Endothelial Glycocalyx Degradation in Critical Illness and Injury

The endothelial glycocalyx is a gel-like layer on the luminal side of blood vessels that is composed of glycosaminoglycans and the proteins that tether them to the plasma membrane. Interest in its properties and function has grown, particularly in the last decade, as its importance to endothelial barrier function has come to light. Endothelial glycocalyx studies have revealed that many critical illnesses result in its degradation or removal, contributing to endothelial dysfunction and barrier break-down. Loss of the endothelial glycocalyx facilitates the direct access of immune cells and deleterious agents (e.g., proteases and reactive oxygen species) to the endothelium, that can then further endothelial cell injury and dysfunction leading to complications such as edema, and thrombosis. Here, we briefly describe the endothelial glycocalyx and the primary components thought to be directly responsible for its degradation. We review recent literature relevant to glycocalyx damage in several critical illnesses (sepsis, COVID-19, trauma and diabetes) that share inflammation as a common denominator with actions by several common agents (hyaluronidases, proteases, reactive oxygen species, etc.). Finally, we briefly cover strategies and therapies that show promise in protecting or helping to rebuild the endothelial glycocalyx such as steroids, protease inhibitors, anticoagulants and resuscitation strategies

Endothelial Glycocalyx Degradation in Critical Illness and Injury

The endothelial glycocalyx is a gel-like layer on the luminal side of blood vessels that is composed of glycosaminoglycans and the proteins that tether them to the plasma membrane. Interest in its properties and function has grown, particularly in the last decade, as its importance to endothelial barrier function has come to light. Endothelial glycocalyx studies have revealed that many critical illnesses result in its degradation or removal, contributing to endothelial dysfunction and barrier break-down. Loss of the endothelial glycocalyx facilitates the direct access of immune cells and deleterious agents (e.g., proteases and reactive oxygen species) to the endothelium, that can then further endothelial cell injury and dysfunction leading to complications such as edema, and thrombosis. Here, we briefly describe the endothelial glycocalyx and the primary components thought to be directly responsible for its degradation. We review recent literature relevant to glycocalyx damage in several critical illnesses (sepsis, COVID-19, trauma and diabetes) that share inflammation as a common denominator with actions by several common agents (hyaluronidases, proteases, reactive oxygen species, etc.). Finally, we briefly cover strategies and therapies that show promise in protecting or helping to rebuild the endothelial glycocalyx such as steroids, protease inhibitors, anticoagulants and resuscitation strategies

Case Report: Inflammation and Endothelial Injury Profiling of COVID-19 Pediatric Multisystem Inflammatory Syndrome (MIS-C)

Introduction: COVID-19 is associated with a novel multi-system inflammatory syndrome that shares some characteristics with Kawasaki\u27s Disease. The syndrome manifestation is delayed relative to COVID-19 onset, with a spectrum of clinical severity. Clinical signs may include persistent fever, gastrointestinal symptoms, cardiac inflammation and/or shock. Case Presentation: We measured 59 inflammatory and endothelial injury plasma analytes in an adolescent girl that presented with malaise, fever, cough, strawberry tongue and jaundice. Her COVID-19 status was positive with detection of 2 SARS-CoV-2 viral genes using polymerase chain reaction. She was treated with intravenous immunoglobulin prior to blood draw, but our plasma measurements suggested a unique analyte expression pattern associated with inflammation, endothelial injury and microvascular glycocalyx degradation. Conclusions: COVID-19 is associated with a multi-system inflammatory syndrome and a unique inflammatory and endothelial injury signature. Summary: Analyte markers of inflammation and endothelial cell injury might serve as putative biomarkers and/or be investigated further as potential therapeutic targets

Pretreatment of Human Cerebrovascular Endothelial Cells with CO-releasing Molecule-3 Interferes with JNK/AP-1 Signaling and Suppresses LPS-induced Proadhesive Phenotype.

OBJECTIVE: Exogenously administered CO interferes with PMN recruitment to the inflamed organs. The mechanisms of CO-dependent modulation of vascular proadhesive phenotype, a key step in PMN recruitment, are unclear. METHODS: We assessed the effects/mechanisms of CO liberated from a water-soluble CORM-3 on modulation of the proadhesive phenotype in hCMEC/D3 in an in vitro model of endotoxemia. To this end, hCMEC/D3 were stimulated with LPS (1 μg/mL) for six hours. In some experiments hCMEC/D3 were pretreated with CORM-3 (200 μmol/L) before LPS-stimulation. PMN rolling/adhesion to hCMEC/D3 were assessed under conditions of laminar shear stress (0.7 dyn/cm(2) ). In parallel, expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 (qPCR), activation of transcription factors, NF-κB and AP-1 (ELISA), and MAPK-signaling (expression/phosphorylation of p38, ERK1/2, and JNK1/2; western blot) were assessed. RESULTS: The obtained results indicate that CORM-3 pretreatment reduces PMN rolling/adhesion to LPS-stimulated hCMEC/D3 (p \u3c 0.05). Decreased PMN rolling/adhesion to hCMEC/D3 was associated with CORM-3-dependent inhibition of MAPK JNK1/2 activation (Tyr-phosphorylation), inhibition of transcription factor, AP-1 (c-Jun phosphorylation), and subsequent suppression of VCAM-1 expression (p \u3c 0.05). CONCLUSIONS: These findings indicate that CORM-3 pretreatment interferes with JNK/AP-1 signaling and suppresses LPS-induced upregulation of the proadhesive phenotype in hCMEC/D3

A proteinase 3 contribution to juvenile idiopathic arthritis-associated cartilage damage

A full understanding of the molecular mechanisms implicated in the etiopathogenesis of juvenile idiopathic arthritis (JIA) is lacking. A critical role for leukocyte proteolytic activity (e.g., elastase and cathepsin G) has been proposed. While leukocyte elastase’s (HLE) role has been documented, the potential contribution of proteinase 3 (PR3), a serine protease present in abundance in neutrophils, has not been evaluated. In this study we investigated: (1) PR3 concentrations in the synovial fluid of JIA patients using ELISA and (2) the cartilage degradation potential of PR3 by measuring the hydrolysis of fluorescently labeled collagen II in vitro. In parallel, concentrations and collagen II hydrolysis by HLE were assessed. Additionally, the levels of the co-secreted primary granule protein myeloperoxidase (MPO) were assessed in synovial fluid of patients diagnosed with JIA. We report the following levels of analytes in JIA synovial fluid: PR3—114 ± 100 ng/mL (mean ± SD), HLE—1272 ± 1219 ng/mL, and MPO—1129 ± 1659 ng/mL, with a very strong correlation between the PR3 and HLE concentrations (rs = 0.898, p \u3c 1 × 10–6 ). Importantly, PR3 hydrolyzed fluorescently labeled collagen II as efficiently as HLE. Taken together, these novel findings suggest that PR3 (in addition to HLE) contributes to JIA-associated joint damage

Proteinase 3 contributes to endothelial dysfunction in an experimental model of sepsis

In sepsis-induced inflammation, polymorphonuclear neutrophils (PMNs) contribute to vascular dysfunction. The serine proteases proteinase 3 (PR3) and human leukocyte elastase (HLE) are abundant in PMNs and are released upon degranulation. While HLE’s role in inflammation-induced endothelial dysfunction is well studied, PR3’s role is largely uninvestigated. We hypothesized that PR3, similarly to HLE, contributes to vascular barrier dysfunction in sepsis. Plasma PR3 and HLE concentrations and their leukocyte mRNA levels were measured by ELISA and qPCR, respectively, in sepsis patients and controls. Exogenous PR3 or HLE was applied to human umbilical vein endothelial cells (HUVECs) and HUVEC dysfunction was assessed by FITC-dextran permeability and electrical resistance. Both PR3 and HLE protein and mRNA levels were significantly increased in sepsis patients (P \u3c 0.0001 and P \u3c 0.05, respectively). Additionally, each enzyme independently increased HUVEC monolayer FITC-dextran permeability (P \u3c 0.01), and decreased electrical resistance in a time- and dose-dependent manner (P \u3c 0.001), an effect that could be ameliorated by novel treatment with carbon monoxide-releasing molecule 3 (CORM-3). The serine protease PR3, in addition to HLE, lead to vascular dysfunction and increased endothelial permeability, a hallmark pathological consequence of sepsis-induced inflammation. CORMs may offer a new strategy to reduce serine protease-induced vascular dysfunction

Replaced by a robot: Service implications in the age of the machine

Service organizations, emboldened by the imperative to innovate, are increasingly introducing robots to frontline service encounters. However, as they augment or substitute human employees with robots, they may struggle to convince a distrusting public of their brand’s ethical credentials. Consequently, this article develops and tests a holistic framework to ascertain a deeper understanding of customer perceptions of frontline service robots (FLSRs) than has previously been attempted. Our experimental studies investigate the effects of the (1) role (augmentation or substitution of human employees or no involvement) and (2) type (humanoid FLSR vs. self-service machine) of FLSRs under the following service contexts: (a) value creation model (asset-builder, service-provider) and (b) service type (experience, credence). By empirically establishing our framework, we highlight how customers’ personal characteristics (openness-to-change and preference for ethical/responsible service provider)  and cognitive evaluations (perceived innovativeness, perceived ethical/societal reputation, and perceived innovativeness-responsibility fit) influence the impact that FLSRs have on service experience and brand usage intent. Our findings operationalize and empirically support seminal frameworks from extant literature, as well as elaborate on the positive and negative implications of using robots to complement or replace service employees. Further, we consider managerial and policy implications for service in the age of machines

Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro.

INTRODUCTION: Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro. METHODS: The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm(2)). RESULTS: Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either β2 (CD18), αL/β2 (CD11α/CD18; LFA-1) or αM/β2 (CD11β/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion. CONCLUSIONS: Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a β2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE

Human severe sepsis cytokine mixture increases beta 2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro

Introduction: Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro. Methods: The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm(2)). Results: Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either beta 2 (CD18), alpha(L)/beta(2) (CD11 alpha/CD18; LFA-1) or alpha(M)/beta(2) (CD11 beta/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion. Conclusions: Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a beta 2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE