new-islet-model-for-3d-study-endothelial-cell-interactions-relevance

New Islet Model for 3D Study of Endothelial and &#946; Cell Interactions: Relevance for the Screening of Cytoprotective Agents

This protocol details an optimized method for the production of small stable spheroids, their culture, and 3D imaging, for the study of the endothelial ...

University of Strasbourg

Besides its well-known functions in hemostasis, thrombin plays a role in various non-hemostatic biological and pathophysiologic processes. We examined the potential of thrombin to promote premature atrial endothelial cells (ECs) senescence.

Thrombin Induces Angiotensin II-Mediated Senescence in Atrial Endothelial Cells: Impact on Pro-Remodeling Patterns.

Whilst the link between aging and thrombogenicity in atrial fibrillation (AF) is well established, the cellular underlying mechanisms are unknown. In AF, the role of senescence in tissue remodeling and prothrombotic state remains unclear.

Atrial Fibrillation Progression Is Associated with Cell Senescence Burden as Determined by p53 and p16 Expression.

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor -4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of -AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by -AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.

The Metabolism of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase Is Protective against the Development of Vascular Calcification.

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39 B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39 plasmablasts and adenosine accumulation. Our study reveals CD39 plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Sepsis expands a CD39 plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity.

The COVID-19 pandemic extended all around the world causing millions of deaths. In addition to acute respiratory distress syndrome, many patients with severe COVID-19 develop thromboembolic complications associated to multiorgan failure and death. Here, we review evidence for the contribution of neutrophils, platelets, and extracellular vesicles (EVs) to the thromboinflammatory process in COVID-19. We discuss how the immune system, influenced by pro-inflammatory molecules, EVs, and neutrophil extracellular traps (NETs), can be caught out in patients with severe outcomes. We highlight how the deficient regulation of the innate immune system favors platelet activation and induces a vicious cycle amplifying an immunothrombogenic environment associated with platelet/NET interactions. In light of these considerations, we discuss potential therapeutic strategies underlining the modulation of purinergic signaling as an interesting target.

Role of neutrophils, platelets, and extracellular vesicles and their interactions in COVID-19-associated thrombopathy.

ABCC6 deficiency and bone loss: A double benefit of etidronate for patient presenting with pseudoxanthoma elasticum?

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na (Na) channels. In arteries, Na channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of Na channels in vascular tone using TTX. We first characterized the expression of Na channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which  and  were the most abundant transcripts, suggesting that murine vascular Na channels consist of Na1.2 channel subtype with Naβ1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks Na channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.

Tetrodotoxin Decreases the Contractility of Mesenteric Arteries, Revealing the Contribution of Voltage-Gated Na Channels in Vascular Tone Regulation.

NTPDase1/CD39, the major vascular ectonucleotidase, exerts thrombo-immunoregulatory function by controlling endothelial P2 receptor activation. Despite the well-described release of ATP from endothelial cells, few data are available regarding the potential role of CD39 as a regulator of arterial diameter. We thus investigated the contribution of CD39 in short-term diameter adaptation and long-term arterial remodeling in response to flow using  male mice. Compared to wild-type littermates, endothelial-dependent relaxation was modified in  mice. Specifically, the vasorelaxation in response to ATP was potentiated in both conductance (aorta) and small resistance (mesenteric and coronary) arteries. By contrast, the relaxing responses to acetylcholine were supra-normalized in thoracic aortas while decreased in resistance arteries from  mice. Acute flow-mediated dilation, measured via pressure myography, was dramatically diminished and outward remodeling induced by in vivo chronic increased shear stress was altered in the mesenteric resistance arteries isolated from  mice compared to wild-types. Finally, changes in vascular reactivity in  mice were also evidenced by a decrease in the coronary output measured in isolated perfused hearts compared to the wild-type mice. Our results highlight a key regulatory role for purinergic signaling and CD39 in endothelium-dependent short- and long-term arterial diameter adaptation to increased flow.

NTPDase1/CD39 Ectonucleotidase Is Necessary for Normal Arterial Diameter Adaptation to Flow.

Physiological calcification of soft tissues is a common occurrence in aging and various acquired and inherited disorders. ABCC6 sequence variations cause the calcification phenotype of pseudoxanthoma elasticum (PXE) as well as some cases of generalized arterial calcification of infancy, which is otherwise caused by defective ENPP1. ABCC6 is primarily expressed in the liver, which has given the impression that the liver is central to the pathophysiology of PXE/generalized arterial calcification of infancy. The emergence of inflammation as a contributor to the calcification in PXE suggested that peripheral tissues play a larger role than expected. In this study, we investigated whether bone marrow-derived ABCC6 contributes to the calcification in PXE. In Abcc6 mice, we observed prevalent mineralization in several lymph nodes and surrounding connective tissues and an extensive network of lymphatic vessels within vibrissae, a calcified tissue in Abcc6 mice. Furthermore, we found evidence of lymphangiogenesis in patients with PXE and mouse skin, suggesting an inflammatory process. Finally, restoring wild-type bone marrow in Abcc6 mice produced a significant reduction of calcification, suggesting that the liver alone is not sufficient to fully inhibit mineralization. With evidence that ABCC6 is expressed in lymphocytes, we suggest that the adaptative immune system and inflammation largely contribute to the calcification in PXE/generalized arterial calcification of infancy.

Bone Marrow-Derived ABCC6 Is an Essential Regulator of Ectopic Calcification In Pseudoxanthoma Elasticum.

Pseudoxanthoma Elasticum (PXE) is an inherited disease characterized by elastic fiber calcification in the eyes, the skin and the cardiovascular system. PXE results from mutations in  that encodes an ABC transporter primarily expressed in the liver and kidneys. It took nearly 15 years after identifying the gene to better understand the etiology of PXE. ABCC6 function facilitates the efflux of ATP, which is sequentially hydrolyzed by the ectonucleotidases ENPP1 and CD73 into pyrophosphate (PPi) and adenosine, both inhibitors of calcification. PXE, together with General Arterial Calcification of Infancy (GACI caused by  mutations) as well as Calcification of Joints and Arteries (CALJA caused by /CD73 mutations), forms a disease continuum with overlapping phenotypes and shares steps of the same molecular pathway. The explanation of these phenotypes place ABCC6 as an upstream regulator of a purinergic pathway (ABCC6 → ENPP1 → CD73 → TNAP) that notably inhibits mineralization by maintaining a physiological Pi/PPi ratio in connective tissues. Based on a review of the literature and our recent experimental data, we suggest that PXE (and GACI/CALJA) be considered as an authentic "purinergic disease". In this article, we recapitulate the pathobiology of PXE and review molecular and physiological data showing that, beyond PPi deficiency and ectopic calcification, PXE is associated with wide and complex alterations of purinergic systems. Finally, we speculate on the future prospects regarding purinergic signaling and other aspects of this disease.

The Purinergic Nature of Pseudoxanthoma Elasticum.

Pathological tissues release a variety of factors, including extracellular vesicles (EVs) shed by activated or apoptotic cells. EVs trapped within the native pathological valves may act as key mediators of valve thrombosis. Human aortic stenosis EVs promote activation of valvular endothelial cells, leading to endothelial dysfunction, and proadhesive and procoagulant responses.

Human Aortic Stenotic Valve-Derived Extracellular Vesicles Induce Endothelial Dysfunction and Thrombogenicity Through AT1R/NADPH Oxidases/SGLT2 Pro-Oxidant Pathway.

Since its emergence in 2019, the study of biomarkers in COVID-19 has focused on predicting the course of this potentially fatal disease. The eosinophil-to-lymphocyte ratio (ELR) appears to be a new indicator of systemic inflammation, morbidity, and mortality in inflammatory, allergic, and cancer. The aim of our study was to determine the prognostic value of ELR and its early variation in predicting in-hospital mortality and severity in emergency department (ED) patients with SARS-CoV-2 infection. Between March 1 and April 30, 2020, we conducted a multicenter, retrospective study in 6 major hospitals in northeastern France. The cohort included patients with a confirmed diagnosis of SARS-CoV-2 infection (moderate-to-severe disease) hospitalized after admission to the ED. A total of 1,035 patients were included in this study. The ELR at 24 h (odds ratio: 0.0054; 95% confidence interval: 0.0001 to 0.2523; P = 0.008) was associated with severity of infection. The only biochemical factor significantly associated with mortality was ΔELR at 24 h (i.e. the difference between ELR values at 24 h and at admission) (odds ratio: 0.0305; 95% confidence interval: 0.0026 to 0.3626; P = 0.006) in univariate analysis. The best ELR threshold for predicting severity of infection was found with the ELR at 24 h with a result of 0.0007 (sensitivity 63.8%, specificity 61%), and for predicting mortality, ΔELR at 24 h found significant values with a threshold of -0.013 (sensitivity 77.4%, specificity 8.48%). In conclusion, ELR at 24 h after ED admission may be a potentially useful biomarker for early prediction of severity of SARS-CoV-2 infection, but its isolated use remains limited for mortality prediction.

Eonisophil-to-lymphocyte ratio and early variation as predictors of severity and in-hospital mortality in patients admitted to the emergency department for SARS-CoV-2 infection.

While the hydrolase activity of soluble epoxide hydrolase (sEH) reduces vascular calcification, it is not known whether the phosphatase activity of sEH (sEH-P) is also involved. Pharmacological and genetic inhibition of sEH-P reduced the increased calcium deposition in rat aortic rings cultured under high-phosphate conditions. This was associated with decreased mRNA expression of the osteochondrogenic markers Msx2 and Sox9. Deendothelialization of the aortic rings abolished this anticalcifying effect, while the calcification of human aortic smooth muscle cells was unaffected by sEH-P inhibition, suggesting a predominant role of the endothelium. Endothelial NO release did not appear to contribute, but an increased level of the calcification inhibitor pyrophosphate anions (PPi) was observed in the culture supernatant of aortic rings when sEH-P was inhibited. In vitro experiments demonstrated that PPi is a substrate of sEH-P, and that inhibiting sEH-P prevented the high-phosphate induced decrease of PPi in human aortic endothelial cells. Furthermore, the aortic calcification related to chronic kidney disease induced by subtotal nephrectomy was reduced in sEH-P-deficient rats compared to wild-type rats. This was associated with an improvement in flow-induced isolated mesenteric artery dilatation and a reduction of cardiac hypertrophy and fibrosis. Vascular calcification is regulated by sEH-P through the metabolism of endothelial PPi. The prevention of vascular calcification, together with the reduction in vascular dysfunction and cardiac remodeling, suggests that inhibiting sEH-P may help to prevent the cardiovascular complications associated with chronic kidney disease.

The phosphatase activity of soluble epoxide hydrolase regulates vascular calcification through the metabolism of pyrophosphate anions.

Ectonucleotidases regulate extracellular purine signaling and play a key role in inflammation and thrombosis, two major components of COVID-19 pathophysiology. We investigated the enzymatic activities of CD39 (NTPDase1) and CD73 (ecto-5’-nucleotidase) as potential biomarkers of disease severity. Patients with RT-PCR-confirmed COVID-19 were enrolled between August 1st, 2020, and August 1st, 2021, and stratified into mild to moderate (MM,  = 55) or severe to critical (SC,  = 30) groups according to WHO criteria. Serum samples were collected on days 1, 3, and 7 following Emergency Department (ED) admission. CD39 and CD73 activities were measured by HPLC using etheno-fluorescent nucleotide substrates and compared to healthy volunteers (HV,  = 30) matched for age and sex. Serum CD73 activity was significantly elevated in COVID-19 patients compared to HV (MM: 76 ± 9.4; SC: 99 ± 21 vs. HV: 25 ± 2.7 pmol/min/µL;  < 0.0001), particularly in patients requiring ≥ 6 L/min oxygen ( = 0.005) or with a respiratory rate > 20/min ( = 0.02). CD73 activity remained elevated throughout hospitalization. Conversely, febrile patients (≥ 38 °C) and those with CRP ≥ 100 mg/L exhibited significantly lower CD73 activity ( = 0.023 and  = 0.004, respectively). CD39 activity was undetectable in 28.2% of patients and showed no association with disease severity or outcome. The lowest CD73 levels were observed in patients requiring oxygen therapy for ≥ 30 days and in those who died before day 7. Serum CD73 activity is associated with hypoxemia and oxygen requirements upon ED admission. Conversely, low CD73 activity reflects a hyperinflammatory state and worse outcomes, supporting its potential use in biomarker-based risk stratification for COVID-19.

Article	Year
Thrombin Induces Angiotensin II-Mediated Senescence in Atrial Endothelial Cells: Impact on Pro-Remodeling Patterns. Journal of clinical medicine\| PubMed ID: 31581517	2019
Atrial Fibrillation Progression Is Associated with Cell Senescence Burden as Determined by p53 and p16 Expression. Journal of clinical medicine\| PubMed ID: 31878008	2019
The Metabolism of Epoxyeicosatrienoic Acids by Soluble Epoxide Hydrolase Is Protective against the Development of Vascular Calcification. International journal of molecular sciences\| PubMed ID: 32560362	2020
Sepsis expands a CD39 plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity. Immunity\| PubMed ID: 34473957	2021
Role of neutrophils, platelets, and extracellular vesicles and their interactions in COVID-19-associated thrombopathy. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 34672094	2022
ABCC6 deficiency and bone loss: A double benefit of etidronate for patient presenting with pseudoxanthoma elasticum? Experimental dermatology\| PubMed ID: 35771123	2022
Tetrodotoxin Decreases the Contractility of Mesenteric Arteries, Revealing the Contribution of Voltage-Gated Na Channels in Vascular Tone Regulation. Marine drugs\| PubMed ID: 36976245	2023
NTPDase1/CD39 Ectonucleotidase Is Necessary for Normal Arterial Diameter Adaptation to Flow. International journal of molecular sciences\| PubMed ID: 37894719	2023
Bone Marrow-Derived ABCC6 Is an Essential Regulator of Ectopic Calcification In Pseudoxanthoma Elasticum. The Journal of investigative dermatology\| PubMed ID: 38367909	2024
The Purinergic Nature of Pseudoxanthoma Elasticum. Biology\| PubMed ID: 38392293	2024
Human Aortic Stenotic Valve-Derived Extracellular Vesicles Induce Endothelial Dysfunction and Thrombogenicity Through AT1R/NADPH Oxidases/SGLT2 Pro-Oxidant Pathway. JACC. Basic to translational science\| PubMed ID: 39170957	2024
Eonisophil-to-lymphocyte ratio and early variation as predictors of severity and in-hospital mortality in patients admitted to the emergency department for SARS-CoV-2 infection. Journal of leukocyte biology\| PubMed ID: 40679357	2025
The phosphatase activity of soluble epoxide hydrolase regulates vascular calcification through the metabolism of pyrophosphate anions. Cell death & disease\| PubMed ID: 41455787	2025
Serum CD73 activity as a biomarker of hypoxemia in COVID-19 patients. Scientific reports\| PubMed ID: 41748828	2026

Gilles Kauffenstein

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