Lung Fibrosis Is Linked to Increased Endothelial Cell Activation and Dysfunctional Vascular Barrier Integrity.

Pulmonary fibrosis (PF) can be a fatal disease characterized by progressive lung scarring. It is still poorly understood how the pulmonary endothelium is involved in the disease pathogenesis. Differences of the pulmonary vasculature between patients and donors were analyzed using transmission electron microscopy, immunohistochemistry, and single-cell RNA sequencing. Vascular barrier resistance, endothelial-immune cell adhesion, and sensitivity to an inflammatory milieu were studied in vitro. Integrity and activation markers were measured by ELISA in human plasma. Transmission electron microscopy demonstrated abnormally swollen endothelial cells (ECs) in fibrotic lungs compared with donors. A more intense CD31 and von Willebrand Factor (vWF) and patchy vascular endothelial (VE)-Cadherin staining in fibrotic lungs supported the presence of a dysregulated endothelium. Integrity markers CD31, VE-Cadherin, Thrombomodulin, and VEGFR-2 (vascular endothelial growth factor receptor-2) and activation marker vWF gene expression was increased in different endothelial subpopulations (e.g., arterial, venous, general capillary, aerocytes) in PF. This was associated with a heightened sensitivity of fibrotic ECs to TNF-α or IFN-γ and elevated immune cell adhesion. The barrier strength was overall reduced in ECs from fibrotic lungs. vWF and IL-8 were increased in the plasma of patients, whereas VE-Cadherin, Thrombomodulin, and VEGFR-2 were decreased. VE-Cadherin staining was also patchy in biopsy tissue and was decreased in plasma samples of patients with PF 6 months after the initial diagnosis. Our data demonstrate highly abnormal ECs in PF. The vascular compartment is characterized by hyperactivation and increased immune cell adhesion, as well as dysfunctional endothelial barrier function. Reestablishing EC homeostasis and function might represent a new therapeutic option for fibrotic lung diseases.

The role of neutrophil extracellular trap formation in kidney transplantation: Implications from donors to the recipient.

Kidney transplantation remains the gold standard for patients with end-stage renal disease, but severe donor organ shortage has led to long waiting lists. The utilization of expanded criteria donor kidneys within the category of deceased donors has enlarged the pool of available kidneys for transplantation; however, these grafts often have an increased risk for delayed graft function or reduced graft survival following transplantation. During brain or circulatory death, neutrophils are recruited to the vascular beds of kidneys where a proinflammatory microenvironment might prime the formation of neutrophil extracellular traps (NETs), web-like structures, containing proteolytic enzymes, DNA, and histones. NETs are known to cause tissue damage and specifically endothelial damage while activating other systems such as coagulation and complement, contributing to tissue injury and an unfavorable prognosis in various diseases. In lung transplantation and kidney transplantation studies, NETs have also been associated with primary graft dysfunction or rejection. In this review, the role that NETs might play across the different phases of transplantation, already initiated in the donor, during preservation, and in the recipient, will be discussed. Based on current knowledge, NETs might be a promising therapeutic target to improve graft outcomes.

Differentiating idiopathic inflammatory myopathies by automated morphometric analysis of MHC-1, MHC-2 and ICAM-1 in muscle tissue.

AIMS: Diagnosis of idiopathic inflammatory myopathies (IIM) is based on morphological characteristics and the evaluation of disease-related proteins. However, although broadly applied, substantial bias is imposed by the respective methods, observers and individual staining approaches. We aimed to quantify the protein levels of major histocompatibility complex (MHC)-1, (MHC)-2 and intercellular adhesion molecule (ICAM)-1 using an automated morphometric method to mitigate bias. METHODS: Double immunofluorescence staining was performed on whole muscle sections to study differences in protein expression in myofibre and endomysial vessels. We analysed all IIM subtypes including dermatomyositis (DM), anti-synthetase syndrome (ASyS), inclusion body myositis (IBM), immune-mediated-necrotising myopathy (IMNM), dysferlinopathy (DYSF), SARS-CoV-2 infection and vaccination-associated myopathy. Biopsies with neurogenic atrophy (NA) and normal morphology served as controls. Bulk RNA-Sequencing (RNA-Seq) was performed on a subset of samples. RESULTS: Our study highlights the significance of MHC-1, MHC-2 and ICAM-1 in diagnosing IIM subtypes and reveals distinct immunological profiles. RNASeq confirmed the precision of our method and identified specific gene pathways in the disease subtypes. Notably, ASyS, DM and SARS-CoV-2-associated myopathy showed increased ICAM-1 expression in the endomysial capillaries, indicating ICAM-1-associated vascular activation in these conditions. In addition, ICAM-1 showed high discrimination between different subgroups with high sensitivity and specificity. CONCLUSIONS: Automated morphometric analysis provides precise quantitative data on immune-associated proteins that can be integrated into our pathophysiological understanding of IIM. Further, ICAM-1 holds diagnostic value for the detection of IIM pathology.

Imbalance in the vWF - ADAMTS13 axis exists early in acute pancreatitis and predicts persistent organ failure and pancreatic necrosis-a prospective study.

BACKGROUND: The pathophysiology of Acute Pancreatitis (AP) may be complicated by endothelial activation. von Willebrand Factor (vWF)- ADAMTS13 axis is a marker of endothelial activation. The study aimed to investigate the axis in AP, comparing it in patients with and without persistent organ failure (OF), with and without pancreatic necrosis, and correlating it with the standard severity scores (CRP, APACHE II, BISAP, SOFA, and qSOFA) METHODS: vWF-Antigen (vWF:Ag), vWF-Collagen-Binding-Assay (vWF:CBA), and ADAMTS13 activity (ADAMTS13:act) levels were measured within 5 days of symptom onset in consecutive patients (n = 98), who were admitted with a first episode of AP (Dec 2021-May 2023). RESULTS: Of the 98 patients admitted with AP, 78(79.6 %) had no or transient OF; 20(20.4 %) had persistent OF. Age was comparable (43.73 ± 15.36 vs 38.65 ± 13.69) [mean ± SD](years), and males were predominant in both groups (70.5 % vs 80 %). Patientswith persistent OF had higher vWF:CBA(%)[323(279-486.5) vs 199.5(159.1-295.75)] and lower ADAMTS13:act(%)[35.4(23.8-56.85) vs 56.35(44.1-71.9)][median (25th - 75th percentile)](P = 0.001) than those with no or transient OF. Patients with pancreatic necrosis (n = 19) had lower ADAMTS13:act(%)[42.79 ± 18.69] than those without pancreatic necrosis (n = 18) [62.49 ± 22.64] (P < 0.01). ADAMTS13:act had a negative correlation(r = -0.2), whereas vWF:Ag and vWF:CBA had a positive correlation (r = 0.2) with the standard severity scores (P < 0.05). ADAMTS13:act could predict pancreatic necrosis [AUROC-0.737, P < 0.05] and persistent OF [AUROC-0.746, P < 0.001], while vWF:CBA could predict persistent OF [AUROC- 0.73, P < 0.001]. CONCLUSION: vWF-ADAMTS13 axis helps to predict severe disease and is associated with poor outcomes in acute pancreatitis.

Hemoglobin increases leukocyte adhesion and initiates lung microvascular endothelial activation via Toll-like receptor 4 signaling.

Cell-free hemoglobin is a pathophysiological driver of endothelial injury during sepsis and acute respiratory distress syndrome (ARDS), but the precise mechanisms are not fully understood. We hypothesized that hemoglobin (Hb) increases leukocyte adhesion and endothelial activation in human lung microvascular endothelial cells (HLMVEC). We stimulated primary HLMVEC, or leukocytes isolated from healthy human donors, with Hb (0.5 mg/mL) and found that leukocyte adhesion to lung endothelium in response to Hb is an endothelial-dependent process. Next, we stimulated HLMVEC with Hb over time (1, 3, 6, and 24 h) and found increased transcription and release of inflammatory cytokines (IL-1ß, IL-8, and IL-6). In addition, Hb exposure variably upregulated transcription, total protein expression, and cell-surface localization of adhesion molecules E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). Since VCAM-1 was most upregulated by Hb, we further tested mechanisms for Hb-mediated upregulation of VCAM-1 in HLMVEC. Although upregulation of VCAM-1 was not prevented by hemoglobin scavenger haptoglobin, heme scavenger hemopexin, or inhibition of nod-like receptor protein 3 (NLRP3) signaling, blocking Toll-like receptor 4 (TLR4) with small molecule inhibitor TAK-242 (1 µM) prevented upregulation of VCAM-1 in response to Hb. Consistently, Hb increased nuclear factor-κB (NF-κB) activation and intracellular reactive oxygen species (ROS), which were both prevented by TLR4 inhibition. Together, these data demonstrate that Hb increases leukocyte-endothelial adhesion and activates HLMVEC through TLR4 signaling, indicating a potential mechanism for Hb-mediated pulmonary vascular injury during inflammatory and hemolytic conditions.

Microvascular blood-brain barrier alterations in isolated brain capillaries of mice over-expressing alpha-synuclein (Thy1-aSyn line 61).

Dysfunction of the blood-brain barrier (BBB) is suggested to play a critical role in the pathological mechanisms of Parkinson's disease (PD). PD-related pathology such as alpha-synuclein accumulation and inflammatory processes potentially affect the integrity of the BBB early in disease progression, which in turn may alter the crosstalk of the central and peripheral immune response. Importantly, BBB dysfunction could also affect drug response in PD. Here we analyzed microvascular changes in isolated brain capillaries and brain sections on a cellular and molecular level during disease progression in an established PD mouse model that overexpresses human wild-type alpha-synuclein (Thy1-aSyn, line 61). BBB alterations observed in Thy1-aSyn mice included reduced vessel density, reduced aquaporin-4 coverage, reduced P-glycoprotein expression, increased low-density lipoprotein receptor-related protein 1 expression, increased pS129-alpha-synuclein deposition, and increased adhesion protein and matrix metalloprotease expression together with alterations in tight junction proteins. Striatal capillaries presented with more dysregulated BBB integrity markers compared to cortical capillaries. These alterations of BBB integrity lead, however, not to an overt IgG leakage in brain parenchyma. Our data reveals intricate alterations in key proteins of BBB function together with histological evidence for altered structure of the brain vasculature. Thy1-aSyn mice represent a useful model to investigate therapeutic targeting of BBB alterations in synucleinopathies.

Amyloid beta peptides (Aß) from Alzheimer's disease neuronal secretome induce endothelial activation in a human cerebral microvessel model.

In Alzheimer's disease (AD), secretion and deposition of amyloid beta peptides (Aß) have been associated with blood-brain barrier dysfunction. However, the role of Aß in endothelial cell (EC) dysfunction remains elusive. Here we investigated AD mediated EC activation by studying the effect of Aß secreted from human induced pluripotent stem cell-derived cortical neurons (hiPSC-CN) harboring a familial AD mutation (Swe+/+) on human brain microvascular endothelial cells (HBMECs) in 2D and 3D perfusable microvessels. We demonstrated that increased Aß levels in Swe+/+ conditioned media (CM) led to stress fiber formation and upregulation of genes associated with endothelial inflammation and immune-adhesion. Perfusion of Aß-rich Swe+/+ CM induced acute formation of von Willebrand factor (VWF) fibers in the vessel lumen, which was attenuated by reducing Aß levels in CM. Our findings suggest that Aß peptides can trigger rapid inflammatory and thrombogenic responses within cerebral microvessels, which may exacerbate AD pathology.

Endothelial activation and stress index as a prognostic factor of diffuse large B-cell lymphoma: the report from the nationwide multi-center Thai Lymphoma Study Group.

Several prognostic models have been introduced to predict outcomes of patients with diffuse large B-cell lymphoma (DLBCL). Endothelial activation and stress index (EASIX) is a surrogate of endothelial dysfunction which has been shown to predict outcomes of patients with various hematologic malignancies. However, the prognostic implication of EASIX for DLBCL is limited and warrants exploration. We conducted a retrospective study enrolling adult DLBCL patients including a discovery cohort from the single-centered university hospital database and a validation cohort from the independent nationwide multi-center registry. EASIX scores were calculated using creatinine, lactate dehydrogenase, and platelet levels. The receiver operating characteristic curve analysis was used to determine optimal cutoff. Statistical analysis explored the impact of EASIX on survival outcomes. A total of 323 patients were included in the discovery cohort. The optimal EASIX cutoff was 1.07 stratifying patients into low (53.9%) and high EASIX (46.1%) groups. Patients with high EASIX had worse 2-year progression-free survival (PFS) (53.4% vs. 81.5%, p<0.001) and overall survival (OS) (64.4% vs. 88.7%, p<0.001) than patients with low EASIX. Multivariate analysis revealed that older age, bulky disease, impaired performance status, and high EASIX were associated with an unfavorable OS. In the validation cohort of 499 patients, the optimal EASIX cutoff was 1.04. Similar to the discovery cohort, high EASIX score was associated with high-risk diseases, worse PFS, and inferior OS. In conclusion, EASIX score was significantly associated with survival outcomes and may be used as a simple prognostic tool to better risk-classify DLBCL.

IL-10 plus the EASIX score predict bleeding events after anti-CD19 CAR T-cell therapy.

Chimeric antigen receptor (CAR) T-cell-associated coagulopathy can cause bleeding events. To explore risk factors for hemorrhage after CAR T-cell therapy, we retrospectively analyzed routine indicators in 56 patients with non-Hodgkin lymphoma and B-cell acute lymphoblastic leukemia who received anti-CD19 CAR T-cell therapy. Disturbance of coagulation occurred mainly within one month post infusion, especially on day 7 and 14. The cumulative incidence of bleeding events within one month was 32.8%, with the median onset of 7 (range, 0-28) days. All bleeding events were grade 1-3. Patients who experienced bleeding events within one month had longer prothrombin time, higher IL-6, higher IL-10, and lower platelets before lymphodepletion. There were also correlations among coagulation-, inflammatory-, and tumor burden-related markers. Multi-variate analysis showed IL-10 (> 7.98 pg/mL; adjusted odds ratio [OR], 13.84; 95% confidence interval [CI], 2.03-94.36; P = 0.007) and the endothelial activation and stress index (EASIX, defined as dehydrogenase [U/L] × creatinine [mg/dL] / platelets [×109 cells/L]; >7.65; adjusted OR, 7.06; 95% CI, 1.03-48.23; P = 0.046) were significant risk factors for bleeding events. IL-10 plus the EASIX defined three risk groups for bleeding events with cumulative incidence of 100% (hazard ratio [HR], 14.47; 95% CI, 2.78-75.29; P < 0.0001), 38.5% (HR, 3.68; 95% CI, 0.82-16.67; P = 0.089), and 11.8% (reference), respectively. Future studies are needed to verify the risk assessment models for bleeding events after CAR T-cell treatment in larger cohorts.

Association Between Endothelial Activation and Stress Index and 28-Day Mortality in Septic ICU patients: a Retrospective Cohort Study.

Background: Endothelial Activation and Stress Index (EASIX) is a reliable alternative biomarker of endothelial dysfunction. Because endothelial activation is involved in sepsis pathophysiology, we aimed to investigate the association between EASIX and prognosis in septic patients. Methods: Data were extracted from the Medical Information Mart for Intensive Care (MIMIC) IV database. EASIX scores were calculated using the formula: lactate dehydrogenase (U/L) × creatinine (mg/dL)/platelet count (109/L). Patients were grouped into tertiles according to log2 transformed EASIX. The primary and secondary outcomes were 28-day and 90-day mortality. Cox proportional hazards models, Kaplan-Meier curves, restricted cubic spline curves, and subgroup analyses were conducted to evaluate the association between EASIX and prognosis in septic patients. Results: A total of 7504 patients were included. Multivariable Cox proportional hazards analyses showed that higher log2-EASIX was associated with increased risk of 28-day mortality (HR, 1.10; 95% CI, 1.07-1.13; P < 0.001). Compared with tertile 1, the tertile 2 and 3 groups had higher risk of 28-day mortality [HR (95% CI) 1.24 (1.09-1.41); HR (95% CI) 1.51 (1.31-1.74)]; P for trend < 0.001). Similar results were found for 90-day mortality. Kaplan-Meier curves showed that patients with higher EASIX had lower 28-day and 90-day survival rates. A linear relationship was found between log2-EASIX and 28-day and 90-day mortality. Conclusion: High EASIX was significantly associated with an increased risk of 28-day and 90-day all-cause mortality in patients with sepsis.

Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells.

Weibel-Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation.

Serum E-selectin and endothelial cell-specific Molecule-1 levels among people living with HIV on long term ART in Uganda: a pilot cross-sectional study.

BACKGROUND: Prolonged exposure to HIV and anti-retroviral therapy (ART) has been linked with endothelial cell activation which subsequently predisposes people living with HIV (PLWH) to cardiovascular diseases. Serum biomarkers of endothelial cell activation such as E-Selectin and endothelial cell-specific molecule-1 (ESM-1) could aid in early detection of PLWH at a risk of cardiovascular diseases. However, there is a paucity of data on these biomarkers like E-selectin and endothelial cell-specific molecule-1 (ESM-1) among PLWH on long term ART (≥ 10 years) in Uganda. The aim of this study is to determine the serum levels of these biomarkers in this population. METHODS: This was a cross-sectional study where we randomly sampled 73 stored serum samples of PLWH who were enrolled in the Infectious Diseases Institute (IDI) ART long term (ALT cohort). We measured serum levels of E-selectin and ESM-1 by ELISA. Data was summarized using median and interquartile range. Inferential statistics were performed to determine predictors of elevated levels of E-selectin. RESULTS: Of the 73 samples analyzed, 38 (52.1%) were from female participants. The mean age was 54 ± 9.0 years. Twenty participants (27.4%) had a history of smoking while 52 (71.2%) had a history of alcohol intake. Twenty-five (34.3%) of the participants were overweight whereas 4 (5.6%) were obese. Fifty-four (74%) had an undetectable viral load (≤ 0 copies/ml) and the mean duration of ART at the time of sampling (2014/2015) was 10.4 ± 0.4 years. While serum levels of ESM-1 were not detectable in any of our samples, the median E-selectin levels was 147.6 µm/L ranging from 8.44 µm/L and 1,979.36 µm/L. Sixty-seven participants (91.8%) had elevated levels of E-selectin (> 39 µm/L). CD4 count > 500 cells/µl compared to lower counts was a predictor of elevated levels of E-Selectin (adjusted Odd Ratio 12.5, 95% CI (1.03 - 149.95, p < 0.05). CONCLUSIONS: The majority (91.8%) of PLWH on long term ART had elevated levels of E-selectin. Having high CD4 count (> 500 cells/µl) was predictive of elevated levels of E-Selectin. Future work should longitudinally assess the trend of levels of E-selectin and ESM-1 while assessing for cardiovascular diseases endpoint.

High Plasma Angiopoietin-2 Levels Predict the Need to Initiate Dialysis within Two Years in Patients with Chronic Kidney Disease.

Volume status, congestion, endothelial activation, and injury all play roles in glomerular filtration rate (GFR) decline. In this study, we aimed to determine whether the plasma endothelial and overhydration markers could serve as independent predictors for dialysis initiation in patients with chronic kidney disease (CKD) 3b-5 (GFR < 45 mL/min/1.72 m2) and preserved ejection fraction. A prospective, observational study in a single academic center was conducted from March 2019 to March 2022. Plasma levels of angiopoietin (Ang)-2, Vascular Endothelial Growth Factor-C (VEGF-C), Vascular Cell Adhesion Molecule-1 (VCAM-1), Copeptin (CPP), beta-trace protein (BTP), brain natriuretic peptide (BNP), and cardiac troponin I (cTnI) were all measured. Lung ultrasound (US) B-lines, bioimpedance, and echocardiography with global longitudinal strain (GLS) were recorded. The study outcome was the initiation of chronic dialysis (renal replacement therapy) during 24 months of follow-up. A total of 105 consecutive patients with a mean eGFR of 21.3 mL/min/1.73 m were recruited and finally analyzed. A positive correlation between Ang-2 and VCAM-1 and BTP was observed. Ang-2 correlated positively with BNP, cTnI, sCr, E/e', and the extracellular water (ECW)/intracellular water (ICW) ratio (ECW/ICW). After 24 months, a deterioration in renal function was observed in 47 patients (58%). In multivariate regression analysis, both VCAM-1 and Ang-2 showed independent influences on risk of renal replacement therapy initiation. In a Kaplan-Meier analysis, 72% of patients with Ang-2 concentrations below the median (3.15 ng/mL) survived without dialysis for two years. Such an impact was not observed for GFR, VCAM, CCP, VEGF-C, or BTP. Endothelial activation, quantified by plasma levels of Ang-2, may play a key role in GFR decline and the need for dialysis initiation in patients with CKD 3b, 4, and 5.

A Scoping Review on Biomarkers of Endothelial Dysfunction in Small Vessel Disease: Molecular Insights from Human Studies.

Small vessel disease (SVD) is a highly prevalent disorder of the brain's microvessels and a common cause of dementia as well as ischaemic and haemorrhagic strokes. Though much about the underlying pathophysiology of SVD remains poorly understood, a wealth of recently published evidence strongly suggests a key role of microvessel endothelial dysfunction and a compromised blood-brain barrier (BBB) in the development and progression of the disease. Understanding the causes and downstream consequences associated with endothelial dysfunction in this pathological context could aid in the development of effective diagnostic and prognostic tools and provide promising avenues for potential therapeutic interventions. In this scoping review, we aim to summarise the findings from clinical studies examining the role of the molecular mechanisms underlying endothelial dysfunction in SVD, focussing on biochemical markers of endothelial dysfunction detectable in biofluids, including cell adhesion molecules, BBB transporters, cytokines/chemokines, inflammatory markers, coagulation factors, growth factors, and markers involved in the nitric oxide cascade.

In vitro study of Nigella sativa and thymoquinone activity on endothelial activation and monocyte adhesion.

INTRODUCTION: Thymoquinone (TQ) is one of the bioactive compounds in Nigella sativa (NS). Also known as black seeds/cumin, it has been postulated to possess anti-atherogenic properties. However, research on the effects of NS oil (NSO) and TQ on atherogenesis remain scarce. The aim of this study is to determine gene and protein expression of Intercellular Adhesion Molecule-1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), and Endothelial-eukocyte adhesion molecule (E-selectin) in Human Coronary Artery Endothelial Cells (HCAECs). METHODS: HCAECs were stimulated for 24 hours (h) with 200 µg/ml of Lipopolysaccharides (LPS) and different concentrations of NSO (55, 110, 220, 440 µg/ml) or TQ (4.5, 9.0, 18.0, 36.0 µm). The effects of NSO and TQ on gene and protein expressions were measured using multiplex gene assay and ELISA assay, respectively. Rose Bengal assay was used to analyse monocyte binding activity. RESULTS: NSO and TQ significantly reduced ICAM-1 and VCAM-1 gene and protein expressions. TQ showed significant reduction activity of the biomarkers in dose dependent manner. HCAECs pre-treated with NSO and TQ for 24 h significantly lowered monocytes adherence compared to non-treated HCAECs. CONCLUSIONS: NSO and TQ supplementation have anti-atherogenic properties and inhibit monocytes' adherence to HCAECs via down-regulation of ICAM-1 expression. NSO could potentially be incorporated in standard treatment regimens to prevent atherosclerosis and its related complications.

Direct Activation of Endothelial Cells by SARS-CoV-2 Nucleocapsid Protein Is Blocked by Simvastatin.

Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiorgan failure in patients with coronavirus disease 2019 (COVID-19). However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved in endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkably, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HUB1-CoV, and influenza virus H1N1 did not activate endothelial cells. These findings are consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy and suggests that simvastatin, an FDA-approved lipid-lowering drug, may help prevent the pathogenesis and improve the outcome of COVID-19 patients. IMPORTANCE Coronavirus disease 2019 (COVID-19), caused by the betacoronavirus SARS-CoV-2, is a worldwide challenge for health care systems. The leading cause of mortality in patients with COVID-19 is hypoxic respiratory failure from acute respiratory distress syndrome (ARDS). To date, pulmonary endothelial cells (ECs) have been largely overlooked as a therapeutic target in COVID-19, yet emerging evidence suggests that these cells contribute to the initiation and propagation of ARDS by altering vessel barrier integrity, promoting a procoagulative state, inducing vascular inflammation and mediating inflammatory cell infiltration. Therefore, a better mechanistic understanding of the vasculature is of utmost importance. In this study, we screened the SARS-CoV-2 viral proteins that potently activate human endothelial cells and found that nucleocapsid protein (NP) significantly activated human endothelial cells through TLR2/NF-κB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Our results provide insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.

Biomarkers of endothelial activation and dysfunction in cardiovascular diseases.

Endothelial activation and dysfunction is an important contributor to atherosclerosis, cardiovascular diseases and cardiorenal syndrome. Endothelial dysfunction is also linked with metabolic syndrome and type II diabetes. The search for specific and sensitive biomarkers of endothelial activation and dysfunction may have important clinical implications. This review pinpoints the differences in biomarkers between endothelial activation and endothelial dysfunction in cardiovascular diseases, and then briefly describes the most relevant biomarkers of endothelial activation. Biomarkers of endothelial activation include endothelial adhesion molecules, cytokines, C-reactive protein, CD62E+/E-selectin activated endothelial microparticles, oxidation of low density lipoproteins, asymmetric dimethylarginine and endocan. This review also presents an update on the novel biomarkers of endothelial dysfunction, such as matrix metalloproteinases (e.g., MMP-7, MMP-9), ANGPTL2, endogdlin, annexin V+ endothelial apoptotic microparticles, and serum homocysteine. Finally, this review emphasizes the limitations of biomarkers of endothelial activation and dysfunction in clinical setting.

The Endothelial Activation and Stress Index (EASIX) score is an independent prognostic factor in patients with diffuse large B-cell lymphoma.

BACKGROUND: The endothelial activation and stress index (EASIX) score has been reported to predict overall survival (OS) in hematological cancers. However, it has not been validated as a prognostic marker for diffuse large B-cell lymphoma (DLBCL) to date. METHODS: The records of 265 patients who presented with DLBCL in the Republic of Korea between January 07, 2004, and March 05, 2020 were retrospectively reviewed. For all included patients, EASIX scores were calculated using serum lactate dehydrogenase (LDH) and creatinine levels and the platelet count measured at diagnosis as follows: LDH (U/L) × creatinine (mg/dL)/platelet count (109/L). RESULTS: The median age of the patients was 64 years. The optimal cutoff value of EASIX according to the receiver operating characteristic analysis for OS was 1.33. All the patients were treated with cyclophosphamide, doxorubicin, vincristine, and prednisone combined with rituximab. The 1-year OS and progression-free survival (PFS) rates were lower in the high-EASIX group than in the low EASIX group (63.8% vs. 84.4%, p < 0.001 and 54.0% vs. 79.6%, p < 0.001, respectively). A high EASIX was an independent poor prognostic factor for OS and PFS (hazard ratio, 1.606; 95% CI, 1.077-2.395; p = 0.020 and hazard ratio, 1.621; 95% CI, 1.066-2.464; p = 0.024, respectively). CONCLUSIONS: EASIX is a readily available and cheaply obtainable parameter in clinical studies and shows considerable potential as a new prognostic marker for patients with newly diagnosed DLBCL.

Effect of red blood cell transfusion on inflammation, endothelial cell activation and coagulation in the critically ill.

BACKGROUND AND OBJECTIVES: Red blood cell (RBC) transfusion is a frequently applied intervention in an intensive care unit. However, transfusion is associated with adverse outcomes including organ failure and thrombo-embolic events. Mechanisms of these effects are not known but may be related to activation of the endothelium or of the coagulation or inflammatory system. We hypothesized that a RBC transfusion in the critically ill would result in further activation of these systems. MATERIALS AND METHODS: In 74 non-bleeding critically ill patients receiving one RBC unit, markers of inflammation, endothelial cell activation and coagulation were measured before transfusion, at 1 h after transfusion and 24 h after transfusion. The impact of disease severity of the recipient on these changes was assessed by comparing septic and non-septic patients (according to sepsis-3 definition) and by correlation of biomarkers with the sequential organ failure assessment (SOFA) score. RESULTS: Levels of von Willebrand Factor (vWF), soluble ICAM-1, soluble thrombomodulin, fibrinogen and d-dimer were already high at baseline, whereas ADAMTS13 levels were low. VWF levels increased significantly 24 h after RBC transfusion (median 478% (338-597) vs. 526% (395-623), p = 0.009). The other biomarkers did not change significantly. Post transfusion change was not dependent on the presence of sepsis and was not correlated with SOFA score. CONCLUSION: RBC transfusion in critically ill patients was associated with an increase in circulating vWF levels, suggesting a further increase in activation of the endothelium, a finding that was independent of the presence of sepsis or organ injury level.

The Effect of Sex-Mismatched Red Blood Cell Transfusion on Endothelial Cell Activation in Critically Ill Patients.

Background: Observational studies suggest that sex-mismatched transfusion is associated with increased mortality. Mechanisms driving mortality are not known but may include endothelial activation. The aim of this study is to investigate the effects of sex-mismatched red blood cell (RBC) transfusions on endothelial cell activation markers in critically ill patients. Study Design and Methods: In patients admitted to the intensive care unit who received a single RBC unit, blood samples were drawn before (T0), 1 h after (T1), and 24 h after transfusion (T24) for analysis of soluble syndecan-1, soluble intercellular adhesion molecule-1, soluble thrombomodulin (sTM), von Willebrand factor antigen, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα). Changes in the levels of these factors were compared between sex-matched and sex-mismatched groups. Results: Of 69 included patients, 32 patients were in the sex-matched and 37 patients were in the sex-mismatched group. Compared to baseline, sex-matched transfusion was associated with significant reduction in sTM level (p value = 0.03). Between-group comparison showed that levels of syndecan-1 and sTM were significantly higher in the sex-mismatched group compared to the sex-matched group at T24 (p value = 0.04 and 0.01, respectively). Also, TNFα and IL-6 levels showed a statistically marginal significant increase compared to baseline in the sex-mismatched group at T24 (p value = 0.06 and 0.05, respectively), but not in the sex-matched group. Discussion: Transfusion of a single sex-mismatched RBC unit was associated with higher syndecan-1 and sTM levels compared to transfusion of sex-matched RBC unit. These findings may suggest that sex-mismatched RBC transfusion is associated with endothelial activation.