Alterations in heparan sulfate proteoglycan synthesis and sulfation and the impact on vascular endothelial function.

The glycocalyx attached to the apical surface of vascular endothelial cells is a rich network of proteoglycans, glycosaminoglycans, and glycoproteins with instrumental roles in vascular homeostasis. Given their molecular complexity and ability to interact with the intra- and extracellular environment, heparan sulfate proteoglycans uniquely contribute to the glycocalyx's role in regulating endothelial permeability, mechanosignaling, and ligand recognition by cognate cell surface receptors. Much attention has recently been devoted to the enzymatic shedding of heparan sulfate proteoglycans from the endothelial glycocalyx and its impact on vascular function. However, other molecular modifications to heparan sulfate proteoglycans are possible and may have equal or complementary clinical significance. In this narrative review, we focus on putative mechanisms driving non-proteolytic changes in heparan sulfate proteoglycan expression and alterations in the sulfation of heparan sulfate side chains within the endothelial glycocalyx. We then discuss how these specific changes to the endothelial glycocalyx impact endothelial cell function and highlight therapeutic strategies to target or potentially reverse these pathologic changes.

Positive Role of Delta Neutrophil Index (DNI) as a Prodiagnostic Marker in Cecal Ligation and Puncture (CLP)-Induced Sepsis Murine Model.

Sepsis is an emergent infectious disease and a leading cause of death despite immediate intervention. While Delta neutrophil index (DNI) and myeloperoxidase (MPO) are known as a prodiagnostic marker of sepsis, the preclinical evidence of the best marker of sepsis is unclear. For this, using a well-designed cecal ligation and puncture (CLP)-induced sepsis mouse model, we comparatively measured the level and cost-effectiveness of sepsis biomarkers such as DNI, myeloperoxidase (MPO), procalcitonin (PCT), and tumor necrosis factor-alpha (TNF-α). First, we found that the optimal time point for early detection is at 6 h, 24 h post-CLP. Strikingly, the peak level and fold change of DNI was revealed at 24 h, further showing the best fold change as compared with other biomarker levels. Given the fold change at 6, 24 h, PCT was next to DNI. Third, a cost-effectiveness survey showed that DNI was the best, with PCT next. Further, DNI level was moderate positively associated with PCT (ρ = 0.697, p = 0.012) and TNF-α (ρ = 0.599, p = 0.040). Collectively, these data indicate that DNI in CLP-induced sepsis mice is as effective as the existent inflammatory biomarkers such as MPO, PCT and TNF-α to predict the prognosis of sepsis. This might have clinically important implications that DNI is cost effective, thus quickly and rationally applying to diverse types of imminent sepsis regardless of species. This might be the first report on the validity of DNI in preclinical CLP-induced murine sepsis.

Platelets factor 4 (PF4) binding enhances in vitro neutrophil extracellular traps (NET) capture of SARs-CoV- 2: Therapeutic implications

Background: SARs-CoV- 2 infection recruits high numbers of neutrophils that extrude neutrophil extracellular traps (NETs), webs of extracellular DNA coated with citrullinated histones (cit-His) and antimicrobial proteins. NETs have also been shown to entrap virions, concentrate antiviral proteins, and inactivate viruses. However, when NETs are degraded, they release NET degradation products (NDPs) such as cit-His, cell-free (cf) DNA, myeloperoxidase (MPO) and neutrophil elastase (NE) that can be toxic to the host. Our group and others have found that NETs and NDPs are highly prominent in patients with severe COVID-19 and are associated with the development of respiratory failure (Figure 1). Platelet factor 4 (PF4) is a highly-positively charged, platelet-specific chemokine that aggregates polyanionic molecules like heparin and DNA. We have shown that PF4 binds to NETs, reducing the release of NDPs by preventing NET digestion by circulating nucleases. Importantly, PF4-NET complexes markedly enhance gram-positive and -negative bacterial entrapment, likely by bridging the negatively charged polyanionic phosphoribose backbone of the NET DNA scaffold to polyanionic surface molecules in the bacterial cell wall. Treatment with PF4 improved outcomes in lipopolysaccharide endotoxemia and cecal ligation and puncture models of murine sepsis. Objectives: The objective of this study was to investigate whether PF4 binding to NETs is similarly protective in SARs-CoV- 2 infection by preventing the degradation of NETs and by enhancing NET-mediated viral capture. Methods: We generated NET-lined microfluidic channels. Neutrophils were isolated from healthy human donors, adhered to fibronectin-coated channels, and incubated with phorbol myristate acetate (PMA) to induce the release of NETs. Channels were then treated with buffer alone or PF4 (100 μg/ml) to compact NETs, after which gamma-irradiated SARS-CoV- 2 (1 x 107 PFU) were infused at 2 dynes/cm2 for 1 hour. Viral particles were then labeled with SARS-CoV- 2 guinea pig antiserum and visualized with a fluorescently-labeled secondary antibody. Viral binding to NETs was quantified using confocal microscopy. Results: Similar to that seen with bacterial attachment to NETs, we observed scant viral binding to non-compact NETs. In contrast, there was abundant binding of SARs-CoV- 2 aggregates to PF4 compacted NETs (Figure 2). Conclusions: These findings demonstrate that PF4 plays a crucial role in NET-mediated viral capture and suggest that PF4-NET complexes may be part of the physiologic mechanism by which viral spread is contained in the host. Moreover, we have previously shown that an Fc-modified version of KKO, a monoclonal antibody directed against complexes of PF4 and polyanions, markedly enhanced the protective effects of PF4 in vitro and in murine models of sepsis. Therefore, we will examine whether PF4 plus modified KKO infusions are able to limit SARS-CoV- 2 viremia, preventing the pneumonitis and multi-system organ dysfunction of severe COVID-19. (Figure Presented).