-1) x min(-1). The nearest practical rate is 120 microg x kg(-1) x min(-1), producing a concentration of 5.39 (1.45) microg x mL(-1). The values for CI, SVRI, and LVSWI were similar between groups at corresponding time periods. CONCLUSIONS: An infusion rate of 120 microg x kg(-1) x min(-1) is clinically practical and capable of achieving experimental cardioprotective propofol concentrations at reperfusion.

Rationale, design and baseline characteristics of the PRO-TECT II study: PROpofol CardioproTECTion for Type II diabetics: a randomized, controlled trial of high-dose propofol versus isoflurane preconditioning in patients undergoing on-pump coronary artery bypass graft surgery.

Diabetes mellitus is a leading cause of death globally and results in significant morbidity and mortality following surgery. After cardiac surgery, diabetic patients are especially at risk for low cardiac output syndrome, which can quadruple the risk for postoperative death. Attempts to prevent low cardiac output syndrome have focused on increasing myocardial tolerance to ischemia (preconditioning), which involves the myocardial mitochondrial ATP-regulated K(ATP) channel, G-protein initiation, nitric oxide synthase, and protein kinase C. Unfortunately, the signal transduction pathways required for preconditioning are corrupted in diabetes. Effective antioxidant intervention during ischemia-reperfusion appears important for preserving myocardial function; thus, alleviating oxidant-mediated post-ischemic injury by increasing antioxidant defenses (cardioprotection) is an alternative to preconditioning. Our previous work suggests that propofol (2,6-diisopropylphenol), an intravenous anesthetic with antioxidant potential, may confer cardioprotection. In this paper, we describe the rationale and methodology of the Pro-TECT II Study, a Phase II randomized controlled trial designed to explore the relationships of biomarkers of oxidative or nitrosative stress in diabetes, to determine the effect of propofol cardioprotection to counteract these effects in patients undergoing elective primary coronary bypass graft surgery with cardiopulmonary bypass, and to provide feasibility and sample size data needed to conduct Phase III trials.