Carboxypeptidase B Inhibits Over-activation of Anaphylatoxin-neutrophil Extracellular Trap Axis in COVID-19 Patients

Abstract Background: Thrombosis and coagulopathy are highly prevalent in severe patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. Neutrophil extracellular traps (NETs) are primary components of immunothrombosis, whereas the mechanism of NET formation remains unclear. We aim to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19.Methods: We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase-DNA. We detected complement-induced NET formation by immunofluorescent staining and evaluated the cytotoxicity to vascular endothelial HUVEC cells by CCK-8 assay.Results: We found that the plasma levels of complements (C3 and C5) and NETs were closely related to coagulopathy and multiple organ dysfunction in patients with COVID-19. By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products.Conclusions: Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.

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Carboxypeptidase B blocks ex vivo activation of the anaphylatoxin-neutrophil extracellular trap axis in neutrophils from COVID-19 patients

Critical Care ◽

10.1186/s13054-021-03482-z ◽

2021 ◽

Vol 25 (1) ◽

Author(s):

Yue Zhang ◽

Kai Han ◽

Chunjing Du ◽

Rui Li ◽

Jingyuan Liu ◽

...

Keyword(s):

Plasma Levels ◽

Degradation Products ◽

Ex Vivo ◽

Neutrophil Extracellular Trap ◽

Immunofluorescent Staining ◽

Vascular Endothelial ◽

Pathological Effect ◽

Carboxypeptidase B ◽

Risk Of Death ◽

Proinflammatory Mediators

Abstract Background Thrombosis and coagulopathy are highly prevalent in critically ill patients with COVID-19 and increase the risk of death. Immunothrombosis has recently been demonstrated to contribute to the thrombotic events in COVID-19 patients with coagulopathy. As the primary components of immunothrombosis, neutrophil extracellular traps (NETs) could be induced by complement cascade components and other proinflammatory mediators. We aimed to explore the clinical roles of NETs and the regulation of complement on the NET formation in COVID-19. Methods We recruited 135 COVID-19 patients and measured plasma levels of C5, C3, cell-free DNA and myeloperoxidase (MPO)-DNA. Besides, the formation of NETs was detected by immunofluorescent staining and the cytotoxicity to vascular endothelial HUVEC cells was evaluated by CCK-8 assay. Results We found that the plasma levels of complements C3 and MPO-DNA were positively related to coagulation indicator fibrin(-ogen) degradation products (C3: r = 0.300, p = 0.005; MPO-DNA: r = 0.316, p = 0.002) in COVID-19 patients. Besides, C3 was positively related to direct bilirubin (r = 0.303, p = 0.004) and total bilirubin (r = 0.304, p = 0.005), MPO-DNA was positively related to lactate dehydrogenase (r = 0.306, p = 0.003) and creatine kinase (r = 0.308, p = 0.004). By using anti-C3a and anti-C5a antibodies, we revealed that the complement component anaphylatoxins in the plasma of COVID-19 patients strongly induced NET formation. The pathological effect of the anaphylatoxin-NET axis on the damage of vascular endothelial cells could be relieved by recombinant carboxypeptidase B (CPB), a stable homolog of enzyme CPB2 which can degrade anaphylatoxins to inactive products. Conclusions Over-activation in anaphylatoxin-NET axis plays a pathological role in COVID-19. Early intervention in anaphylatoxins might help prevent thrombosis and disease progression in COVID-19 patients.

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The bone marrow niche components are adversely affected in sepsis

Molecular Biomedicine ◽

10.1186/s43556-020-00010-3 ◽

2020 ◽

Vol 1 (1) ◽

Author(s):

Fan Yin ◽

Han Qian ◽

Caiwen Duan ◽

Botao Ning

Keyword(s):

Bone Marrow ◽

Vascular Endothelial Cells ◽

Cecal Ligation ◽

Treg Cells ◽

Multiple Organ ◽

Bone Marrow Niche ◽

Vascular Endothelial ◽

Bm Niche ◽

Cell Components ◽

The Impact

Abstract Multiple organ dysfunction is an important cause of death in patients with sepsis. Currently, few studies have focused on the impact of sepsis on bone marrow (BM), especially on the cell components of BM niche. In this study, we performed mouse sepsis models by intraperitoneal injection of LPS and cecal ligation and puncture (CLP). The changes of niche major components in the mouse BM among vascular structures, mesenchymal stem cells and Treg cells were observed and analyzed. The results showed that pathological changes in BM was earlier and more prominent than in other organs, and various cell components of the BM niche changed significantly, of which vascular endothelial cells increased transiently with vascular remodeling and the regulatory T cells decreased over a long period of time. These results indicated that the components of the BM niche underwent series of adaptive changes in sepsis.

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Cardiovascular Effects of Endothelin

Physiology ◽

10.1152/physiologyonline.1997.12.3.113 ◽

1997 ◽

Vol 12 (3) ◽

pp. 113-117

Author(s):

JD Grossman ◽

JP Morgan

Keyword(s):

Endothelial Cells ◽

Heart Disease ◽

Vascular Endothelial Cells ◽

Cardiovascular Effects ◽

Multiple Organ ◽

Vascular Endothelial ◽

Cellular Calcium ◽

Organ Systems

Endothelin is a potent vasoconstricting agent released by vascular endothelial cells. Endothelin affects multiple organ systems and occurs at high levels with heart disease. Changes in cellular calcium levels and Ca2+ sensitization of myofilaments mediate the endothelin pathway. The ability to modulate endothelin levels may lead to many effective therapies.

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Localization of factor-VIII-related antigen in human vascular subendothelium

Blood ◽

10.1182/blood.v55.5.752.bloodjournal555752 ◽

1980 ◽

Vol 55 (5) ◽

pp. 752-756

Author(s):

JH Rand ◽

II Sussman ◽

RE Gordon ◽

SV Chu ◽

V Solomon

Keyword(s):

Electron Microscopy ◽

Endothelial Cells ◽

Blood Vessel ◽

Factor Viii ◽

Platelet Adhesion ◽

Vascular Endothelial Cells ◽

Staining Technique ◽

Immunofluorescent Staining ◽

Vascular Endothelial ◽

Factor Viii Related Antigen

Factor-VIII-related antigen has previously been shown to be synthesized by vascular endothelial cells. Using both an immunofluorescent staining technique and electron microscopy, we have demonstrated the presence of factor-VIII-related antigen in human vascular subendothelium. This finding may have implications in the mechanism of platelet adhesion to deendothelialized blood vessel surfaces.

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Localization of factor-VIII-related antigen in human vascular subendothelium

Blood ◽

10.1182/blood.v55.5.752.752 ◽

1980 ◽

Vol 55 (5) ◽

pp. 752-756 ◽

Cited By ~ 83

Author(s):

JH Rand ◽

II Sussman ◽

RE Gordon ◽

SV Chu ◽

V Solomon

Keyword(s):

Electron Microscopy ◽

Endothelial Cells ◽

Blood Vessel ◽

Factor Viii ◽

Platelet Adhesion ◽

Vascular Endothelial Cells ◽

Staining Technique ◽

Immunofluorescent Staining ◽

Vascular Endothelial ◽

Factor Viii Related Antigen

Abstract Factor-VIII-related antigen has previously been shown to be synthesized by vascular endothelial cells. Using both an immunofluorescent staining technique and electron microscopy, we have demonstrated the presence of factor-VIII-related antigen in human vascular subendothelium. This finding may have implications in the mechanism of platelet adhesion to deendothelialized blood vessel surfaces.

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Low Concentration of S100A8/9 Promotes Angiogenesis-Related Activity of Vascular Endothelial Cells: Bridges among Inflammation, Angiogenesis, and Tumorigenesis?

Mediators of Inflammation ◽

10.1155/2012/248574 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 12

Author(s):

Changyou Li ◽

Siyuan Li ◽

Changkai Jia ◽

Lingling Yang ◽

Zicheng Song ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Vascular Endothelial Cells ◽

Vascular Endothelial Cell ◽

Tumor Development ◽

Inflammatory Cells ◽

Dependent Manner ◽

Vascular Endothelial ◽

Low Concentration ◽

Huvec Cells

Previous studies showed that several members of the S100A family are involved in neovascularization and tumor development. This study checked whether low concentrations of S100A8 or S100A9 has any effect on the behaviour of vascular endothelial cells. A human umbilical vascular endothelial cell (HUVEC) line was used to measure vascular endothelial cell bioactivity related to angiogenesis, such as cell proliferation, migration, and vessel formation. In the low concentration range up to 10 μg/mL, either each alone or in combination, S100A8 and S100A9 proteins promoted proliferation of HUVEC cells in a dose-dependent manner. The presence of both proteins in culture showed additive effects over each single protein. Both proteins enhanced HUVEC cells to migrate across the transwell membrane and to form tube-like structures on the Matrigel surface. When mixed in Matrigel and injected subcutaneously in Balb/c mice, both proteins increased vessel development in the gel plugs. Microarray assay of HUVEC cells treated with 10 μg/mL S100A8 revealed that ribosome pathway, pathogenicEscherichia coliinfection pathway, apoptosis, and stress response genes were modulated by S100A8 treatment. We propose that S100A8 and S100A9 proteins from either infiltrating inflammatory cells or tumor cells play an important role in the interplay among inflammation, angiogenesis, and tumorigenesis.

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Identification of a New Way to Induce Differentiation of Dermal Fibroblasts Into Vascular Endothelial Cells

10.21203/rs.3.rs-216763/v1 ◽

2021 ◽

Author(s):

Xiaoling Cui ◽

Jie Wen ◽

Xiao Li ◽

Nan Li ◽

Xuxiao Hao ◽

...

Keyword(s):

Endothelial Cells ◽

Western Blot ◽

Vascular Function ◽

Vascular Endothelial Cells ◽

Dermal Fibroblasts ◽

Angiogenic Factors ◽

Immunofluorescent Staining ◽

Vascular Endothelial ◽

Qrt Pcr ◽

Small Chemical

Abstract Background: Human dermal fibroblasts (HDFs) have the potential to differentiate into vascular endothelial cells (VECs), but their differentiation rate is low and the mechanism involved is unclear. The small molecule pathway controls the phenotype of fibroblasts by activating cellular signaling pathways, which is a more convenient method in the differentiation strategy of dermal fibroblasts into vascular endothelial cells.Methods: In this study, dermal fibroblasts were treated with the different doses of CPP, and the mRNA level and protein level were detected by qPCR, Western blot and immunofluorescent staining. Matrigel assays also were used to teste the angiogenic ability of vascular endothelial cells derived from dermal fibroblasts.Results: Here, we report that a small chemical molecule, CPP ((E)-4-(4-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl) piperazin-1-yl) styryl)-1-methylpyridin-1-ium iodide), efficiently induces the differentiation of dermal fibroblasts into Vascular endothelial cells. First, we observed that the morphology of CPP-treated dermal fibroblasts elongated, curved and formed circular patterns. Western blot and qRT-PCR analyses revealed that CPP effectively reduced the level of the dermal fibroblasts-marker Vimentin and increased levels of the vascular endothelial cells -markers CD31 and CD133. Detection of the percentage of CD31-positive cells from immunofluorescent staining confirmed that CPP efficiently induces dermal fibroblasts to differentiate into vascular endothelial cells. Matrigel assays showed that CPP-treated dermal fibroblasts have the functions of vascular endothelial cells. Western blot and qRT-PCR analyses of pro-angiogenic factors (VEGF, FGF-2 and PDGF-BB) showed that CPP induces dermal fibroblasts to vascular endothelial cells by promoting the expression of pro-angiogenic factors (VEGF, FGF-2 and PDGF-BB). Conclusions: Our results indicate that the small chemical molecule CPP efficiently induces the differentiation of dermal fibroblasts into vascular endothelial cells. Simultaneously, this new inducer provides a potential to develop new approaches to restore vascular function for the treatment of ischemic vascular diseases.

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Inflammatory Cytokine-Producing Cells and Inflammation Markers in the Synovium of Osteoarthritis Patients Evidenced in Human Herpesvirus 7 Infection

International Journal of Molecular Sciences ◽

10.3390/ijms21176004 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6004

Author(s):

Valerija Groma ◽

Mihails Tarasovs ◽

Sandra Skuja ◽

Sofija Semenistaja ◽

Zaiga Nora-Krukle ◽

...

Keyword(s):

Endothelial Cells ◽

Plasma Levels ◽

Synovial Membrane ◽

Vascular Endothelial Cells ◽

Human Herpesvirus ◽

Joint Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Low Grade ◽

Vascular Endothelial ◽

Tissue Samples

A direct association between joint inflammation and the progression of osteoarthritis (OA) has been proposed, and synovitis is considered a powerful driver of the disease. Among infections implicated in the development of joint disease, human herpesvirus 7 (HHV-7) infection remains poorly characterized. Therefore, we assessed synovitis in OA patients; determined the occurrence and distribution of the HHV-7 antigen within the synovial membrane of OA-affected subjects; and correlated plasma levels of the pro-inflammatory cytokines tumor necrosis factor (TNF), interleukin-6 (IL-6), and TNF expressed locally within lesioned synovial tissues with HHV-7 observations, suggesting differences in persistent latent and active infection. Synovial HHV-7, CD4, CD68, and TNF antigens were detected immunohistochemically. The plasma levels of TNF and IL-6 were measured by an enzyme-linked immunosorbent assay. Our findings confirm the presence of persistent HHV-7 infection in 81.5% and reactivation in 20.5% of patients. In 35.2% of patients, virus-specific DNA was extracted from synovial membrane tissue samples. We evidenced the absence of histopathologically detectable synovitis and low-grade changes in the majority of OA patients enrolled in the study, in both HHV-7 PCR+ and HHV-7 PCR‒ groups. The number of synovial CD4-positive cells in the HHV-7 polymerase chain reaction (PCR)+ group was significantly higher than that in the HHV-7 PCR‒ group. CD4- and CD68-positive cells were differently distributed in both HHV-7 PCR+ and HHV-7 PCR‒ groups, as well as in latent and active HHV-7 infection. The number of TNF+ and HHV-7+ lymphocytes, as well as HHV-7+ vascular endothelial cells, was strongly correlated. Vascular endothelial cells, especially in the case of infection reactivation, appeared vulnerable. The balance between virus latency and reactivation is a long-term relationship between the host and infectious agent, and the immune system appears to be involved in displaying overreaction when a shift in the established equilibrium develops.

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Multi-Marker Immunofluorescent Staining and PD-L1 Detection on Circulating Tumour Cells from Ovarian Cancer Patients

Cancers ◽

10.3390/cancers13246225 ◽

2021 ◽

Vol 13 (24) ◽

pp. 6225

Author(s):

Du-Bois Asante ◽

Michael Morici ◽

Ganendra R. K. A. Mohan ◽

Emmanuel Acheampong ◽

Isaac Spencer ◽

...

Keyword(s):

Ovarian Cancer ◽

Vascular Endothelial Cells ◽

Tumour Cells ◽

Immunofluorescent Staining ◽

Vascular Endothelial ◽

Circulating Tumour Cells ◽

Antibody Staining ◽

Epithelial Marker ◽

Staining Protocol ◽

Insight Into

Detection of ovarian cancer (OC) circulating tumour cells (CTCs) is primarily based on targeting epithelial markers, thus failing to detect mesenchymal tumour cells. More importantly, the immune checkpoint inhibitor marker PD-L1 has not been demonstrated on CTCs from OC patients. An antibody staining protocol was developed and tested using SKOV-3 and OVCA432 OC cell lines. We targeted epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers for detection of CTCs, and CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. CTCs were enriched using the ParsortixTM system from 16 OC patients. Results revealed the presence of CTCs in 10 (63%) cases. CTCs were heterogeneous, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) for both (hybrid). PAX8 was only found in 11/157 (7%) CTCs. In addition, 62/157 (39%) CTCs were positive for PD-L1. Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.007) and mesenchymal (p = 0.0009) expressing CTCs. Characterisation of CTC phenotypes in relation to clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity plays in OC and its relationship with PD-L1.

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Elevated Plasma Levels of Soluble Platelet Glycoprotein VI (GPVI) in Patients with Thrombotic Patients

Blood ◽

10.1182/blood.v120.21.5145.5145 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5145-5145

Author(s):

Hideo Wada ◽

Tsutomu Nobori ◽

Katsuki Naitoh

Keyword(s):

Healthy Volunteers ◽

Plasma Levels ◽

Vascular Endothelial Cells ◽

Early Stage ◽

Platelet Glycoprotein ◽

Vascular Endothelial ◽

Glycoprotein Vi ◽

Th 16 ◽

Von Willebrand ◽

Willebrand Factor

Abstract Abstract 5145 Introduction Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets and it must be diagnosed at the early stage. This study, examined the activation of platelets by measuring of soluble platelet glycoprotein VI (sGPVI) level in patients with TMA. Materials and Methods The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in 40 healthy volunteers, 46 patients without thrombosis (TH), 15 postoperative patients, 13 with disseminated intravascular coagulation (DIC) and 70 with TMA. The plasma levels of GPVI and VWFpp were measured by ELISA and ADAMTS13 was measured by the FRETS assay. There were 27 TMA with markedly decreased ADAMTS13 levels (TMA-ADAMTS13) and 43 TMA without (TMA-Other). Results The plasma levels of sGPVI (median; 25. 0–75. 0%tile) were significantly higher in patients without TH (16. 2 ng/mL 12. 6–22. 5 ng/mL), postoperative patients (31. 6 ng/mL; 28. 3–35. 1 ng/mL), DIC (44. 5 ng/mL; 36. 6– 60. 8 ng/mL), TMA (40. 8 ng/mL; 32. 9–56. 7 ng/mL) than in healthy volunteers (11. 4 ng/mL; 9. 1–14. 8 ng/mL). In addition, the plasma levels of sGPVI post in postoperative patients were significantly higher than in patients without TH (p< 0. 001), and these levels were also significantly higher in those with TMA and DIC than in without TH (p< 0. 001, respectively). The plasma levels of sGPVI were significantly higher in patients with TTP-ADAMTS13 (36. 8 ng/mL; 30. 5–49. 0 ng/mL) and TMA-Other (51. 9 ng/mL; 37. 4–66. 3 ng/mL) than in patients without TH (p< 0. 001, respectively). Conclusion The measurement of sGPVI is therefore considered to be important for the diagnosis of TMA. Disclosures: Wada: Mochida pharmaceutical CO., LTD: Suporting GPVI assay Other.

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