Abstract P808: The Main Peptide Ssubstrates of Neurolysin Enhance Brain Microvascular Permeability in a Human in vitro Model

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Abraham Al-Ahmad ◽  
Vardan T Karamyan
Keyword(s):  
Direct Action ◽  
Induced Pluripotent Stem Cell ◽  
Microvascular Permeability ◽  
Dependent Manner ◽  
Direct Effects ◽  
Edema Formation ◽  
Concentration Dependent Manner ◽  
Therapeutic Implications ◽  
Bbb Permeability

Increased brain microvascular permeability and disruption of blood-brain barrier (BBB) function are among hallmarks of ischemic stroke. Recently, peptidase neurlysin (Nln) has been identified as a compensatory and cerebroprotective mechanism in the post-stroke brain that functions to process a diverse group of extracellular neuropeptides, including bradykinin (BK), neurotensin (NT) and substance P (SP). A number of studies suggest involvement of BK, NT and SP in BBB impairment and edema formation after stroke, however there is paucity of data in regards to the direct effects of these peptides on the brain microvascular endothelial cells (BMECs) and BBB. The purpose of this study was to evaluate the direct effects of BK, NT and SP on permeability of BBB in an in vitro model based on human, induced pluripotent stem cell (iPSC)-derived BMECs. Our data indicate that all three peptides increase BBB permeability in a concentration-dependent manner in an in vitro model formed from two different iPSC lines (CTR90F and CTR65M) and widely used hCMEC/D3 human BMECs. Combination of BK, NT and SP at a sub-effective concentration also resulted in increased BBB permeability in the iPSC-derived model. Furthermore, we observed abrogation of BK, NT and SP effects with pretreatment of pharmacological blockers targeting their specific receptors or in presence of recombinant neurolysin (Nln). This is the first experimental study to document increased permeability of BBB in response to direct action of NT in an in vitro model. In addition, our study confirms the expected but not well-documented, direct effect of SP on BBB permeability and adds to the well-recognized actions of BK on BBB. Lastly, we demonstrate that peptidase Nln can neutralize the effects of these peptides on BBB, suggesting potential therapeutic implications.

Endothelium-independent Vasoconstricting and Vasodilating Actions of Halothane on Rat Mesenteric Resistance Blood Vessels 

1995 ◽  
Vol 82 (1) ◽  
pp. 221-235 ◽  
Author(s):  
W. A. Boyle ◽  
G. M. Maher
Keyword(s):  
Blood Vessels ◽  
Vascular Tissue ◽  
Cardiovascular Effects ◽  
Image Analysis System ◽  
Dependent Manner ◽  
Direct Effects ◽  
Concentration Dependent Manner ◽  
Rat Mesenteric Artery

Background Whether volatile anesthetics produce changes in vascular resistance and blood flow because of direct effects on vascular tissue is unclear. Direct vasoconstricting and vasodilating actions have been demonstrated in isolated conductance arteries in vitro, but there is little information regarding direct effects on the small vessels that mediate resistance and flow changes in vivo. Methods We investigated the actions of halothane on 50-200 microM branches of the rat mesenteric artery that were cannulated and studied in vitro. The vessels were pressurized to 60 mmHg, and vascular dimensions were continuously monitored using a computer-based real-time image analysis system. The vessel bath was perfused with HCO3(-)-buffered saline (37 degrees C) equilibrated with 95% O2/5% CO2 (+/- halothane). The vascular endothelium was mechanically removed before cannulation in some vessels. Results In unstimulated vessels, halothane had a concentration-dependent vasoconstricting action (EC50 = 0.45 mM approximately 1.5 vol% at 37 degrees C) that was largely transient and was similar to that produced by caffeine. Both halothane and caffeine constrictions were unaffected by bath [Ca2+], nifedipine (1 microM) or Cd2+ (100 microM) and were abolished by ryanodine (10 microM). In addition, caffeine responses were attenuated by halothane in a concentration-dependent manner (EC50 = 1.6 mM). In vessels preconstricted with KCl (40 mM) or phenylephrine (10(-6) M), halothane produced transient constriction followed by concentration-dependent vasodilation. Ryanodine, which abolished halothane constrictions, had little effect on the amplitude of KCl- or phenylephrine-induced constrictions or the vasodilating action of halothane. Removal of the endothelium likewise had little effect on the vasoconstricting or the vasodilating actions of halothane in unstimulated, KCl- or phenylephrine-constricted vessels. Halothane completely relaxed KCl and phenylephrine constrictions with EC50 values of 0.36 mM (1.2% at 37 degrees C) and 0.75 mM (2.5%), respectively, in intact vessels before ryanodine; 0.25 mM (0.8%) and 0.59 mM (1.9%) in intact vessels after ryanodine; and 0.52 mM (1.7%) and 0.67 mM (2.2%) in endothelium-denuded vessels. Conclusions Halothane has endothelium-independent vasoconstricting and vasodilating actions in isolated mesenteric resistance blood vessels. The vasoconstricting action appears to involve halothane-induced Ca2+ release from caffeine/ryanodine-sensitive intracellular store(s). The vasodilating action in phenylephrine- or KC1-constricted vessels is independent of the Ca(2+)-releasing action and most likely involves an effect(s) on sarcolemmal-dependent Ca2+ signaling (e.g., extracellular Ca2+ influx) and/or Ca2+ activation of contractile proteins. The magnitude of both the vasoconstricting and the vasodilating actions of halothane in these vessels at clinically relevant concentrations suggests these direct actions contribute to the overall cardiovascular effects of halothane in vivo.

scholarly journals Anesthesia triggers drug delivery to experimental glioma in mice by hijacking caveolar transport

2021 ◽  
Author(s):  
Lena Spieth ◽  
Stefan A Berghoff ◽  
Sina K Stumpf ◽  
Jan Winchenbach ◽  
Thomas Michaelis ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Endothelial Cells ◽  
Membrane Lipid ◽  
Therapeutic Drug ◽  
Dependent Manner ◽  
General Anesthetics ◽  
Concentration Dependent Manner ◽  
Simultaneous Exposure ◽  
Therapeutic Implications ◽  
Bbb Permeability

Abstract Background Pharmaceutical intervention in the CNS is hampered by the shielding function of the blood-brain barrier (BBB). To induce clinical anesthesia, general anesthetics such as isoflurane readily penetrate the BBB. Here, we investigated whether isoflurane can be utilized for therapeutic drug delivery. Methods Barrier function in primary endothelial cells was evaluated by transepithelial/transendothelial electrical resistance, and nanoscale STED and SRRF microscopy. In mice, BBB permeability was quantified by extravasation of several fluorescent tracers. Mouse models including the GL261 glioma model were evaluated by MRI, immunohistochemistry, electron microscopy, western blot, and expression analysis. Results Isoflurane enhances BBB permeability in a time- and concentration-dependent manner. We demonstrate that, mechanistically, isoflurane disturbs the organization of membrane lipid nanodomains and triggers caveolar transport in brain endothelial cells. BBB tightness re-establishes directly after termination of anesthesia, providing a defined window for drug delivery. In a therapeutic glioblastoma trial in mice, simultaneous exposure to isoflurane and cytotoxic agent improves efficacy of chemotherapy. Conclusions Combination therapy, involving isoflurane-mediated BBB permeation with drug administration has far-reaching therapeutic implications for CNS malignancies.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Improved Method for Obtaining of Arctigenin from Arctium Lappa L. and its Antiproliferative Effect on Human Hepatocarcinoma HepG2 Cells

2020 ◽  
Vol 16 (3) ◽  
pp. 358-362
Author(s):  
Renan S. Teixeira ◽  
Paulo H.D. Carvalho ◽  
Jair A.K. Aguiar ◽  
Valquíria P. Medeiros ◽  
Ademar A. Da Silva Filho ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Crude Extract ◽  
Hepg2 Cells ◽  
Liver Carcinoma ◽  
Adhesion Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Arctium Lappa ◽  
Nih 3T3

Background: Arctigenin is a lignan found in Arctium lappa L. (Asteraceae) that displays anti-inflammatory activities. Previous studies showed that the crude extract of A. Lappa has antitumor activity in human liver carcinoma, lung and stomach cancer cells. The aim of this study was to obtain arctigenin from A. lappa L., as well as to evaluate its antiproliferative effects in cells of liver carcinoma (HepG2) and fibroblasts (NIH/3T3). Methods: Arctigenin was obtained from the hydrolysis of arctiin, which was isolated from the crude extract of A. lappa. The effects of arctigenin and arctiin on HepG2 cell viability and cell adhesion were analyzed by MTT method. Adhesion assay was also carried out to evaluate the antitumor activity. Results: Our results showed that the analytical process to obtain arctigenin was fast and easy. In vitro experiments showed that arctigenin (107-269 μM) decreased HepG2 cells viability and did not cause cytotoxicity on NIH/3T3 cells. Arctigenin (27-269 μM) demonstrated anti-adhesion in HepG2 cells in a concentration-dependent manner, when compared with control. Conclusion: These results suggest a promising pharmacological activity for arctigenin as an antiproliferative compound.

scholarly journals Human Neuronal Cell Lines as An In Vitro Toxicological Tool for the Evaluation of Novel Psychoactive Substances

2021 ◽  
Vol 22 (13) ◽  
pp. 6785
Author(s):  
Valeria Sogos ◽  
Paola Caria ◽  
Clara Porcedda ◽  
Rafaela Mostallino ◽  
Franca Piras ◽  
...  
Keyword(s):  
Cell Death ◽  
Neuronal Cell ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Novel Psychoactive Substances ◽  
Psychoactive Substances ◽  
Concentration Dependent Manner ◽  
Mechanisms Of Toxicity ◽  
Neuronal Cell Lines

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4′-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.

scholarly journals The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide, an In Vitro Study

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3886
Author(s):  
Stefania Sut ◽  
Irene Ferrarese ◽  
Maria Giovanna Lupo ◽  
Nicola De Zordi ◽  
Elisa Tripicchio ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Hepatoma Cell ◽  
Density Lipoprotein ◽  
In Vitro Study ◽  
Volatile Constituent ◽  
Dependent Manner ◽  
Human Hepatoma Cell ◽  
Concentration Dependent Manner

In the present study the ability of supercritical carbon dioxide (SCO2) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO2 extracts, one oil (ML-SCO2) and a semisolid (MW-SCO2), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO2 was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO2 reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO2 (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO2 extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.

scholarly journals Cannabidiol (CBD) Alters the Functionality of Neutrophils (PMN). Implications in the Refractory Epilepsy Treatment

2021 ◽  
Vol 14 (3) ◽  
pp. 220
Author(s):  
Claudia Taborda Gómez ◽  
Fabiana Lairion ◽  
Marisa Repetto ◽  
Miren Ettcheto ◽  
Amalia Merelli ◽  
...  
Keyword(s):  
Refractory Epilepsy ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Nuclear Condensation ◽  
Concentration Dependent Manner ◽  
Superoxide Anion Production ◽  
Excretory Function ◽  
Psychoactive Effects ◽  
Stress Damage

Cannabidiol (CBD), a lipophilic cannabinoid compound without psychoactive effects, has emerged as adjuvant of anti-epileptic drugs (AEDs) in the treatment of refractory epilepsy (RE), decreasing the severity and/or frequency of seizures. CBD is considered a multitarget drug that could act throughout the canonical endocannabinoid receptors (CB1-CB2) or multiple non-canonical pathways. Despite the fact that the CBD mechanism in RE is still unknown, experiments carried out in our laboratory showed that CBD has an inhibitory role on P-glycoprotein excretory function, highly related to RE. Since CB2 is expressed mainly in the immune cells, we hypothesized that CBD treatment could alter the activity of polymorphonuclear neutrophils (PMNs) in a similar way that it does with microglia/macrophages and others circulating leukocytes. In vitro, CBD induced PMN cytoplasmatic vacuolization and proapoptotic nuclear condensation, associated with a significantly decreased viability in a concentration-dependent manner, while low CBD concentration decreased PMN viability in a time-dependent manner. At a functional level, CBD reduced the chemotaxis and oxygen consumption of PMNs related with superoxide anion production, while the singlet oxygen level was increased suggesting oxidative stress damage. These results are in line with the well-known CBD anti-inflammatory effect and support a potential immunosuppressor role on PMNs that could promote an eventual defenseless state during chronic treatment with CBD in RE.

scholarly journals Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Nutrients ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 123
Author(s):  
Natalia K. Kordulewska ◽  
Justyna Topa ◽  
Małgorzata Tańska ◽  
Anna Cieślińska ◽  
Ewa Fiedorowicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Reaction ◽  
Wide Spectrum ◽  
Cell Monolayer ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tnf Α

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150–450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

Vasorelaxing Activity of Stilbenoid and Phenanthrene Derivatives from Brasiliorchis porphyrostele: Involvement of Smooth Muscle CaV1.2 Channels

Planta Medica ◽  
2020 ◽  
Vol 86 (09) ◽  
pp. 631-642
Author(s):  
Watcharee Waratchareeyakul ◽  
Fabio Fusi ◽  
Miriam Durante ◽  
Amer Ahmed ◽  
Walter Knirsch ◽  
...  
Keyword(s):  
Channel Blocker ◽  
Antihypertensive Agents ◽  
Dependent Manner ◽  
Vascular Effects ◽  
Concentration Dependent Manner ◽  
Spasmolytic Activity ◽  
Depth Analysis ◽  
Cav1.2 Channels ◽  
Vasorelaxant Activity

AbstractFive compounds, 3,4′-dihydroxy-3′,5,5′-trimethoxydihydrostilbene, 1; 3,4′-ihydroxy-3′,5′-dimethoxydihydrostilbene, 2; 3,4′-dihydroxy-5,5′-dimethoxydihydrostilbene, 3; 9,10-dihydro-2,7-dihydroxy-4,6-dimethoxyphenanthrene, 4; and the previously unreported 1,2,6,7-tetrahydroxy-4-methoxyphenanthrene, 5 were isolated from the South American orchid, Brasiliorchis porphyrostele. An in-depth analysis of their vascular effects was performed on in vitro rat aorta rings and tail main artery myocytes. Compounds 1 – 4 were shown to possess vasorelaxant activity on rings pre-contracted by the α 1 receptor agonist phenylephrine, the CaV1.2 stimulator (S)-(−)-Bay K 8644, or depolarized with high K+ concentrations. However, compound 5 was active solely on rings stimulated by 25 mM but not 60 mM K+. The spasmolytic activity of compounds 1 and 4 was significantly affected by the presence of an intact endothelium. The KATP channel blocker glibenclamide and the KV channel blocker 4-aminopyridine significantly antagonized the vasorelaxant activity of compounds 4 and 1, respectively. In patch-clamp experiments, compounds 1 – 4 inhibited Ba2+ current through CaV1.2 channels in a concentration-dependent manner, whereas neither compound 4 nor compound 1 affected K+ currents through KATP and KV channels, respectively. The present in vitro, comprehensive study demonstrates that Brasiliorchis porphyrostele may represent a source of vasoactive agents potentially useful for the development of novel antihypertensive agents that has now to be validated in vivo in animal models of hypertension.

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