Evaluation of Endocan as a Treatment for Acute Inflammatory Respiratory Failure.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a life-threatening condition resulting from acute pulmonary inflammation. However, no specific treatment for ARDS has yet been developed. Previous findings suggest that lung injuries related to ARDS could be regulated by endocan (Esm-1). The aim of this study was to evaluate the potential efficiency of endocan in the treatment of ARDS. METHODS: We first compared the features of acute pulmonary inflammation and the severity of hypoxemia in a tracheal LPS-induced acute lung injury (ALI) model performed in knockout (Esm1-/-) and wild type (WT) littermate C57Bl/6 mice. Next, we assessed the effects of a continuous infusion of glycosylated murine endocan in our ALI model in Esm1-/- mice. RESULTS: In our ALI model, we report higher alveolar leukocytes (p < 0.001), neutrophils (p < 0.001), and MPO (p < 0.001), and lower blood oxygenation (p < 0.001) in Esm1-/- mice compared to WT mice. Continuous delivery of glycosylated murine endocan after LPS-induced ALI resulted in decreased alveolar leukocytes (p = 0.012) and neutrophils (p = 0.012), higher blood oxygenation levels (p < 0.001), and reduced histological lung injury (p = 0.04), compared to mice treated with PBS. CONCLUSIONS: Endocan appears to be an effective treatment in an ARDS-like model in C57Bl/6 mice.

Endothelial Cell-Specific Molecule-1 Inhibits Albuminuria in Diabetic Mice.

Background: Diabetic kidney disease (DKD) is the most common cause of kidney failure in the world, and novel predictive biomarkers and molecular mechanisms of disease are needed. Endothelial cell-specific molecule-1 (Esm-1) is a secreted proteoglycan that attenuates inflammation. We previously identified that a glomerular deficiency of Esm-1 associates with more pronounced albuminuria and glomerular inflammation in DKD-susceptible relative to DKD-resistant mice, but its contribution to DKD remains unexplored. Methods: Using hydrodynamic tail-vein injection, we overexpress Esm-1 in DKD-susceptible DBA/2 mice and delete Esm-1 in DKD-resistant C57BL/6 mice to study the contribution of Esm-1 to DKD. We analyze clinical indices of DKD, leukocyte infiltration, podocytopenia, and extracellular matrix production. We also study transcriptomic changes to assess potential mechanisms of Esm-1 in glomeruli. Results: In DKD-susceptible mice, Esm-1 inversely correlates with albuminuria and glomerular leukocyte infiltration. We show that overexpression of Esm-1 reduces albuminuria and diabetes-induced podocyte injury, independent of changes in leukocyte infiltration. Using a complementary approach, we find that constitutive deletion of Esm-1 in DKD-resistant mice modestly increases the degree of diabetes-induced albuminuria versus wild-type controls. By glomerular RNAseq, we identify that Esm-1 attenuates expression of kidney disease-promoting and interferon (IFN)-related genes, including Ackr2 and Cxcl11. Conclusions: We demonstrate that, in DKD-susceptible mice, Esm-1 protects against diabetes-induced albuminuria and podocytopathy, possibly through select IFN signaling. Companion studies in patients with diabetes suggest a role of Esm-1 in human DKD.

Cleaved endocan acts as a biologic competitor of endocan in the control of ICAM-1-dependent leukocyte diapedesis.

Dysregulated leukocyte diapedesis is a major contributor to acute severe inflammatory states like sepsis and acute respiratory distress syndrome, which are common conditions in critically ill subjects. Endocan is a circulating proteoglycan that binds to the leukocyte integrin LFA-1 and blocks its interaction with its endothelial ligand ICAM-1, subsequently leading to the inhibition of leukocyte recruitment. Recent data have highlighted the hypothetic role of p14, endocan's major catabolite found in the bloodstream of septic patients, as a potential antagonist of endocan, thus participating in the regulation of acute inflammation. We hereby characterize the role of p14 as a biologic competitor of endocan, through assessment of its molecular interactions with LFA-1, endocan, and ICAM-1, as well as its effects on human leukocyte trafficking. Using immunodetection assay, we report that p14 can bind to LFA-1, thus inhibiting the interaction between LFA-1 and endocan, which in turn leads to the restoration of the ICAM-1/LFA-1 interaction. In primary human T cells trafficking assays, we underline the absence of effect of p14 on ICAM-1-dependent adhesion and migration, as well as on transendothelial migration. However, in those models, p14 reverses the antimigratory effect of endocan. To conclude, our study supports the hypothesis of an antagonistic role of p14 versus endocan in its effect on the LFA-1/ICAM-1-dependent human leukocyte recruitment.

Endocan regulates acute lung inflammation through control of leukocyte diapedesis.

Acute respiratory distress syndrome is a severe form of respiratory failure, occurring in up to 20% of patients admitted to the intensive care unit with sepsis. Dysregulated leukocyte diapedesis is a major contributor to acute respiratory distress syndrome. Endocan is a circulating proteoglycan that binds to the leukocyte integrin leukocyte functional antigen-1 and blocks its interaction with its endothelial ligand, ICAM-1. The objective of this study was to evaluate the role of endocan in the control of acute lung inflammation. In vitro, endocan inhibited human leukocyte transendothelial migration as well as ICAM-1-dependent migration but had a very mild effect on ICAM-1-dependent adhesion. Endocan also acted as an inhibitor of transendothelial migration of mouse leukocytes. The effect of systemic administration of recombinant human endocan was assessed in a model of acute lung inflammation in BALB/c mice. Treatment with endocan 1 h after intratracheal LPS challenge reduced the alveolar inflammatory response, diminished histological features of acute lung injury, and improved respiratory function. These results highlight the anti-inflammatory role of human endocan and its protective effect against acute lung injury.NEW & NOTEWORTHY We show here that endocan inhibits ICAM-1-dependent human leukocyte transendothelial migration and ICAM-1-dependent adhesion. We also found that in BALB/c mice with tracheal LPS-induced acute lung injury treatment with recombinant human endocan reduces lung inflammation, notably through reduction of neutrophilic recruitment, and restores normal lung function. These results confirm the hypothesis that human endocan may have a protective effect against acute lung inflammation.

Endocan, sepsis, pneumonia, and acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) and hospital-acquired pneumonia (HAP) are major problems of public health in intensive care units (ICUs), occurring in 15% of critically ill patients. Among the factors explaining ARDS development, sepsis is known as a frequent cause. Sepsis, ARDS, and HAP increase morbidity, mortality, length of stay in the ICU, and the overall costs of healthcare. The major challenge remains to identify accurately among critically ill patients those at risk of poor outcomes who could benefit from novel therapies. Endocan is released by the pulmonary endothelium in response to local or systemic injury. It inhibits mainly leukocyte diapedesis rather than leukocyte rolling or adhesion to the endothelial cells both in vitro and in vivo. Endocan was evaluated in 25 clinical reports, including 2454 critically ill patients and 452 healthy controls. The diagnostic value of endocan for sepsis or sepsis severity was equal to procalcitonin but its prognostic value was better. A predictive value for postoperative pneumonia was evidenced in two studies, and a predictive value for ARDS in four studies from three independent centers. This review presents an overview of the structure, expression, and functions of endocan. We also hereby summarize the potential applications of endocan in the prediction and prognosis of ARDS and HAP, as well as in the prognosis of sepsis.

Low endocan levels are predictive of Acute Respiratory Distress Syndrome in severe sepsis and septic shock.

PURPOSE: Endocan is a circulating proteoglycan measured at high blood levels during severe sepsis, with a likely lung anti-inflammatory function. The aim of this study was to assess whether paradoxically low endocan levels at Intensive Care Unit (ICU) admission could predict Acute Respiratory Distress Syndrome (ARDS) within 72 h in severe septic patients. MATERIALS AND METHODS: Patients admitted for severe sepsis in the ICU of a French University Hospital were included in a prospective single-center observational study between October 2014 and March 2016. RESULTS: 72 patients admitted in ICU for severe sepsis were included. Endocan blood values at inclusion were significantly lower in patients who developed an ARDS at 72 h (p < 0.001). For endocan blood values > 5.36 ng/mL, the adjusted OR for development of ARDS at 72 h was of 0.001 (95% CI 0-0.215; p = 0.011). In our cohort, an endocan value < 2.54 ng/mL predicted ARDS at 72 h with a positive predictive value of 1 (Sp = 1 (95% CI 0.94-1)). CONCLUSIONS: In a cohort of severe septic patients, we observed that low blood levels of endocan at ICU admission were predictive of ARDS at 72 h.

Endothelial Cell-Specific Molecule-1 in Critically Ill Patients With Hematologic Malignancy.

OBJECTIVES: To assess whether serum concentration of endothelial cell-specific molecule-1 (Endocan) at ICU admission is associated with the use of ICU resources and outcomes in critically ill hematology patients. DESIGN: Prospective multicenter cohort study. SETTING: Seventeen ICUs in France and Belgium. PATIENTS: Seven hundred forty-four consecutive critically ill hematology patients; 72 critically ill septic patients without hematologic malignancy; 276 healthy subjects. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Median total endocan concentrations were 4.46 (2.7-7.8) ng/mL. Endocan concentrations were higher in patients who had received chemotherapy before ICU admission (4.7 [2.8-8.1] ng/mL vs. 3.7 [2.5-6.3] ng/mL [p = 0.002]). In patients with acute respiratory failure, endocan levels were increased in patients with drug-induced pulmonary toxicity compared with other etiologies (p = 0.038). Total endocan levels higher than 4.46 ng/mL were associated with a higher cumulative probability of renal replacement therapy requirement (p = 0.006), a higher requirement of mechanical ventilation (p = 0.01) and a higher requirement of vasopressors throughout ICU stay (p < 0.0001). By multivariate analysis, total endocan levels at admission were independently associated with ICU mortality (odds ratios, 1.39; 95% CI, 1.06-1.83; p = 0.018). The predictive value of endocan peptide fragments of 14 kDa in terms of mortality and life-sustaining therapies requirement was inferior to that of total endocan. Endocan levels were higher in critically ill hematology patients compared with healthy subjects (p < 0.0001) but lower than endocan values in critically ill septic patients without hematologic malignancy (p = 0.005) CONCLUSIONS:: Serum concentrations of endocan at admission are associated with the use of ICU resources and mortality in critically ill hematology patients. Studies to risk-stratify patients in the emergency department or in the hematology wards based on endocan concentrations to identify those likely to benefit from early ICU management are warranted.

Endocan is a stable circulating molecule in ICU patients.

BACKGROUND: Endocan is a lung endothelial cell secreted proteoglycan, possessing multiple physiological roles and potential therapeutic and diagnostic utility as biomarker in pneumonia and acute respiratory distress syndrome. Endocan synthesis and secretion can be induced by proinflammatory cytokines such as TNF-α, but can also be subject of proteolytic degradation causing preanalytical variation. METHODS: We investigated the stability of endocan in conventional serum, plasma, anticoagulated whole blood, as well as whole blood and plasma stabilized with protease inhibitors. RESULTS: Among the recipient tubes for blood collection, those with EDTA gave minimal interference. No dilution effect was observed on recovery tests from 1:2 to 1:16 (v:v). The recovery test in 10 plasma EDTA samples from healthy subjects or septic patients indicated a median recovery of 104.5% [104%-107.5%], and 97% [88.5%; 102.5%], respectively. Patient's plasma endocan remains stable when stored at room temperature till 72h, or following 3 freeze thaw cycles. Finally, no interference was observed with hemolytic, icteric or turbidic plasma samples. CONCLUSION: These results are consistent with the view that endocan measured in ICU patients is intact, stable, and accurate. Then, the low endocan level observed in ICU patients who developed ARDS is likely to be reliable.

Evaluation of endothelial biomarkers as predictors of organ failures in septic shock patients.

BACKGROUND: Endothelial injury is recognized to trigger organ failures during the first 48h of septic shock. We evaluate endothelial biomarkers at ICU admission in their ability to predict severity, outcome, and organ failures in septic shock patients. METHODS: This prospective observational pilot study was conducted in a medical intensive care unit of a university hospital. Plasma levels of endothelial biomarkers as angiopoietin-2, sE-selectin or endocan were measured at ICU admission of 20 patients presenting with septic shock. Clinical and biological data were recorded at inclusion and each day during the first week. RESULTS: Significant correlations were found between angiopoietin-2 and severity scores at Day 1: SAPS2 (r(2)=0.620; p=0.004) and LOD score (r(2)=0.681; p=0.001). The angiopoietin-2 level was significantly higher in patients presenting with organ failure such as hemodynamic, renal or hepatic failure. It correlated with catecholamine infusion dose and was higher in non survivors compared with survivors (33.5 [28.9-51.4] vs. 12.4 [6.4-14.7]ng/ml; p=0.001). In contrast, in that population presenting with septic shock, endocan level at inclusion was not related to any organ failure at inclusion or Day 1 but appeared lower in patients presenting with respiratory failure at Day 3 compared to those who do not (1.9 [0.99-3.1] vs 5.2 [3.1-17.2]ng/ml; p=0.032). The endocan level at inclusion was correlated with the decrease in PaO2/FiO2 ratio at Day 2 (r(2)=0.628; p=0.0004) and Day 3 (r(2)=0.645; p=0.005). Endocan level <2.54ng/ml at admission is predictive of a respiratory failure presence at Day 3. CONCLUSION: In septic shock patients, angiopoietine-2 is related with clinical severity during the first 24h but only endocan is able to predict the presence of respiratory failure at Day 3.

The non glycanated endocan polypeptide slows tumor growth by inducing stromal inflammatory reaction.

Endocan expression is increasingly studied in various human cancers. Experimental evidence showed that human endocan, through its glycan chain, is implicated in various processes of tumor growth. We functionally characterize mouse endocan which is also a chondroitin sulfate proteoglycan but much less glycanated than human endocan. Distant domains from the O-glycanation site, located within exons 1 and 2 determine the glycanation pattern of endocan. In opposite to the human homologue, overexpression of mouse endocan in HT-29 cells delayed the tumor appearance and reduced the tumor growth rate. This tumor growth inhibition is supported by non glycanated form of mouse endocan. Non glycanated human endocan overexpressed in HT-29, A549 or K1000 cells also exhibited an anti-tumor effect. Moreover, systemic delivery of non glycanated human endocan also results in HT-29 tumor growth delay. In vitro, endocan polypeptide did not affect HT-29 cell proliferation, nor cell viability. In tumor tissue sections, a stromal inflammatory reaction was observed only in tumors overexpressing endocan polypeptide, and depletion of CD122+ cells was able to delete partially the anti-tumor effect of endocan polypeptide. These results reveal a novel pathway for endocan in the control of tumor growth, which involves inflammatory cells of the innate immunity.

The disulfide bond between cysteine 10 and cysteine 34 is required for CCL18 activity.

Asthma is a Th2-mediated disease that involves Th2 cell and eosinophil migration into the bronchial mucosa which is dependent upon the expression of a specific set of chemokines within the lung. Among them, CCL18 seems to play a key role because of its preferential expression in the lung, and its up-regulation by Th2 cytokines. Here, we show that the optimal naïve T cell and basophil chemotaxis, and basophil histamine release induced by rhCCL18 occurred at a 100 time lower concentration with CHO-derived rhCCL18 than with E. coli-derived rhCCL18. FT-ICR mass spectrometry of the intact chemokines showed that the rhCCL18 produced by CHO cells contained the 2 disulfide bonds Cys10-Cys34 and Cys11-Cys50, in clear contrast to the rhCCL18 derived from E. coli where the Cys10-Cys34 bond was absent. We found that reduction of the Cys10-Cys34 of the CHO-derived rhCCL18 resulted in a shift of its activity, reaching the same level as the E. coli-derived rhCCL18. These results demonstrate that the Cys10-Cys34 disulfide bond is involved in the function of CCL18.

Identification of a 14 kDa endocan fragment generated by cathepsin G, a novel circulating biomarker in patients with sepsis.

Severe septic syndrome, which is the most prevalent and lethal cause of acute respiratory distress syndrome, remains one of the most frequent causes of admission and death in intensive care units (ICU). Inflammatory phenomenon leading to severe sepsis are multiple and not yet completely understood. The main target damage during severe sepsis is the endothelium. Endocan, specifically secreted by activated-pulmonary vascular endothelial cells, is thought to play a key role in the control of the lung inflammatory reaction. A recent clinical investigation found that a low plasma endocan level was predictive of respiratory failure. In this study, the hypothesis that low levels of endocan may result from proteolysis was tested. We demonstrate that cathepsin G (CG), neutrophil elastase (NE), and to a lesser extent proteinase 3 (PR3), degrade endocan. Interestingly, a novel endocan peptide fragment of 14 kDa, named p14, was identified, resulting from the specific cleavage of endocan by CG, corresponding to the N-terminal 111-116 amino acids of the endocan polypeptide. An immunoassay specific for p14 endocan fragment was then developed, and revealed increased plasma levels of p14 in 20 out of 55 severe septic patients, ranging from 0.52 to 10.40 ng/mL versus undetectable p14 in plasma from 32 control subjects (p=0.0011). No correlations were found between p14 and endocan blood levels in severe septic patients. Taken together, the p14 endocan fragment represents a novel interesting biomarker which could participate to the pathogenesis of sepsis.

Development of monoclonal antibodies and ELISA specific for the mouse vascular endocan.

Human vascular endocan is a proteoglycan exhibiting tumorigenic activity through both its glycan and protein cores. Endocan mRNA is identified as being one of the most significant molecular signatures defining a poor prognosis in lung, breast, kidney, prostate, and thyroid malignancies. The survival inversely correlates with endocan expression in tumor tissue from hepatocarcinoma, and in serum from lung cancer. In mouse, endocan mRNA is also increased in tumor vessels. However, mouse endocan has not yet been fully characterized. Here, we produced a panel of rat monoclonal antibodies directed against mouse endocan, leading to the development of a specific mouse/rat endocan ELISA. Mouse endocan serum level was measured at a median of 0.96 ng/mL and 1.08 ng/mL in 129Sv mice and C57bl6, respectively. These results also provide new tools to characterize and explore the role of endocan in mouse and rat models of human diseases. These results present mouse vascular endocan as a circulating molecule similar to human endocan.