Preliminary Evidence for IL-10-Induced ACE2 mRNA Expression in Lung-Derived and Endothelial Cells: Implications for SARS-Cov-2 ARDS Pathogenesis

Angiotensin-converting enzyme 2 (ACE2) is a receptor for the spike protein of SARS-COV-2 that allows viral binding and entry and is expressed on the surface of several pulmonary and non-pulmonary cell types, with induction of a “cytokine storm” upon binding. Other cell types present the receptor and can be infected, including cardiac, renal, intestinal, and endothelial cells. High ACE2 levels protect from inflammation. Despite the relevance of ACE2 levels in COVID-19 pathogenesis, experimental studies to comprehensively address the question of ACE2 regulations are still limited. A relevant observation from the clinic is that, besides the pro-inflammatory cytokines, such as IL-6 and IL-1β, the anti-inflammatory cytokine IL-10 is also elevated in worse prognosis patients. This could represent somehow a “danger signal”, an alarmin from the host organism, given the immuno-regulatory properties of the cytokine. Here, we investigated whether IL-10 could increase ACE2 expression in the lung-derived Calu-3 cell line. We provided preliminary evidence of ACE2 mRNA increase in cells of lung origin in vitro, following IL-10 treatment. Endothelial cell infection by SARS-COV-2 is associated with vasculitis, thromboembolism, and disseminated intravascular coagulation. We confirmed ACE2 expression enhancement by IL-10 treatment also on endothelial cells. The sartans (olmesartan and losartan) showed non-statistically significant ACE2 modulation in Calu-3 and endothelial cells, as compared to untreated control cells. We observed that the antidiabetic biguanide metformin, a putative anti-inflammatory agent, also upregulates ACE2 expression in Calu-3 and endothelial cells. We hypothesized that IL-10 could be a danger signal, and its elevation could possibly represent a feedback mechanism fighting inflammation. Although further confirmatory studies are required, inducing IL-10 upregulation could be clinically relevant in COVID-19-associated acute respiratory distress syndrome (ARDS) and vasculitis, by reinforcing ACE2 levels.

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Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

10.1055/s-0039-1684767 ◽

1979 ◽

Author(s):

S. Korach ◽

D. Ngo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Vascular Endothelial Cells ◽

Intercellular Junctions ◽

Cell Types ◽

Endothelial Damage ◽

Antibody Concentration ◽

High Yields ◽

Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

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Venous and Arterial Endothelial Cells from Human Umbilical Cords: Potential Cell Sources for Cardiovascular Research

International Journal of Molecular Sciences ◽

10.3390/ijms22020978 ◽

2021 ◽

Vol 22 (2) ◽

pp. 978

Author(s):

Skadi Lau ◽

Manfred Gossen ◽

Andreas Lendlein ◽

Friedrich Jung

Keyword(s):

Endothelial Cells ◽

Umbilical Cord ◽

Membrane Integrity ◽

Cell Types ◽

Cell Number ◽

Human Umbilical Cord ◽

Cardiovascular Research ◽

Cardiovascular Devices ◽

Arterial Endothelial Cells

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.

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Histone/protein deacetylases and T-cell immune responses

Blood ◽

10.1182/blood-2011-10-292003 ◽

2012 ◽

Vol 119 (11) ◽

pp. 2443-2451 ◽

Cited By ~ 85

Author(s):

Tatiana Akimova ◽

Ulf H. Beier ◽

Yujie Liu ◽

Liqing Wang ◽

Wayne W. Hancock

Keyword(s):

T Cell ◽

Cell Biology ◽

T Regulatory Cells ◽

Hdac Inhibitors ◽

Experimental Studies ◽

Cell Subset ◽

Histone Protein ◽

Anti Inflammatory

Abstract Clinical and experimental studies show that inhibition of histone/protein deacetylases (HDAC) can have important anti-neoplastic effects through cytotoxic and proapoptotic mechanisms. There are also increasing data from nononcologic settings that HDAC inhibitors (HDACi) can exhibit useful anti-inflammatory effects in vitro and in vivo, unrelated to cytotoxicity or apoptosis. These effects can be cell-, tissue-, or context-dependent and can involve modulation of specific inflammatory signaling pathways as well as epigenetic mechanisms. We review recent advances in the understanding of how HDACi alter immune and inflammatory processes, with a particular focus on the effects of HDACi on T-cell biology, including the activation and functions of conventional T cells and the unique T-cell subset, composed of Foxp3+ T-regulatory cells. Although studies are still needed to tease out details of the various biologic roles of individual HDAC isoforms and their corresponding selective inhibitors, the anti-inflammatory effects of HDACi are already promising and may lead to new therapeutic avenues in transplantation and autoimmune diseases.

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Abstract 116: Slit-Robo Signaling Regulates Lipopolysaccharide-induced Endothelial Inflammation and Expression of Slit/Robo is Dysregulated During Endotoxemia

Circulation Research ◽

10.1161/res.113.suppl_1.a116 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Helong Zhao ◽

Appakkudal Anand ◽

Ramesh Ganju

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Inflammatory Responses ◽

Umbilical Vein ◽

Model Studies ◽

Endothelial Inflammation ◽

Anti Inflammatory ◽

Whole Heart

Abstract Introduction: Lipopolysaccharide (LPS) is one of the critical factors which induce endothelial inflammation during the pathogenesis of atherosclerosis, endocarditis and sepsis shock induced heart injury. The secretory Slit2 protein and its endothelial receptors Robo1 and Robo4 have been shown to regulate mobility and permeability of endothelial cells, which could be functional in regulating LPS induced endothelial inflammation. Hypothesis: We hypothesized that in addition to regulating permeability and migration of endothelial cells, Slit2-Robo1/4 signaling might regulate other LPS-induced endothelial inflammatory responses. Methods and Results: Using Human Umbilical Vein Endothelial Cells (HUVEC) culture, we observed that Slit2 treatment suppressed LPS-induced secretion of pro-inflammatory cytokines (including GM-CSF), cell adhesion molecule upregulation and monocyte (THP-1 cell) adhesion. With siRNA knock down techniques, we further confirmed that this anti-inflammatory effect is mediated by the interaction of Slit2 with its dominant receptor in endothelial cells, Robo4, though the much lesser expressed minor receptor Robo1 is pro-inflammatory. Our signaling studies showed that downstream of Robo4, Slit2 suppressed inflammatory gene expression by inhibiting the Pyk2 - NF-kB pathway following LPS-TLR4 interaction. In addition, Slit2 can induce a positive feedback to its expression and downregulate the pro-inflammatory Robo1 receptor via mediation of miR-218. Moreover, both in in vitro studies using HUVEC and in vivo mouse model studies indicated that LPS also causes endothelial inflammation by downregulating the anti-inflammatory Slit2 and Robo4 and upregulating the pro-inflammatory Robo1 during endotoxemia, especially in mouse arterial endothelial cells and whole heart. Conclusions: Slit2-Robo1/4 signaling is important in regulation of LPS induced endothelial inflammation, and LPS in turn causes inflammation by interfering with the expression of Slit2, Robo1 and Robo4. This implies that Slit2-Robo1/4 is a key regulator of endothelial inflammation and its dysregulation during endotoxemia is a novel mechanism for LPS induced cardiovascular pathogenesis.

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Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation

Frontiers in Immunology ◽

10.3389/fimmu.2021.789142 ◽

2021 ◽

Vol 12 ◽

Author(s):

Solvey Pollmann ◽

David Scharnetzki ◽

Dominique Manikowski ◽

Malte Lenders ◽

Eva Brand

Keyword(s):

Endothelial Cells ◽

Endothelial Dysfunction ◽

Endothelial Function ◽

Fabry Disease ◽

Specific Inhibitor ◽

Small Vessel ◽

Endothelial Glycocalyx ◽

Enzyme Replacement ◽

Anti Inflammatory

Fabry disease (FD) is an X-linked multisystemic lysosomal storage disease due to a deficiency of α-galactosidase A (GLA/AGAL). Progressive cellular accumulation of the AGAL substrate globotriaosylceramide (Gb3) leads to endothelial dysfunction. Here, we analyzed endothelial function in vivo and in vitro in an AGAL-deficient genetic background to identify the processes underlying this small vessel disease. Arterial stiffness and endothelial function was prospectively measured in five males carrying GLA variants (control) and 22 FD patients under therapy. AGAL-deficient endothelial cells (EA.hy926) and monocytes (THP1) were used to analyze endothelial glycocalyx structure, function, and underlying inflammatory signals. Glycocalyx thickness and small vessel function improved significantly over time (p<0.05) in patients treated with enzyme replacement therapy (ERT, n=16) and chaperones (n=6). AGAL-deficient endothelial cells showed reduced glycocalyx and increased monocyte adhesion (p<0.05). In addition, increased expression of angiopoietin-2, heparanase and NF-κB was detected (all p<0.05). Incubation of wild-type endothelial cells with pathological globotriaosylsphingosine concentrations resulted in comparable findings. Treatment of AGAL-deficient cells with recombinant AGAL (p<0.01), heparin (p<0.01), anti-inflammatory (p<0.001) and antioxidant drugs (p<0.05), and a specific inhibitor (razuprotafib) of angiopoietin-1 receptor (Tie2) (p<0.05) improved glycocalyx structure and endothelial function in vitro. We conclude that chronic inflammation, including the release of heparanases, appears to be responsible for the degradation of the endothelial glycocalyx and may explain the endothelial dysfunction in FD. This process is partially reversible by FD-specific and anti-inflammatory treatment, such as targeted protective Tie2 treatment.

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Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

Cell Transplantation ◽

10.3727/000000002783985837 ◽

2002 ◽

Vol 11 (4) ◽

pp. 369-377 ◽

Cited By ~ 24

Author(s):

Makarand V. Risbud ◽

Erdal Karamuk ◽

René Moser ◽

Joerg Mayer

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Three Dimensional ◽

Mesh Size ◽

Cell Types ◽

Cell Number ◽

Human Umbilical Vein ◽

Hydrogel Matrix ◽

Umbilical Vein Endothelial Cells

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan–collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require pre-coating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

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Abstract 11710: Poloxamer 188 Protects Coronary Artery Endothelial Cells After Extended Hypoxia and Reoxygenation in an in vitro Model of Simulated Ischemia/Reperfusion Injury

Circulation ◽

10.1161/circ.144.suppl_2.11710 ◽

2021 ◽

Vol 144 (Suppl_2) ◽

Author(s):

zhu li ◽

Matthew J Hampton ◽

Matthew B Barajas ◽

Matthias L Riess

Keyword(s):

Endothelial Cells ◽

Coronary Artery ◽

Myocardial Ischemia ◽

Ischemia Reperfusion Injury ◽

Calcium Influx ◽

Ischemia Reperfusion ◽

Cell Types ◽

Membrane Function ◽

Ldh Release

Reperfusion restores blood flow after myocardial ischemia but can cause additional cellular injury by the sudden reintroduction of oxygen and nutrients. There is still no effective remedy for myocardial ischemia/reperfusion (IR) injury. Our previous study using cardiomyocytes (CMs) found that, after 3 hrs hypoxia followed by 2 hrs reoxygenation, viability decreased, and release of lactate dehydrogenase (LDH), calcium influx, membrane leakage (insertion of fluorescent probe FM1-43) significantly increased, indicating that cell membrane function was negatively affected. This was attenuated by the triblock copolymer Poloxamer (P)188. Here, we first hypothesized that endothelial cells are also susceptible to simulated IR injury, albeit requiring longer hypoxia times. We further hypothesized that P188 can also attenuate simulated IR injury in endothelial cells when given upon reoxygenation. Mouse coronary artery endothelial cells (MCAECs) were exposed to different durations of hypoxia (2, 3, 12 and 24 hrs) in serum- and glucose-free media +/- reoxygenation for 2 hrs in regular media. P188 was administered upon reoxygenation at 0, 100, 300 or 1,000 μM in experiments of 24 hrs hypoxia / 2 hrs reoxygenation. LDH release was measured and compared to appropriately timed normoxic control experiments. Reoxygenation and hypoxia times significantly longer than 3 hrs were required to elicit sufficient injury (panel A). When P188 was given upon reoxygenation after 24 hrs hypoxia, it dose-dependently attenuated LDH release (panel B). These findings contrast to the higher susceptibility of CMs to IR injury that only allowed shorter hypoxia durations. They also confirm a protective effect of P188 on the endothelium, not just on CMs. These findings have important implications for co-culture models with MCAECs and CMs to elucidate the interplay of both cell types on each other when studying mechanisms of cardioprotective strategies and compounds like P188.

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TMOD-21. A NOVEL IN-VITRO METHOD TO MODEL MACROPHAGES IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.882 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi220-vi220

Author(s):

Hasan Alrefai ◽

Andee Beierle ◽

Lauren Nassour ◽

Nicholas Eustace ◽

Zeel Patel ◽

...

Keyword(s):

Cell Types ◽

In Vitro Models ◽

Tumor Initiating Cells ◽

Serum Free ◽

Brain Tumor Initiating Cells ◽

Multiple Cell ◽

Anti Inflammatory ◽

Free Media ◽

Inflammatory Macrophages

Abstract BACKGROUND The GBM tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemoresistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Others have addressed this problem by employing 3D bioprinting to incorporate astrocytes and macrophages in an extracellular matrix; however, they used serum-containing media and classically polarized anti-inflammatory macrophages. Serum has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, highlighting the importance of excluding it from these models. Additionally, tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. METHODS THP-1 monocytes and normal human astrocytes (NHAs) were transitioned into serum-free HL-1 and neurobasal-based media, respectively. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft(PDX) lines, using a transwell insert. CD206 expression was used to validate polarization and a cytokine array was used to characterize the cells. RESULTS There was no difference in proliferation rates at 72 hours for THP-1 monocytes grown in serum-free HL-1 media compared to serum-containing RPMI 1640 (p > 0.95). Macrophages polarized via transwell inserts expressed the lymphocyte chemoattractant protein, CCL2, whereas resting(M0), pro-inflammatory(M1), and anti-inflammatory(M2) macrophages did not. Additionally, these macrophages expressed more CXCL1 and IL-1ß relative to M1 macrophages. We have also demonstrated a method to maintain a tri-culture model of GBM PDX cells, NHAs, and TAMs in a serum-free media that supports the growth/maintenance of all cell types. CONCLUSIONS We have demonstrated a novel method by which we can polarize macrophages towards a tumor-supportive phenotype that differs in cytokine expression from traditionally polarized macrophages. This higher-fidelity method of modeling TAMs in GBM can aid in the development of targeted therapeutics that may one day enter the clinic in hopes of improving outcomes in GBM.

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Identification of a Kavain Analog with Efficient Anti-inflammatory Effects

Scientific Reports ◽

10.1038/s41598-019-49383-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Olivier Huck ◽

Xiaxian Han ◽

Hannah Mulhall ◽

Iryna Gumenchuk ◽

Bin Cai ◽

...

Keyword(s):

Joint Destruction ◽

Cell Types ◽

Interferon Γ ◽

Piper Methysticum ◽

Tnf Α ◽

Osteoclast Activation ◽

Anti Inflammatory ◽

Soft Tissue Inflammation

Abstract Kavain, a compound derived from Piper methysticum, has demonstrated anti-inflammatory properties. To optimize its drug properties, identification and development of new kavain-derived compounds was undertaken. A focused library of analogs was synthesized and their effects on Porphyromonas gingivalis (P. gingivalis) elicited inflammation were evaluated in vitro and in vivo. The library contained cyclohexenones (5,5-dimethyl substituted cyclohexenones) substituted with a benzoate derivative at the 3-position of the cyclohexanone. The most promising analog identifed was a methylated derivative of kavain, Kava-205Me (5,5-dimethyl-3-oxocyclohex-1-en-1-yl 4-methylbenzoate.) In an in vitro assay of anti-inflammatory effects, murine macrophages (BMM) and THP-1 cells were infected with P. gingivalis (MOI = 20:1) and a panel of cytokines were measured. Both cell types treated with Kava-205Me (10 to 200 μg/ml) showed significantly and dose-dependently reduced TNF-α secretion induced by P. gingivalis. In BMM, Kava-205Me also reduced secretion of other cytokines involved in the early phase of inflammation, including IL-12, eotaxin, RANTES, IL-10 and interferon-γ (p < 0.05). In vivo, in an acute model of P. gingivalis-induced calvarial destruction, administration of Kava-205Me significantly improved the rate of healing associated with reduced soft tissue inflammation and osteoclast activation. In an infective arthritis murine model induced by injection of collagen-antibody (ArthriomAb) + P. gingivalis, administration of Kava-205Me was able to reduce efficiently paw swelling and joint destruction. These results highlight the strong anti-inflammatory properties of Kava-205Me and strengthen the interest of testing such compounds in the management of P. gingivalis elicited inflammation, especially in the management of periodontitis.

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Levosimendan exerts anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro

Thrombosis and Haemostasis ◽

10.1160/th14-06-0549 ◽

2015 ◽

Vol 113 (02) ◽

pp. 350-362 ◽

Cited By ~ 13

Author(s):

Konstantin A. Krychtiuk ◽

Lukas Watzke ◽

Christoph Kaun ◽

Elisabeth Buchberger ◽

Renate Hofer-Warbinek ◽

...

Keyword(s):

Myocardial Infarction ◽

Endothelial Cells ◽

Cardiac Myocytes ◽

Acute Decompensated Heart Failure ◽

Umbilical Vein ◽

Polymorphonuclear Neutrophils ◽

Intercellular Adhesion Molecule ◽

Induced Expression ◽

Anti Inflammatory

SummaryLevosimendan is a positive inotropic drug for the treatment of acute decompensated heart failure (HF). Clinical trials showed that levosimendan was particularly effective in HF due to myocardial infarction. Myocardial necrosis induces a strong inflammatory response, involving chemoattractants guiding polymorphonuclear neutrophils (PMN) into the infarcted myocardial tissue. Our aim was to examine whether levosimendan exhibits anti-inflammatory effects on human adult cardiac myocytes (HACM) and human heart microvascular endothelial cells (HHMEC). Cardiac myocytes and endothelial cells were stimulated with interleukin-1β (IL)-1β (200 U/ml) and treated with levosimendan (0.1–10 μM) for 2–48 hours. IL-1β strongly induced expression of IL-6 and IL-8 in HACM and E-selectin and intercellular adhesion molecule-1 (ICAM-1) in HHMEC and human umbilical vein endothelial cells (HUVEC). Treatment with levosimendan strongly attenuated IL-1β-induced expression of IL-6 and IL-8 in HACM as well as E-selectin and ICAM-1 in ECs. Levosimendan treatment further reduced adhesion of PMN to activated endothelial cells under both static and flow conditions by approximately 50 %. Incubation with 5-hydroxydecanoic acid, a selective blocker of mitochondrial ATP-dependent potassium channels, partly abolished the above seen anti-inflammatory effects. Additionally, levosimendan strongly diminished IL-1β-induced reactive oxygen species and nuclear factor-κB (NF-κB) activity through inhibition of S536 phosphorylation. In conclusion, levosimendan exhibits anti-inflammatory effects on cardiac myocytes and endothelial cells in vitro. These findings could explain, at least in part, the beneficial effects of levosimendan after myocardial infarction.

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