Regulation of ecto‐apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3)

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154432/1/fsb2027011011.pd

In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody–Mediated Venous Thrombosis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136296/1/art39938_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136296/2/art39938.pd

Patients with COVID-19: in the dark-NETs of neutrophils.

SARS-CoV-2 infection poses a major threat to the lungs and multiple other organs, occasionally causing death. Until effective vaccines are developed to curb the pandemic, it is paramount to define the mechanisms and develop protective therapies to prevent organ dysfunction in patients with COVID-19. Individuals that develop severe manifestations have signs of dysregulated innate and adaptive immune responses. Emerging evidence implicates neutrophils and the disbalance between neutrophil extracellular trap (NET) formation and degradation plays a central role in the pathophysiology of inflammation, coagulopathy, organ damage, and immunothrombosis that characterize severe cases of COVID-19. Here, we discuss the evidence supporting a role for NETs in COVID-19 manifestations and present putative mechanisms, by which NETs promote tissue injury and immunothrombosis. We present therapeutic strategies, which have been successful in the treatment of immunο-inflammatory disorders and which target dysregulated NET formation or degradation, as potential approaches that may benefit patients with severe COVID-19

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Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/174763/1/art42141_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/174763/2/art42141.pd

Ectonucleotidase-Mediated Suppression of Lupus Autoimmunity and Vascular Dysfunction

ObjectivesCD39 and CD73 are surface enzymes that jut into the extracellular space where they mediate the step-wise phosphohydrolysis of the autocrine and paracrine danger signals ATP and ADP into anti-inflammatory adenosine. Given the role of vascular and immune cells’ “purinergic halo” in maintaining homeostasis, we hypothesized that the ectonucleotidases CD39 and CD73 might play a protective role in lupus.MethodsLupus was modeled by intraperitoneal administration of pristane to three groups of mice: wild-type (WT), CD39−/−, and CD73−/−. After 36 weeks, autoantibodies, endothelial function, kidney disease, splenocyte activation/polarization, and neutrophil activation were characterized.ResultsAs compared with WT mice, CD39−/− mice developed exaggerated splenomegaly in response to pristane, while both groups of ectonucleotidase-deficient mice demonstrated heightened anti-ribonucleoprotein production. The administration of pristane to WT mice triggered only subtle dysfunction of the arterial endothelium; however, both CD39−/− and CD73−/− mice demonstrated striking endothelial dysfunction following induction of lupus, which could be reversed by superoxide dismutase. Activated B cells and plasma cells were expanded in CD73−/− mice, while deficiency of either ectonucleotidase led to expansion of TH17 cells. CD39−/− and CD73−/− mice demonstrated exaggerated neutrophil extracellular trap release, while CD73−/− mice additionally had higher levels of plasma cell-free DNA.ConclusionThese data are the first to link ectonucleotidases with lupus autoimmunity and vascular disease. New therapeutic strategies may harness purinergic nucleotide dissipation or signaling to limit the damage inflicted upon organs and blood vessels by lupus

Circulating Calprotectin as a Predictive and Severity Biomarker in Patients with COVID-19

Background: New tools for the assessment and prediction of the severity of hospitalized COVID-19 patients can help direct limited resources to patients with the greatest need. Circulating levels of calprotectin (S100A8/S100A9) reflect inflammatory activity in multiple conditions, and have been described as being elevated in COVID-19 patients, but their measurement is not routinely utilized. The aim of our study was to assess the practical and predictive value of measuring circulating calprotectin levels in patients at admission and during their hospitalization. Methods: Circulating calprotectin levels were measured in 157 hospitalized patients with COVID-19 using an automated quantitative chemiluminescent assay. Results: Circulating calprotectin levels were strongly correlated with changing respiratory supplementation needs of patients. The overall trajectory of circulating calprotectin levels generally correlated with patient improvement or deterioration. Conclusions: Routine measurement of circulating calprotectin levels may offer a valuable tool to assess and monitor hospitalized patients with COVID-19, as well as other acute inflammatory conditions

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism

Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p < 0.05), and increased THP-1 monocyte adhesion to ECs (p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism

Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19

Regulation of ecto‐apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3)

The ectoenzyme CD39 suppresses thrombosis and inflammation by suppressing ATP and ADP to AMP. However, mechanisms of CD39 transcriptional and post-translational regulation are not well known. Here we show that CD39 levels are modulated by inhibition of phosphodiesterase 3 (PDE3). RAW macrophages and human umbilical vein endothelial cells (HUVECs) were treated with the PDE3 inhibitors cilostazol and milrinone, then analyzed using qRT-PCR, immunoprecipitation/Western blot, immunofluorescent staining, radio-thin-layer chromatography, a malachite green assay, and ELISA. HUVECs expressed elevated CD39 protein (2-fold [P<0.05] for cilostazol and 2.5-fold [P<0.01] for milrinone), while macrophage CD39 mRNA and protein were both elevated after PDE3 inhibition. HUVEC ATPase activity increased by 25% with cilostazol and milrinone treatment (P<0.05 and P<0.01, respectively), as did ADPase activity (47% and 61%, P<0.001). There was also a dose-dependent elevation of soluble CD39 after treatment with 8-Br-cAMP, with maximal elevation of 60% more CD39 present compared to controls (1 mM, P<0.001). Protein harvested after 8-Br-cAMP treatment showed that ubiquitination of CD39 was decreased by 43% compared to controls. A DMSO or PBS vehicle control was included for each experiment based on solubility of cilostazol, milrinone, and 8-Br-cAMP. These results indicate that PDE3 inhibition regulates endothelial CD39 at a post-translational level.—Baek, A. E., Kanthi, Y., Sutton, N. R., Liao, H., Pinsky, D. J. Regulation of ecto-apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3)