Evans Blue Dye Research Articles

Endogenous FGF21 attenuates blood-brain barrier disruption in penumbra after delayed recanalization in MCAO rats through FGFR1/PI3K/Akt pathway.

Restoration of blood circulation within "time window" is the principal treating goal for treating acute ischemic stroke. Previous studies revealed that delayed recanalization might cause serious ischemia/reperfusion injury. However, plenty of evidences showed delayed recanalization improved neurological outcomes in acute ischemic stroke. This study aims to explore the role of delayed recanalization on blood-brain barrier (BBB) in the penumbra (surrounding ischemic core) and neurological outcomes after middle cerebral artery occlusion (MCAO). Recanalization was performed on the 3rd day after MCAO. BBB disruption was tested by Western blotting, Evans blue dye, and immunofluorescence staining. Infarct volume and neurological outcomes were evaluated on the 7th day after MCAO. The expression of fibroblast growth factor 21 (FGF21), fibroblast growth factor receptor 1 (FGFR1), phosphatidylinositol-3-kinase (PI3K), and serine/threonine kinase (Akt) in the penumbra were observed by immunofluorescence staining and/or Western blotting. The extraversion of Evans blue, IgG, and albumin increased surrounding ischemic core after MCAO, but significantly decreased after recanalization. The expression of Claudin-5, Occludin, and zona occludens 1 (ZO-1) decreased surrounding ischemic core after MCAO, but significantly increased after recanalization. Infarct volume reduced and neurological outcomes improved following recanalization (on the 7th day after MCAO). The expressions of Claudin-5, Occludin, and ZO-1 decreased surrounding ischemic core following MCAO, which were up-regulated corresponding to the increases of FGF21, p-FGFR1, PI3K, and p-Akt after recanalization. Intra-cerebroventricular injection of FGFR1 inhibitor SU5402 down-regulated the expression of PI3K, p-Akt, Occludin, Claudin-5, and ZO-1 in the penumbra, which weakened the beneficial effects of recanalization on neurological outcomes after MCAO. Delayed recanalization on the 3rd day after MCAO increases endogenous FGF21 in the penumbra and activates FGFR1/PI3K/Akt pathway, which attenuates BBB disruption in the penumbra and improves neurobehavior in MCAO rats.

A Green Approach of Utilising Banana Peel (Musa paradisiaca) as Adsorbent Precursor for an Anionic Dye Removal: Kinetic, Isotherm and Thermodynamics Analysis

Methods for removing pollutants include membrane isolation, ion exchange, precipitation, transformation, and biosorption. Adsorption is a cost-effective method of treating industrial wastewater and a common commercial method for concentrating valuable molecules or eliminating contaminants. Banana peel is one of the largest underutilized agricultural wastes in Malaysia. A novel method of using a low-cost biosorbent made from banana peel and Evans blue (EB) dye as a target is the target of this study. The optimal conditions for EB dye adsorption occurred at a dye concentration of 200 mg/L, adsorbent dosages between 10 and 20 g/L, temperature of 25 °C, incubation time of 180 min, and agitation speeds of 100 rpm. Statistical discriminatory analysis showed that the pseudo-second-order kinetic model and the Redlich–Petersen isotherm model were the best models. The maximum adsorption capacity based on Langmuir’s isotherm prediction was 58.51 mg g−1. A non–linear regression of the thermodynamic van’t Hoff plot based on a dimensionless equilibrium constant resulted in negative values for Gibb’s free energy and enthalpy, indicating that the adsorption process is spontaneous and exothermic.

Comparative study of the effectiveness of phlebotonic drugs in the experiment

Introduction. Chronic venous diseases are a common group of diseases with a significant risk of complications requiring timely correction. As a rule, phlebotonic drugs based on flavonoid complexes are used for the treatment and prevention of venous diseases.Aim. To evaluate the effectiveness of the use of various phlebotonic drugs in the conditions of experimental chronic venous insufficiency.Materials and methods. Varicose veins were modeled in Wistar rats by partial stricture of the deep femoral vein. The studied medicines were administered orally in a course of 30 days from the moment of surgery. During the work, the change in the following parameters was evaluated: the rate oflocal blood flow in the skin in dynamics, the degree of vascular permeability, the concentration of proinflammatory cytokines (TNF-α, IL-6) and matrix metalloproteinase 9 (MMP9) in the vascular wall. The rate of local blood flow was assessed by ultrasound Dopplerography. The change of vascular permeability was studied by the degree of extravasation of the Evans blue dye in the Miles test. The content of proinflammatory cytokines and MMR9 was determined by enzyme-linked immunoassay. The results were statistically processed.Results. The study showed that the course oral administration of all the studied venotonizing drugs led to the restoration of hemodynamics and a significant (p˂ 0.05) decrease in the degree of vascular permeability in relation to untreated animals. It is worth noting that the use of a micronised purified flavonoid fraction 1 contributed to the development of a more pronounced vasal effect, which was reflected in an increase in blood flow velocity and a decrease in vascular permeability compared to the rest of the studied drugs. At the same time, the administration of micronised purified flavonoid fraction 1 to animals led to a statistically significant decrease in the concentration of proinflammatory cytokines, which was not observed when using other drugs.Conclusion. The course administration of the micronised purified flavonoid fraction 1leads to the development of a pronounced phlebotonic effect, expressed in the restoration of microcirculation, a decrease of the inflammation in the vascular wall.

Adsorption kinetics, equilibrium, and thermodynamic studies to understand adsorption behavior of Evans blue dye by durian husk

Adsorption kinetics, equilibrium, and thermodynamic studies to understand adsorption behavior of Evans blue dye by durian husk

Inhibition of the intracellular domain of Notch1 results in vascular endothelial cell dysfunction in sepsis.

Notch signaling is critical for regulating the function of vascular endothelial cells (ECs). However, the effect of the intracellular domain of Notch1 (NICD) on EC injury in sepsis remains unclear. We established a cell model of vascular endothelial dysfunction and induced sepsis in a mouse model via lipopolysaccharide (LPS) injection and cecal ligation and puncture (CLP). Endothelial barrier function and expression of endothelial-related proteins were determined using CCK-8, permeability, flow cytometry, immunoblot, and immunoprecipitation assays. The effect of NICD inhibition or activation on endothelial barrier function was evaluated in vitro. Melatonin was used for NICD activation in sepsis mice. The survival rate, Evans blue dye of organs, vessel relaxation assay, immunohistochemistry, ELISA, immunoblot were used to explore the specific role of melatonin for sepsis induced vascular dysfunction in vivo. We found that LPS, interleukin 6, and serum collected from septic children could inhibit the expression of NICD and its downstream regulator Hes1, which impaired endothelial barrier function and led to EC apoptosis through the AKT pathway. Mechanistically, LPS decreased the stability of NICD by inhibiting the expression of a deubiquitylating enzyme, ubiquitin-specific proteases 8 (USP8). Melatonin, however, upregulated USP8 expression, thus maintaining the stability of NICD and Notch signaling, which ultimately reduced EC injury in our sepsis model and elevated the survival rate of septic mice. We found a previously uncharacterized role of Notch1 in mediating vascular permeability during sepsis, and we showed that inhibition of NICD resulted in vascular EC dysfunction in sepsis, which was reversed by melatonin. Thus, the Notch1 signaling pathway is a potential target for the treatment of sepsis.

Neuregulin-1 reduces vascular leak and airway epithelium permeability caused by respiratory viral infection

Abstract Respiratory viruses, such as Respiratory Syncytial Virus (RSV), are associated with significant morbidity and mortality. Mice with pre-existing atopy are protected from mortality to Sendai virus (SeV), a rodent virus related to RSV. We found that lungs of atopic mice had increased levels of neuregulin-1(NRG1), and hypothesized that SeV induces death via lung vascular leak, and that NRG1 might inhibit vascular leakage. Non-atopic C57Bl/6 mice were treated with NRG1 (500ng/30 mcl, i.n) or PBS for five days before infection with SeV (2×10 5pfu) or UV-inactivated SeV (UV-SeV) and 8 days post viral inoculation, Evan’s Blue Dye (EBD) was injected i.v with EBD concentration determined spectrophotometrically one hour later in homogenized lungs. Vascular leak in SeV-infected mice was 65.25% and 46.62% more than in UV-SeV-treated or SeV-infected mice pre-treated with NRG1, respectively. We also found that RSV infection of well-differentiated human bronchial epithelial cells (hBEC) grown at the air-liquid interface caused 70KDa FITC-Dextran leakage 8 days post-inoculation. NRG1 pretreatment for 5 days before RSV inoculation reduced epithelial leakage 8 days post-inoculation by 2.5 fold (p<0.001). These results suggest NRG1 has the potential to improve epithelial barrier function in rodent and human systems and may explain the increased survival following a severe respiratory viral infection. Abigail Wexner Research Institute at Nationwide Children’s Hospital, Start-up fund to MHG

Abstract 712: IL-19 Is A Lymphokine That Induces Prox1-dependent Lymphangiogenesis And Permeability

Introduction: Atherosclerosis is a vascular inflammatory disease associated with lymphangiogenesis, but whether lymphangiogenesis contributes to disease progression or is simply a compensatory attempt at disease resolution is currently unresolved. Recent studies are revealing a role for lymphatic vessels in reverse cholesterol transport (RCT), which could attenuate atherosclerosis. We have previously reported that Interleukin-19 (IL-19), an anti-inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro-angiogenic. This drives our hypothesis that one mechanism whereby IL-19 is atheroprotective is by promoting functional lymphangiogenesis and lymphatic flux. Methods & Results: IL-19 can significantly induce human dermal lymphatic endothelial cell (hdLEC) proliferation, migration, and tube formation, to the same degree as VEGFC, a potent inducer of lymphangiogenesis. RNA sequencing analysis indicates that IL-19 induces an angiogenic transcriptional program through Prox1, a master regulator of lymphangiogenesis. Knockdown of Prox1 led to a significant decrease in IL-19-associated proliferation and migration. IL-19 also increased expression of Angpt2, an endothelial cell protein that induces permeability through regulation of tight junctions, through a Prox1-dependent mechanism. Electric Cell-substrate Impedance Sensing assays showed that IL-19 treatment increased hdLEC permeability and mitigated oxLDL-associated decreased permeability, suggestive of maintaining lymphatic barrier function during atherosclerosis. IL-19 treatment also increased dil-HDL transport across an hdLEC barrier. Preliminary in vivo data suggests injection of rmIL-19 in Il19 -/- mice increases lymphatic permeability and flux through increased lymphatic uptake of Evans blue dye. Conclusion: These data suggest that IL-19 increases lymphangiogenesis, permeability, and may increase RCT to plausibly decrease atherosclerosis. These results can influence our understanding of the association between the lymphatic system and atherosclerosis.

Dasatinib Attenuates Inflammatory Outcomes of the Sepsis Induced Acute Lung Injury Through mTOR Pathway

This study aimed to investigate the therapeutic potential of dasatinib, an FDA-approved drug for chronic myeloid leukemia, in sepsis-induced acute lung injury (ALI). In the in vitro part of the study, RAW 264.7 macrophages were stimulated with lipopolysaccharide (LPS), and the anti-inflammatory effects of dasatinib were assessed by measuring pro-inflammatory mediators and cytokines. In the in vivo part, ALI was induced in mice through intraperitoneal LPS injection. The researchers investigated alveolar-capillary permeability, the inflammatory response, and the therapeutic efficacy of dasatinib in ALI. Dasatinib administration was found to attenuate inflammation by inhibiting the mTOR pathway. In the in vivo experiments, dasatinib reduced the levels of pro-inflammatory cytokines and chemokines in bronchoalveolar lavage fluid (BALF). It also decreased lung tissue permeability, as evidenced by a lower amount of Evans blue dye detected after ALI. Furthermore, dasatinib alleviated lung histological damage and reduced the lung injury grade, demonstrating its protective role in ALI. By inhibiting the mTOR pathway in macrophages, dasatinib exhibited a protective and anti-inflammatory effect on ALI. It reduced the intrapulmonary inflammatory response, prevented capillary disruption, and inhibited the accumulation of inflammatory cells. These findings suggest that dasatinib holds promise as a potential treatment option for protecting lung tissue in ALI.

Cytoskeletal Remodeling and Gap Junction Translocation Mediates Blood-Brain Barrier Disruption by Non-invasive Low-Voltage Pulsed Electric Fields.

High-voltage pulsed electric fields (HV-PEF) delivered with invasive needle electrodes for electroporation applications is known to induce off-target blood-brain barrier (BBB) disruption. In this study, we sought to determine the feasibility of minimally invasive PEF application to produce BBB disruption in rat brain and identify the putative mechanisms mediating the effect. We observed dose-dependent presence of Evans Blue (EB) dye in rat brain when PEF were delivered with a skull mounted electrode used for neurostimulation application. Maximum region of dye uptake was observed while using 1500V, 100 pulses, 100µs and 10Hz. Results of computational models suggested that the region of BBB disruption was occurring at thresholds of 63V/cm or higher; well below intensity levels for electroporation. In vitro experiments recapitulating this effect with human umbilical vein endothelial cells (HUVEC) demonstrated cellular alterations that underlie BBB manifests at low-voltage high-pulse conditions without affecting cell viability or proliferation. Morphological changes in HUVECs due to PEF were accompanied by disruption of actin cytoskeleton, loss of tight junction protein-ZO-1 and VE-Cadherin at cell junctions and partial translocation into the cytoplasm. Uptake of propidium iodide (PI) in PEF treated conditions is less than 1% and 2.5% of total number of cells in high voltage (HV) and low-voltage (LV) groups, respectively, implying that BBB disruption to be independent of electroporation under these conditions. 3-D microfabricated blood vessel permeability was found to increase significantly following PEF treatment and confirmed with correlative cytoskeletal changes and loss of tight junction proteins. Finally, we show that the rat brain model can be scaled to human brains with a similar effect on BBB disruption characterized by electric field strength (EFS) threshold and using a combination of two bilateral HD electrode configurations.

Cardioprotective effects of shock wave therapy: A cardiac magnetic resonance imaging study on acute ischemia-reperfusion injury.

Cardioprotection strategies remain a new frontier in treating acute myocardial infarction (AMI), aiming at further protect the myocardium from the ischemia-reperfusion damage. Therefore, we aimed at investigating the mechano-transduction effects induced by shock waves (SW) therapy at time of the ischemia reperfusion as a non-invasive cardioprotective innovative approach to trigger healing molecular mechanisms. We evaluated the SW therapy effects in an open-chest pig ischemia-reperfusion (IR) model, with quantitative cardiac Magnetic Resonance (MR) imaging performed along the experiments at multiple time points (baseline (B), during ischemia (I), at early reperfusion (ER) (∼15 min), and late reperfusion (LR) (3 h)). AMI was obtained by a left anterior artery temporary occlusion (50 min) in 18 pigs (32 ± 1.9 kg) randomized into SW therapy and control groups. In the SW therapy group, treatment was started at the end of the ischemia period and extended during early reperfusion (600 + 1,200 shots @0.09 J/mm2, f = 5 Hz). The MR protocol included at all time points LV global function assessment, regional strain quantification, native T1 and T2 parametric mapping. Then, after contrast injection (gadolinium), we obtained late gadolinium imaging and extra-cellular volume (ECV) mapping. Before animal sacrifice, Evans blue dye was administrated after re-occlusion for area-at-risk sizing. During ischemia, LVEF decreased in both groups (25 ± 4.8% in controls (p = 0.031), 31.6 ± 3.2% in SW (p = 0.02). After reperfusion, left ventricular ejection fraction (LVEF) remained significantly decreased in controls (39.9 ± 4% at LR vs. 60 ± 5% at baseline (p = 0.02). In the SW group, LVEF increased quickly ER (43.7 ± 11.4% vs. 52.4 ± 8.2%), and further improved at LR (49.4 ± 10.1) (ER vs. LR p = 0.05), close to baseline reference (LR vs. B p = 0.92). Furthermore, there was no significant difference in myocardial relaxation time (i.e. edema) after reperfusion in the intervention group compared to the control group: ΔT1 (MI vs. remote) was increased by 23.2±% for SW vs. +25.2% for the controls, while ΔT2 (MI vs. remote) increased by +24.9% for SW vs. +21.7% for the control group. In conclusion, we showed in an ischemia-reperfusion open-chest swine model that SW therapy, when applied near the relief of 50' LAD occlusion, led to a nearly immediate cardioprotective effect translating to a reduction in the acute ischemia-reperfusion lesion size and to a significant LV function improvement. These new and promising results related to the multi-targeted effects of SW therapy in IR injury need to be confirmed by further in-vivo studies in close chest models with longitudinal follow-up.

Role of apigenin in high glucose-induced retinal microvascular endothelial cell dysfunction via regulating NOX4/p38 MAPK pathway in vitro.

To investigate the retinoprotective role of Apigenin (Api) against high glucose (HG)-induced human retinal microvascular endothelial cells (HRMECs), and to explore its regulatory mechanism. HRMECs were stimulated by HG for 48h to establish the in vitro cell model. Different concentrations of Api (2.5, 5, and 10 µmol/L) were applied for treatment. Cell counting kit-8 (CCK-8), Transwell, and tube formation assays were performed to examine the effects of Api on the viability, migration, and angiogenesis in HG-induced HRMECs. Vascular permeability was evaluated by Evans blue dye. The inflammatory cytokines and oxidative stress-related factors were measured using their commercial kits. Protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and p38 mitogen-activated protein kinase (MAPK) was measured by Western blot. Api prevented HG-induced HRMECs viability, migration, angiogenesis, and vascular permeability in a concentration-dependent manner. Meanwhile, Api also concentration-dependently inhibited inflammation and oxidative stress in HRMECs exposed to HG. In addition, HG caused an elevated expression of NOX4, which was retarded by Api treatment. HG stimulation facilitated the activation of p38 MAPK signaling in HRMECs, and Api could weaken this activation partly via downregulating NOX4 expression. Furthermore, overexpression of NOX4 or activation of p38 MAPK signaling greatly weakened the protective role of Api against HG-stimulated HRMECs. Api might exert a beneficial role in HG-stimulated HRMECs through regulating NOX4/p38 MAPK pathway.

PULMONARY VASCULAR ENDOTHELIAL GLYCOCALYX DEGRADATION CONTRIBUTES TO ACUTE LUNG INJURY IN EXPERIENCING HEATSTROKE.

Objectives: This study investigated the role and potential involvement of pulmonary vascular glycocalyx degradation in acute lung injury in rats with severe heatstroke (HS). Methods: Rats in an established HS model were exposed to a heated environment for 60 min in an incubator (temperature, 40°C ± 2°C; humidity, 65% ± 5%). Following pretreatment with heparanase III (HPSE III) or heparin, pathological lung injury, arterial blood gas, alveolar barrier disruption, and hemodynamic changes were evaluated. The vascular endothelial structures of the lungs were examined using electron microscopy. The concentration of Evans blue dye in the lungs and arterial blood gas were assessed. An enzyme-linked immunosorbent assay was used to quantify the plasma concentration of heparan sulfate proteoglycan. The expression of glypican-1 and syndecan-1 in pulmonary vessels was measured using immunofluorescence. Western blots were used to detect the expression of TNF-α, IL-6, and vascular endothelial biomarkers in the rat lungs. Pulmonary apoptosis was assessed using a TUNEL (terminal dUTP nick end labeling) assay, and the concentrations of malondialdehyde were measured. Results: Glycocalyx shedding aggravated lung injuries. Severe histopathological damage was observed, and indexes of lung function deviated from abnormal ranges. In addition, pulmonary vascular endothelial cells were disrupted. Compared with the HS group, the plasma concentration of heparan sulfate proteoglycan significantly increased in the HPSE group ( P < 0.05). The expression of glypican-1 and syndecan-1 decreased, and the extravasation of Evans blue dye increased ( P < 0.01). Endothelial biomarker expression increased in the lung tissue, whereas occludin expression decreased. Moreover, TNF-α and IL-6 were overexpressed following heat stress. Furthermore, apoptosis of pulmonary tissues and the concentration of malondialdehyde in rat lungs increased in the HS and HPSE groups. Conclusions : Heatstroke induced pulmonary glycocalyx degradation, which increased vascular permeability and aggravated vascular endothelial dysfunction, contributing to apoptosis, inflammation, and oxidation in the pulmonary tissues.

Efficacy of CU06-1004 via regulation of inflammation and endothelial permeability in LPS-induced acute lung injury

BackgroundAcute lung injury (ALI) is a life-threatening condition that fundamentally results from inflammation and edema in the lung. There are no effective treatments available for clinical use. Previously, we found that as a leakage blocker CU06-1004 prevents endothelial barrier disruption and enhances endothelial cell survival under inflammatory conditions. In this study, we aimed to elucidate the effect of CU06-1004 in terms of prevention of inflammation and endothelial dysfunction in an ALI mouse model.MethodsAn ALI model was established that included intraperitoneal administration of LPS. Following LPS administration, survival rates and lung wet/dry ratios were assessed. Histological analysis was performed using hematoxylin and eosin staining. Scanning electron microscopy was used to examine alveolar and capillary morphology. Cytokines such as IL-1β, IL-6, and TNF-α were analyzed using an ELISA assay of bronchoalveolar lavage fluid (BALF) and serum. Neutrophil infiltration was observed in BALF using Wright-Giemsa staining, and myeloperoxidase (MPO) activity was assessed. Pulmonary vascular leakage was confirmed using Evans-blue dye, and the expression of junctional proteins was evaluated using immunofluorescent staining. Expression of adhesion molecules was observed using immunofluorescence staining. NF-κB activation was determined using immunohistochemistry and western blot analysis.ResultsSurvival rates and pulmonary edema were ameliorated with CU06-1004 treatment. Administration of CU06-1004 normalized histopathological changes induced by LPS, and alveolar-capillary wall thickening was reduced. Compared with the LPS-challenged group, after CU06-1004 treatment, the infiltration of immune cells was decreased in the BALF, and MPO activity in lung tissue was reduced. Similarly, in the CU06-1004 treatment group, pro-inflammatory cytokines were significantly inhibited in both BALF and serum. Evans-blue leakage was reduced, and the expression of junctional proteins was recovered in the CU06-1004 group. Adhesion molecules were downregulated and NF-κB activation was inhibited after CU06-1004 treatment.ConclusionsThese results suggested that CU06-1004 had a therapeutic effect against LPS-induced ALI via alleviation of the inflammatory response and protection of vascular integrity.

Effect of Sublethal Concentrations of Zinc Oxide Nanoparticles on Bacillus cereus

Zinc oxide nanoparticles (ZnONPs), which are produced on a large scale, pose a potential threat to various environments because they can interact with the microbial populations found in them. Bacteria that are widespread in soil, water, and plant material include the Bacillus cereus group, which plays an important role in biodegradation and the nutrient cycle and is a major factor determining ecological balance. This group includes, among others, the foodborne pathogen B. cereus sensu stricto (herein referred to as B. cereus). The aim of this study was a comprehensive assessment of the effects of commercially available ZnONPs on B. cereus. The MIC (minimum inhibitory concentration) for B. cereus was 1.6 mg/mL, and the MBC (minimum bactericidal concentration) was 1.8 mg/mL. Growth of B. cereus was inhibited by a concentration of ZnONPs lower than or equal to MIC50. Concentrations from 0.2 to 0.8 mg/mL inhibited the growth of these bacteria in liquid media, induced symptoms of oxidative stress, and stimulated an environmental stress response in the form of biofilm and endospore formation. In addition, ZnONPs negatively affected the ability of the bacteria to break down the azo dye Evans Blue but enhanced the antimicrobial properties of phenolic compounds. Sublethal concentrations of ZnONPs generally decreased the activity of B. cereus cells, especially in the presence of phenolics, which indicates their potential toxicological impact, but at the same time they induced universal defence responses in these cells, which in the case of potential pathogens can hinder their removal.

Anethole Pretreatment Modulates Cerebral Ischemia/Reperfusion: The Role of JNK, p38, MMP-2 and MMP-9 Pathways.

Anethole (AN) is one of the major constituents of several plant oils, demonstrating plentiful pharmacological actions. Ischemic stroke is the main cause of morbidity and death worldwide, particularly since ischemic stroke therapeutic choices are inadequate and limited; thus, the development of new therapeutic options is indispensable. This study was planned to explore the preventive actions of AN in ameliorating cerebral ischemia/reperfusion-induced brain damage and BBB permeability leakage, as well as to explore anethole's potential mechanisms of action. The proposed mechanisms included modulating JNK and p38 as well as MMP-2 and MMP-9 pathways. Sprague-Dawley male rats were randomly assigned into four groups: sham, middle cerebral artery occlusion (MCAO), AN125 + MCAO, and AN250 + MCAO. Animals in the third and fourth groups were pretreated with AN 125 or 250 mg/kg orally, respectively, for two weeks before performing middle cerebral artery occlusion (MCAO)-induced cerebral ischemic/reperfusion surgery. Animals that experienced cerebral ischemia/reperfusion exhibited amplified infarct volume, Evans blue intensity, brain water content, Fluoro-Jade B-positive cells, severe neurological deficits, and numerous histopathological alterations. MCAO animals exhibited elevated MMP-9 and MMP-2 gene expressions, enzyme activities, augmented JNK, and p38 phosphorylation. On the other hand, pretreatment with AN diminished the infarct volume, Evans blue dye intensity, brain water content, and Fluoro-Jade B-positive cells, improved the neurological score and enhanced histopathological examination. AN effectively lowered MMP-9 and MMP-2 gene expression and enzyme activities and diminished phosphorylated JNK, p38. AN decreased MDA content, amplified GSH/GSSG ratio, SOD, and CAT, decreased the serum and brain tissue homogenate inflammatory cytokines (TNF-α, IL-6, IL-1β), NF-κB, and deterred the apoptotic status. This study revealed the neuroprotective ability of AN against cerebral ischemia/reperfusion in rats. AN boosted blood-brain barrier integrity via modulating MMPs and diminished oxidative stress, inflammation, and apoptosis through the JNK/p38 pathway.

MicroRNA-501 controls myogenin+/CD74+ myogenic progenitor cells during muscle regeneration

ObjectiveSkeletal muscle regeneration is markedly impaired during aging. How adult muscle stem cells contribute to this decrease in regenerative capacity is incompletely understood. We investigated mechanisms of age-related changes in myogenic progenitor cells using the tissue-specific microRNA 501. MethodsYoung and old C57Bl/6 mice were used (3 months or 24 months of age, respectively) with or without global or tissue-specific genetic deletion of miR-501. Muscle regeneration was induced using intramuscular cardiotoxin injection or treadmill exercise and analysed using single cell and bulk RNA sequencing, qRT-PCR and immunofluorescence. Muscle fiber damage was assessed with Evan`s blue dye (EBD). In vitro analysis was performed in primary muscle cells obtained from mice and humans. ResultsSingle cell sequencing revealed myogenic progenitor cells in miR-501 knockout mice at day 6 after muscle injury that are characterized by high levels of myogenin and CD74. In control mice these cells were less in number and already downregulated after day 3 of muscle injury. Muscle from knockout mice had reduced myofiber size and reduced myofiber resilience to injury and exercise. miR-501 elicits this effect by regulating sarcomeric gene expression through its target gene estrogen-related receptor gamma (Esrrg). Importantly, in aged skeletal muscle where miR-501 was significantly downregulated and its target Esrrg significantly upregulated, the number of myog+/CD74+ cells during regeneration was upregulated to similar levels as observed in 501 knockout mice. Moreover, myog+/CD74+-aged skeletal muscle exhibited a similar decrease in the size of newly formed myofibers and increased number of necrotic myofibers after injury as observed in mice lacking miR-501. ConclusionsmiR-501 and Esrrg are regulated in muscle with decreased regenerative capacity and loss of miR-501 is permissive to the appearance of CD74+ myogenic progenitors. Our data uncover a novel link between the metabolic transcription factor Esrrg and sarcomere formation and demonstrate that stem cell heterogeneity in skeletal muscle during aging is under miRNA control. Targeting Esrrg or myog+/CD74+ progenitor cells might improve fiber size and myofiber resilience to exercise in aged skeletal muscle.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Transplantation of endothelial progenitor cells overexpressing mir-126-3p improves vascular repair in a diabetic rat model.

Abstract P2-21-05: Vascular effects of Platelet-derived growth factor-B (PDGFB) in breast cancer metastasis to the brain

Abstract Brain metastases are associated with shortened survival and poorer quality of life in breast cancer patients. Recently, the importance of platelet-derived growth factor-B (PDGFB) and its stromally localized cognate receptor, platelet-derived growth factor receptor-beta (PDGFRβ) in promoting breast cancer metastasis to the brain was identified by our group. Historically, PDGFB-to-PDGFRβ signaling is known to promote angiogenesis and vessel maturation both in normal physiology and in cancer, but the mechanism(s) behind this tumor stromal interplay in brain metastasis is poorly understood. Based on our previous work and the work of others, we hypothesized that breast tumor-derived PDGFB directly modulates both brain endothelial and blood brain barrier (BBB) function by altering angiogenesis and endothelial permeability. To test this directly, we over-expressed PDGFB ligand in MDA-MB-231 and HCC1187 triple negative breast cancer cell lines and compared the angiogenic potential of the conditioned medium (CM) from these cells. We utilized both a 3D in vitro (human brain microvascular endothelial cell, HBMEC) spheroid angiogenesis model and a tri-culture BBB system (HBMECs in direct interaction with human astrocytes and human pericytes) to test the BBB vasculature. CM harvested from PDGFB overexpressing cells significantly increased the sprouting angiogenesis potential of 3D HBMECs and the migration of 3D tri-culture BBB spheroids compared to CM obtained from parental cells. In addition, CM from PDGFB overexpressing cells caused the surface of BBB spheroids to exhibit significantly increased permeability to high molecular weight dextran compared to CM from parental cells. In agreement with these findings, the tight junction protein ZO1 was diminished. Importantly, in vivo BBB permeability was also tested upon intracardiac injection of MDA-MB-231 cells with and without PDGFB overexpression. These studies revealed that Evans blue dye fluorescence intensity was significantly higher in the brains of mice injected with PDGFB overexpressing cells compared to parental cells. Overall, these studies reveal a previously unrecognized role for PDGFB-to-PDGFRβ signaling in brain vascular remodeling and BBB permeability, which has clinical implications for women with PDGFB-positive breast cancer. Citation Format: Sajita Shah, Rebecca Packard, Sarah Steck, Alexis Mossing, Yalini Ramamoorthy, Katie Thies, Alexander Didier, Jonathan Spehar, Dillon Richardson, Tasneem Arsiwala, Jonathan Godbout, Steven Sizemore, Paul Lockman, Gina Sizemore. Vascular effects of Platelet-derived growth factor-B (PDGFB) in breast cancer metastasis to the brain [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-05.

Topically Administered NOX4 Inhibitor, GLX7013114, Is Efficacious in Treating the Early Pathological Events of Diabetic Retinopathy.

NADPH oxidases (NOXs) are implicated in the early pathological events of diabetic retinopathy (DR). The NOX4 inhibitor GLX7013114, topically administered, reduced oxidative damage and apoptosis in the rat streptozotocin model of DR. GLX7013114 protected retinal neurons and retinal ganglion cell function and reduced the expression of pro-inflammatory cytokines in the diabetic retina. GLX7013114 diminished the diabetes-induced increase in vascular endothelial growth factor levels and Evans blue dye leakage in retinal tissue. GLX7013114 exhibits neuroprotective, anti-inflammatory, and vasculoprotective properties that suggest it may have a role as a putative therapeutic for the early events of DR.

Natural product-based bioactive agents in combination attenuate neuroinflammation in a tri-culture model.

Introduction: Neuroinflammation is an important pathological event contributing to the onset and progression of neurodegenerative diseases. The hyperactivation of microglia triggers the release of excessive proinflammatory mediators that lead to the leaky blood-brain barrier and impaired neuronal survival. Andrographolide (AN), baicalein (BA) and 6-shogaol (6-SG) possess anti-neuroinflammatory properties through diverse mechanisms of action. The present study aims to investigate the effects of the pair-combinations of these bioactive compounds in attenuating neuroinflammation. Methods: A tri-culture model with microglial N11 cells, microvascular endothelial MVEC(B3) cells, and neuroblastoma N2A cells was established in a transwell system. AN, BA and 6-SG used alone (25µM) or in pair-wised combinations (12.5 + 12.5µM) were subjected to the tri-culture system. Upon the stimulation of lipopolysaccharides (LPS) at 1μg/mL, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) levels were determined by ELISA assays. Immunofluorescence staining was applied to investigate the nuclear translocation of nuclear factor kappa B p65 (NF-κB p65) on N11 cells, expressions of protein zonula occludens-1 (ZO-1) on MVEC cells and phosphorylated tau (p-tau) on N2A cells, respectively. The endothelial barrier permeability of MVEC cells was assessed by the Evans blue dye, and the resistance from the endothelial barrier was measured by transepithelial/endothelial electrical resistance (TEER) value. Neuronal survival of N2A cells was determined by Alamar blue and MTT assays. Results: Combinations of AN-SG and BA-SG synergistically lowered the TNF and IL-6 levels in LPS-induced N11 cells. Remarkably, the combined anti-neuroinflammatory effects of AN-SG and BA-SG remained significantly greater compared to their individual components at the same concentration level. The molecular mechanism of the attenuated neuroinflammation was likely to be mediated by downregulation of NF-κB p65 translocation (p < 0.0001 vs. LPS stimulation) in N11 cells. In the MVEC cells, both AN-SG and BA-SG restored TEER values, ZO-1 expression and reduced permeability. Furthermore, AN-SG and BA-SG significantly improved neuronal survival and reduced expressions of p-tau on N2A cells. Discussion: The AN-SG and BA-SG combinations showed greater anti-neuroinflammatory potential than those used alone in mono- and tri-cultured N11 cells, thereby further protecting endothelial tight junction and neuronal survival. Taken together, AN-SG and BA-SG may provide improved anti-neuroinflammatory and neuroprotective activities.