scholarly journals Comprehensive Evidence of Carrier-Mediated Distribution of Amantadine to the Retina across the Blood–Retinal Barrier in Rats

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1339
Author(s):  
Yusuke Shinozaki ◽  
Shin-ichi Akanuma ◽  
Yuika Mori ◽  
Yoshiyuki Kubo ◽  
Ken-ichi Hosoya
Keyword(s):  
Transport Systems ◽  
Blood Retinal Barrier ◽  
Neuroprotective Effects ◽  
Index Method ◽  
Cationic Drugs ◽  
Retinal Distribution ◽  
Vitro Model ◽  
A Minor

Amantadine, a drug used for the blockage of NMDA receptors, is well-known to exhibit neuroprotective effects. Accordingly, assessment of amantadine transport at retinal barriers could result in the application of amantadine for retinal diseases such as glaucoma. The objective of this study was to elucidate the retinal distribution of amantadine across the inner and outer blood–retinal barrier (BRB). In vivo blood-to-retina [3H]amantadine transport was investigated by using the rat retinal uptake index method, which was significantly reduced by unlabeled amantadine. This result indicated the involvement of carrier-mediated processes in the retinal distribution of amantadine. In addition, in vitro model cells of the inner and outer BRB (TR-iBRB2 and RPE-J cells) exhibited saturable kinetics (Km in TR-iBRB2 cells, 79.4 µM; Km in RPE-J cells, 90.5 and 9830 µM). The inhibition of [3H]amantadine uptake by cationic drugs/compounds indicated a minor contribution of transport systems that accept cationic drugs (e.g., verapamil), as well as solute carrier (SLC) organic cation transporters. Collectively, these outcomes suggest that carrier-mediated transport systems, which differ from reported transporters and mechanisms, play a crucial role in the retinal distribution of amantadine across the inner/outer BRB.

scholarly journals Involvement of 27-Hydroxycholesterol in Mitotane Action on Adrenocortical Carcinoma

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 885
Author(s):  
Antonina Germano ◽  
Daniela Rossin ◽  
Valerio Leoni ◽  
Noemi Iaia ◽  
Laura Saba ◽  
...  
Keyword(s):  
Adrenocortical Carcinoma ◽  
Cholesterol Metabolism ◽  
Cell Model ◽  
Gas Chromatography Mass Spectrometry ◽  
Strong Increase ◽  
Vitro Model ◽  
A Minor ◽  
Cholesterol Precursors

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Mitotane, the standard treatment for ACC, impairs adrenocortical steroid biosynthesis and cholesterol metabolism. In the H295R cell line, a standard ACC in vitro model, mitotane was previously reported to enhance the production of some oxysterols. To verify the possible mechanistic involvement of oxysterols in the anti-ACC effect of mitotane, a gas chromatography mass spectrometry (GC-MS) profiling of oxysterols and the main cholesterol precursors was carried out in H295R cells. Among the oxysterols detected in mitotane-treated cells, 27OHC was markedly produced, as well as lanosterol and lathosterol cholesterol precursors. In this cell model, mitotane was confirmed to affect mitochondrial transmembrane potential and induce apoptosis. Such cytotoxic effects were perfectly matched by H295R cell treatment with a single identical micromolar amount of 27OHC. The mitotane-dependent strong increase in 27OHC was confirmed in vivo, in the plasma of ACC patients under treatment with the drug. Moreover, lanosterol, lathosterol, desmosterol and, to a minor extent, 24-hydroxycholesterol and 25-hydroxycholesterol plasma levels were significantly increased in those patients. The cytotoxic effect of mitotane on ACC cells may be partly related to the increased intracellular level of 27OHC induced by the drug itself.

scholarly journals Antagomirs Targeting MicroRNA-134 Increase Limk1 Levels After Experimental Seizures in Vitro and in Vivo

2017 ◽  
Vol 43 (2) ◽  
pp. 636-643 ◽  
Author(s):  
Jiahang Sun ◽  
Xiaoying Gao ◽  
Dawei Meng ◽  
Yang Xu ◽  
Xichun Wang ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Neuroprotective Effects ◽  
Protein Expressions ◽  
Vitro Model ◽  
Pilocarpine Injection

Background: MiR-134 is enriched in dendrites of hippocampal neurons and plays crucial roles in the progress of epilepsy. The present study aims to investigate the effects of antagomirs targeting miroRNA-134 (Ant-134) on limk1 expression and the binding of miR-134 and limk1 in experimental seizure. Methods: Status epilepticus (SE) rat model was established by lithium chloride-pilocarpine injection and was treated with Ant-134 by intracerebroventricular injection. Low Mg2+-exposed primary neurons were used as an in vitro model of SE. The expression of miR-134 was determined using real-time PCR. Protein expressions of limk1 and cofilin were determined by Western blotting. Luciferase reporter assay was used to examine the binding between miR-134 and limk1 3’-untranslated region. Results: The expression of miR-134 was markedly enhanced in hippocampus of the SE rats and low Mg2+-exposed neurons. Ant-134 increased the expression of limk1 and reduced the expression of cofilin in the SE hippocampus and Low Mg2+-exposed neurons. In addition, luciferase reporter assay confirmed that miR-134 bound limk1 3’-UTR. MiR-134 overexpression inhibited limk1 mRNA and protein expressions in neurons. Conclusion: Blockage of miR-134 upregulates limk1 expression and downregulated cofilin expression in hippocampus of the SE rats. This mechanism may contribute to the neuroprotective effects of Ant-134.

scholarly journals Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2530
Author(s):  
Bijean D. Ford ◽  
Diego Moncada Giraldo ◽  
Camilla Margaroli ◽  
Vincent D. Giacalone ◽  
Milton R. Brown ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Bactericidal Activity ◽  
Scavenger Receptor ◽  
Receptor Expression ◽  
Blood Monocytes ◽  
Bacterial Killing ◽  
Blood Neutrophils ◽  
Vitro Model

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

scholarly journals Radiofrequency Irradiation Modulates TRPV1-Related Burning Sensation in Rosacea

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1424
Author(s):  
Seyeon Oh ◽  
Myeongjoo Son ◽  
Joonhong Park ◽  
Donghwan Kang ◽  
Kyunghee Byun
Keyword(s):  
Burning Sensation ◽  
In Vivo Model ◽  
Molecular Evidence ◽  
Exposed Skin ◽  
Heat Treated ◽  
Vitro Model ◽  
Effective Use ◽  
Inflammatory Molecules

Rosacea is a skin inflammatory condition that is accompanied by not only redness and flushing but also unseen symptoms, such as burning, stinging, and itching. TRPV1 expression in UVB-exposed skin can lead to a painful burning sensation. Upregulated TRPV1 expression helps release neuropeptides, including calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide, which can activate macrophage and inflammatory molecules. In this study, we found that radiofrequency (RF) irradiation reduced TRPV1 activation and neuropeptide expression in a UVB-exposed in vivo model and UVB- or heat-treated in an in vitro model. RF irradiation attenuated neuropeptide-induced macrophage activation and inflammatory molecule expression. Interestingly, the burning sensation in the skin of UVB-exposed mice and patients with rosacea was significantly decreased by RF irradiation. These results can provide experimental and molecular evidence on the effective use of RF irradiation for the burning sensation in patients with rosacea.

scholarly journals Generation of a Novel In Vitro Model to Study Endothelial Dysfunction from Atherothrombotic Specimens

2021 ◽  
Author(s):  
Susan Gallogly ◽  
Takeshi Fujisawa ◽  
John D. Hung ◽  
Mairi Brittan ◽  
Elizabeth M. Skinner ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
In Vitro Model ◽  
Umbilical Vein ◽  
Coronary Syndrome ◽  
Coronary Endothelial Cells ◽  
Vitro Model ◽  
Umbilical Vein Endothelial Cells

Abstract Purpose Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. Methods In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. Results CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. Conclusions CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

scholarly journals AZP2006, a new promising treatment for Alzheimer’s and related diseases

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
N. Callizot ◽  
C. Estrella ◽  
S. Burlet ◽  
A. Henriques ◽  
C. Brantis ◽  
...  
Keyword(s):  
Therapeutic Application ◽  
Neuroprotective Effects ◽  
Beneficial Effects ◽  
Potential Therapeutic Application ◽  
Promising Treatment ◽  
Central Synapses ◽  
First Time ◽  
Pgrn Level

AbstractProgranulin (PGRN) is a protein with multiple functions including the regulation of neuroinflammation, neuronal survival, neurite and synapsis growth. Although the mechanisms of action of PGRN are currently unknown, its potential therapeutic application in treating neurodegenerative diseases is huge. Thus, strategies to increase PGRN levels in patients could provide an effective treatment. In the present study, we investigated the effects of AZP2006, a lysotropic molecule now in phase 2a clinical trial in Progressive Supranuclear Palsy patients, for its ability to increase PGRN level and promote neuroprotection. We showed for the first time the in vitro and in vivo neuroprotective effects of AZP2006 in neurons injured with Aβ1–42 and in two different pathological animal models of Alzheimer’s disease (AD) and aging. Thus, the chronic treatment with AZP2006 was shown to reduce the loss of central synapses and neurons but also to dramatically decrease the massive neuroinflammation associated with the animal pathology. A deeper investigation showed that the beneficial effects of AZP2006 were associated with PGRN production. Also, AZP2006 binds to PSAP (the cofactor of PGRN) and inhibits TLR9 receptors normally responsible for proinflammation when activated. Altogether, these results showed the high potential of AZP2006 as a new putative treatment for AD and related diseases.

scholarly journals 3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics

2021 ◽  
Vol 22 (6) ◽  
pp. 2925
Author(s):  
Victor Häussling ◽  
Romina H Aspera-Werz ◽  
Helen Rinderknecht ◽  
Fabian Springer ◽  
Christian Arnscheidt ◽  
...  
Keyword(s):  
Bone Metabolism ◽  
In Vitro Model ◽  
Bone Diseases ◽  
3D Environment ◽  
Type 2 Diabetics ◽  
Mineralized Matrix ◽  
Vitro Model

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

scholarly journals Role of the C5a-C5a receptor axis in the inflammatory responses of the lungs after experimental polytrauma and hemorrhagic shock

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shinjini Chakraborty ◽  
Veronika Eva Winkelmann ◽  
Sonja Braumüller ◽  
Annette Palmer ◽  
Anke Schultze ◽  
...  
Keyword(s):  
Hemorrhagic Shock ◽  
Inflammatory Responses ◽  
Experimental Models ◽  
Lung Damage ◽  
C5a Receptor ◽  
Cytokine Levels ◽  
Vitro Model

AbstractSingular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

scholarly journals Delivery of Thyronamines (TAMs) to the Brain: A Preliminary Study

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1616
Author(s):  
Nicoletta di Leo ◽  
Stefania Moscato ◽  
Marco Borso' ◽  
Simona Sestito ◽  
Beatrice Polini ◽  
...  
Keyword(s):  
High Performance ◽  
In Vitro Model ◽  
New Drugs ◽  
Therapeutic Approaches ◽  
Neuroprotective Effects ◽  
Pharmacological Response ◽  
Preliminary Study ◽  
Vitro Model ◽  
The Brain

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood–brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property.

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