Evans Blue Albumin Research Articles

Kaempferol protects gut-vascular barrier from high glucose-induced disorder via NF-κB pathway

Kaempferol is a natural edible flavonoid reported to treat high-fat diet-induced intestinal inflammation; however, the underlying molecular mechanisms remain unclear. This research aims to investigate the protective effect of kaempferol on the gut-vascular barrier (GVB) induced by high glucose and elucidate the underlying mechanism. Evans blue albumin efflux assay was used to test endothelial cell permeability. The results showed that kaempferol (50 μM) significantly reversed the high glucose-induced monolayer barrier permeability of rat intestinal microvascular endothelial cells (RIMVECs), while kaempferol significantly alleviated the high glucose-induced rarefication of the tight junction protein Claudin-5. Moreover, kaempferol also reduced high glucose-induced angiogenesis and cell migration via inhibiting the VEGFR2/p38 pathway. Kaempferol also protected against high glucose-induced overproduction of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 by inhibiting NF-κB p65 nuclear translocation. In addition, kaempferol had similar effects to the NF-κB inhibitor SN50 in reducing high glucose-induced ICAM-1 expression and endothelial barrier permeabilization. Our findings in part reveal the pathological mechanism of hyperglycemia-related gastrointestinal diseases and underlie the molecular mechanism of kaempferol in inhibiting bowel inflammation from a novel perspective.

Porphyromonas gingivalis bacteremia increases the permeability of the blood-brain barrier via the Mfsd2a/Caveolin-1 mediated transcytosis pathway

Bacteremia induced by periodontal infection is an important factor for periodontitis to threaten general health. P. gingivalis DNA/virulence factors have been found in the brain tissues from patients with Alzheimer’s disease (AD). The blood-brain barrier (BBB) is essential for keeping toxic substances from entering brain tissues. However, the effect of P. gingivalis bacteremia on BBB permeability and its underlying mechanism remains unclear. In the present study, rats were injected by tail vein with P. gingivalis three times a week for eight weeks to induce bacteremia. An in vitro BBB model infected with P. gingivalis was also established. We found that the infiltration of Evans blue dye and Albumin protein deposition in the rat brain tissues were increased in the rat brain tissues with P. gingivalis bacteremia and P. gingivalis could pass through the in vitro BBB model. Caveolae were detected after P. gingivalis infection in BMECs both in vivo and in vitro. Caveolin-1 (Cav-1) expression was enhanced after P. gingivalis infection. Downregulation of Cav-1 rescued P. gingivalis-enhanced BMECs permeability. We further found P. gingivalis-gingipain could be colocalized with Cav-1 and the strong hydrogen bonding between Cav-1 and arg-specific-gingipain (RgpA) were detected. Moreover, P. gingivalis significantly inhibited the major facilitator superfamily domain containing 2a (Mfsd2a) expression. Mfsd2a overexpression reversed P. gingivalis-increased BMECs permeability and Cav-1 expression. These results revealed that Mfsd2a/Cav-1 mediated transcytosis is a key pathway governing BBB BMECs permeability induced by P. gingivalis, which may contribute to P. gingivalis/virulence factors entrance and the subsequent neurological impairments.

Irbesartan eases lipopolysaccharide-induced lung injury In Vitro and In Vivo.

Acute lung injury (ALI) is classified as a devastating pulmonary disorder contributing to significant incidence and fatality rate. Irbesartan (IRB) is an angiotensin II receptor blocker that has been proposed to protect against oleic acid-induced ALI. To this end, the current study is concentrated on ascertaining the role of IRB in ALI and figuring out the probable action mechanism. First, cell counting kit-8 (CCK-8) appraised the viability of human pulmonary microvascular endothelial cells (HPMVECs) exposed to ascending concentrations of IRB. HPMVEC injury model and a mouse model of ALI induced by lipopolysaccharide (LPS) were pretreated by IRB. In vitro, cell viability was estimated by CCK-8 assay, and lactate dehydrogenase (LDH) release was tested by LDH assay kit. Enzyme-linked immunosorbent assay (ELISA) and Western blotting estimated the expression levels of inflammatory factors. Fluorescein isothiocyanate-dextran was used to assess HPMVEC permeability. Western blotting examined the expression of adherent and tight junction proteins. In vivo, hematoxylin and eosin staining evaluated lung tissue damage and lung wet/dry (W/D) weight was measured. ELISA analyzed the levels of inflammatory factors in the serum and bronchoalveolar lavage fluid (BALF), and Western blotting examined the expression of inflammatory factors. The total cell, neutrophil, and macrophage numbers in BALF were determined using a cell counter. Lung capillary permeability was assayed by Evans blue albumin and total protein concentration in BALF was measured using bicinchoninic acid method. Immunofluorescence assay and Western blotting examined the expression of adherent and tight junction proteins in lung tissues. It was observed that IRB dose-dependently enhanced the viability while reduced LDH release, inflammatory response as well as permeability in LPS-challenged HPMVECs in vitro. In addition, LPS-stimulated lung tissue damage, pulmonary edema, inflammatory response as well as lung capillary permeability in vivo were all reversed following IRB treatment. Collectively, IRB treatment might elicit protective behaviors against LPS-triggered ALI.

Nanowired Delivery of Curcumin Attenuates Methamphetamine Neurotoxicity and Elevates Levels of Dopamine and Brain-Derived Neurotrophic Factor.

Curcumin is a well-known antioxidant used as traditional medicine in China and India since ages to treat variety of inflammatory ailments as a food supplement. Curcumin has antitumor properties with neuroprotective effects in Alzheimer's disease. Curcumin elevates brain-derived neurotrophic factor (BDNF) and dopamine (DA) levels in the brain indicating its role in substance abuse. Methamphetamine (METH) is one of the most abused substances in the world that induces profound neurotoxicity by inducing breakdown of the blood-brain barrier (BBB), vasogenic edema and cellular injuries. However, influence of curcumin on METH-induced neurotoxicity is still not well investigated. In this investigation, METH neurotoxicity and neuroprotective effects of curcumin nanodelivery were examined in a rat model. METH (20mg/kg, i.p.) neurotoxicity is evident 4h after its administration exhibiting breakdown of BBB to Evans blue albumin in the cerebral cortex, hippocampus, cerebellum, thalamus and hypothalamus associated with vasogenic brain edema as seen measured using water content in all these regions. Nissl attaining exhibited profound neuronal injuries in the regions of BBB damage. Normal curcumin (50mg/kg, i.v.) 30min after METH administration was able to reduce BBB breakdown and brain edema partially in some of the above brain regions. However, TiO2 nanowired delivery of curcumin (25mg/kg, i.v.) significantly attenuated brain edema, neuronal injuries and the BBB leakage in all the brain areas. BDNF level showed a significant higher level in METH-treated rats as compared to saline-treated METH group. Significantly enhanced DA levels in METH-treated rats were also observed with nanowired delivery of curcumin. Normal curcumin was able to slightly elevate DA and BDNF levels in the selected brain regions. Taken together, our observations are the first to show that nanodelivery of curcumin induces superior neuroprotection in METH neurotoxicity probable by enhancing BDNF and DA levels in the brain, not reported earlier.

Dysfunctional Glymphatic System with Disrupted Aquaporin 4 Expression Pattern on Astrocytes Causes Bacterial Product Accumulation in the CSF during Pneumococcal Meningitis.

ABSTRACTPneumococcal meningitis, inflammation of the meninges due to an infection of the Central Nervous System caused by Streptococcus pneumoniae (the pneumococcus), is the most common form of community-acquired bacterial meningitis globally. Aquaporin 4 (AQP4) water channels on astrocytic end feet regulate the solute transport of the glymphatic system, facilitating the exchange of compounds between the brain parenchyma and the cerebrospinal fluid (CSF), which is important for the clearance of waste away from the brain. Wistar rats, subjected to either pneumococcal meningitis or artificial CSF (sham control), received Evans blue-albumin (EBA) intracisternally. Overall, the meningitis group presented a significant impairment of the glymphatic system by retaining the EBA in the CSF compartments compared to the uninfected sham group. Our results clearly showed that during pneumococcal meningitis, the glymphatic system does not function because of a detachment of the astrocytic end feet from the blood-brain barrier (BBB) vascular endothelium, which leads to misplacement of AQP4 with the consequent loss of the AQP4 water channel's functionality.

Upregulation of FOXO1 contributes to lipopolysaccharide-induced pulmonary endothelial injury by induction of autophagy.

BackgroundAutophagy is activated during the pathogenesis of endothelial dysfunction and sepsis-associated acute lung injury (ALI). This study aimed to investigate whether autophagy affected endothelial barrier dysfunction and lung injury in a murine model of lipopolysaccharide (LPS)-induced ALI, and then further clarify whether forkhead box O1 (FOXO1), an autophagy-related transcriptional factor, contributed to autophagy activation and ALI induced by LPS.MethodsMale C57BL/6 mice were treated with LPS (30 mg/kg), and then were allocated to a control group and an LPS group with or without FOXO1 inhibitor (AS1842856) treatment, respectively. Primary cultured mouse lung vascular endothelial cells (MLVECs) were treated with LPS, autophagy inhibitor 3-methyladenine (3-MA), AS1842856, and small interfering RNA (siRNA) targeting autophagy-related gene 5 (ATG5) or FOXO1. Endothelial autophagic flux was assessed by transfection of MLVECs with red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3) adenovirus. Endothelial permeability was analyzed by the diffusion of fluorescein isothiocyanate-carboxymethyl (FITC)-dextran through the endothelial monolayer. Evans blue albumin tracer was used to measure the pulmonary transvascular permeability, and hematoxylin and eosin (H&E) staining was used to observe pathological changes in the lung tissues. Immunofluorescence staining was also used to detect the expression of zonula occludens-1 (ZO-1) and FOXO1.ResultsThis study found autophagy induction in lung tissues of endotoxemic mice and LPS-treated MLVECs, as evidenced by elevated expression of light chain 3 II (LC3-II) and Unc-51-like kinase (ULK1) and autophagic flux. LPS treatment decreased vascular endothelial (VE)-cadherin and ZO-1 expression and increased endothelial permeability in MLVECs, which were significantly alleviated by autophagy inhibitor 3-MA and ATG5 siRNA. It was found that both phosphorylated FOXO1 and FOXO1 were upregulated in the lung tissues of endotoxemic mice and LPS-treated MLVECs. Both FOXO1 inhibitor AS1842856 and FOXO1 siRNA suppressed LPS-induced autophagy and endothelial cell injury in MLVECs. Moreover, FOXO1 inhibition profoundly alleviated autophagy, lung endothelial hyperpermeability, and ALI in endotoxemic mice.ConclusionsThis work demonstrated that FOXO1 upregulation is an important contributor to LPS-induced autophagy in pulmonary VE cells. The detrimental effects of FOXO1 in endotoxemia-associated endothelial dysfunction and ALI are partly due to its potent pro-autophagic property. Inhibition of FOXO1 may be a potential therapeutic option for the treatment of ALI.

QiShenYiQi Inhibits Tissue Plasminogen Activator-Induced Brain Edema and Hemorrhage after Ischemic Stroke in Mice.

Background: Thrombolysis with tissue plasminogen activator (tPA) remains the only approved drug therapy for acute ischemic stroke. However, delayed tPA treatment is associated with an increased risk of brain hemorrhage. In this study, we assessed whether QiShenYiQi (QSYQ), a compound Chinese medicine, can attenuate tPA-induced brain edema and hemorrhage in an experimental stroke model. Methods: Male mice were subjected to ferric chloride-induced carotid artery thrombosis followed by mechanical detachment of thrombi. Then mice were treated with QSYQ at 2.5 h followed by administration of tPA (10 mg/kg) at 4.5 h. Hemorrhage, infarct size, neurological score, cerebral blood flow, Evans blue extravasation, FITC-labeled albumin leakage, tight and adherens junction proteins expression, basement membrane proteins expression, matrix metalloproteinases (MMPs) expression, leukocyte adhesion, and leukocyte infiltration were assessed 24 h after tPA administration. Results: Compared with tPA alone treatments, the combination therapy of QSYQ and tPA significantly reduced hemorrhage, infarction, brain edema, Evans blue extravasation, albumin leakage, leukocyte adhesion, MMP-9 expression, and leukocyte infiltration at 28.5 h after stroke. The combination also significantly improved the survival rate, cerebral blood flow, tight and adherens junction proteins (occludin, claudin-5, junctional adhesion molecule-1, zonula occludens-1, VE-cadherin, α-catenin, β-catenin) expression, and basement membrane proteins (collagen IV, laminin) expression. Addition of QSYQ protected the downregulated ATP 5D and upregulated p-Src and Caveolin-1 after tPA treatment. Conclusion: Our results show that QSYQ inhibits tPA-induced brain edema and hemorrhage by protecting the blood-brain barrier integrity, which was partly attributable to restoration of energy metabolism, protection of inflammation and Src/Caveolin signaling activation. The present study supports QSYQ as an effective adjunctive therapy to increase the safety of delayed tPA thrombolysis for ischemic stroke.

The β3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury.

BackgroundAcute lung injury (ALI) is a severe respiratory disease with high rates of morbidity and mortality. Many mediators regarding endogenous or exogenous are involved in the pathophysiology of ALI. Here, we have uncovered the involvement of integrins and matrix metalloproteinases, as critical determinants of excessive inflammation and endothelial permeability, in the regulation of ALI.MethodsInflammatory cytokines were measured by quantitative real-time PCR for mRNA levels and ELISA for secretion levels. Endothelial permeability assay was detected by the passage of rhodamine B isothiocyanate-dextran. Mice lung permeability was assayed by Evans blue albumin (EBA). Western blot was used for protein level measurements. The intracellular reactive oxygen species (ROS) were evaluated using a cell-permeable probe, DCFH-DA. Intratracheal injection of lipopolysaccharide (LPS) into mice was conducted to establish the lung injury model.ResultsExogenous MMP-9 significantly aggravated the inflammatory response and permeability in mouse pulmonary microvascular endothelial cells (PMVECs) treated by LPS, whereas knockdown of MMP-9 exhibited the opposite phenotypes. Knockdown of integrin β3 or β5 in LPS-treated PMVECs significantly downregulated MMP-9 expression and decreased inflammatory response and permeability in the presence or absence of exogenous MMP-9. Additionally, the interaction of MMP-9 and integrin β5 was impaired by a ROS scavenger, which further decreased the pro-inflammatory cytokines production and endothelial leakage in PMVECs subjected to co-treatment (LPS with exogenous MMP-9). In vivo studies, exogenous MMP-9 treatment or knockdown β3 integrin significantly decreased survival in ALI mice. Notably, knockdown of β5 integrin alone had no remarkable effect on survival, but which combined with anti-MMP-9 treatment significantly improved the survival by ameliorating excessive lung inflammation and permeability in ALI mice.ConclusionThese findings support the β3/5 integrin-MMP-9 axis as an endogenous signal that could play a pivotal role in regulating inflammatory response and alveolar-capillary permeability in ALI.

Mechanical Ventilation Exacerbates Poly (I:C) Induced Acute Lung Injury: Central Role for Caspase-11 and Gut-Lung Axis.

BackgroundThe mechanisms by which moderate tidal volume ventilation (MTV) exacerbates preexisting lung injury are unclear. We hypothesized that systemic endotoxemia via the gut-lung axis would lead to non-canonical and canonical inflammasome activation and pyroptosis in a two-hit model involving polyinosinic-polycytidylic acid (Poly(I:C)), a synthetic analog of dsRNA and MTV and that this would associate with acute lung injury (ALI).MethodsAnesthetized mice were administered Poly(I:C) intratracheally and then 6 h later, they were mechanically ventilated for 4 h with otherwise non-injurious MTV (10ml/kg). Changes in intestinal and alveolar capillary permeability were measured. Further documentation of ALI was assessed by evans blue albumin permeability, protein and IL-1 family concentration in bronchoalveolar lavage fluid (BALF) or plasma, and histopathology in cohorts of wildtype (WT), whole body genetically ablated caspase-11 (caspase-11-/-), caspase-1/caspase-11 double knockout (caspase-1/11-/-), gasdermin D (GSDMD)-/-, nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3)-/- and advanced glycosylation end product-specific receptor (RAGE) -/- mice.ResultsNon-injurious MTV exacerbated the mild lung injury associated with Poly(I:C) administration. This included the disruption of alveolar-capillary barrier and increased levels of interleukin (IL)-6, high mobility group proteins 1 (HMGB-1), IL-1β in BALF and IL-18 in plasma. Combined (Poly(I:C)-MTV) injury was associated with increase in gastrointestinal permeability and endotoxin in plasma and BALF. Poly(I:C)-MTV injury was sensitive to caspase-11 deletion with no further contribution of caspase-1 except for maturation and release of IL-18 (that itself was sensitive to deletion of NLRP3). Combined injury led to large increases in caspase-1 and caspase-11. Genetic ablation of GSDMD attenuated alveolar-capillary disruption and release of cytokines in combined injury model.ConclusionsThe previously noted exacerbation of mild Poly(I:C)-induced ALI by otherwise non-injurious MTV is associated with an increase in gut permeability resulting in systemic endotoxemia. The gut-lung axis resulted in activation of pulmonary non-canonical (cytosolic mediated caspase-11 activation) and canonical (caspase-1) inflammasome (NLRP3) mediated ALI in this two-hit model resulting in GSDMD sensitive alveolar capillary barrier disruption, pyroptosis (alveolar macrophages) and cytokine maturation and release (IL-1β; IL-18). Pharmacologic strategies aimed at disrupting communication between gut and lung, inhibition of inflammasomes or GSDMD in pyroptosis may be useful in ALI.

Dexamethasone downregulates the expressions of MMP-9 and oxidative stress in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

Steroids have been shown to be beneficial in patients and mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection; however, the mechanism for this beneficial effect is unknown. We speculated that the effect of steroids in eosinophilic meningitis caused by A. cantonensis infection may be mediated by the downregulation of matrix metallopeptidase-9 (MMP-9) and oxidative stress pathways via glucocorticoid receptors (GRs). We found blood-brain barrier (BBB) dysfunction in mice with eosinophilic meningitis 2-3 weeks after infection as evidenced by increased extravasation of Evans blue and cerebrospinal fluid (CSF) albumin levels. The administration of dexamethasone significantly decreased the amount of Evans blue and CSF albumin. The effect of dexamethasone was mediated by GRs and heat shock protein 70, resulting in subsequent decreases in the expressions of nuclear factor kappa B (NF-κB), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) in the CSF and brain parenchymal after 2 weeks of steroid administration. Steroid treatment also decreased CSF/brain homogenate MMP-9 concentrations, but had no effect on CSF MMP-2 levels, indicating that MMP-9 rather than MMP-2 played a major role in BBB dysfunction in mice with eosinophilic meningitis. The concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) gradually increased after 1-3 weeks of infection, and the administration of dexamethasone significantly downregulated the concentration of oxidized derivative 8-OHdG in CSF. In conclusion, increased 8-OHdG and MMP-9 concentrations were found in mice with eosinophilic meningitis caused by A. cantonensis infection. The effect of dexamethasone was mediated by GRs and significantly decreased not only the levels of 8-OHdG and MMP-9 but also NF-κB, JNK and ERK.

Impairment and restoration of the blood-nerve barrier and its correlation with pain following gradual nerve elongation of the rat sciatic nerve

Purpose: This study aimed to evaluate the time course of impairment and restoration of the blood-nerve barrier (BNB) following gradual elongation of the sciatic nerve and to clarify its association with nociception.Materials and Methods: The right femur was lengthened at a rate of 1.5 mm/day for 10 days. Von Frey tests were performed until 50 days after lengthening. Compound muscle action potentials (CMAPs) were measured to assess gross dysfunction of the elongated nerve. Evans blue-albumin tracing and immunohistochemistry for endothelial barrier antigen (EBA), rat endothelial cell antigen-1 (RECA-1), and CD68 for qualitative and quantitative analysis of the BNB and macrophage infiltration were performed for up to 50 days after cessation of lengthening in three segments of the sciatic nerves.Results: Paw-withdrawal threshold was significantly decreased at 7 days from initiation and began to recover from day 25 after lengthening. CMAPs showed delayed latency and attenuated amplitude but recovered at day 30 after cessation. On days 10 and 30 after cessation, spotted leakage of Evans blue-albumin in the endoneurium was observed, and the ratio of EBA/RECA-1-positive microvessels was significantly decreased, which subsequently recovered simultaneously in all segments on day 50 after cessation. Macrophages did not infiltrate the BNB at any time point.Conclusion: The restoration of BNB function following gradual nerve elongation was associated with the resolution of mechanical allodynia. Our findings provide insight into the association between nerve stretch injury and chronic nociception in adult male rats, which are potentially relevant to human orthopedic procedures and chronic neuropathic pain.

Photodynamic Opening of the Blood–Brain Barrier Using Different Photosensitizers in Mice

In a series of previous studies, we demonstrated that the photodynamic therapy (PDT), as a widely used tool for treatment of glioblastoma multiforme (GBM), also site-specifically opens the blood–brain barrier (BBB) in PDT-dose and age-related manner via reversible disorganization of the tight junction machinery. To develop the effective protocol of PDT-opening of the BBB, here we answer the question of what kind of photosensitizer (PS) is the most effective for the BBB opening. We studied the PDT-opening of the BBB in healthy mice using commercial photosensitizers (PSs) such as 5-aminolevulenic acid (5-ALA), aluminum phthalocyanine disulfonate (AlPcS), zinc phthalocyanine (ZnPc) and new synthetized PSs such as galactose functionalized ZnPc (GalZnPc). The spectrofluorimetric assay of Evans Blue albumin complex (EBAC) leakage and 3-D confocal imaging of FITC-dextran 70 kDa (FITCD) extravasation clearly shows a revisable and dose depended PDT-opening of the BBB to EBAC and FITCD associated with a decrease in presence of tight junction (TJ) in the vascular endothelium. The PDT effects on the BBB permeability, TJ expression and the fluorescent signal from the brain tissues are more pronounced in PDT-GalZnPc vs. PDT-5-ALA/AlPcS/ZnPc. These pre-clinical data are the first important informative platform for an optimization of the PDT protocol in the light of new knowledge about PDT-opening of the BBB for drug brain delivery and for the therapy of brain diseases.

Cellular source of hypothalamic macrophage accumulation in diet-induced obesity

BackgroundObese mice on a high-fat diet (HFD) display signs of inflammation in the hypothalamic arcuate nucleus (ARC), a critical area for controlling systemic energy metabolism. This has been suggested as a key mechanism of obesity-associated hypothalamic dysfunction. We reported earlier that bone marrow-derived macrophages accumulate in the ARC to sustain hypothalamic inflammation upon chronic exposure to an HFD. However, the mechanism underlying hypothalamic macrophage accumulation has remained unclear.MethodsWe investigated whether circulating monocytes or myeloid precursors contribute to hypothalamic macrophage expansion during chronic HFD feeding. To trace circulating myeloid cells, we generated mice that express green fluorescent protein (GFP) in their lysozyme M-expressing myeloid cells (LysMGFP mice). We conducted parabiosis and bone marrow transplantation experiments using these animals. Mice received an HFD for 12 or 30 weeks and were then sacrificed to analyze LysMGFP cells in the hypothalamus. Hypothalamic vascular permeability in the HFD-fed obese mice was also tested by examining the extravascular leakage of Evans blue and fluorescence-labeled albumin. The timing of LysMGFP cell entry to the hypothalamus during development was also evaluated.ResultsOur parabiosis and bone marrow transplantation experiments revealed a significant infiltration of circulating LysMGFP cells into the liver, skeletal muscle, choroid plexus, and leptomeninges but not in the hypothalamic ARC during chronic HFD feeding, despite increased hypothalamic vascular permeability. These results suggested that the recruitment of circulating monocytes is not a major mechanism for maintaining and expanding the hypothalamic macrophage population in diet-induced obesity. We demonstrated instead that LysMGFP cells infiltrate the hypothalamus during its development. LysMGFP cells appeared in the hypothalamic area from the late embryonic period. This cellular pool suddenly increased immediately after birth, peaked at the postnatal second week, and adopted an adult pattern of distribution after weaning.ConclusionsBone marrow-derived macrophages mostly populate the hypothalamus in early postnatal life and may maintain their pool without significant recruitment of circulating monocytes throughout life, even under conditions of chronic HFD feeding.

Bradykinin receptor-1 activation induces inflammation and increases the permeability of human brain microvascular endothelial cells.

Neuroinflammatory disorders such as Alzheimer's and Parkinson's diseases are characterised by chronic inflammation and loss of vascular integrity. Bradykinin 1 receptor (B1R) activation has been implicated in many neuroinflammatory diseases, but the contribution of B1R to inflammation and vascular breakdown is yet to be determined. As a result, the present study evaluated the effect of B1R stimulation using Des-Arg-9-BK on the cytokine profile and junctional properties of human cerebral microvascular endothelial cells (hCMVECs). Results showed that stimulation of B1R receptors increased secretion of pro-inflammatory cytokines, interleukin-6 (IL-6), IL-8, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1), but decreased the expression of vascular endothelial growth factor (VEGF), a cytokine and growth factor required for maintenance of the vasculature. B1R stimulation also resulted in the loss of occludin expression at tight junctions with no change in VE-cadherin expression. There was also a significant increase in permeability to Evans blue albumin, suggesting an increase of vascular permeability. Taken together, these results suggest that B1R activation that occurs in neuroinflammatory diseases may contribute to both the inflammation and loss of blood-brain barrier integrity that is characteristic of these diseases.

Propylene glycol alginate sodium sulphate attenuates LPS-induced acute lung injury in a mouse model.

Propylene glycol alginate sodium sulphate, a sulphated polysaccharide, has been used to treat hyperlipidaemia and ischaemia–reperfusion injury of liver. This study aimed to investigate the effect of propylene glycol alginate sodium sulphate on LPS-induced acute lung injury. Propylene glycol alginate sodium sulphate was injected intraperitoneally into male C57BL/6 mice with or without LPS administration. Survival rates were calculated. Serum, bronchoalveolar lavage fluid and lung tissues were collected to determine lung histology, wet/dry ratio, Evans blue albumin permeability, protein levels, the counts of immune cells and the levels of inflammatory cytokines and chemokines. Serum alanine aminotransferase, aspartate transaminase, creatinine and blood urea nitrogen levels were also measured. Additionally, NF-κB signalling was detected in the lung. Propylene glycol alginate sodium sulphate treatment significantly improved the survival of mice suffering from LPS. Lung histological injury, wet/dry ratio, Evans blue albumin permeability, neutrophils and the inflammatory cytokines and chemokines were significantly reduced by propylene glycol alginate sodium sulphate treatment. NF-κB signalling was significantly inhibited by propylene glycol alginate sodium sulphate in the lung of mice subjected to LPS. Furthermore, serum alanine aminotransferase, aspartate transaminase, creatinine and blood urea nitrogen levels were also significantly decreased after propylene glycol alginate sodium sulphate administration. This study suggests that NF-κB signalling and inhibition of pro-inflammatory cytokines, chemokines and neutrophil accumulation may be involved in the process of acute lung injury attenuation by propylene glycol alginate sodium sulphate.

EphA2 antagonism alleviates LPS-induced acute lung injury via Nrf2/HO-1, TLR4/MyD88 and RhoA/ROCK pathways

Eph receptor tyrosine kinases have a wide range of biological functions and have gradually been recognized increasingly as key regulators of inflammation and injury diseases. Although previous studies suggested that EphA2 receptor may be involved in the regulation of inflammation and vascular permeability in injured lung, the detailed effects of EphA2 on LPS-induced acute lung injury (ALI) are still inadequate and the underlying mechanism remains poorly understood. In this study, we detected the effects of EphA2 antagonism on inflammation, pulmonary vascular permeability and oxidative stress in LPS-induced ALI and investigate the potential mechanism. Our results showed that EphA2 antagonism markedly inhibited the cytokines release and inflammatory cells infiltration in BALF, prevented the LPS-induced elevations of MPO activity and MDA level in lung tissues. Our study also found that EphA2 antagonism significantly decreased the wet/dry ratios, reduced the Evans blue albumin extravasation in lung tissues and obviously alleviated the LPS-induced increment of pulmonary vascular permeability. Mechanistically, EphA2 antagonism significantly increased the activation of Nrf2 along with its target antioxidant enzyme HO-1 and inhibited the expressions of TLR4/MyD88 in lung tissues and A549 alveolar epithelial cells. Furthermore, EphA2 antagonism dramatically inhibited the LPS-evoked activations of RhoA/ROCK in lung tissues. In conclusion, our data indicate that EphA2 receptor plays an essential role in LPS-induced ALI and EphA2 antagonism has protective effects against LPS-induced ALI via Nrf2/HO-1, TLR4/MyD88 and RhoA/ROCK pathways. These results suggest that antagonism of EphA2 may be an effective therapeutic strategy for the treatment of ALI.

Intravascular Delivery of TAT‐conjugated Focal Adhesion Kinase Protects against Acute Lung Injury

Acute lung injury (ALI), a major cause of high mortality in all age groups, occurs because of endothelial barrier failure. But it lacks a cure. We showed that endothelial focal adhesion kinase (FAK) is barrier protective (PMID: 23033075), suggesting FAK may be a cure for ALI. To test this hypothesis, we purified wild type FAK (FAKWT) and FAK with YàF mutation at tyrosine 925 (FAKMUT), which is expressed in FAK's focal adhesion targeting domain. Then we non‐covalently conjugated the proteins with the cell‐permeable peptide TAT (HIV‐1 transactivator of transcription). We intranasally instilled LPS in anesthetized mice. After 24h, we intravenously infused TAT‐FAKp or TAT‐FAKpY925F. After 4 h, we gave intravenous Evans Blue‐albumin (EBA). After a further 4 h, we assayed ALI in terms of EBA concentration and leukocyte counts in the bronchoalveolar lavage (BAL). Also, by live confocal microscopy of isolated, blood‐perfused lungs, we assayed endothelial F‐actin in terms of the lung endothelial fluorescence of Rhodamin Phalloidin. LPS increased EBA and leukocytes two‐times above baseline (p<0.05 each). TAT‐FAKWT, but not TAT‐FAKpMUT inhibited both responses (p<0.05). Endothelial F‐actin was markedly higher after TAT‐FAKWT than after TAT‐FAKpMUT infusion (p<0.05). LPS‐induced mortality in 5 days was 80% for untreated or TAT‐FAKpMUT infused mice. However, TAT‐FAKpWT infusion decreased the mortality to 25% (p<0.05). Our findings indicate that purified FAK can be effectively given by intravenous injection. TAT‐FAKWT, given well after the initiation of LPS‐induced ALI, markedly increased survival. The protective effect was due to FAK's Y925 residue that is likely responsible for enhancing cortical F‐actin, thereby strengthening the endothelial barrier (PMID:23033075). We propose that intravenous delivery of TAT‐FAKpWT might be effective therapy for ALI (HL36024).Support or Funding InformationNIH: HL36024This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Non‑canonical Wnt signaling contributes to ventilator‑induced lung injury through upregulation of WISP1 expression.

Mechanical ventilation may cause ventilator-induced lung injury (VILI). Canonical Wnt signaling has been reported to serve an important role in the pathogenesis of VILI. Bioinformatics analysis revealed that canonical and non-canonical Wnt signaling pathways were activated in VILI. However, the role of non-canonical Wnt signaling in the pathogenesis of VILI remains unclear. The present study aimed to analyze the potential role of non-canonical Wnt signaling in VILI pathogenesis. Lung injury was assessed via Evans blue albumin permeability and histological scoring, as well as by inflammatory cytokine expression and total protein concentration in bronchoalveolar lavage fluid. The relative protein expression of canonical and non-canonical Wnt signaling pathway components were examined via western blotting and immunohistochemistry. The results demonstrated that 6 h of mechanical ventilation at low tidal volume (LTV; 6 ml/kg) or moderate tidal volume (MTV; 12 ml/kg) induced lung injury in sensitive A/J mice. Ventilation with MTV increased the protein levels of Wnt-induced secreted protein 1 (WISP1), Rho-associated protein kinase 1 (ROCK1), phosphorylated (p)-Ras homolog gene family, member A and p-C-Jun N-terminal kinase (JNK). Inhibition of ROCK1 by Y27632 and JNK by SP600125 attenuated MTV-induced lung injury and decreased the expression of proteins involved in non-canonical Wnt signaling, including WISP1. In conclusion, non-canonical Wnt signaling participates in VILI by modulating WISP1 expression, which has been previously noted as critical for VILI development. Therefore, the non-canonical Wnt signaling pathway may provide a preventive and therapeutic target in VILI.

5-Hydroxytryptophan: A precursor of serotonin influences regional blood-brain barrier breakdown, cerebral blood flow, brain edema formation, and neuropathology.

5-Hydroxytryptophan (5-HTP), a precursor of serotonin, is therapeutically used for several psychiatric disorders such as anxiety and depression in the clinic. However, severe side effects, including abnormal mental functions, behavioral disturbances and intolerance are associated with this treatment. 5-HTP-induced elevation of plasma and brain serotonin levels may affect blood-brain barrier (BBB) breakdown, edema formation and regional cerebral blood flow (CBF) disturbances. Breakdown of BBB to serum proteins leads to vasogenic brain edema formation and cellular injuries. However, 5-HTP-neurotoxicity is still not well known. In this investigations 5-HTP induced elevation of endogenous plasma and brain serotonin levels and its effect on BBB breakdown, edema formation neuronal injuries was examined in a rat model. Furthermore, potential role of oxidative stress and nitric oxide (NO) was evaluated. In addition, several neurochemical agents such as p-CPA (5-HT synthesis inhibitor) indomethacin (prostaglandin synthase inhibitor), diazepam (ant stress drug), cyproheptadine, ketanserin (5-HT2 receptor antagonists) and vinblastine (inhibitor of microtubule function) were examined on 5-HT neurotoxicity. Our observations suggest that 4h after 5-HTP administrations, the endogenous serotonin levels increased by fourfold (150mg/kg) in the plasma and brain associated with profound hyperthermia (+3.86±0.24°C, oxidative stress and NO upregulation. Breakdown of the BBB to Evans blue albumin (EBA) in 8 brain regions and to [131]Iodine in 14 brain regions was observed. The CBF exhibited marked reduction in all the brain regions examined. Brain edema and cellular injuries are present in the areas associated with BBB disruption. Drug treatments reduced the BBB breakdown, edema formation NO production and brain pathology. These observations are the first to point out that 5-HTP-neurotoxicity caused by BBB breakdown, edema formation and NO production is instrumental in causing adverse mental and behavioral abnormalities, not reported earlier.

Abstract 16654: The Role of Glycolytic ATP Generation in the Restoration of Vascular Endothelial Barrier Function

Introduction: Acute Lung Injury (ALI) is characterized by endothelial barrier dysfunction of the lung vasculature, leading to disassembly of endothelial adherens junctions, increased vascular permeability and fluid accumulation in the lungs. Little is known about the bioenergetics of the barrier disruption and repair processes. The glycolysis regulatory enzyme PFKFB3 has recently been identified as a key mediator of endothelial glucose metabolism and could thus serve as a potential mediator of barrier repair. Methods and Results: To quantitatively measure metabolic changes in response to an inflammatory stimulus, we performed a Seahorse bioenergetic flux assay using human lung microvascular endothelial cells (HLMVECs). Glycolysis, as measured by the extracellular acidification rate, increased acutely by 36% (from 9.1 to 12.4 mpH/min) following treatment with the inflammatory mediator TNFα. This upregulation was abolished in the presence of PFK15, a specific inhibitor of PFKFB3. PFKFB3 activity was also necessary for barrier restoration of HLMVECs following injury, as measured by a transendothelial electrical resistance (TER) assay (p<0.0001). Live cell microscopy of endothelial cells expressing the ATP biosensor Perceval HR revealed that ATP is generated at cell junctions following TNFα treatment. Additionally, controlled translocation of PFKFB3 to the plasma membrane resulted in enhanced barrier restoration after injury, both in terms of the extent of barrier integrity as well as the rate of recovery (p<0.0001). Finally, PFKFB3 was pharmacologically inhibited in vivo following lung injury with the bacterial toxin LPS in mice. This inhibition resulted in significantly impaired recovery of lung vascular barrier function as measured by an Evans blue albumin (EBA) permeability assay (p<0.001). Endothelial cell-specific overexpression of PFKFB3 in mice by liposomal delivery resulted in an enhanced rate of recovery from LPS mediated injury. Lung vascular leakiness decreased from 29.0 to 18.3 ng EBA/g body weight at two days following the LPS injury (p<0.001), thus achieving pre-injury levels of barrier function. Conclusions: Glycolytic ATP generation regulated by PFKFB3 is essential for restoration of the endothelial barrier after inflammatory injury. A better understanding of the endothelial barrier bioenergetics could allow for the development of novel therapeutic approaches which enhance barrier function in severe diseases such as acute lung injury.