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

IntroductionOsteopontin (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.MethodsHere, 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 discussionWe 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

A clinical appraisal of propofol-mediated, antioxidant-based cardioprotection during coronary artery bypass grafting with cardiopulmonary bypass

Coronary artery disease is the leading cause of death in North America. The invasiveness of its treatment depends on its severity; less severe disease can be treated pharmacologically or surgically without significantly different outcomes, but coronary artery bypass grafting (CABG) clearly reduces mortality among medium- and high-risk patients compared to percutaneous and non-surgical intervention. Although the majority of patients undergoing surgical revascularization emerge without severe postoperative complications, a significant portion of patients encounter a postoperative complication known as low cardiac output syndrome which can quadruple the overall mortality rate for CABG. Intraoperative ischemia reperfusion injury is a major factor in the development of low cardiac output syndrome; so effective intraoperative myocardial protection is central to reducing its incidence, and represents an opportunity to considerably improve patient outcomes. The introductory chapter of this thesis describes the origin and role of reactive oxygen species (ROS) in myocardial ischemia-reperfusion injury. In addition, it introduces key strategies targeted to reduce ROS-mediated myocardial ischemia-reperfusion injury, highlighting key clinical studies that translated these strategies to reduce the severity of ischemia-reperfusion injury during CABG. The central hypothesis of the clinical project on which this thesis is based states that propofol reduces the incidence of low cardiac output syndrome subsequent to CABG with CPB by decreasing the magnitude of 15-F₂t-isoprostane generation during ischemia-reperfusion. The second chapter introduces propofol, and will review previous studies that explore its cardioprotective potential. The experimental section of this thesis describes the development of a quantitative technique for propofol analysis in whole blood, and its application in a dose finding study that define the parameters for achieving experimentally relevant concentrations of propofol during cardiopulmonary bypass. These two studies were fundamental to the development of a clinical study evaluating ROS generation and the incidence low cardiac output syndrome in patients undergoing CABG surgery. Preliminary results that address the central hypothesis are subsequently presented, along with an alternative proposed mechanism for propofol-mediated cardioprotection. This thesis will conclude with a summary of findings and a description of several future studies aimed at testing, generating, and evaluating new hypotheses.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

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 BNP_1–32 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

Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan.

Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, we observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, we sought to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan

Extracellular matrix downregulation in the Drosophila heart preserves contractile function and improves lifespan.

Aging is associated with extensive remodeling of the heart, including basement membrane (BM) components that surround cardiomyocytes. Remodeling is thought to impair cardiac mechanotransduction, but the contribution of specific BM components to age-related lateral communication between cardiomyocytes is unclear. Using a genetically tractable, rapidly aging model with sufficient cardiac genetic homology and morphology, e.g. Drosophila melanogaster, we observed differential regulation of BM collagens between laboratory strains, correlating with changes in muscle physiology leading to cardiac dysfunction. Therefore, we sought to understand the extent to which BM proteins modulate contractile function during aging. Cardiac-restricted knockdown of ECM genes Pericardin, Laminin A, and Viking in Drosophila prevented age-associated heart tube restriction and increased contractility, even under viscous load. Most notably, reduction of Laminin A expression correlated with an overall preservation of contractile velocity with age and extension of organismal lifespan. Global heterozygous knockdown confirmed these data, which provides new evidence of a direct link between BM homeostasis, contractility, and maintenance of lifespan

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