Oxidized LDL regulates efferocytosis through the CD36-PKM2-mtROS pathway.

Macrophage efferocytosis, the process by which phagocytes engulf and remove apoptotic cells (ACs), plays a critical role in maintaining tissue homeostasis. Efficient efferocytosis prevents secondary necrosis, mitigates chronic inflammation, and impedes atherosclerosis progression. However, the regulatory mechanisms of efferocytosis under atherogenic conditions remain poorly understood. We previously demonstrated that oxidized LDL (oxLDL), an atherogenic lipoprotein, induces mitochondrial reactive oxygen species (mtROS) in macrophages via CD36. In this study, we demonstrate that macrophage mtROS facilitate continual efferocytosis through a positive feedback mechanism. However, oxLDL disrupts continual efferocytosis by dysregulating the internalization of ACs. This disruption is mediated by an overproduction of mtROS. Mechanistically, oxLDL/CD36 signaling promotes the translocation of cytosolic PKM2 to mitochondria, facilitated by the chaperone GRP75. Mitochondrial PKM2 then binds to Complex III of the electron transport chain, inducing mtROS production. This study elucidates a novel regulatory mechanism of efferocytosis in atherosclerosis, providing potential therapeutic targets for intervention. SUMMARY: Macrophages clear apoptotic cells through a process called efferocytosis, which involves mitochondrial ROS. However, the atherogenic oxidized LDL overstimulates mitochondrial ROS via the CD36-PKM2 pathway, disrupting continual efferocytosis. This finding elucidates a novel molecular mechanism that explains defects in efferocytosis, driving atherosclerosis progression.

Protocol to identify flavonoid antagonists of the SARS-CoV-2 main protease.

Flavonoids are naturally occurring metabolites of plants that can inhibit the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), which is required for viral replication. Here, we present a protocol to identify flavonoid antagonists of the SARS-CoV-2 Mpro. We describe steps for the expression and purification of Mpro and a kinetic enzymatic assay for Mpro activity using a dequenching fluorescence resonance energy transfer peptide substrate. We then detail procedures for using this enzymatic assay to test flavonoid antagonism and reversible inhibition. For complete details on the use and execution of this protocol, please refer to Lin et al.1.

Lower glycemia levels in subjects with excessive erythrocytosis during the oral glucose tolerance test living in conditions of severe hypoxia.

Background: Previous studies showed that residents of higher elevations have lower glucose levels. Our objective in this study is to determine the basal and postprandial glucose levels in apparently healthy permanent residents of the miner population center of La Rinconada located 5100 meters (m) above sea level. Method: Forty male permanent residents of the Rinconada miner population center were studied. The oral glucose tolerance test was used to evaluate basal and postprandial glycemia levels at 1, 2, and 3 h. Results: The individuals had a mean age of 43.95 ± 8.54 years. Basal glycemia in subjects without excessive erythrocytosis (EE) was 73.3 ± 7.9 mg/dL, while levels in patients with EE were 57.98 ± 7.38 mg/dL. In the postprandial period, at 1 h after oral glucose overload, a mean value of 76.35 ± 13.53 mg/dL was observed in subjects with EE compared to 94.68 ± 9.98 mg/dL in subjects without EE. After 2 h, subjects with EE had a glycemia level of 72.91 ± 9.17 mg/dL EE compared to 90.73 ± 13.86 mg/dL without EE. At 3 h, the average glycemia level in subjects with EE was 70.77 ± 8.73 mg/dL compared to 87.79 ± 14.16 mg/dL in those without EE. Conclusion: These findings suggest that under hypoxic conditions, glycemia levels are lower in both subjects with and without EE, having obtained lower levels in subjects with EE in relation to those with normal values of Hb and Hct. The results of this study indicate that in the conditions of severe hypoxia, blood glucose levels are below the values considered normal for sea level.

Targeting Cysteine Oxidation in Thrombotic Disorders.

Oxidative stress increases the risk for clinically significant thrombotic events, yet the mechanisms by which oxidants become prothrombotic are unclear. In this review, we provide an overview of cysteine reactivity and oxidation. We then highlight recent findings on cysteine oxidation events in oxidative stress-related thrombosis. Special emphasis is on the signaling pathway induced by a platelet membrane protein, CD36, in dyslipidemia, and by protein disulfide isomerase (PDI), a member of the thiol oxidoreductase family of proteins. Antioxidative and chemical biology approaches to target cysteine are discussed. Lastly, the knowledge gaps in the field are highlighted as they relate to understanding how oxidative cysteine modification might be targeted to limit thrombosis.

Low-frequency inherited complement receptor variants are associated with purpura fulminans.

ABSTRACT: Extreme disease phenotypes can provide key insights into the pathophysiology of common conditions, but studying such cases is challenging due to their rarity and the limited statistical power of existing methods. Herein, we used a novel approach to pathway-based mutational burden testing, the rare variant trend test (RVTT), to investigate genetic risk factors for an extreme form of sepsis-induced coagulopathy, infectious purpura fulminans (PF). In addition to prospective patient sample collection, we electronically screened over 10.4 million medical records from 4 large hospital systems and identified historical cases of PF for which archived specimens were available to perform germline whole-exome sequencing. We found a significantly increased burden of low-frequency, putatively function-altering variants in the complement system in patients with PF compared with unselected patients with sepsis (P = .01). A multivariable logistic regression analysis found that the number of complement system variants per patient was independently associated with PF after controlling for age, sex, and disease acuity (P = .01). Functional characterization of PF-associated variants in the immunomodulatory complement receptors CR3 and CR4 revealed that they result in partial or complete loss of anti-inflammatory CR3 function and/or gain of proinflammatory CR4 function. Taken together, these findings suggest that inherited defects in CR3 and CR4 predispose to the maladaptive hyperinflammation that characterizes severe sepsis with coagulopathy.

Plant flavonoid inhibition of SARS-CoV-2 main protease and viral replication.

Plant-based flavonoids have been evaluated as inhibitors of ß-coronavirus replication and as therapies for COVID-19 on the basis of their safety profile and widespread availability. The SARS-CoV-2 main protease (Mpro) has been implicated as a target for flavonoids in silico. Yet no comprehensive in vitro testing of flavonoid activity against SARS-CoV-2 Mpro has heretofore been performed. We screened 1,019 diverse flavonoids for their ability to inhibit SARS-CoV-2 Mpro. Multiple structure-activity relationships were identified among active compounds such as enrichment of galloylated flavonoids and biflavones, including multiple biflavone analogs of apigenin. In a cell-based SARS-CoV-2 replication assay, the most potent inhibitors were apigenin and the galloylated pinocembrin analog, pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). Molecular dynamic simulations predicted that PGHG occludes the S1 binding site via a galloyl group and induces a conformational change in Mpro. These studies will advance the development of plant-based flavonoids-including widely available natural products-to target ß-coronaviruses.

The unfolded protein response links ER stress to cancer-associated thrombosis.

Thrombosis is a common complication of advanced cancer, yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasma from patients with gastric and non-small cell lung cancer who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasma of patients who developed clots compared with those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK). Release of extracellular vesicles bearing tissue factor (EVTFs) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase tissue factor (TF) synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by ADP-ribosylation factor 1 knockdown or GBF1 antagonism, verifying the role of vesicular trafficking. Our findings show that UPR activation resulted in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.

Cysteine and methionine oxidation in thrombotic disorders.

Thrombosis is the leading cause of death in many diseased conditions. Oxidative stress is characteristic of these conditions. Yet, the mechanisms through which oxidants become prothrombotic are unclear. Recent evidence suggests protein cysteine and methionine oxidation as prothrombotic regulators. These oxidative post-translational modifications occur on proteins that participate in the thrombotic process, including Src family kinases, protein disulfide isomerase, ß2 glycoprotein I, von Willebrand factor, and fibrinogen. New chemical tools to identify oxidized cysteine and methionine proteins in thrombosis and hemostasis, including carbon nucleophiles for cysteine sulfenylation and oxaziridines for methionine, are critical to understanding why clots occur during oxidative stress. These mechanisms will identify alternative or novel therapeutic approaches to treat thrombotic disorders in diseased conditions.

Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation.

BACKGROUND: Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines. OBJECTIVES: We hypothesized that PDI is sulfenylated during oxidative stress, contributing to the prothrombotic potential of PDI. METHODS: Biochemical and enzymatic assays using purified proteins, platelet and endothelial cell assays, and in vivo murine thrombosis studies were used to evaluate the role of oxidative stress in PDI sulfenylation and prothrombotic activity. RESULTS: PDI exposure to oxidants resulted in the loss of PDI reductase activity and simultaneously promoted sulfenylated PDI generation. Following exposure to oxidants, sulfenylated PDI spontaneously converted to disulfided PDI. PDI oxidized in this manner was able to transfer disulfides to protein substrates. Inhibition of sulfenylation impaired disulfide formation by oxidants, indicating that sulfenylation is an intermediate during PDI oxidation. Agonist-induced activation of platelets and endothelium resulted in the release of sulfenylated PDI. PDI was also sulfenylated by oxidized low-density lipoprotein (oxLDL). In an in vivo model of thrombus formation, oxLDL markedly promoted platelet accumulation following an arteriolar injury. PDI oxidoreductase inhibition blocked oxLDL-mediated augmentation of thrombosis. CONCLUSION: PDI sulfenylation is a critical posttranslational modification that is an intermediate during disulfide PDI formation in the setting of oxidative stress. Oxidants generated by vascular cells during activation promote PDI sulfenylation, and interference with PDI during oxidative stress impairs thrombus formation.

Redox stress in COVID-19: Implications for hematologic disorders.

COVID-19 is the respiratory illness caused by the beta coronavirus SARS-CoV-2. COVID-19 is complicated by an increased risk for adverse thrombotic events that promote organ failure and death. While the mechanism of action for SARS-CoV-2 is still being understood, how SARS-CoV-2 infection impacts the redox environment in hematologic conditions is unclear. In this review, the redox mechanisms contributing to SARS-CoV-2 infection, coagulopathy and inflammation are briefly discussed. Specifically, sources of oxidant generation by hematopoietic and non-hematopoietic cells are identified with special emphasis on leukocytes, platelets, red cells, and endothelial cells. Furthermore, reactive cysteines in SARS-CoV-2 are also discussed with respect to oxidative cysteine modification and current therapeutic implications. Lastly, sickle cell disease will be discussed as a hematologic disorder with a pre-existing prothrombotic redox condition that complicates treatment strategies for COVID-19. An understanding of the redox mechanism may identify potential targets for COVID-19-mediated thrombosis in hematologic disorders.

Shining a light on SSP4: A comprehensive analysis and biological applications for the detection of sulfane sulfurs.

Fluorescent probes are useful tools for the detection of sulfane sulfurs in biological systems. In this work, we report the development of SSP4, a widely used probe generated in our laboratory. We describe its evolution, preparation, and physical/chemical properties. Fluorescence analyses of SSP4 determined its high selectivity and sensitivity to sulfane sulfurs, even with the interfering presence of other species, such as amino acids and metal ions. Protocols for using SSP4 in a relatively quick and simple manner for the detection of persulfidated proteins, including papain, BSA, and GAPDH were developed. The method was then applied to human protein disulfide isomerase (PDI), leading to the discovery that persulfidation can occur at PDI's non-active site cysteines, and that PDI reductase activity is affected by sulfane sulfur treatment. Protocols for using SSP4 for the bioimaging of exogenous and endogenous sulfane sulfurs in different -cell lines were also established. These results should guide further applications of SSP4.

Role of Angiopoietin-Tie axis in vascular and lymphatic systems and therapeutic interventions.

The Angiopoietin (Ang)-Tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie) axis is an endothelial cell-specific ligand-receptor signaling pathway necessary for vascular and lymphatic development. The Ang-Tie axis is involved in regulating angiogenesis, vascular remodeling, vascular permeability, and inflammation to maintain vascular quiescence. Disruptions in the Ang-Tie axis are involved in many vascular and lymphatic diseases and play an important role in physiological and pathological vascular processes. Given recent advances in the Ang-Tie axis in the vascular and lymphatic systems, this review focuses on the multiple functions of the Ang-Tie axis in inflammation-induced vascular permeability, vascular remodeling, atherosclerosis, ocular angiogenesis, tumor angiogenesis, and metastasis. A summary of relevant therapeutic approaches to the Ang-Tie axis, including therapeutic antibodies, recombinant proteins and small molecule drugs are also discussed. The purpose of this review is to provide new hypotheses and identify potential therapeutic strategies based on the Ang-Tie signaling axis for the treatment of vascular and lymphatic-related diseases.

Oxidative Cysteine Modification of Thiol Isomerases in Thrombotic Disease: A Hypothesis.

Significance: Oxidative stress is a characteristic of many systemic diseases associated with thrombosis. Thiol isomerases are a family of oxidoreductases important in protein folding and are exquisitely sensitive to the redox environment. They are essential for thrombus formation and represent a previously unrecognized layer of control of the thrombotic process. Yet, the mechanisms by which thiol isomerases function in thrombus formation are unknown. Recent Advances: The oxidoreductase activity of thiol isomerases in thrombus formation is controlled by the redox environment via oxidative changes to active site cysteines. Specific alterations can now be detected owing to advances in the chemical biology of oxidative cysteine modifications. Critical Issues: Understanding of the role of thiol isomerases in thrombus formation has focused largely on identifying single disulfide bond modifications in isolated proteins (e.g., αIIbß3, tissue factor, vitronectin, or glycoprotein Ibα [GPIbα]). An alternative approach is to conceptualize thiol isomerases as effectors in redox signaling pathways that control thrombotic potential by modifying substrate networks. Future Directions: Cysteine-based chemical biology will be employed to study thiol-dependent dynamics mediated by the redox state of thiol isomerases at the systems level. This approach could identify thiol isomerase-dependent modifications of the disulfide landscape that are prothrombotic.

Platelet reactivity in dyslipidemia: atherothrombotic signaling and therapeutic implications.

The risks for adverse thrombotic events, including myocardial infarction, stroke, and deep vein thrombosis, are markedly increased in dyslipidemia and other metabolic disorders and are the major cause of death worldwide. Recent evidence points out that increased thrombotic risk in dyslipidemia is mediated by platelets circulating in a pre-activated state. The mechanisms of platelet reactivity in this setting are multifaceted including platelet activation by classic agonist receptor signaling as well as platelet sensitization by pattern recognition receptors. Elevated platelet counts in dyslipidemia due to dysregulation in hematopoiesis also contribute to the overall thrombotic phenotype. Despite recent advancements in antiplatelet and anticoagulation therapies, recurrences of adverse thrombotic events remain to be a large clinical burden. In the light of new knowledge, understanding mechanisms that drive pathologic thrombosis in dyslipidemia, the antithrombotic approach shall be revisited. Here, we discuss potential therapeutic avenues based on the overview of platelet signaling mechanisms that contribute to a prothrombotic phenotype in dyslipidemia.

Regulation of pro-σK activation: a key checkpoint in Bacillus subtilis sporulation.

The Gram-positive bacterium Bacillus subtilis initiates the sporulation process under conditions of nutrient limitation. Here, we review related work in this field, focusing on the protein processing of the pro-σK activation. The purpose of this review is to illustrate the mechanism of pro-σK activation and provide structural insights into the regulation of spore production. Sporulation is not only important in basic science but also provides mechanistic insight for bacterial control in applications in, e.g., food industry.

Cysteine sulfenylation by CD36 signaling promotes arterial thrombosis in dyslipidemia.

Arterial thrombosis in the setting of dyslipidemia promotes clinically significant events, including myocardial infarction and stroke. Oxidized lipids in low-density lipoproteins (oxLDL) are a risk factor for athero-thrombosis and are recognized by platelet scavenger receptor CD36. oxLDL binding to CD36 promotes platelet activation and thrombosis by promoting generation of reactive oxygen species. The downstream signaling events initiated by reactive oxygen species in this setting are poorly understood. In this study, we report that CD36 signaling promotes hydrogen peroxide flux in platelets. Using carbon nucleophiles that selectively and covalently modify cysteine sulfenic acids, we found that hydrogen peroxide generated through CD36 signaling promotes cysteine sulfenylation of platelet proteins. Specifically, cysteines were sulfenylated on Src family kinases, which are signaling transducers that are recruited to CD36 upon recognition of its ligands. Cysteine sulfenylation promoted activation of Src family kinases and was prevented by using a blocking antibody to CD36 or by enzymatic degradation of hydrogen peroxide. CD36-mediated platelet aggregation and procoagulant phosphatidylserine externalization were inhibited in a concentration-dependent manner by a panel of sulfenic acid-selective carbon nucleophiles. At the same concentrations, these probes did not inhibit platelet aggregation induced by the purinergic receptor agonist adenosine diphosphate or the collagen receptor glycoprotein VI agonist collagen-related peptide. Selective modification of cysteine sulfenylation in vivo with a benzothiazine-based nucleophile rescued the enhanced arterial thrombosis seen in dyslipidemic mice back to control levels. These findings suggest that CD36 signaling generates hydrogen peroxide to oxidize cysteines within platelet proteins, including Src family kinases, and lowers the threshold for platelet activation in dyslipidemia.

Platelet Dysfunction and Thrombosis in JAK2V617F-Mutated Primary Myelofibrotic Mice.

OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.

Injury measurements improve interpretation of thrombus formation data in the cremaster arteriole laser-induced injury model of thrombosis.

Background: The cremaster arteriole laser-induced injury model is a powerful technique with which to investigate the molecular mechanisms that drive thrombus formation. This model is capable of direct visualization and quantification of accumulation of thrombus constituents, including both platelets and fibrin. However, a large degree of variability in platelet accumulation and fibrin formation is observed between thrombi. Strategies to understand this variability will enhance performance and standardization of the model. We determined whether ablation injury size contributes to variation in platelet accumulation and fibrin formation and, if so, whether incorporating ablation injury size into measurements reduces variation. Methods: Thrombus formation was initiated by laser-induced injury of cremaster arterioles of mice (n=59 injuries). Ablation injuries within the vessel wall were consistently identified and quantified by measuring the length of vessel wall injury observed immediately following laser-induced disruption. Platelet accumulation and fibrin formation as detected by fluorescently-labeled antibodies were captured by digital intra-vital microscopy. Results: Laser-induced disruption of the vessel wall resulted in ablation injuries of variable length (18-95 µm) enabling interrogation of the relationship between injury severity and thrombus dynamics. Strong positive correlations were observed between vessel injury length and both platelet and fibrin when the data are transformed as area under the curve (Spearman r = 0.80 and 0.76 respectively). Normalization of area under the curve measurements by injury length reduced intraclass coefficients of variation among thrombi and improved hypothesis testing when comparing different data sets. Conclusions: Measurement of vessel wall injury length provides a reliable and robust marker of injury severity. Injury length can effectively normalize measurements of platelet accumulation and fibrin formation improving data interpretation and standardization.

Modification of HDL by reactive aldehydes alters select cardioprotective functions of HDL in macrophages.

While increased levels of high-density lipoprotein (HDL)-cholesterol correlate with protection against cardiovascular disease, recent findings demonstrate that HDL function, rather than HDL-cholesterol levels, may be a better indicator of cardiovascular risk. One mechanism by which HDL function can be compromised is through modification by reactive aldehydes such as acrolein (Acro), 4-hydroxynonenal, and malondialdehyde (MDA). In this study, we tested the hypothesis that modification of HDL with reactive aldehydes would impair HDL's athero-protective functions in macrophages. Compared to native HDL, Acro- and MDA-modified HDL have impaired abilities to promote migration of primary peritoneal macrophages isolated from C57BL6/J mice. Incubation of macrophages with MDA-HDL also led to an increased ability to generate reactive oxygen species. Our studies revealed that the changes in HDL function following aldehyde modification are likely not through activation of canonical nuclear factor-kappa B signaling pathways. Consistent with this finding, treatment of either noncholesterol-loaded macrophages or foam cells with modified forms of HDL does not lead to significant changes in expression levels of inflammatory markers. Importantly, our data also demonstrate that changes in HDL function are dependent on the type of modification present on the HDL particle. Our findings suggest that modification of HDL with reactive aldehydes can impair some, but not all, of HDL's athero-protective functions in macrophages.